眼刺激性試験代替法フルオレセイン漏出試験法

目次
評価会議報告書 ------------------------------------------------------------------------------

1

第三者評価報告書 ---------------------------------------------------------------------------

7

STATEMENT ON THE SCIENTIFIC VALIDITY OF
CYTOTOXICITY/CELL FUNCTION BASED IN VITRO ASSAYS FOR
EYE IRRITATION TESTING ------------------------------------------------------ 17
OECD GUIDELINE FOR THE TESTING OF CHEMICALS No.460,
Fluorescein Leakage Test Method for Identifying Ocular Corrosives
and Severe Irritants---------------------------------------------------------------------- 23

眼刺激性評価のためのフルオレセイン漏出試験法の評価会議報告

JaCVAM 評価会議

平成 24 年（2012 年）10 月 1 日

1JaCVAM 評価会議
吉田武美（日本毒性学会）：座長
浅野哲秀（日本環境変異原学会）
五十嵐良明（国立医薬品食品衛生研究所 生活衛生化学部）
大島健幸（日本化学工業協会）
小笠原弘道（独立行政法人

医薬品医療機器総合機構）

小野寺博志（独立行政法人

医薬品医療機器総合機構）

黒澤 努（日本動物実験代替法学会）
杉山真理子（日本化粧品工業連合会）
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）
長谷川隆一（独立行政法人
牧

製品評価技術基盤機構）

栄二（日本免疫毒性学会）

増田光輝（座長推薦）
横関博雄（日本皮膚アレルギー・接触皮膚炎学会）
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）
吉村 功（座長推薦）
渡部一人（日本製薬工業協会）
任期：平成 24 年 4 月 1 日～平成 26 年 3 月 31 日

以上2

本報告の評価対象は、OECD の Test Guideline 460（TG 460）で説明されている眼刺激性評
価のための Fluorescein leakage test method である 1, 2)。
以下ではこの試験法を当該試験法と記すが、具体名が必要な場合は、「フルオレセイン漏出試験法」
あるいは「FL 試験法」と記すことにする。
当該試験法は、単層の細胞層を通過する sodium fluorescein (Na-FL) 量を測ることで，被験物質による強度の眼刺激性と眼腐食性を代替的に評価するものである 3-5)。今回、眼刺激性試験評価委員
会からの報告 2)を受け、以下の 10 項目について評価したので報告する。
＜審議内容＞
1.

当該試験法は、どのような従来試験法を代替するものか。または、どのような毒性を評価ある
いは予測するものか。
当該試験法は、ウサギを用いたドレイズ眼刺激性試験法で評価されてきた毒性の一部である、強度

眼刺激性と眼腐食性を検出するための代替法である。
TG 460 では、当該試験法で陽性とされた被験物質は、強度眼刺激性あるいは眼腐食性を示すが、当該試験法での陰性結果は、被験物質が強度眼刺激性あるいは眼腐食性物質でないことを示すものではないとしている。
当該試験法は、トップダウン方式（末尾の用語集参照）による強度眼刺激性あるいは眼腐食性を検出する試験法である 6)。

2.

当該試験法と従来試験法の間にどのような科学的なつながりがあるか。
当該試験法は、インサート(別図参照)の内側に単層の細胞層を構築し、細胞層の上部に被験物質を曝露した後、細胞層の上部からフルオレセインを添加する。このフルオレセインの細胞層下部への漏出を測定することによって、被験物質による細胞への傷害や細胞間結合の脆弱化を評価するものである。
これに対してウサギを用いたドレイズ試験法は、被験物質の角膜、結膜、虹彩への傷害を、角膜 80
点、結膜 20 点、虹彩 10 点、最大が 110 点のスコアで評価している。
すなわち、ドレイズ試験法は、細胞間結合や細胞自身への傷害以外の損傷も評価しているという点で、当該試験法より多様な損傷を評価している。

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3.

当該試験法とそのデータは、透明で独立な科学的評価を受けているか。
当該試験法の妥当性を示すデータは、J. Gartlon と R. Clothier によって background review

document (BRD) としてまとめられており、そのデータの妥当性は、ECVAM によって透明で独立した評価を受けている。
4.

当該試験法は、従来試験法の代替法として、どのような物質又は製品を評価することを目的としているか。
当該試験法は、医薬品､医療機器、化粧品、医薬部外品、農薬など、ドレイズ眼刺激性試験法が適用される物質または製品の眼刺激性・眼腐食性を評価することを目的としている。
5.

当該試験法は、ハザード評価あるいはリスク評価のどちらに有用であるか。
当該試験法は、強度眼刺激性または眼腐食性を検出するものであるから、ハザード評価に有用であるが、リスク評価には利用できない。
6.

当該試験法は、目的とする物質又は製品の毒性を評価できるか。その場合、当該試験法の適用条件が明確になっているか。
当該試験法は、強度眼刺激性あるいは眼腐食性をトップダウン方式で検出する際に、最初の段階で実施される試験法である。
当該試験法で陽性の場合、毒性があると判断できるが、陰性の場合には、毒性がないと判断することはできない。陰性の場合に、その毒性がないことを確かめるには、他の試験法でさらに検討をする
ことが必要である。
適用可能な物質は水溶性物質に限られている。強酸、強塩基、組織固定液、強揮発性物質は適用外である。
7.

当該試験法はプロトコルの微細な変更に対して頑健であるか。
以下のことから、当該試験法は、プロトコルの微細な変更に対して頑健と考えられる。
１）培養細胞として広く使用されている MDCK 細胞株を用いている。
２）単層の細胞層が適切に構成されていることがフルオレセイン漏出の有無で容易に確認できる。
３）TG460 では特別な条件設定は見当らず、BRD にも特段の注意がない。

8.

当該試験法の技術習得は、適切な訓練と経験を経ている担当者にとって容易なものであるか。
試験法の実施に特殊な設備が必要か。
単層の細胞層の構築以外は特殊技能が不要であり､特殊設備も不要である。
単層の細胞層の構築についても、TG 460 に詳細な説明があるので、適切な訓練と経験を経ている担当者であれば技術習得が容易である。
9.

当該試験法は、従来試験法と比べて時間的経費的に優れているか。
当該試験法は、ドレイズ眼刺激性試験法に比べて、時間的経費的に優れている。
4

10. 当該試験法は、動物福祉の観点及び科学的見地から、目的とする物質又は製品の毒性を評価する代替法として、行政上利用することは可能か。
当該試験法は、動物を用いずに強度眼刺激性・眼腐食性を評価できるので、動物福祉の観点から有用である。
目的とする物質又は製品の毒性を評価する代替法として、科学的見地から行政上利用することが可能である。
しかし、トップダウン方式でのみ利用可能であること、水溶性物質のみが適用可能であること、からその有用性は限定的である。
参考文献
1) 吉村功，山本直樹: FL 試験法（Fluorescein leakage test method；フルオレセイン漏出試験法）の概要, JaCVAM 評価会議 (4, June, 2012)
2) Proposal for a New Test Guideline 460: Fluorescein Leakage Test Method for Identifying
Ocular Corrosives and Severe Irritants.
3) Gartlon, J., Clothier, R.: Fluorescein Leakage Assay Background Review Document as an
Alternative Method for Eye Irritation Testing. Available under Validation Study
Documents, Section Eye Irritation at [http://ecvam.jrc.it]4) Gartlon, J., Clothier, R.: Fluorescein Leakage Assay Background Review Document as an
Alternative Method for Eye Irritation Testing: Appendices and Annexes. Available under
Validation Study Documents, Section Eye Irritation at [http://ecvam.jrc.it]5) Fluorescein Leakage (FL) Test DB-ALM Protocol no.71. Available under Validation Study
Documents, Section Eye Irritation at [http://ecvam.jrc.it]
6) ECVAM Retrospective Validation Study on Cytotoxicity/Cell-Function Based in vitro
Assays for the prediction of Eye Irritation: ESAC Peer Review. Available under Validation
Study Documents, Section Eye Irritation at [http://ecvam.jrc.it]

用語集
トップダウン方式
一つの被験物質に対して複数の試験法を逐次適用する試験方式の一つ。前段階の試験法で毒性があると判定された場合は､それで試験を終了し、毒性が無いと判断された場合は､次段階の試験法を適用して､無毒性が真であるかどうかを確認するという毒性判定法。

5

眼刺激性試験代替法フルオレセイン漏出試験法（Fluorescein leakage test method；FL 試験法）の第三者評価報告書

2012 年 6 月

眼刺激性試験代替法評価委員会

1眼刺激性試験代替法評価委員会
吉村 功（東京理科大学）
山本 直樹（藤田保健衛生大学）
小坂 忠司（残留農薬研究所）
竹内 小苗（P&G）
細井 一弘（参天製薬株式会社）
加藤 雅一（株式会社 J-TEC）
簾内 桃子（国立医薬品食品衛生研究所）
増田 光輝（国立医薬品食品衛生研究所）

2

まえおき
OECD は公に認めた毒性試験法について，試験ガイドライン（Test Guideline: TG）
を作成し公開している．TG 編纂の主体は Working Group of National Coordinatorsof the Test Guideline Programme (WNT) である．
FL 試験法にはいくつかのプロトコルがある．WNT が焦点を当てて検討したのは，階層的試験方針で眼腐食性・強度眼刺激性を確認するトップダウン方式の
INVITTOX Protocol No. 71 （プロトコル 71）と，階層的試験方式で眼刺激性が無いことを確認するボトムアップ方式の INVITTOX Protocol No. 120（プロトコル120）である．2011 年 4 月 12～14 日にパリの OECD 本部で行われた WNT 第 23 回
会合に提出・検討されたのは，トップダウン方式の プロトコル 71 の TG 案（Draft
OECD Guideline for the Testing of Chemicals : Fluorescein Leakage Test Method
for Identifying Ocular Corrosives and Severe Irritants）であった．この TG 案は，
同年 11 月 8 日の会合で修正され，12 月 5 日締め切りで意見公募が行われ，2012 年
4 月に改訂が検討され，6 月に最終案が提出された．
本報告では，主としてこの TG 案に基づいたフルオレセイン漏出試験法（FL 試験法）の概要を紹介し，評価を行う．
１． 試験法の位置づけ
FL 試験法は，眼腐食性・強度眼刺激性物質，すなわち UN-GHS，EU-CLP，U.S.-EPA
でカテゴリーI の化合物・複合物（以下，化学物質）を検出するためのインビトロ試験法である．強度眼刺激性物質とは，それに触れることで，21 日経っても回復しない眼組織傷害，あるいは，強度の視力損傷，を引き起こすものである．眼腐食性物質とは，眼に回復不能な組織傷害を与えるものである．上記 TG 案では，FL 試験法を，
眼腐食性・強度眼刺激性を持つ水溶性物質をトップダウン方式で検出する際に，最初に用いる試験法としている．
２． 試験法の原理
いろいろな物質が眼の中に入るのを阻止するのは，角膜と結膜の重要な役割である．
これは細胞間結合で制御されている．FL 試験法は，その傷害を測るために，透過性インサートの薄膜上に Madin-Darby Canine Kidney (MDCK) 細胞，すなわちMDCK-CB997 尿細管上皮細胞を単層培養して，コンフルエント状態になっているモデルを用いる．試験では，フルオレセインナトリウム（sodium fluorescein; Na-F）が単層培養した細胞の間をどれだけ通過するかを測って，短時間の被験物質曝露による毒性発現を評価する．この Na-F 漏出量は，化学的に引き起こされた細胞間結合の損傷に比例するから，これで被験物質の眼刺激性が評価できる．
インサート薄膜上の MDCK 細胞の状態は下図の通りである．単層培養でコンフル
エント状態になった MDCK はインサート内の半透過性薄膜上に生成されていて，インサートは 24 穴プレートの各ウエルに置かれている．
3

試験では，被験物質をインサートの薄膜上に形成された単層培養でコンフルエント
状態の細胞に滴下する．滴下 1 分後に被験物質を払拭し，明るい蛍光を発する無毒性染料の Na-F を 30 分間，単層細胞の上に適用する．被験物質による細胞間結合の損
傷は，この時間の間に単層細胞とインサートの薄膜を通過するフルオレセイン量（fluorescein leakage; FL）で同定される．
単層細胞とインサートの薄膜を通過してウエル内の一定量溶液（Basal chamber
media）に届いた Na-F 量は，ウエル内のフルオレセイン濃度を分光蛍光光度計で測定することで得られる．すなわち，FL は，ブランク対照と最大通過対照（maximum
leakage control）におけるフルオレセイン強度値（fluorescein intensity; FI）を参照
して計算できる．被験物質の用量ごとに，対照との比較で，漏出率（%），すなわち細胞間結合の損傷を測定する．無処理の単層培養でコンフルエント状態のインサートと無細胞のインサートにおける値と対比して 20%の FL が得られる濃度 FL20
(mg/mL)を計算する．この FL20 値を眼腐食・強度眼刺激の確認に用いる．
３． 試験手順と結果の判定
3.1 細胞単層の作成
MDCK-CB997 細胞の単層培養は，DMEM/Nutrient Mix F12 を入れた細胞培
養フラスコ内で培養されたサブコンフルエント状態の細胞を使って作成する．細胞間結合を完全にするために，FL 試験法での溶媒/溶液では，カルシウム濃度を常に1.8mM（200mg/L）から 1.0mM（111mg/L）の間に収めておくことが重要である．
均一かつ再現可能な細胞間結合を生成するには，継代数をある範囲にしておくべきである．試験結果の再現性を確保するために，できれば，その継代数の範囲は解凍後
4

3～30 継代にするべきである．この範囲であれば細胞が類似した機能性を保つからである．
FL 試験法の実施に先立って，トリプシンを適用してフラスコから細胞を外し，解凍し，一定量を 24 穴プレート内のインサートに播種する．細胞を播種するインサー
トとしては，厚み 80 ～150 m，孔サイズ（pore size）0.45 m の混合セルロースエ
ステル薄膜を持つ直径 12 mm のものを用いるべきである．バリデーションでは，
Millicell-HA 12mm インサートが用いられた．インサートと膜タイプの性質は，細胞成長と化学結合に影響するので，他のインサートを用いるときは，§7.2 に示す熟達度確認用化合物（proficiency chemicals）を使って同等性を確かめるべきである．
ある種の化学物質は Millicell-HA インサートの薄膜と結合して試験結果を説明しがたいものにする．たとえば塩化ベンザルコニウムなどのカチオンは，荷電薄膜と化学結合する傾向がある．インサート薄膜との化学結合は，化学的な曝露時間を増やして化学物質の毒性を過大に見せかける．しかし同時に，インサートの薄膜へのカチオ
ン的化学結合が FL を物理的に減らし，化学物質の毒性を過小に見せかけることもある．いずれも結果の説明を困難にする．
化学結合の発現は，無細胞のインサートの薄膜を最大濃度の被験物質に曝露させた後で，標準時間，標準濃度の Na-F 染色を行うことで監視できる．Na-F 染色が起こったら，被験物質を払拭した後でインサート薄膜が黄色になるからである．細胞に被験物質を適用した結果が適切に説明できるためには，被験物質の化学結合の性質を知っておくことが必須である．
インサート上に播種した細胞は，化学物質曝露の際に，単層のコンフルエント状態を作っていなければならない．一つのインサートに 1.6×105 個の細胞があるように，細胞には，濃度 4×105 cells/mL の懸濁液 400μL を加えなければならない．この条件であれば，播種後 96 時間でコンフルエント状態の細胞単層が構成できる．
MDCK 細胞培養は，CO2 濃度が 5±1%，温度が 37±1℃で，湿度が保たれている
培養器で行わなければならない．細胞がバクテリア，ウイルス，マイコプラズマ，真菌類に汚染されないように注意すべきである．
3.2 被験物質の適用と対照物質
被験物質の新鮮な保存溶液は実験ごとに用意し，30 分以内に使用しなければならない．被験物質は，血清蛋白結合を避けるために，1.0～1.8mM 濃度でカルシウムを含みフェノールレッドを含まない Hanks’ Buffered Saline Solution (HBSS) 内に用意しなければならない．また，被験物質が HBSS に 250mg/mL まで溶解することを実験前に確かめておかなければならない．化学物質が，この濃度で，均一で 2 層には分かれていない安定した懸濁液・乳液状態を 30 分以上維持するのであれば，HBSS
を溶剤として使うことができる．
化学物質がこの濃度で HBSS に可溶でないならば，FL 試験法とは異なる試験法を使うべきである．HBSS に不溶であるためにミネラルオイル（light mineral oil）を5

溶剤とすると，FL 試験法が適切な結果をもたらすかどうか分からないからである．
被験物は，カルシウム（1.0～1.8mM）を含みフェノールレッドを含まない HBSS
で 1, 25, 100, 250 mg/mL の 5 用量に調製された溶液と，原液又は飽和溶液である．
固体物質の場合は，750 mg/mL という高濃度も含める．この濃度のときは，ポジ
チブ・ディスプレースメント・ピペットで細胞に適用してもよい．25 mg/mL と100mg/mL で毒性が発現したときは，1, 25, 50, 75, 100 mg/mL で 2 回再実験をしなければならない．1 mg/mL で毒性が発現したときは，0.10, 0.1, 0.25, 1 , 10 mg/mL
で 2 回再実験をしなければならない．
被験物は，培養液を除去した後で，除菌され，37℃に温められている，カルシウムを含みフェノールレッドを含まない HBSS で 2 回洗浄したコンフルエント状態の細胞単層に適用する．実験では，用意された各濃度が，少なくとも 3 回反復して実験されなければならない．
室温で 1 分間曝露した後，注意して被験物を吸引除去し，滅菌して 37℃に温められた，カルシウムを含みフェノールレッドを含まない HBSS を用いてコンフルエン
ト状態の細胞単層を 2 回洗浄し，すぐ FL を測定する．
インサート上の単層培養された細胞の整合性と，実験での細胞の感度が過去の実績
範囲内にあることを確かめるために，各実験実行（run）には陰性対照（NC）と陽性対照（PC）を含めなければならない．
陽性対照としては，Brij 35 （CAS No. 9002-92-0）の 100 mg/mL の使用が勧められている．この濃度は，ほぼ 30%（20%～40%でよい）の FL をもたらすはずである．
陰性対照としては，カルシウムを含みフェノールレッドを含まない HBSS が勧められている．FL20 を計算するには，最大漏出対照も各実験実行に含めることが必要である．最大漏出は無細胞インサート対照を用いて求める．
3.3 フルオレセイン通過性の検量
被験物質と対照物質を除去したら，直ちに十分な量の 0.1%（w/v）Na-F 液をMillicell-HA インサートに添加し，細胞を室温に 30 分間置いておく．フルオレセイ
ンを添加した培養の最後に，注意して各ウエルからインサートを取り除く．各フィル
タを視認で検査し操作中に生じた損傷があったら記録する．
細胞単層とインサートを通過したフルオレセイン量はインサートを取り除いた後のウエルに残っている溶液で定量する．測定は，波長 485 nm と 530 nm の励起・放出波長の各々で，分光蛍光光度計を用いて行う．分光蛍光光度計の感度は最大 FL
値（無細胞インサート）と最小 FL 値（陰性対照）の差の最大値が含まれるようにしておかなければならない．使用する分光蛍光光度計の違いが影響しないように，最大漏出対照に対して，FI が 4000 を超えるように感度を定めておく．ただし，最大 FL
値は 9999 を超えないようにする．

6

3.4

結果の説明と予測モデル

被験物質の各濃度での相対的な FL 値，すなわち%FL 値は，各実験実行における陰性対照での FI 値と，最大漏出対照での FI 値を参照して，被験物質の FI 値から次のようにして計算する．
すなわち，反復実行に対する，最大漏出 FI の平均値を x とし，陰性対照での FIの平均値を y とすると，100%漏出の平均値 z は，z = x – y となる．各用量で，単層を通過する FL の相対値，%FL は，%FL = [(m  y) / z] ×100% で計算する．ここでm は，各濃度での FI の 3 反復測定の平均値である．
20%漏出に対応する用量 FL20 は，用量反応曲線の直線補間で求める．すなわち，20%より小さい%FL 値 B とそれをもたらしている用量 MB，及び 20%より大きい%FL 値 C とそれをもたらしている用量 MC を調べ，次式で計算する．
FL20 = [(20 B) / (C B)] × (MC  MB) + MB
眼腐食性・強度眼刺激性の予測においては，FL20 ≦ 100 mg/mL のとき，被験物
質をカテゴリーI と判定する．すなわち，臨界値（cut-off value）は 100 である．
3.5 結果の承認
最大漏出値（x）は 4000 以上，0%漏出値（y）は 300 以下，100%漏出値（z）
は 3700 と 6000 の間でなければならない．陽性対照の%FL が 20%から 40%の間であれば試験結果は承認できる．
４． 試験法の正確性
文献１によれば，水溶性で眼腐食性・強度眼刺激性の化学物質の偽陽性率は 7%（GHS と CLP で 7/103）から 9%（EPA で 9/99），偽陰性率は 54%（EPA で 15/28）
から 56%（GHS と CLP で 27/48）である．括弧内の比率表示における分母は，プロ
トコル 71 に基づくバリデーション研究に用いられた被験物質数，分子は判定が誤りであった被験物質数である．これより，バリデーション研究で得られた予測の正確度（concordance rate）は，77.5%（GHS と CLP で 117/151）から 82.7%（EPA で 105/127）となる．
眼腐食性・強度眼刺激性を確かめるという目的に限定すれば，注目すべき値は，正確度や偽陰性率ではなく偽陽性率である．正確度が大きくないことや，偽陰性率が大きいことは重大な弱点でない．階層的試験方式と重み付け評価方式を用いる場合，後に続くインビトロ代替法で，FL 試験法で見逃された偽陰性物質が確認できるからである．
注：上記の数値は，文献 2, 3 に基づいて得られたものの筈である．ところがこれらの文献にあるデータ（文献 2 の Table 6.2.4.2.3）は，次のものである．EPA での7

偽陽性率に「9% (9/99)」と「8.3% (9/109)」という食い違いがある．本評価委員会はまだその理由を確かめることができないでいる．文献 2 には他にも集計ミスがあるので，文献１の数値が正しいと感じられる．
分類システム

正確度

偽陽性率

偽陰性率

77.9% (113/145)

7.1% (7/99)

54.3% (25/46)

77.5% (117/151)

6.8% (7/103)

56.3% (27/48)

82.5% (113/137)

8.3% (9/109)

53.6% (15/28)

EU
GHS
EPA

上の表の数値は，次に示す 3 分類表（文献 2 の Tables 6.2.4.2.1 a, b, c）を後付け
的に 2 分類に縮約したものである．後付けの宿命として，過大評価になっている可能性を認識しておくべきであろう．
EU 分類

判定

GHS 分類判定

NC R36 R41

判定

NC Cat2 Cat1

EPA 分類
III/IV

II

I

NC

41

19

21

NC

36

24

14

III/IV

36

16

10

R36

12

20

13

Cat2

4

32

13

II

23

15

5

R41

4

3

21

Cat1

3

4

21

I

3

6

13

計

57

42

46

計

43

60

48

計

62

37

28

どのような化学物質で偽陽性が出やすいかということについては，データが見あた
らない．
５． 試験結果の再現性
文献 2, 3 では，施設内再現性，技術移転性，施設間再現性を，過去の文献で得られるデータで多種多様な観点から検討をしている．しかし検討しているデータは，プ
ロトコルの違いがあったり，検討対象の被験物質が界面活性剤に偏っているというような特性があったり，技術移転性と施設間再現性が交絡していたりしていて，総体としての結論が明らかでない．ここでは，プロトコル 71 に適用できると思われる結果を参考として示すだけにする．
5.1 施設内再現性施設内で，単純反復の生データ（raw data）が入手できて，実験作業者や反復について施設内再現性が検討できる場合について調べた結果では，FL20 (mg/mL) の変動係数（coefficient variation）の中央値及び平均値は，56.5%～63.2%であった．
8

5.2 技術移転性
プルトコル 71 に関して技術移転性を検討できるデータは，文献 4 の 60 化合物
4 施設の実験データである．そこでは FL20 についての実験間ピアソン相関係数（計
量値の相関係数）が報告されている．報告されている値は 0.167～0.778 であるが，相関係数の値が低いのは特定のある施設と他の施設の間だけである．問題の施設では，プロトコルが忠実に守られていなかったようである．再現性のある結果を得るには，プロトコルの曖昧さを減らすと同時に，プロトコルを忠実に守るトレーニングが必須である．細胞を処理する技術はそれほど困難なものではないからである．
5.3

施設間再現性前記の文献 4 では FL20 についての施設間の結果の乖離をピアソン相関係数で評価している．得られている値は 0.214～0.841 である．これについても前項で述べたことと同じで，特定のある施設が低い相関係数の源になっている．これをもって施設間再現性が悪いというのか，プロトコルが悪いというのか，それとも技術移転性が悪いというのか，結論は明らかでない．
６． 試験法の適用範囲
この試験法の適用対象は水溶性化合物のみである．文献 1 では，水溶性で，希釈によって毒性が変わらない強度眼刺激性物質は，この試験法で正確に確かめることができるとしている．
強酸・強塩基・定着薬・強揮発性物質はこの試験法の適用範囲外である．これらの化学物質は FL 試験法で評価できない仕組み，例えば，広範な凝固，鹸化・加水分解，ある種の特殊な化学反応等，を伴うからである．
着色性や粘着性のある被験物質も予測性を侵すので適用が妥当でない．これらのタイプの物質は，短時間曝露後に細胞単層から取り除くのが困難である．
液体中に懸濁している固体は，急速に沈殿する傾向があるので適用濃度を正確に定めることが困難である．化学的あるいは物理的にこれらの性質を持つ物質を評価から除外すると， EU，EPA，GHS のどのクラス分けシステムで評価しても，FL 試験
法の性能は非常によい．
７．

その他の注意7.1 適切な技術移転が達成できたことを確かめるには，表１の熟達度確認用化合
物を使う．

9

表１

熟達度確認用化合物

化合物

CAS 番号

化合物分類

物理性状

ﾄﾞﾚｲｽﾞ試験の結果

FL 試験の結果#1

8001-54-5

オニウム 化合物

液体

カテゴリー1

腐食性/強刺激性#258-33-3アミン/アミディン…

固体

カテゴリー1

腐食性/強刺激性#31310-73-2

アルカリ

液体

カテゴリー1

腐食性/強刺激性#4151-21-3カルボキシル酸（塩）液体カテゴリー1

腐食性/強刺激性#5619-66-9カルボキシル酸, …

固体

カテゴリー2(A)

非腐食性/強刺激性#66484-52-2

無機塩

固体カテゴリー2(A)

非腐食性/強刺激性#7609-14-3ケトン，エステル

液体

カテゴリー2(B)

非腐食性/強刺激性#856-81-5

アルコール

液体

カテゴリー無非腐食性/強刺激性#1: Benzalkonium chloride (5%)，#2: Promethazine hydrochloride，#3: Sodium hydroxide (10%)，#4: Sodium lauryl sulfate (15%)，#5: 4-carboxy-benzaldehyde，#6: Ammonium nitrate，#7: Ethyl-2-methylaceto-acetate，#8: Glycerol

CAS 番号：Chemical Abstract Service Registry Number
7.2 実験での注意点
MDCK 細胞の培養には特別な技術的制約がある．Na-F が単層培養でコンフルエ
ント状態になった細胞間の通過を阻止する細胞間結合は，培養継代数が増えると弱まる傾向がある．不完全に形成された細胞間接合は無処置対照で FL を増加させる．細胞は時間経過と共に変異するので，実験では，許容できる培養継代数を定めて置くことが必要である．
８． FL 試験法の概要の本委員会としての結論
日本の施設で確認した実験データがないので外国文献上のデータを信用して評価すると，FL 試験法の偽陽性率は，水溶性で眼腐食性・強度眼刺激性の化合物に限定したとき，7%（GHS と CLP で 7/103）から 9%（EPA）である．FL 試験法はこの性能で十分と思われる目的に対して，トップダウン方式の最初の段階で用いることが許される試験法である．
トップダウン方式で用いるのであれば，偽陽性率は小さい方が望ましい．陽性であるという判定の臨界値を小さくすることで，偽陽性率を小さくすることは今後の検討課題である．
本委員会は，FL 試験法の日本語訳として，フルオレセイン漏出試験法（FL 試験法と略記）を提案する．

10

９． 文献1. OECD WNT, 2011. Revised draft OECD Guideline for the Testing of Chemicals:
Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants.
2.

Gartlon, J., Clothier, R., 2008. Fluorescein Leakage Assay Background Review
Document as an Alternative Method for Eye Irritation Testing.

3.

Gartlon, J., Clothier, R., Fluorescein Leakage Assay Background Review Document as an
Alternative Method for Eye Irritation Testing: Appendices and Annexes.
4. Balls, M., Botham, P.A., Bruner, L.H., Spielmann, H., 1995. The EC/HO international
validation study on alternatives to the Draize eye irritation test. Toxicology In Vitro, 9(6):871-929.

１０． 用語
UN-GHS ： Globally Harmonized System of Classification and Labeling ofChemicals by the United Nation （国連による化学物質の分類とラベル付けの世界統一システム）の省略形である．これは，有害作用情報を伝えることで，雇用者，労働者，輸送者，消費者，事故被害者等の人々と環境を護ろうとして，図表示・用語警
告・危険声明・警戒宣言・安全情報シートなどの伝達手段を用いて，物理的・健康的・
環境的危険の，標準化されたタイプとレベルに応じて，化学物質をクラス分けするシ
ステムである．このシステムのカテゴリー I（GHS Category I）は，これに属する被験物質を眼の表面に適用したとき，21 日経っても完全には回復しない眼組織損傷，あるいは視力障害をもたらすことで特徴付けられる．
EU-CLP： European Commission Regulation on the Classification, Labellingand Packaging of Substance and Mixtures の省略形である．化学物質分類のUN-GHS システムを欧州に導入したものである．
EPA Category I：米国環境保護庁（Environmental Protection Agency）が定めているクラス分けのカテゴリーI のことで，その内容は，21 日以上にわたって，腐食（眼組織を回復できないように破壊すること），角膜関連損傷，あるいは刺激をもたらす化学物質である．

11

EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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STATEMENT ON THE SCIENTIFIC VALIDITY OF CYTOTOXICITY/CELLFUNCTION BASED IN VITRO ASSAYS FOR EYE IRRITATION TESTING
At its 31st meeting, held on 7 and 8 July, 2009 at the European Centre for the Validation of
Alternative Methods (ECVAM), Ispra, Italy, the non-Commission members of the ECVAM
Scientific Advisory Committee (ESAC)1 unanimously endorsed the following statement:
The replacement of traditional animal-based test methods by alternative ones should ideallybe obtained by one-to-one replacements: to keep the testing regime simple and economical
one single alternative method should, wherever feasible, be sufficient to generate data ofequal or better quality than the traditional test.
However, in the case of eye irritation it is currently generally accepted that, in the foreseeable
future, no single in vitro eye irritation test will be able to replace the in vivo Draize eye test to
predict across the full range of irritation for different chemical classes. However, strategic
combinations of several alternative test methods within a (tiered) testing strategy may be able
to replace the Draize eye test.
A possible conceptual framework for such a (tiered) testing strategy has been developed
within an ECVAM workshop (Ref. 1). The framework is based on alternative eye irritation
methods that vary in their capacity to detect either severe irritant substances (EU R41; GHS'Category 1') or substances considered non-irritant (EU 'Non-Classified'; GHS 'No Category').
According to this framework the entire range of irritancy may be resolved by arranging tests
in a tiered strategy that may be operated from either end: to detect first severe irritants andresolve absence of irritancy ("Top-Down Approach") or to proceed inversely, starting with theidentification of non-irritants first ("Bottom-Up Approach"). Mild irritancy will be resolved ina last tier in both approaches.
To evaluate the scientific validity of possible building blocks of such a test strategy and toassess their possible placement within a Bottom-Up and Top-Down Approach, ECVAM has
undertaken a retrospective validation study of four cell-based in vitro methods.
The test methods evaluated were:
a.
b.
c.
d.

Cytosensor Microphysiometer (INVITTOX Protocols 97 and 102 modified)2
Fluorescein Leakage (INVITTOX Protocols 71, 82, 86 and120);
Neutral Red Release (INVITTOX Protocol 54 and PREDISAFETM);
Red Blood Cell haemolysis (INVITTOX Protocols 37 and 99),

The four test methods, including ten protocol variations, were subjected to independent,
expert review with respect to their use to either
1

Details can be found in the PRP report
Invittox protocols can be downloaded from ECVAM's database service on Alternative Methods to Animal
Experimentation, DBALM: http://ecvam-dbalm.jrc.ec.europa.eu
2Page 1 of 6
ESAC31_CBA_eye-irritation_20091005.doc

EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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a) initiate a Bottom-Up Approach, for consideration for regulatory use to identify nonirritants (EU: 'Non Classified'; GSH: 'No Category'; EPA: 'Category IV') from all otherclasses as part of a tiered testing strategy, orb) to initiate a Top-Down Approach, to identify ocular corrosives and severe irritants(EU R41, GHS 'Category 1', and EPA 'Category I') from all other classes as part of atiered testing strategy.
In the absence of internationally agreed performance criteria for either approach, the PRP of
the ESAC applied the following criteria:• any test used to initiate a Top-Down Approach must balance specificity and sensitivity
to correctly identify a substantial proportion of severe irritants, with a false positive
rate that would not lead to the over-classification of an unreasonable number ofmaterials of lower ocular irritancy potential – an over-classification rate (false
positives) of <10% was considered acceptable• any test used to initiate a Bottom-Up Approach should ideally give no false negatives
with respect to human safety, and no false negative should be produced by highmoderate or severe irritants.
Following independent ESAC peer review of this retrospective validation study andconsidering the potential test strategies in which the tests may be used, the ESAC concluded
the following:
1. CYTOSENSOR MICROPHYSIOMETER TEST METHODThe Cytosensor Microphysiometer test method can be used for two of the three EU and GHS
classification categories used for the endpoint of ocular irritation:
A. The Cytosensor Microphysiometer test method (INVITTOX Protocol 102 modified) is
considered to have been scientifically validated and to be ready for consideration forregulatory use as an initial step within a Top-Down Approach to identify ocular corrosives
and severe irritants (EU R41, GHS Category 1, and EPA Category I) from all other classes forthe chemical applicability domain of water-soluble chemicals (substances and mixtures).
B. Furthermore, the Cytosensor Microphysiometer test method (INVITTOX Protocol 102
modified) is considered to have been scientifically validated and to be ready for consideration
for regulatory use as an initial step within a Bottom-Up Approach to identify non-irritants(EU:NC; GHS: NC; EPA: cat IV) from all other classes only for water-soluble surfactants andwater-soluble surfactant-containing mixtures.
C. On the basis of a thorough evaluation of the data compiled in the course of the ECVAM
validation study, the ESAC concludes that the Cytosensor Microphysiometer test method
does NOT correctly identify moderate and mild ocular irritants (EU: R36; GHS: Cat 2A/B;
EPA: Cat II/III). Therefore, the test method can only be employed to make decisions on twoof the three categories of the eye irritation classification scheme (see A and B). Consequently,
ESAC does NOT recommend this test method as a full replacement method. It should be
noted in this context that the Top-Down and Bottom-Up Approach foresees the theoreticalpossibility of a default mild/moderate categorization (e.g. EU R36 or GHS Cat 2) of all those
substances neither identified as ocular corrosives and severe irritants (see A) nor as "nonPage 2 of 6
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In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

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classified" substances (see B) in the first two tiers of the strategy. However, the test method's
high false negative rate (9-55%) when initiating a top-down approach and high false positive
rate (50-69%) when initiating a bottom-up approach exclude the possibility to use the methodfor default categorization. The test methods can thus not be considered a full-replacement
method on its own using the Top-Down and Bottom-Up approach.
Although these recommendations are based on the evaluation of data sets obtained using
specific hard- and software, it is anticipated that other Cytosensor Microphysiometerequipment and software may become available with either equivalent or better performanceand will need to be efficiently validated. Depending on the similarity of new equipment withrespect to the validated one, this may be performed as a Similar Method Validation ('me-too')or an Update Validation. ESAC therefore recommends the development of PerformanceStandards for the Cytosensor Microphysiometer test method.
The current chemical applicability domain is limited: whilst in some cases this might beincreased by expanding the data set of studied compounds, the test method is not amenable totesting non-water soluble solids, suspensions, or viscous materials.

2. FLUORESCEIN LEAKAGE TEST METHODThe Fluorescein Leakage test method (INVITTOX Protocol 71) is considered to have beenscientifically validated and to be ready for consideration for regulatory use as an initial step
within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41,
GSH Category 1, and EPA Category I) from all other classes for water-soluble chemicals(substances and mixtures).
Additional testing and further refinement, in particular with respect to variability anddefinition of the applicability domain, by expanding the dataset of tested chemicals and directcomparison with in vivo data is recommended and should be kept under review.
With regard to the• Neutral Red Release (INVITTOX Protocol 54 and PREDISAFETM);• Fluorescein Leakage (INVITTOX Protocols 82, 86 and120);• Red Blood Cell haemolysis (INVITTOX Protocols 37 and 99),
ESAC considers that the available evidence is insufficient3 to support a recommendation thatthey are ready for consideration for regulatory use.
Similarly, the available evidence for Fluorescein Leakage INVITTOX Protocol 71 does notsupport a recommendation for its use to initiate a Bottom-Up Approach for regulatory use.

3

Details can be found in the PRP report
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European Centre for the Validation of Alternative Methods (ECVAM)

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This statement takes account of the dossiers prepared for peer review; the views of
independent experts of the ESAC Peer Review Panel (PRP) who evaluated the dossiers
against defined validation criteria as well as supplementary submissions made by the
Validation Management Group.

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In agreement with common practice upon completion of a validation study, ESAC
recommends the development of Performance Standards for the Cytosensor
Microphysiometer and the Fluorescein Leakage assays to allow the validation of similar test
methods or modifications of the validated test methods based on pre-defined evaluation andacceptance criteria.

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Joachim Kreysa

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Head of Unit

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In vitro methods Unit

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European Centre for the Validation of Alternative Methods

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Ispra, 10th July 2009

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In vitro methods Unit
European Centre for the Validation of Alternative Methods (ECVAM)

153

REFERENCECS

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1.

Scott, L. et al. (2009) A proposed eye irritation testing strategy to reduce and replacein vivo studies using Bottom-Up and Top-Down approaches. Toxicol In Vitro. May
31. [Epub ahead of print]

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European Centre for the Validation of Alternative Methods (ECVAM)

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The ESAC was established by the European Commission, and is composed of nominees fromthe EU Member States, industry, academia and animal welfare organisations, together withrepresentatives of the relevant Commission services.

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This statement was endorsed by the following members of the ESAC:

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Ms Argelia Castaño(Spain)
Ms Maija Dambrova (Latvia)
Ms Alison Gray (ESTIV)
Ms Katalin Horvath (Hungary)
Ms Dagmar Jírová (Czech Republic)
Mr Roman Kolar (Eurogroup for Animals)
Ms Elisabeth Knudsen (Denmark - acting as moderator at the meeting)
Mr Manfred Liebsch (Germany)
Mr Gianni Dal Negro (EFPIA)
Mr. Walter Pfaller (Austria)
Mr Tõnu Püssa (Estonia)
Mr Dariusz Sladowski (Poland)
Mr Jon Richmond (UK)Ms Vera Rogiers (ECOPA)
Mr Michael Ryan (Ireland)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Carl Westmoreland (COLIPA)
Mr Timo Ylikomi (Finland)

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The following Commission Services and Observer Organisations were involved in the
consultation process, but not in the endorsement process itself:

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Commission services
Mr Joachim Kreysa (DG JRC, Head of In vitro methods Unit/ECVAM, chairman)
Mr Claudius Griesinger (DG JRC, ESAC secretariat)
Ms Susanne Hoke (DG ENTR)
Ms Susanna Louhimies (DG ENV)
Mr Juan Riego Sintes (DG JRC)
The following observers were present
Mr Hajime Kojima (JaCVAM)
Mr William Stokes (NICEATM)
Ms Marilyn Wind (ICCVAM)

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Adopted:
2 October 2012

OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Fluorescein Leakage Test Method for Identifying Ocular Corrosives and Severe Irritants
INTRODUCTION
1.
The Fluorescein Leakage (FL) test method is an in vitro test method that can be used
under certain circumstances and with specific limitations to classify chemicals (substances andmixtures) as ocular corrosives and severe irritants, as defined by the United Nations (UN)Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (Category 1),
the European Union (EU) Regulation on Classification, Labelling and Packaging of Substancesand Mixtures (CLP) (Category 1), and the U.S. Environmental Protection Agency (EPA)(Category I) (1) (2) (3). For the purpose of this Test Guideline, severe irritants are defined as
chemicals that cause tissue damage in the eye following test substance administration that is notreversible within 21 days or causes serious physical decay of vision, while ocular corrosives are
chemicals that cause irreversible tissue damage to the eye. These chemicals are classified as UN
GHS Category 1, EU CLP Category 1, or U.S. EPA Category I.
2.
While the FL test method is not considered valid as a complete replacement for the invivo rabbit eye test, the FL is recommended for use as part of a tiered testing strategy for
regulatory classification and labelling. Thus, the FL is recommended as an initial step within a
Top-Down approach to identify ocular corrosives/severe irritants, specifically for limited types ofchemicals (i.e. water soluble substances and mixtures) (4)(5).
3.
It is currently generally accepted that, in the foreseeable future, no single in vitro eye
irritation test will be able to replace the in vivo eye test (TG 405 (6)) to predict across the fullrange of irritation for different chemical classes. However, strategic combinations of several
alternative test methods within a (tiered) testing strategy may be able to replace the in vivo eye
test (5). The Top-Down approach (5) is designed to be used when, based on existing information,a chemical is expected to have high irritancy potential.
4.
Based on the prediction model detailed in paragraph 35, the FL test method can identify
substances within a limited applicability domain as ocular corrosives/severe irritants (UN GHS
Category 1; EU CLP Category 1; U.S. EPA Category I) without any further testing. The same isassumed for mixtures although mixtures were not used in the validation. Therefore, the FL test
method may be used to determine the eye irritancy/corrosivity of chemicals, following the© OECD, (2012)
You are free to use this material for personal, non-commercial purposes without seeking priorconsent from the OECD, provided the source is duly mentioned. Any commercial use of thismaterial is subject to written permission from the OECD.

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sequential testing strategy of TG 405 (6). However, a chemical that is not predicted as ocularcorrosive or severe irritant with the FL test method would need to be tested in one or more
additional test methods (in vitro and/or in vivo) that are capable of accurately identifying i)
chemicals that are in vitro false negative ocular corrosives/severe irritants in the FL (UN GHS
Category 1; EU CLP Category 1; U.S. EPA Category I); ii) chemicals that are not classified foreye corrosion/irritation (UN GHS No Category; EU CLP No Category; U.S. EPA Category IV);
and/or iii) chemicals that are moderate/mild eye irritants (UN GHS Categories 2A and 2B; EU
CLP Category 2; U.S. EPA Categories II and III).
5.
The purpose of this Test Guideline is to describe the procedures used to evaluate thepotential ocular corrosivity or severe irritancy of a test substance as measured by its ability to
induce damage to an impermeable confluent epithelial monolayer. The integrity of trans-epithelial
permeability is a major function of an epithelium such as that found in the conjunctiva and thecornea. Trans-epithelial permeability is controlled by various tight junctions. Increasing thepermeability of the corneal epithelium in vivo has been shown to correlate with the level ofinflammation and surface damage observed as eye irritation develops.
6.
In the FL test method, toxic effects after a short exposure time to the test substance aremeasured by an increase in permeability of sodium fluorescein through the epithelial monolayerof Madin-Darby Canine Kidney (MDCK) cells cultured on permeable inserts. The amount offluorescein leakage that occurs is proportional to the chemical-induced damage to the tightjunctions, desmosomal junctions and cell membranes, and can be used to estimate the ocular
toxicity potential of a test substance. Annex I provides a diagram of MDCK cells grown on an
insert membrane for the FL test method.
7.

Definitions are provided in Annex II.

INITIAL CONSIDERATIONS AND LIMITATIONS
8.
This Test Guideline is based on the INVITTOX protocol No. 71 (7) that has been
evaluated in an international validation study by the European Centre for the Validation of
Alternative Methods (ECVAM) (8), in collaboration with the US Interagency Coordinating
Committee on the Validation of Alternative Methods (ICCVAM) and the Japanese Center for the
Validation of Alternative Methods (JaCVAM).
9.
The FL test method is not recommended for the identification of chemicals whichshould be classified as mild/moderate irritants or of chemicals which should not be classified forocular irritation (substances and mixtures) (i.e. GHS Cat. 2A/2B, no category; EU CLP Cat. 2, nocategory; US EPA Cat. II/III/IV), as demonstrated by the validation study (4) (8).
10.
The test method is only applicable to water soluble chemicals (substances andmixtures). The ocular severe irritation potential of chemicals that are water soluble and/or where
the toxic effect is not affected by dilution is generally predicted accurately using the FL test
method (8). To categorise a chemical as water soluble, under experimental conditions, it should
be soluble in sterile calcium-containing (at a concentration of 1.0-1.8 mM), phenol red-free,Hanks’ Buffered Salt Solution (HBSS) at a concentration ≥ 250 mg/mL (one dose above the cutoff of 100 mg/mL). However, if the test substance is soluble below the concentration 100 mg/mL,
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but already induces a FL induction of 20 % at that concentration (meaning FL20 < 100 mg/mL), it
can still be classified as GHS Cat. 1 or EPA Cat. 1.
11.
The identified limitations for this test method exclude strong acids and bases, cell
fixatives and highly volatile chemicals from the applicability domain. These chemicals havemechanisms that are not measured by the FL test method, e.g. extensive coagulation, saponificationor specific reactive chemistries. Other identified limitations for this method are based upon the
results for the predictive capacity for coloured and viscous test substance (8). It is suggested thatboth types of chemicals are difficult to remove from the monolayer following the short exposure
period and that predictivity of the test method could be improved if a higher number of washingsteps was used. Solid chemicals suspended in liquid have the propensity to precipitate out and thefinal concentration to cells can be difficult to determine. When substances within these chemicaland physical classes are excluded from the database, the accuracy of FL across the EU, EPA, andGHS classification systems is substantially improved (8).
12.
Based on the purpose of this test method (i.e. to identify ocular corrosives/severe irritants
only), false negative rates (see Paragraph 13) are not critical since such substances would be
subsequently tested with other adequately validated in vitro tests or in rabbits, depending on
regulatory requirements, using a sequential testing strategy in a weight of evidence approach (6)(see also paragraphs 3 and 4).
13.
Other identified limitations of the FL test method are based on false negative and false
positive rates. When used as an initial step within a Top-Down approach to identify water soluble
ocular corrosive/severe irritant substances and mixtures (UN GHS Category 1; EU CLP Category
1; U.S. EPA Category I), the false positive rate for the FL test method ranged from 7% (7/103;
UN GHS and EU CLP) to 9% (9/99; U.S. EPA) and the false negative rate ranged from 54%(15/28; U.S. EPA) to 56% (27/48; UN GHS and EU CLP) when compared to in vivo results.
Chemical groups showing false positive and/or false negative results in the FL test method are notdefined here.
14.
Certain technical limitations are specific to the MDCK cell culture. The tight junctions
that block the passage of the sodium-fluorescein dye through the monolayer are increasingly
compromised with increasing cell passage number. Incomplete formation of the tight junctions
results in increased FL in the non-treated control. Therefore, a defined permissible maximal
leakage in the non-treated controls is important (see paragraph 38: 0% leakage). As with all in
vitro assays there is the potential for the cells to become transformed over time, thus it is vital that
passage number ranges for the assays are stated.
15.
The current applicability domain might be increased in some cases, but only after
analyzing an expanded data set of studied test substances, preferably acquired through testing (4).
This Test Guideline will be updated accordingly as new information and data are considered.
16.
For any laboratory initially establishing this assay, the proficiency chemicals provided
in Annex III should be used. Laboratories can use these chemicals to demonstrate their technicalcompetence in performing the FL test method prior to submitting FL assay data for regulatory
hazard classification purposes.
PRINCIPLE OF THE TEST
3
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The FL test method is a cytotoxicity and cell-function based in vitro assay that is
performed on a confluent monolayer of MDCK CB997 tubular epithelial cells that are grown on
semi-permeable inserts and model the non-proliferating state of the in vivo corneal epithelium.
The MDCK cell line is well established and forms tight junctions and desmosomal junctions
similar to those found on the apical side of conjunctival and corneal epithelia. Tight anddesmosomal junctions in vivo prevent solutes and foreign materials penetrating the corneal
epithelium. Loss of trans-epithelial impermeability, due to damaged tight junctions anddesmosomal junctions, is one of the early events in chemical-induced ocular irritation.
18.
The test substance is applied to the confluent layer of cells grown on the apical side of
the insert. A short 1 min exposure is routinely used to reflect the normal clearance rate in human
exposures. An advantage of the short exposure period is that water-based substances and mixtures
can be tested neat, if they can be easily removed after the exposure period. This allows more
direct comparisons of the results with the chemical effects in humans. The test substance is thenremoved and the non-toxic, highly fluorescent sodium-fluorescein dye is added to the apical sideof the monolayer for 30 minutes. The damage caused by the test substance to the tight junctions isdetermined by the amount of fluorescein which leaks through the cell layer within a defined
period of time.
19.
The amount of sodium-fluorescein dye that passes through the monolayer and the insert
membrane into a set volume of solution present in the well (to which the sodium-fluorescein dye
leaks in) is determined by measuring spectrofluorometrically the fluorescein concentration in thewell. The amount of fluorescein leakage (FL) is calculated with reference to fluoresence intensity(FI) readings from two controls: a blank control, and a maximum leakage control. The percentage
of leakage and therefore amount of damage to the tight junctions is expressed, relative to thesecontrols, for each of the set concentrations of the test substance. Then the FL20 (i.e. concentrationthat causes 20% FL relative to the value recorded for the untreated confluent monolayer andinserts without cells), is calculated. The FL20 (mg/mL) value is used in the prediction model for
identification of ocular corrosives and severe irritants (see paragraph 35).
20.
Recovery is an important part of a test substance’s toxicity profile that is also assessedby the in vivo ocular irritation test. Preliminary analyses indicated that recovery data (up to 72 hfollowing the chemical exposure) could potentially increase the predictive capacity of
INVITTOX Protocol 71 but further evaluation is needed and would benefit from additional data,
preferably acquired by further testing (7). This Test Guideline will be updated accordingly as new
information and data are considered.
PROCEDURE
Preparation of the cellular monolayer
21.
The monolayer of MDCK CB997 cells is prepared using sub-confluent cells growing incell culture flasks in DMEM/Nutrient Mix F12 (1x concentrate with L-glutamine, 15 mM
HEPES, calcium (at a concentration of 1.0-1.8 mM) and 10% heat-inactivated FCS/FBS).
Importantly, all media/solutions used throughout the FL assay should contain calcium at aconcentration between 1.8 mM (200 mg/L) and 1.0 mM (111 mg/L) to ensure tight junction
formation and integrity. Cell passage number range should be controlled to ensure even and
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reproducible tight junctions formation. Preferably, the cells should be within the passage range 330 from thawing because cells within this passage range have similar functionality, which aids
assay results to be reproducible.
22.
Prior to performing the FL test method, the cells are detached from the flask bytrypsinisation, centrifuged and an appropriate amount of cells is seeded into the inserts placed in
24-well plates (see Annex I). Twelve mm diameter inserts with membrane of mixed cellulose
esters, a thickness of 80-150 µm and a pore size of 0.45 µm, should be used to seed the cells. In
the validation study, Millicell-HA 12 mm inserts were used. The properties of the insert andmembrane type are important as these may affect cell growth and chemical binding. Certain typesof chemicals may bind to the Millicell-HA insert membrane, which could affect the interpretationof results. Proficiency chemicals (see Annex III) should be used to demonstrate equivalency ifother membranes are used.
23.
Chemical binding to the insert membrane is more common for cationic chemicals, suchas benzalkonium chloride, which are attracted to the positively charged membrane (8). Chemicalbinding to the insert membrane may increase the chemical exposure period, leading to an overestimation of the toxic potential of the chemical, but can also physically reduce the leakage offluorescein through the insert by binding of the dye to the cationic chemical bound to the insert
membrane, leading to an under-estimation of the toxic potential of the chemical. This can bereadily monitored by exposing the membrane alone to the top concentration of the chemicaltested and then adding sodium-fluorescein dye at the normal concentration for the standard time
(no cell control). If binding of the sodium-fluorescein dye occurs, the insert membrane appears
yellow after the test material has been washed-off. Thus, it is essential to know the binding
properties of the test substance in order to be able to interpret the effect of the chemical on thecells.
24.
Cell seeding on inserts should produce a confluent monolayer at the time of chemical
exposure. 1.6 x 105 cells should be added per insert (400 µL of a cell suspension with a density of
4 x 105 cells / mL). Under these conditions, a confluent monolayer is usually obtained after 96
hours in culture. Inserts should be examined visually prior to seeding, so as to ensure that any
damages recorded at the visual control described at paragraph 30 is due to handling.
25.
The MDCK cell cultures should be kept in incubators in a humidified atmosphere, at
5% ± 1% CO2 and 37 ± 1 ºC. The cells should be free of contamination by bacteria, viruses,
mycoplasma and fungi.
Application of the Test and Control Chemicals
26.
A fresh stock solution of test substance should be prepared for each experimental run
and used within 30 minutes of preparation. Test substances should be prepared in calciumcontaining (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS to avoid serum protein
binding. Solubility of the chemical at 250 mg/mL in HBSS should be assessed prior to testing. If
at this concentration the chemical forms a stable suspension or emulsion (i.e. maintains
uniformity and does not settle or separate into more than one phase) over 30 minutes, HBSS canstill be used as solvent. However, if the chemical is found to be insoluble in HBSS at thisconcentration, the use of other test methods instead of FL should be considered. The use of lightmineral oil as a solvent, in cases where the chemical is found to be insoluble in HBSS, should be
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considered with caution as there is not enough data available to conclude on the performance of
the FL assay under such conditions.
27.
All chemicals to be tested are prepared in sterile calcium-containing (at a concentrationof 1.0-1.8 mM), phenol red-free, HBSS from the stock solution, at five fixed concentrations
diluted on a weight per volume basis: 1, 25, 100, 250 mg/mL and a neat or a saturated solution.
When testing a solid chemical, a very high concentration of 750 mg/mL should be included. This
concentration of chemical may have to be applied on the cells using a positive displacement
pipette. If the toxicity is found to be between 25 and 100 mg/mL, the following additional
concentrations should be tested twice: 1, 25, 50, 75, 100 mg/mL. The FL20 value should bederived from these concentrations provided the acceptance criteria were met.
28.
The test substances are applied to the confluent cell monolayers after removal of the cell
culture medium and washing twice with sterile, warm (37ºC), calcium-containing (at aconcentration of 1.0-1.8 mM), phenol red-free, HBSS. Previously, the filters have been visuallychecked for any pre-existing damages that could be falsely attributed to potential
incompatibilities with test chemicals. At least three replicates should be used for eachconcentration of the test substance and for the controls in each run. After 1 min of exposure atroom temperature, the test substance should be carefully removed by aspiration, the monolayer
should be washed twice with sterile, warm (37ºC), calcium-containing (at a concentration of 1.01.8 mM), phenol red-free, HBSS, and the fluorescein leakage should be immediately measured.
29.
Concurrent negative (NC) and positive controls (PC) should be used in each run todemonstrate that monolayer integrity (NC) and sensitivity of the cells (PC) are within a defined
historical acceptance range. The suggested PC chemical is Brij 35 (CAS No. 9002-92-0) at 100
mg/mL. This concentration should give approximately 30% fluorescein leakage (acceptable range
20-40% fluorescein leakage, i.e. damage to cell layer). The suggested NC chemical is calciumcontaining (at a concentration of 1.0-1.8 mM), phenol red-free, HBSS (untreated, blank control).
A maximum leakage control should also be included in each run to allow for the calculation ofFL20 values. Maximum leakage is determined using a control insert without cells.
Determination of fluorescein permeability
30.
Immediately after removal of the test and control substances, 400μL of 0.1 mg/mL
sodium-fluorescein solution (0.01% (w/v) in calcium-containing [at a concentration of 1.0-1.8mM], phenol red-free, HBSS) is added to the Millicell-HA inserts. The cultures are kept for 30
minutes at room temperature. At the end of the incubation with fluorescein, the inserts arecarefully removed from each well. Visual check is performed on each filter and any damage
which may have occurred during handling is recorded.
31.
The amount of fluorescein that leaked through the monolayer and the insert isquantified in the solution which remained in the wells after removal of the inserts. Measurements
are done in a spectrofluorometer at excitation and emission wavelengths of 485 nm and 530 nm,
respectively. The sensitivity of the spectrofluorometer should be set so that there is the highest
numerical difference between the maximum FL (insert with no cells) and the minimum FL (insert
with confluent monolayer treated with NC). Because of the differences in the used
spectrofluorometer, it is suggested that a sensitivity is used which will give fluorescence intensity> 4000 at the maximum fluorescein leakage control. The maximum FL value should not be
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greater than 9999. The maximum fluorescence leakage intensity should fall within the linear
range of the spectrofluorometer used.
Interpretation of results and Prediction model
32.
The amount of FL is proportional to the chemical-induced damage to the tight junctions.
The percentage of FL for each tested concentration of chemical is calculated from the FL values
obtained for the test substance with reference to FL values from the NC (reading from theconfluent monolayer of cells treated with the NC) and a maximum leakage control (reading forthe amount of FL through an insert without cells).
The mean maximum leakage fluorescence intensity = x
The mean 0% leakage fluorescence intensity (NC) = y
The mean 100% leakage is obtained by subtracting the mean 0% leakage from the mean
maximum leakage,i.e. x - y = z
33.
The percentage leakage for each fixed dose is obtained by subtracting the 0% leakage tothe mean fluorescence intensity of the three replicate readings (m), and dividing this value by the
100% leakage, i.e. %FL = [(m-y) / z] x 100%, where:m = the mean fluorescence intensity of the three replicate measurements for theconcentration involved% FL = the percent of the fluorescein which leaks through the cell layer
34.
The following equation for the calculation of the chemical concentration causing 20%
FL should be applied:
FLD = [(A-B) / (C-B)] x (MC –MB) + MB
Where:
D = % of inhibition
A = % damage (20% fluorescein leakage)
B = % fluorescein leakage < A
C = % fluorescein leakage > A
MC = Concentration (mg/mL) of C
MB = Concentration (mg/mL) of B
35.
The cut-off value of FL20 for predicting chemicals as ocular corrosives/severe irritants isgiven below:
FL20 (mg/mL)

UN GHS C&L

EU CLP C&L

U.S. EPA C&L

Category 1

Category 1

Category I≤ 100
C&L: classification and labelling

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The FL test method is recommended only for the identification of water soluble ocularcorrosives and severe irritants (UN GHS Category 1, EU CLP Category 1, U.S. EPA Category I)(see paragraphs 1 and 10).
37.
In order to identify water soluble chemicals (substances and mixtures) (4) (7) (8) as"inducing serious eye damage" (UN GHS/EU CLP Category 1) or as an "ocular corrosive or
severe irritant" (U.S. EPA Category I), the test substance should induce an FL20 value of ≤ 100
mg/mL.
Acceptance of results
38.
The mean maximum fluorescein leakage value (x) should be higher than 4000 (seeparagraph 31), the mean 0% leakage (y) should be equal or lower than 300, and the mean 100%
leakage (z) should fall between 3700 and 6000.
39.
A test is considered acceptable if the positive control produced 20% to 40% damage tothe cell layer (measure as % fluorescein leakage).
DATA AND REPORTING
Data
40.
For each run, data from individual replicate wells (e.g. fluorescence intensity values andcalculated percentage FL data for each test substance, including classification) should be reported
in tabular form. In addition, means ± SD of individual replicate measurements in each run should
be reported.
Test Report
41.

The test report should include the following information:

Test and Control Substances- Chemical name(s) such as the structural name used by the Chemical Abstracts Service(CAS), followed by other names, if known;- Chemical CAS number, if known;- Purity and composition of the substance or mixture (in percentage(s) by weight), to theextent this information is available;- Physical-chemical properties relevant to the conduct of the study (e.g. physical state,
volatility, pH, stability, water solubility, chemical class);- Treatment of the test/control substance prior to testing, if applicable (e.g. warming,
grinding);- Storage conditions;
Justification of the Test Method and Protocol Used
- Should include considerations regarding applicability domain and limitations of the test
method;Test Conditions
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Description of cell system used, including certificate of authenticity and the mycoplasma
status of the cell line;
Details of test procedure used;
Test substance concentration(s) used;
Duration of exposure to the test substance;
Duration of incubation with fluorescein;
Description of any modifications of the test procedure;
Description of evaluation criteria used;
Reference to historical data of the model (e.g. negative and positive controls, benchmark
chemicals, if applicable);
Information on the technical proficiency demonstrated by the laboratory;

Results- Tabulation of data from individual test substances and controls for each run and each
replicate measurement (including individual results, means and SDs);- The derived classification(s) with reference to the prediction model and/or decision
criteria used;- Description of other effects observed;
Discussion of the Results- Should include considerations regarding a non-conclusive outcome (paragraph 35: FL20 >100 mg/mL) and further testing;Conclusions

9© OECD, (2012)

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OECD/OCDELITERATURE
1. UN (2009), United Nations Globally Harmonized System of Classification and Labelling ofChemicals (GHS), Third revised edition, New York & Geneva: United Nations Publications.
ISBN: 978-92-1-117006-1. Available at:[http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html]
2. EC (2008), Regulation (EC) No 1272/2008 of the European Parliament and of the Council of
16 December 2008 on classification, labelling and packaging of substances and mixtures,
amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation(EC) No 1907/2006, Official Journal of the European Union L353, 1-1355.
3. U.S. EPA (1996), Label Review Manual: 2nd Edition, EPA737-B-96-001, Washington DC:
U.S. Environmental Protection Agency.
4. EC-ECVAM (2009), Statement on the scientific validity of cytotoxicity/cell-function based invitro assays for eye irritation testing. Available under Publications at:[http://ecvam.jrc.it/index.htm]
5. Scott, L. et al. (2010), A proposed eye irritation testing strategy to reduce and replace in vivo
studies using Bottom-Up and Top-Down approaches, Toxicol. In Vitro 24, 1-9.
6. OECD (2002), Test No. 405: Acute Eye Irritation/Corrosion, OECD Guidelines for the
Testing of Chemicals, Section 4, OECD Publishing. doi: 10.1787/9789264070646-en
7. EC-ECVAM (1999), INVITOX Protocol 71: Fluorescein Leakage Test, Ispra, Italy:
European Centre for the Validation of Alternative Methods (ECVAM). Available at:[http://ecvam-dbalm.jrc.ec.europa.eu]
8. EC-ECVAM (2008), Fluorescein Leakage Assay Background Review Document as an
Alternative Method for Eye Irritation Testing. Available under Validation Study Documents,
Section Eye Irritation at: [http://ecvam.jrc.it/index.htm]
9. OECD (2005), Guidance Document on the Validation and International Acceptance of New
or Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment
No. 34. OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]

10© OECD, (2012)

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ANNEX IDIAGRAM OF MDCK CELLS GROWN ON AN INSERT MEMBRANE FOR THE FL TESTMETHODA confluent layer of MDCK cells is grown on the semi-permeable membrane of an insert. The inserts areplaced into the wells of 24 well plates.

Figure taken from: Wilkinson, P.J. (2006), Development of an in vitro model to investigate repeat ocular
exposure, Ph.D. Thesis, University of Nottingham, UK.

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OECD/OCDEANNEX II
DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of “relevance.” The term is often used interchangeably
with “concordance”, to mean the proportion of correct outcomes of a test method.
EPA Category I: Chemicals that produce corrosive (irreversible destruction of ocular tissue) or corneal
involvement or irritation persisting for more than 21 days (4).
EU CLP (European Commission Regulation on the Classification, Labelling and Packaging of
Substances and Mixtures): Implements in the European Union (EU) the UN GHS system for theclassification of chemicals (substances and mixtures) (3).
False negative rate: The proportion of all positive chemicals falsely identified by a test method as
negative. It is one indicator of test method performance.
False positive rate: The proportion of all negative chemicals that are falsely identified by a test method as
positive. It is one indicator of test method performance.
FL20: Can be estimated by the determination of the concentration at which the tested chemical causes 20%of the fluorescein leakage through the cell layer.
Fluorescein leakage: the amount of fluorescein which passes through the cell layer, measured
spectrofluorometrically.
GHS (Globally Harmonized System of Classification and Labeling of Chemicals by the United
Nation (UN)): A system proposing the classification of chemicals (substances and mixtures) according tostandardized types and levels of physical, health and environmental hazards, and addressing corresponding
communication elements, such as pictograms, signal words, hazard statements, precautionary statements
and safety data sheets, so that to convey information on their adverse effects with a view to protect people(including employers, workers, transporters, consumers and emergency responders) and the environment(2).
GHS Category 1: Production of tissue damage in the eye, or serious physical decay of vision, following
application of a test substance to the anterior surface of the eye, which is not fully reversible within 21
days of application.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
organism, system or (sub) population is exposed to that agent.
Mixture: Used in the context of the UN GHS (2) as a mixture or solution composed of two or more
substances in which they do not react.
Negative control: An untreated replicate containing all components of a test system. This sample isprocessed with test substance-treated samples and other control samples to determine whether the solvent
interacts with the test system.
Not-classified: Chemicals that are not classified as UN GHS Categories 1, 2A, or 2B; EU CLP Categories
1 or 2; or U.S. EPA Categories I, II, or III ocular irritants (2) (3) (4).
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Ocular corrosive: (a) A chemical that causes irreversible tissue damage to the eye. (b) Chemicals that areclassified as UN GHS Category 1; EU CLP Category 1; or U.S. EPA Category I ocular irritants (2) (3) (4).
Ocular irritant: (a) A chemical that produces a reversible change in the eye following application to theanterior surface of the eye; (b) Chemicals that are classified as UN GHS Categories 2A, or 2B; EU CLP
Category 2; or U.S. EPA Categories II or III ocular irritants (2)(3)(4).
Ocular severe irritant: (a) A chemical that causes tissue damage in the eye following application to theanterior surface of the eye that is not reversible within 21 days of application or causes serious physicaldecay of vision. (b) Chemicals that are classified as UN GHS Category 1; EU CLP Category 1; or U.S.
EPA Category I ocular irritants (2) (3) (4).
Positive control: A replicate containing all components of a test system and treated with a chemical
known to induce a positive response. To ensure that variability in the positive control response across time
can be assessed, the magnitude of the positive response should not be extreme.
Proficiency Chemicals: A sub-set of the list of Reference Chemicals that can be used by a naïve
laboratory to demonstrate proficiency with the validated reference test method.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method (9).
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility and intra-laboratory repeatability.
Replacement test: A test which is designed to substitute for a test that is in routine use and accepted forhazard identification and/or risk assessment, and which has been determined to provide equivalent or
improved protection of human or animal health or the environment, as applicable, compared to theaccepted test, for all possible testing situations and chemicals.
Sensitivity: The proportion of all positive/active chemicals that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method (9).
Serious eye damage: Is the production of tissue damage in the eye, or serious physical decay of vision,
following application of a test substance to the anterior surface of the eye, which is not fully reversible
within 21 days of application.
Solvent/vehicle control: An untreated sample containing all components of a test system, including thesolvent or vehicle that is processed with the test substance-treated and other control samples to establish
the baseline response for the samples treated with the test substance dissolved in the same solvent orvehicle. When tested with a concurrent negative control, this sample also demonstrates whether the solvent
or vehicle interacts with the test system.
Specificity: The proportion of all negative/inactive chemicals that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results and is an important consideration inassessing the relevance of a test method.
13© OECD, (2012)

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OECD/OCDE

Substance: Used in the context of the UN GHS as chemical elements and their compounds in the naturalstate or obtained by any production process, including any additive necessary to preserve the stability ofthe product and any impurities deriving from the process used, but excluding any solvent which may beseparated without affecting the stability of the substance or changing its composition.
Tiered testing strategy: A stepwise testing strategy where all existing information on a test substance isreviewed, in a specified order, using a weight-of-evidence process at each tier to determine if sufficient
information is available for a hazard classification decision, prior to progression to the next tier. If theirritancy potential of a test substance can be assigned based on the existing information, no additional
testing is required. If the irritancy potential of a test substance cannot be assigned based on the existing
information, a step-wise sequential animal testing procedure is performed until an unequivocal
classification can be made.
Validated test method: A test method for which validation studies have been completed to determine therelevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be foundacceptable for the proposed purpose (9).
Weight-of-evidence: The process of considering the strengths and weaknesses of various pieces of
information in reaching and supporting a conclusion concerning the hazard potential of a chemical.

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ANNEX IIIPROFICIENCY CHEMICALS FOR THE FL TEST METHOD
Prior to routine use of a test method that adheres to this Test Guideline, laboratories should demonstratetechnical proficiency by correctly identifying the ocular corrosivity classification of the 8 chemicals
recommended in Table 1. These chemicals were selected to represent the range of responses for local eye
irritation/corrosion, which is based on results in the in vivo rabbit eye test (TG 405) (i.e., Categories 1, 2A,
2B, or No Category according to the UN GHS and EU CLP (1)(2)(6). However, considering the validated
usefulness of the FL assay (i.e., to identify ocular corrosives/severe irritants only), there are only two test
outcomes for classification purposes (corrosive/severe irritant or non-corrosive/non-severe irritant) todemonstrate proficiency. Other selection criteria were that chemicals are commercially available, there arehigh quality in vivo reference data available, and there are high quality data from the FL test method. For
this reason, the proficiency chemicals were selected from the "Fluorescein Leakage Assay Background
Review Document as an Alternative Method for Eye Irritation Testing" (8), which was used for the
retrospective validation of the FL test method.

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OECD/OCDE

Table 1: Recommended chemicals for demonstrating technical proficiency with FL
Chemical

CAS NR

Benzalkonium
chloride (5%)

8001-54-5

Promethazinehydrochloride

58-33-3

Sodiumhydroxide (10%)
Sodium laurylsulfate (15%)

Chemical Class1
Onium
compoundAmine/Amidine,
Heterocyclic,
Organic sulphur
compoundPhysicalForm

In Vivo
Classification2

LiquidCategory 1

Solid

Category 1

1310-73-2

Alkali

LiquidCategory 1

151-21-3

Carboxylic acid(salt)

LiquidCategory 1

4-carboxybenzaldehyde

619-66-9

Carboxylic acid,
Aldehyde

Solid

Category 2(A)Ammonium
nitrate6484-52-2

Inorganic salt

Solid

Category 2(A)Ethyl-2methylacetoacetate609-14-3

Ketone, Ester

LiquidCategory 2(B)Glycerol

56-81-5

AlcoholLiquidNo Category

In Vitro
Classification3
Corrosive/
Severe IrritantCorrosive/
Severe IrritantCorrosive/
Severe IrritantCorrosive/
Severe IrritantNon-corrosive/
Non-severe
irritant
Noncorrosive/
Non-severe
irritant
Noncorrosive/
Non-severe
irritant
Noncorrosive/
Non-severe
irritant

Abbreviations: CAS NR = Chemical Abstracts Service Registry Number
1
Chemical classes were assigned to each test substance using a standard classification scheme, based on the
National Library of Medicine Medical Subject Headings (MeSH) classification system (available at
http//www.nlm.nih.gov/mesh)
2
Based on results from the in vivo rabbit eye test (OECD TG 405) and using the UN GHS and EU CLP(1)(2)(6).
3
Based on results obtained with FL (INVITTOX Protocol No. 71).

© OECD, (2012)16

Fluorescein Leakage Assay
Background Review Document As
An Alternative Method for Eye
Irritation Testing
Dr Joanne Gartlon
Dr Richard Clothier
CCR.IHCP.C431273.X0
January 2008

Fluorescein Leakage Assay Background Review Document

Acknowledgements
The generous contributions from the following companies and individuals who provided
in vitro and/or in vivo data considered in this Background Review Document are
gratefully acknowledged:
Avon Products Inc., Suffern, USA
Uma Santhanam, Steve Gettings
Cosmetic, Toiletry, and Fragrance Association, Washington, USA
Carol Eisenmann
European Centre for the Validation of Alternative Methods, Ispra, Italy
Chantra Eskes, Valerie Zuang, Pilar Prieto
European Chemicals Bureau, Ispra, Italy
Andrew WorthFund for the Replacement of Animals in Medical Experiments (FRAME)
Michael Balls
Institute for In Vitro Sciences, Inc., Gaithersburg, USA
Roger Curren, John Harbell
J&J Consumer Products Worldwide R&D&E, Skillman, USA
Lauren Bernhofer, Lily Agarwal
L’Oreal Research, Aulnay Sous Bois, France
Christine van den Berghe, Odile de Silvaii.

Fluorescein Leakage Assay Background Review Document.

Fluorescein Leakage Assay Background Review Document
List of contents
Based on the Modular Approach to Validation and on the ECVAM
Validation Workshop
Page
Number1.

Data collection

1

1.1.

Description of the methods used to collect data, including
literature searches or other sources, and number of studies collected

1

1.2.

Brief description of data collected on overall study management

62.

Test Definition (Module 1)

14

2.1.

Rationale for the proposed test method

14

2.1.1. Intended uses/ purpose

14

2.1.2. Regulatory rationale and applicability

15

2.1.3. Scientific basis for the test (mechanistic)15

2.1.4. Similarities and differences of modes of action in the test method
2.2.

and the reference species

19

Test method protocols

27

2.2.1. Description of protocol components and rationale for differences, if
available

51

2.2.2. Proposed critical components of the protocol that impact on reproducibility
and/or predictive capacity of the assay include PM60

2.2.3. List of studies with similar protocols (no protocol differences, or no
impact of protocol differences on predictive capacity)61

2.2.4. Known applicability and limitations of the assay (including ranges of
irritancy, types of substances, technical limitations)2.2.5. Others

63
67

iii

Fluorescein Leakage Assay Background Review Document.

3.

Within-laboratory reproducibility (Module 2)

70

3.1.

Table presenting the results and relevant information for studies

71

3.2.

Compilation of results

74

3.2.1. Statistical approach(es) used: description & rationale for theapproach used

74

3.2.2. Results and discussion

76

3.2.3. Additional analyses of operator and time variability

96

3.3

Additional studies where raw data are not available: attempt to combine thedata using weight-of-evidence approaches

4.

Transferability (Module 3)

4.1.

Brief description of study results on transferability and availability of

104
107

Standardised Operating Procedures (SOPs)

107

4.2.

Facilities and major fixed equipment needed

109

4.3.

Required level of training, expertise, and demonstrated proficiency

5.
5.1.

needed109

Between-laboratory reproducibility (Module 4)

111

Table presenting the relevant results and information for studies
where raw data are available

112

5.2.

Discussions from the literature

113

5.3.

Compilation of results

115

5.3.1. Statistical approach(es) used: description & rationale for the approach
used to determine between-laboratory reproducibility
5.3.2. Relevant results and information for each study

115
116

5.4.1. Additional studies where raw data are not available: attempt to combine thedata using weight-of-evidence approaches

122

5.4.2. Discussions from the literature

123

5.4.3. Compilation of results

125

5.4.3.1. Statistical approach(es) used: description & rationale for the approach
used to determine between-laboratory reproducibility

iv

125

Fluorescein Leakage Assay Background Review Document.

5.4.3.2 Compilation of results

126

5.4.3.3. Attempt to combine the data using weight-of-evidence approaches

138

6.

Predictive Capacity (Module 5)

140

6.1.

Studies with available raw data

141

6.1.1. Table presenting the relevant information for each study where raw
in vitro and in vivo data were available

141

6.1.2. In vivo reference data used to assess the performance of the FL assay
for studies where raw in vitro and in vivo data were available
6.1.3. Brief description of the studies with available raw in vitro and in vivo data
a.
b.

142
143

Statistical approaches detailed in the CTFA publications(Gettings et al., 1996)

143

Principal results reported in the literature143

6.1.4. Compilation of data on predictive capacity of the test method fromstudies with raw data available

145

6.1.4.1. Description & rationale for the PM applied and statistical approaches used

145

a.

Classification systems

145

b.

Prediction models (PMs)145

6.1.4.2. Description of the performance compared to the reference and eventually
the human situation for each study

146

6.1.4.3. Discussions
a.
b.

151

Descriptions of limitations of the test method (i.e. applicability domain basedon the results of the compiled data)

151

Possible rationale for the differences observed

152

6.2.1. Table presenting the relevant information for each study where raw
data were not available

153

6.2.2. In vivo reference data used to assess the performance of the FL assay

157

6.2.3. Brief description of the studies without raw data available

161

a.

Statistical Approaches

161

b.

Main results

164

6.2.4. Compilation of data on predictive capacity of the test method from studiesv

Fluorescein Leakage Assay Background Review Document

without raw data.

170

6.2.4.1. Description & rationale for the prediction model(s) applied and statistical
approach(es) used

170

6.2.4.2. Description of performance compared to reference and eventually,to the human situation for each study
6.2.4.3. Discussions
a.
b.

176
201

Description of the limitations of the test method (e.g., applicability domain
based on results from compilation of data)

201

Possible rationale for differences observed

206

6.3.1. Attempt to combine the data using weight-of-evidence approaches

208

6.3.2. Modifications to the assay if designed today

213

7. Applicability Domain

215

8. Supporting materials

222

8.1. Relevant publications, other scientific reports and reviews

222

8.2. Relevant unpublished data

223

9.

224

Referencesvi

Fluorescein Leakage Assay Background Review Document.

Appendices
Appendix I

Standard letter inviting companies to contribute to the FL assay
BRD, and accompanying FL assay questionnaire

Appendix II Complete list of protocol steps

2
6

Annexes
Annex I

Standard FL Protocols: a. The Fluorescein Leakage Test,
INVITTOX Protocol No. 71; b. Fluorescein Leakage Test
- SOP of Company # 4, INVITTOX Protocol No. 120;c. Trans-epithelial Permeability (TEP) Assay, INVITTOX
Protocol No. 86; d. Fixed Dose Procedure For The
Fluorescein Leakage Test, INVITTOX Protocol No. 82;
e. Company # 3 Robotic Trans-Epithelial Permeability (TEP) Assay;f. ECVAM Prevalidation Study Phase II Protocol;
g. ECVAM Prevalidation Study Phase III Protocol

Annex IIa

Raw data used in the statistical analyses for intra-laboratory
variability

Annex IIb

144

Raw data used in the statistical analyses for intra-laboratory variability– CTFA Phase III TEP Assay data

Annex III

63

145

Between-laboratory FL assay data for a. COLIPA test materials:i) chemicals ii) formulations b. EC/HO chemicals; c. ECVAM
Prevalidation Phase II; d. ECVAM Prevalidation Phase III

Annex IVa

Information available on the chemicals tested to assess FL assay
predictivity of in vivo eye irritation

Annex IVb

148

Information available on the products/formulations tested toassess FL assay predictivity of in vivo eye irritation

Annex Vai

147

CTFA Study Phase III data set containing predicted and actual EU,
GHS and EPA classifications for each formulation tested using the
vii

188

Fluorescein Leakage Assay Background Review Document.

TEP assay/ INVITTOX Protocol No. 86. Raw in vitro and in vivo
data were available
Annex Vaii

203

Data sets containing predicted and actual EU, GHS and EPA
classifications for each test material. Data sets contained summarised
in vitro data.

Annex Vb

208

In vivo reference data as reported in the literature for each substancetested using the FL assay.

Annex Vc

209

In vitro and in vivo reference data as submitted by Company # 3
Consumer Products Worldwide R&D&E for 17 formulations.

Annex VI
Annex VII

Table presenting the studies where FL assay results were used toassess the assay’s predictive capacity for in vivo ocular irritation

211

Raw in vivo data as entered into ECVAM v6 template

222

Formulation Annexes
Annex A

223

CTFA Study Phase III formulation compositions (from draft HET-CAM
BRD: Appendix C2 (ICCVAM/NICEATM, 2004)

Annex B
Annex C

210

224

COLIPA Study test chemicals and formulations compositions (fromCOLIPA)

233

Formulation compositions from Company # 3

246

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Fluorescein Leakage Assay Background Review Document.

List of Tables
1.1. Results of the different search terms in the various databases

1

1.2 Description of studies featuring FL assay protocol information
and/or data, in chronological order.

6

2.1.3. Summary of the physicochemical properties that the FL assay is(in)capable of modelling.

18

2.1.4.1 Summary of the events involved in chemical-induced eye irritationin vivo which are (not) modelled by the FL assay.

23

2.1.4.2. Examples of chemical classes and their effects through thedifferent mechanisms categorised (Scott et al., (under preparation)).

24

2.2. Test Method Protocols

27

2.2.1.1. Insert properties (modified from Ward et al., 1997a)

55

2.2.1.2. PM for data generated using INVITTOX Protocol No. 71 aspresented in the EC/HO Study publication (Balls et al., 1995).

58

2.2.1.3. The PM for INVITTOX Protocol No. 82 (taken from thepublication of Clothier et al., (1994)).

59

2.2.1.4. PM from INVITTOX Protocol No. 120

59

2.2.1.5 PM from INVITTOX Protocol No. 86 (TEP assay)

59

3.1.

71

Table presenting the results and relevant information for studies

3.2.2. Summary table of the overall reproducibility per study. There were
25 data sets (compared to 14 in table 3.1) as some studies
contained multiple data sets (e.g. ECVAM Prevalidation study(Southee, 1998)).

77

3.2.2.1. The main protocol differences between the FL assay protocols forwhich data were available to assess intra-laboratory variability.

84

3.2.2.2. Relevant in vitro and in vivo information for the types of test
materials and potencies.

90

3.2.2.3. PM from the COLIPA study for a version of INVITTOX Protocol
No. 120.

94

3.2.2.4. Variation in classifications assigned according to the COLIPA
PM for the three laboratories that participated in the ECVAM

ix

Fluorescein Leakage Assay Background Review Document

Prevalidation study Phase III (n ≥2).

.

95

3.2.3.1. Results and relevant information for each study

97

3.2.3.2. Operator variability for all the data sets featured in table 3.2.3.1

100

3.2.3.3. Impact of failed experimental runs on overall reproducibility.

101

3.2.3.4. The number of materials for which two or more outliers wereidentified in relation to the total number of materials within the data
set

102

3.3. Presents the results and relevant information for each study containing
non-raw data.

104

4.2. The fixed equipment required to carry out the FL assay
5.1.

109

Table presenting the relevant results and information for studies
where raw data are available

112

5.3.2.1.a. Summarised ECVAM Prevalidation Phase II FL20 (mg/ml) data
(All raw data for Phase II, for each laboratory provided in Annex III on
CD)

116

5.3.2.1.b. Summarised ECVAM Prevalidation Phase III FL20 (mg/ml) data
(All raw data for Phase III, for each laboratory provided in Annex III
on CD)

116

5.3.2.2. PM applied to ECVAM Prevalidation study Phase III data

120

5.3.2.3. Mean FL20 (mg/ml) T4 data and classification for each test material ineach laboratory.

120

5.3.2.4. Summary table of proportion of identical predicted classifications
from each laboratory

121

5.4.1. Relevant results and information for studies where raw data were
not available

122

5.4.3.2.1. Between-laboratory FL20 (mg/ml) data for COLIPA study test
materials.

126

5.4.3.2.2. Between-laboratory FL20 (mg/ml) data for EC/HO study test
chemicals

128

5.4.3.2.3. Inter-laboratory correlation of the FL20 (mg/ml) data fromthe four laboratories (from Balls et al., 1995).

x

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Fluorescein Leakage Assay Background Review Document.

5.4.3.2.4. Modified PM from EC/HO study used to assign irritant andnon-irritant classifications.

130

5.4.3.2.5. Predicted classifications for EC/HO study data generated using
the PM featured in table 5.4.3.2.4.

131

5.4.3.2.6. Summary table of proportion of identical predicted classifications
from each laboratory.

134

5.4.3.2.7. Predicted classifications for COLIPA Study data generated using
the PM featured in table 5.3.2.2.

135

5.4.3.2.8. Summary table of proportion of identical predicted classifications
from each laboratory.

137

5.4.3.2.9. FL20 (mg/ml) data from the EC/HO study and the COLIPA study forthe nine common test chemicals (from Zanvit et al., 1999). A
version of INVITTOX Protocol No. 120 was used.

138

6.1.1. Table presenting the relevant information for each study where raw
in vitro and in vivo data were available

141

6.1.2. In vivo reference data used to assess the performance of the FL assay
for studies where raw in vitro and in vivo data were available

142

6.1.3. The findings reported in the literature for those studies which used
PMs to assess the predictive capacity of the TEP assay.
6.1.4.1.1. PM from INVITTOX Protocol No. 86

144
145

6.1.4.1.2. Modified TEP assay PM for predicting EU, GHS and EPA
classifications.

146

6.1.4.2.1.a. Contingency tables for CTFA Phase III data generated using
INVITTOX Protocol No. 86 and the PM featured in table
6.1.4.1.2. -EU classification system

147

6.1.4.2.1.b. Contingency tables for CTFA Phase III data generatedusing INVITTOX Protocol No. 86 and the PM featured intable 6.1.4.1.2. -GHS classification system

147

6.1.4.2.1.c. Contingency tables for CTFA Phase III data generatedusing INVITTOX Protocol No. 86 and the PM featured in table
6.1.4.1.2. -EPA classification system

xi

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Fluorescein Leakage Assay Background Review Document.

6.1.4.2.2.a. Evaluation of the performance of INVITTOX Protocol No. 86
for predicting ocular irritation according to the EU classification
system –Non-irritants versus the rest

150

6.1.4.2.2.b. Evaluation of the performance of INVITTOX Protocol No. 86
for predicting ocular irritation according to the GHS
classification system –Non-irritants versus the rest.

150

6.1.4.2.2.c. Evaluation of the performance of INVITTOX Protocol No. 86
for predicting ocular irritation according to the EPA
classification system –Non-irritants versus the rest150

6.2.1. Table presenting the relevant information for each study where raw
data were not available.

153

6.2.2. In vivo reference data used to assess the performance of the FL
assay

157

6.2.3. The findings reported in the literature for those studies which used
PMs to assess the predictive capacity of the various FL assay
protocols.

164

6.2.4.1.1. PM submitted to ECVAM for FL20 (mg/ml) values withrecovery data, based upon the PM featured in the EC/HO study(Balls et al., 1995).

171

6.2.4.1.2. ICCVAM/NICEATM criteria for assigning GHS classifications
to Draize data (from draft HET-CAM BRD (2004)).

172

6.2.4.1.3. ICCVAM/NICEATM criteria for assigning EU classifications
to Draize data (from draft HET-CAM BRD (2004)).

173

6.2.4.1.4. EPA classification system for ocular irritation (from draft
HET-CAM BRD (2004)).

173

6.2.4.1.5. PM for FL20 (mg/ml) values.

174

6.2.4.1.6. PM for FL20 (mg/ml) values produced 4h following a 15 minutechemical exposure period (modified from Brantom et al., 1997).

174

6.2.4.1.7. Modified PM for INVITTOX Protocol No. 82 data (fromClothier et al., (1994)).

175

6.2.4.2.1.a. Contingency table for EC/HO study data generated usingxii

Fluorescein Leakage Assay Background Review Document.

INVITTOX Protocol No. 71 and the PM featured in table
6.2.4.1.5. -EU classification system

177

6.2.4.2.1.b. Contingency table for EC/HO study data generated using
INVITTOX Protocol No. 71 and the PM featured in table
6.2.4.1.5. -GHS classification system

178

6.2.4.2.1.c. Contingency table for EC/HO study data generated using
INVITTOX

Protocol No. 71 and the PM featured in table

6.2.4.1.5. -EPA classification system

179

6.2.4.2.2.a Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EU classification
system –Non-irritants versus the rest.

180

6.2.4.2.2.b. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the GHS
classification system –Non-irritants versus the rest.

180

6.2.4.2.2.c. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EPA
classification system –Non-irritants versus the rest.

180

6.2.4.2.3.a Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EU classification
system –Severe irritants versus the rest.

182

6.2.4.2.3.b. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the GHS
classification system –Severe irritants versus the rest.

182

6.2.4.2.3.c. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EPA classification
system –Severe irritants versus the rest.

182

6.2.4.2.4.a. Contingency table for data generated using INVITTOX
Protocol No. 71 with recovery time-point and the PM featured
in table 6.2.4.1.1. -EU classification system.
6.2.4.2.4.b. Contingency table for data generated using INVITTOX
Protocol No. 71 with recovery time-point and the PM featured in

xiii

183

Fluorescein Leakage Assay Background Review Documenttable 6.2.4.1.1. -GHS classification system.

.

184

6.2.4.2.4.c. Contingency table for data generated using INVITTOX
Protocol No. 71 with recovery time-point and the PM featured intable 6.2.4.1.1. -EPA classification system.

184

6.2.4.2.5.a Evaluation of the performance of INVITTOX Protocol No. 71
with recovery data for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest

186

6.2.4.2.5.b. Evaluation of the performance of INVITTOX Protocol No. 71
with recovery data for predicting ocular irritation according to the
GHS classification system –Non-irritants versus the rest.

186

6.2.4.2.5.c. Evaluation of the performance of INVITTOX Protocol No. 71
with recovery data for predicting ocular irritation according to the
EPA classification system –Non-irritants versus the rest.

186

6.2.4.2.6. Evaluation of the performance of INVITTOX Protocol No. 71
with recovery data for predicting ocular irritation according to the
GHS classification system –Severe irritants versus the rest.

187

6.2.4.2.7.a. Contingency table for data generated using INVITTOX
Protocol No. 120 and the PM featured in table 6.2.4.1.6.
–EU classification system.

188

6.2.4.2.7.b. Contingency table for data generated using INVITTOX
Protocol No. 120 and the PM featured in table 6.2.4.1.6.
–GHS classification system.

189

6.2.4.2.7.c. Contingency table for data generated using INVITTOX
Protocol No. 120 and the PM featured in table 6.2.4.1.6.
–EPA classification system.

189

6.2.4.2.8.a Evaluation of the performance of INVITTOX
Protocol No. 120 for predicting ocular irritation according to
the EU classification system –Non-irritant versus the rest.

190

6.2.4.2.8.b. Evaluation of the performance of INVITTOX
Protocol No. 120 for predicting ocular irritation according to
the GHS classification system –Non-irritant versus the rest.

xiv

190

Fluorescein Leakage Assay Background Review Document.

6.2.4.2.8.c. Evaluation of the performance of INVITTOX
Protocol No. 120 for predicting ocular irritation according to
the EPA classification system –Non-irritant versus the rest.

190

6.2.4.2.9.a. Evaluation of the performance of INVITTOX
Protocol No. 120 for predicting ocular irritation according to
the GHS classification system –Severe irritants versus the rest.

191

6.2.4.2.9.b. Evaluation of the performance of INVITTOX
Protocol No. 120 for predicting ocular irritation according to
the EPA classification system –Severe irritants versus the rest.

191

6.2.4.2.10. Contingency table for data generated using INVITTOX
Protocol No. 82 and the PM featured in table 6.2.4.1.7.
–EU classification system.

192

6.2.4.2.11. Evaluation of the performance of INVITTOX
Protocol No. 82 for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest.

193

6.2.4.2.12.a. Contingency table for data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5.
–EU classification system.

194

6.2.4.2.12.b. Contingency table for data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5.
–GHS classification system.

194

6.2.4.2.12.c. Contingency table for data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5.
–EPA classification system.

195

6.2.4.2.13.a Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EU classification
system –Non-irritants versus the rest.

196

6.2.4.2.13.b. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the GHS
classification system –Non-irritants versus the rest.
6.2.4.2.13.c. Evaluation of the performance of INVITTOX Protocol No. 71

xv

196

Fluorescein Leakage Assay Background Review Document.

for predicting ocular irritation according to the EPA
classification system –Non-irritants versus the rest.

196

6.2.4.2.14.a. Contingency table for data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5.
–EU classification system.

197

6.2.4.2.14.b. Contingency table for data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5.
–GHS classification system.

197

6.2.4.2.14.c. Contingency table for data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5.
–EPA classification system.

198

6.2.4.2.15.a Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EUclassification system –Non-irritants versus the rest.

199

6.2.4.2.15.b. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the GHS
classification system –Non-irritants versus the rest.

199

6.2.4.2.15.c. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EPA
classification system –Non-irritants versus the rest.

199

6.2.4.2.16.a Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EUclassification system –Severe irritants versus the rest.

200

6.2.4.2.16.b. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the GHS
classification system –Severe irritants versus the rest.

200

6.2.4.2.16.c. Evaluation of the performance of INVITTOX Protocol No. 71
for predicting ocular irritation according to the EPA
classification system –Severe irritants versus the rest.

xvi

200

Fluorescein Leakage Assay Background Review Document.

List of Figures

2.1.3.1. Diagram of MDCK cells grown on an insert membrane for
the FL assay (from Wilkinson, 2006).

16

2.1.3.2. Equation for the calculation of thechemical/formulation concentration causing 20 percent
FL (FL20) (from INVITTOX Protocol No. 71).
2.1.4.

17

Weighted scoring system for ocular lesions(from Draize et al., 1944).

21

2.2.2.1. Critical elements of FL assay protocols

60

3.2.1. ◊ SD and ♦ CV values plotted against the mean FL assay results;
a. FL20 (mg/ml)

74

b. FL (%)

75c. FL (mM) (n≥2)

75

3.2.2.1. The CV values for INVITTOX Protocol No. 71 ♦, and INVITTOX
Protocol No. 120 ◊ plotted against FL assay results for all
available raw data.

87

3.2.2.2. The CV values for TEP assay data plotted against the mean
TEP assay values (n=2) for Company # 3 formulations

88

3.2.2.3. The effect of time on the ECVAM Prevalidation Phase II
SLS positive control FL% values for the four participating
laboratories at a.) T0; b.) T4; □, FAL; ■, Company # 7; ▲, Company # 3; ∆,ECVAM
89
3.2.2.4. The CV values plotted against mean FL20 (mg/ml) T4 values
for the ten surfactants tested in three laboratories;□, FAL; ▲, Company # 3; ∆, ECVAM
91
3.2.2.5. The CVs plotted against the mean T4 FL20 (mg/ml) values
for surfactants and surfactant-based formulations tested by the
FAL as part of the COLIPA study.

92

3.2.2.6. The CVs plotted against the mean EC50 (%) values fora. formulations tested by Company # 3 Consumer Products WorldwidexviiFluorescein Leakage Assay Background Review Document

R&D&E; b. CTFA Phase III data.

.

93

5.3.2.1. Mean FL% ±SD values for Phase II positive control data
(0.16mg/ml SLS) from each laboratory at T0 (pattern) and T4(blank).

118

5.3.2.2. Mean FL% ±SD values for Phase III positive control data
(0.16mg/ml SLS) from each laboratory at T0 (pattern) and T4(blank).

119

6.2.4. Correlation of FR85 (%) data with EU classifications.

xviii

175

Fluorescein Leakage Assay Background Review Document

Abbreviations
AB
ANOVA
ATCC
BMedSci
BRD
CAPB
CI
CMC
COLIPA
CTAB
CTFA
CV
DDSW
DMEM
DMSO
EC
EC50
ECACC
ECETOC
EC/HO
ECVAM
EGF
EGTA
EMEM
EPA
EU
FAL
FBS
FCS
FD
FHSA
FL
FLT
FL%
FR
FRAME
GLP
h
HBSS
HCE-T
hi
HRIPT
IC

alamar blue
analysis of variance
American Type Culture Collection
Bachelor of medical science placement student
background review document
cocamidopropylbetaine
confidence interval
critical micelle concentration
European Cosmetic, Toiletry and Perfumery Association
cetyl trimethyl ammonium bromide
Cosmetics, Toiletries and Fragrance Association
coefficient of variancedouble distilled sterile water
Dulbecco’s modified eagle medium
dimethyl sulphoxide
European Commission
effective concentration causing 50% fluorescein leakage
European Collection of Cell Cultures
European Centre for Ecotoxicology and Toxicology of Chemicals
European Community/ Home Office
European Centre for the Validation of Alternative Methods
epidermal growth factor
ethylenebis(oxyethylenenitrilo)tetraacetic acid
Eagle’s modified essential medium
Environmental Protection Agency
European Union
FRAME Alternatives Laboratory
foetal bovine serum
foetal calf serum
fluorescein isothiocyanate dextran
Federal Hazardous Substances Act
fluorescein leakage
fluorescein leakage test
percentage of fluorescein leakage for a set chemical concentration
fluorescein retention
Fund for the Replacement of Animals in Medical Experiments
good laboratory practicehourhanks balanced salt solution
human cornel epithelium -transfectedheat inactivatedhuman repeated insult patch testing
inhibitory concentrationxix.

Fluorescein Leakage Assay Background Review Document

IIVS
IRAG
J-HCET
KB
kD
LVET
MAS
MDCK
MEM
min
MMAS
MTT
Na
NR
NRR
OD
OECD
OIC
PBS
PI
PM
PTFE
RT
RTECS
SCNM
SD
SDS
secs
SEM
SIS
SV40
T
TEP
TG
w/o
w/v
w/w

Institute for In Vitro Sciences
US Interagency Regulatory Alternatives Group
Japanese human corneal epithelium transfectedkenacid blue
kilodalton
low volume eye test
maximum average score
Madin-Darby Canine Kidney
mininum essential medium
minutemodified maximum average score[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
sodium chloride
3-amino-7-di-methylamino-2-methylphenazinehydrochloride
neutral red release
optical density
Organisation for Economic and Community Development
ocular irritation categories
phosphate buffered solution
prediction interval
prediction model
PolyTetraFluoroEthylene
room temperature
Registry of Toxic Effects of Chemical Substances
study criteria not met
standard deviation
sodium dodecyl sulphate
seconds
standard error of the meanscience information service
simian virus 40
time-pointtrans-epithelial permeability
test guideline
without
weight/volume
weight/weightxx.

Fluorescein Leakage Assay Background Review Document

1. Data collection
1.1. Description of the methods used to collect data, including literature searches
or other sources, and number of studies collected
Initially,
threeinternet-based
scientific
literature
databases
PubMed(www.ncbi.nlm.nih.gov/entrez/query.fcgi), Toxnet (www.toxnet.nlm.nih.gov), andScience Direct (www.sciencedirect.com), were searched in order to attain an initial
overview of the work carried out using the fluorescein leakage (FL) assay. The
following search terms were used; “fluorescein leakage assay,” “fluorescein leakage
test,” and “trans-epithelium permeability assay.” The trans-epithelium permeability(TEP) assay and the fluorescein leakage assay are essentially the same andprotocol information and results from the TEP assay are included in this backgroundreview document (BRD), i.e. general remarks pertaining to the FL assay are also
applicable for the TEP assay unless otherwise stated. The number of publications
from the different search terms in the various databases are shown (table 1.1.).
Table 1.1. Results of the different search terms in the various databases
Database
Search term
fluorescein leakage
assay
fluorescein leakage
test
trans-epithelium
permeability assay

PubMedToxnetScience
Direct3 (129)

5 (6)

4 (4)

3 (132)

6 (8)

4 (4)

0 (3)

0 (5)

0 (0)

Bold figures refer to the actual number of relevant papers, results in brackets refers
to number of hits per search term. NB. It is to be noted that the Science Direct database required speech-marks around the search term in order to produce the relevant
results.
In general, these databases led to credible scientific publications regarding thefollowing types of FL assay information; assay development, protocol variations, anduse of the assay for predicting in vivo eye irritation. The search terms failed to find anumber of papers which were known feature the FL assay, e.g. the EC/HOInternational validation study on alternatives to the Draize eye irritation test (Balls etal., 1995), and the USA Cosmetics, Toiletries and Fragrance Association (CTFA)
Evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize
primary eye irritation test, Phase III (Gettings et al., 1996).
The relevant papers found using the search terms featured in table 1.1. are listed foreach database in chronological order:1

Fluorescein Leakage Assay Background Review Document

Fluorescein leakage assay:-PubmedBotham, P., Osborne, R., Atkinson, K., Carr, G., Cottin, M., van Buskirk, R.G., 1997.
IRAG working group 3. Cell function-based assays. Interagency Regulatory
Alternatives Group. Food and Chemical Toxicology, 35(1):67-77.
Jones, P. A., Budynsky, E., Cooper, K. J., Decker, D., Griffiths, H. A., Fentem, J. H.,
2001. Comparative evaluation of five in vitro tests for assessing the eye irritation
potential of hair-care products. ATLA, 29(6):669-92.
Clothier, R., Starzec, G., Pradel, L., Baxter, V., Jones, M., Cox, H., Noble, L., 2002.
The prediction of human skin responses by using the combined in vitro fluoresceinleakage/Alamar Blue (resazurin) assay. ATLA, 30(5):493-504.
-Toxnet
Gautheron, P., Duprat, P., Hollander, C.F., 1994. Investigations of the MDCK
permeability assay as an in vitro test of ocular irritancy. In Vitro Toxicology, 7:33-43.
Botham, P., Osborne, R., Atkinson, K., Carr, G., Cottin, M., van Buskirk, R.G., 1997.
IRAG working group 3. Cell function-based assays. Interagency Regulatory
Alternatives Group. Food and Chemical Toxicology, 35(1):67-77.
Ward, R.K., Mungall, S., Carter, J., Clothier, R.H., 1997. Evaluation of tissue culture
insert membrane compatibility in the fluorescein leakage assay. Toxicology In Vitro,
11:761-768.
Clothier, R.H., Starzec, G., Stipho, S., Kwong, Y.C., 1999. Assessment of initialdamage and recovery following exposure of MDCK cells to an irritant. Toxicology In
Vitro, 13:713-717.
Zanvit, A., Meunier, P. A., Clothier, R., Ward, R., Buiatti-Tcheng, M., 1999. Ocular
irritancy assessment of cosmetics formulations and ingredients: fluorescein leakage
test. Toxicology In Vitro, 13:385-391.
-Science Direct
Shaw, A. J., Balls, M., Clothier, R.H., Bateman, N.D., 1991. Predicting ocular
irritancy and recovery for injury using MDCK cells. Toxicology In Vitro, 5:569-571
Botham, P., Osborne, R., Atkinson, K., Carr, G., Cottin, M., van Buskirk, R.G., 1997.
IRAG working group 3. Cell function-based assays. Interagency Regulatory
Alternatives Group. Food and Chemical Toxicology, 35(1):67-77.
Ward, R.K., Mungall, S., Carter, J., Clothier, R.H., 1997. Evaluation of tissue culture
insert membrane compatibility in the fluorescein leakage assay. Toxicology In Vitro,
11:761-768.

2

Fluorescein Leakage Assay Background Review Document

Clothier, R.H., Starzec, G., Stipho, S., Kwong, Y.C., 1999. Assessment of initialdamage and recovery following exposure of MDCK cells to an irritant. Toxicology In
Vitro, 13:713-717.
Fluorescein leakage test:-PubmedBotham, P., Osborne, R., Atkinson, K., Carr, G., Cottin, M., van Buskirk, R.G., 1997.
IRAG working group 3. Cell function-based assays. Interagency Regulatory
Alternatives Group. Food and Chemical Toxicology, 35(1):67-77.
Jones, P.A., Budynsky, E., Cooper, K. J., Decker, D., Griffiths, H.A., Fentem, J.H.,
2001. Comparative evaluation of five in vitro tests for assessing the eye irritation
potential of hair-care products. ATLA, 29(6):669-92.
Clothier, R., Starzec, G., Pradel, L., Baxter, V., Jones, M., Cox, H., Noble, L., 2002.
The prediction of human skin responses by using the combined in vitro fluoresceinleakage/Alamar Blue (resazurin) assay. ATLA, 30(5):493-504.
-ToxnetClothier, R.H., Morgan, S.J., Atkinson, K.A., Garle, M.J., Balls, M., 1994.
Development of a fixed-dose approach for the fluorescein leakage test. Toxicology In
Vitro, 8: 883-884.
Gautheron, P., Duprat, P., Hollander, C.F., 1994. Investigations of the MDCK
permeability assay as an in vitro test of ocular irritancy. In Vitro Toxicology, 7:33-43.
Botham, P., Osborne, R., Atkinson, K., Carr, G., Cottin, M., van Buskirk, R.G., 1997.
IRAG working group 3. Cell function-based assays. Interagency Regulatory
Alternatives Group. Food and Chemical Toxicology, 35(1):67-77.
Ward, R.K., Mungall, S., Carter, J., Clothier, R.H., 1997. Evaluation of tissue culture
insert membrane compatibility in the fluorescein leakage assay. Toxicology In Vitro,
11:761-768.
Cottin, M., Zanvit, A., 1999. Fluorescein leakage test: A useful tool in ocular safetyassessment. Toxicology In Vitro, 11:399–405.
Zanvit, A., Meunier, P. A., Clothier, R., Ward, R., Buiatti-Tcheng, M., 1999. Ocular
irritancy assessment of cosmetics formulations and ingredients: fluorescein leakage
test. Toxicology In Vitro, 13: 385-391.
-Science Direct
Clothier, R.H., Morgan, S.J., Atkinson, K.A., Garle, M.J., Balls, M., 1994.
Development of a fixed-dose approach for the fluorescein leakage test. Toxicology In
Vitro, 8: 883-884.

3

Fluorescein Leakage Assay Background Review Document

Brantom, P.G., Bruner, L.H., Chamberlain, M., deSilva, O., Dupuis, J., Earl, L.K.,
Lovell, D.P., Pape, W.J.W., Uttley, M., Bagley, D.M., Baker, F.W., Brachter, M.,
Courtellemont, P., Declercq, L., Freeman, S., Steiling, W., Walker, A.P., Carr, G.J.,
Dami, N., Thomas, G., Harbell, J., Jones, P.A., Pfannenbecker, U., Southee, J.A.,
Tcheng, M., Argembeaux, H., Castelli, D., Clothier, R., Esdaile, D.J., Itigaki, H., Jung,
K., Kasai, Y., Kojima, H., Kristen, U., Larnicol, M., Lewis, R.W., Marenus, K., Moreno,O., Peterson, A., Rasmussen, E. S., Robles, C., Stern, M., 1997. A summary reportof the COLIPA international validation study on alternatives to the Draize rabbit eye
irritation test. Toxicology In Vitro, 11(N1-2): 141-179.
Cottin, M., Zanvit, A., 1999. Fluorescein leakage test: A useful tool in ocular safetyassessment. Toxicology In Vitro, 11:399–405.
Zanvit, A., Meunier, P. A., Clothier, R., Ward, R., Buiatti-Tcheng, M., 1999. Ocular
irritancy assessment of cosmetics formulations and ingredients: fluorescein leakage
test. Toxicology In Vitro, 13: 385-391.
Trans-epithelium permeability assay:-no relevant publications found in any of the databases
An internet search using the ‘Google’ search engine was performed in order toaccess a greater range of material concerning the FL assay, such as conference
abstracts and journal comments. From these pieces a list of companies andorganisations which use, or have used the FL assay were compiled. The Google
search engine was also helpful in acquiring information pertaining to the field of invitro testing for ocular irritation. Protocol information and/or data acquired using theGoogle search engine were only used in this BRD if they led to credible sources, e.g.
journal websites or official company/institute websites known in the field of in vitro
science. For example, one TEP assay INVITTOX Protocol and three FL assay
INVITTOX Protocols were taken from the ECVAM Science Information System (SIS)
website (http://ecvam-sis.jrc.it) which required registration to access the on-line
protocols.
The internet searches were very useful for quickly gaining an overview of the
publications featuring the FL assay. The journal articles were particularly useful for
acquiring protocol information. In comparison, the FL assay data featured in journals
had a tendency to be summarised to varying degrees, e.g. from mean ± SD of
repeated experiments, to statistical correlations regarding performance. Where
contact details were available and current, the authors of all FL assay related
publications were approached and invited to submit ‘complete’ protocol information
and raw data. A standard letter and questionnaire was sent to the various
companies and research institutes (Appendix I).
The majority of raw data featured in this BRD were primarily taken from the Fund for
the Replacement of Alternative Medical Experiments (FRAME) in-house database.
This raw data were produced as a result of the FRAME Alternatives Laboratory’s(FAL) participation in FL assay validation and evaluation studies. Additional raw data
were given by those companies which submitted data and protocol information to be
used in this BRD. As internet searches only revealed a small number of companies
and institutes that have reported their use of the FL assay, many other research4

Fluorescein Leakage Assay Background Review Document

divisions of cosmetic and pharmaceutical companies were contacted. Of 17
companies contacted, two responded as not having FL assay protocol information ordata to submit, 14 did not respond at all, and two agreed to participate in the study.
Protocol information and data were provided by Company # 3 and Company # 4.
Additional TEP assay data from Company # 3 which was part of the CTFA study
Phase III was also submitted through collaboration with the CTFA, 1101 17th Street,
NW Suite 300, Washington D.C., 20036-4702, USA. From all the available sources,
33 data sets generated using various FL assay protocols were collected and featurein this BRD.
The publications that were obtained and reviewed for this BRD are listed in table 1.2.
It is acknowledged that many of these publications were not discovered as a result of
the search terms shown in table 1.1.. Many of the publications were acquired byreviewing the reference sections of those papers obtained as a result of the initial
searches as described in table 1.1.. Additional papers were also obtained due to theauthors, of this BRD, awareness of relevant publications regarding the FL assay.

5

Fluorescein Leakage Assay Background Review Document

1.2. Brief description of data collected on overall study management
Table 1.2. Description of studies featuring FL assay protocol information and/or data, in chronological orderStudy name

Trans-epithelial Permeabilityof Fluorescein In Vitro as an
Assay to Determine Eye
Irritants (Tchao, 1988). Poster
LossofTrans-epithelial
Impermeability of a Confluent
Layer of Madin-Darby Canine
Kidney (MDCK) Cells as a
Determinantof
OcularIrritancy Potential (Shaw etal., 1990)
Predicting Ocular Irritancy
and Recovery from Injury
using Madin-Darby Canine
Kidney Cells (Shaw et al.,
1991)
Human Corneal Epithelial
Primary Cultures and Cell
Lines with Extended Life
Span: In Vitro Model for
Ocular Studies (Kahn et al.,
1993)

Study
management

Chemicalselection,
chemicalcoding?

Process of
datacollection
andstatisticalanalysis

Avail. ofstandard
prediction
model

Quality of data (invivo and in vitro
GLP compliant)Avail. ofstandard
protocol*In-house

In -house.
NochemicalcodingIn-house

N

In vitro: non-GLP

N

Data format in
publication
Tween
concentrations
producingsimilar FL;
amount of FL
not stated. FL
summarised ingraphs T0

In-house

In-house.
No
chemicalcodingN

In vitro: non-GLP;
In vivo: GLP

N (INVITTOX
Protocol No.
71)

Mean FL20,
FL50 (mg/ml)±SEM T0

In-house

In-house.
No
chemicalcoding

In vitro: non-GLP

N (INVITTOX
Protocol No.
71)

Mean FL20(mg/ml) ±SEM
T0, T72In-house

N/A –nochemicals
testedN

Mean FR% ±SD or SEM T0summarised ingraphsIn-house

In-houseNIn-house

N

6

In vitro: non-GLP,
In vivo: GLP

Fluorescein Leakage Assay Background Review Document

Study name

Investigations of the MDCKPermeability Assay as an
In Vitro Test of Ocular
Irritancy (Gautheron et al.,
1994)
Use
of
In
Vitro
Methodology to Predict
Irritancy
PotentialofSurfactants (Hubbard et al.,
1994)
Development of a Fixed
Dose Approach for The
Fluorescein Leakage Test
(Clothier et al., 1994)

The Evaluation of Pesticide
Ingredients
andFormulations In Vitro andCorrelations with In Vivo
Data (Clothier et al., 1995)

Study
management

In-house

In-house

Chemicalselection,
chemicalcoding?

In-house. No
chemicalcoding
Surfactants
in-house,
formulations
independent;
formulations
coded

Process of
datacollection
andstatisticalanalysis

In-house

In-house; nostatisticalanalysis

In-house

In-house.
ChemicalcodingIn-house

In-house

In-house. No
chemicalcodingIn-house; nostatisticalanalysis

7

Avail. ofstandard
prediction
model

N

N

N

N

Quality of data(in vivo and invitro GLP
compliant)
In vitro: non-GLP;
In vivo: GLP(historical datafrom Gautheron
et al., (1994);
Kennah et al.,(1989) Grant ,1986)

In vitro: non-GLP;
In vivo: GLP notstatedIn vitro: non-GLP;
In vivo: GLP(historical datafrom Botham etal., (1989))
In vitro: non-GLP;
In vivo: GLP(OECD
Guidelines for
Testing ofChemicals (1987)
No. 45 "Acute
Eye Irritation/
Corrosion)

Avail. ofstandard
protocol*Data formatinpublication

N

Mean FL20,
FL50 (mg/ml)
w/o SD or
SEM T0N (INVITTOX
Protocol No.
71)

Mean FL(mg/ml), (%)± SEM T0,
T24, T48 andraw data

N (INVITTOX
Protocol No.
82)

Mean FL% ±SEM T0, T72

INVITTOX
Protocol No.
71

Mean FL20(mg/ml) ±SEM
T0, T72

Fluorescein Leakage Assay Background Review Document

Study name
The EC/HO International
Validation
Studyon
Alternatives to the Draize
Eye Irritation Test (Balls etal., 1995)
CTFA
Evaluationof
Alternatives Program: An
Evaluation of In Vitro
Alternatives to the Draize
Primary Eye Irritation Test:
Phase III (Gettings et al.,
1996)
Effects of Surfactant Retreatment In Vitro: A Method
to Evaluate Changes in Cell
Junctions and Cell Viability(ClothierandSansom,1996)
Evaluation of Tissue Culture
InsertMembraneCompatibility
intheFluorescein Leakage Assay(Ward et al., 1997a)
Evaluation of Chemically
Induced Toxicity Using an
In Vitro Human Corneal
Epithelium (Ward et al.,1997b)

Study
management

Independent

Independent

Chemicalselection,
chemicalcoding?

Process of
datacollection
andstatisticalanalysis

Independent.
ChemicalcodingIndependent-analyses
performed by
BIBRA.

Independent.
ChemicalcodingIndependent

In-house

In-house. No
chemicalcodingIn-house.
Chemicalcoding for
EC/HO test
chemicals
only

In-house

In-house

In-house. No
chemicalcodingIn-house; nostatisticalanalysis

In-house

In-house

8

Avail. ofstandard
prediction
model

Quality of data(in vivo and invitro GLP
compliant)Avail. ofstandard
protocol*Data format in
publication

Y

In vitro: non-GLP;
In vivo: GLP

Y -not strictly
followed(INVITTOX
Protocol No.
71)

FL20 (mg/ml)
correlations
from 4 labs within vivo MMAS
data T0N

In vitro: non-GLP;
In vivo: GLP,
concurrent in vivo
and in vitro
testingN(INVITTOX
Protocol No.
86)

Mean EC50(%) w/o SD or
SEM T0N (adaptation of
INVITTOX
Protocol No.
71 and No.
80)

Mean FL%±SD T0, T1,
T24, T72
summarised ingraphsN

N

In vitro: non-GLP
In vitro: non-GLP;
In vivo: GLP(historical datafrom Balls et al.,(1995); ECETOC(1992))

N

In vitro: non-GLP,
In vivo: GLP notstatedN (INVITTOX
Protocol No.
71)

N

Mean FL20(mg/ml) ±SD
T0, T72
Mean TEP
relativefluorescein
retention (%) ±SD on graphs
T0, T24, T48

Fluorescein Leakage Assay Background Review Document

Studymanagement

Chemicalselection,
chemicalcoding?

In-house

In-house. No
chemicalcodingLAB A IRAG Working
Group 3: Cell Functionbased Assays (Botham etal., 1997)

Independent

Unknown.
Chemicalcodingunknown

LAB B IRAG Working
Group 3: Cell Functionbased Assays (Botham etal., 1997)

Independent(one data set
from CTFA
Phase III
(Gettings et al.,1996)

Independent
for CTFA dataset, unknown
for 2nd data
set. Chemicalcoding

A Summary Report of the
COLIPA
International
Validation
Studyon
Alternatives to the Draize
Rabbit Eye Irritation Test
(Brantom et al., 1997)

Independentincluding
selection ofparticipating
laboratories.

Independent.
ChemicalcodingStudy name
Fluorescein Leakage Test:a Useful Tool in Ocular
Safety Assessment (Cottin
and Zanvit, 1997)

Process of
datacollection
andstatisticalanalysis

In-houseN

Quality of data(in vivo and invitro GLP
compliant)
In vitro: non-GLP;
In vivo: according
to Officiel de la
Republique
Francais,
24/10/1984.
In vitro: GLP
stated inpublication as
unknown
In vivo: GLP(historical data)
In vitro: GLP notstated;In vivo: GLP for
CTFA data; 2nddata set GLP
stated inpublication as
unknown.

Independent

Y protocolcommon to
all labs

In vitro: non-GLP;
In vivo: GLP

In-house

In-house

9

Avail. ofstandard
prediction
model

N

N

Avail. ofstandard
protocol*Data format in
publication

N

Mean FL10
FL20 (mg/ml),
w/o SD or SEM
T0, T24

N

Pearson's
correlation
coefficients forin vitro-in vivo
data T0N

N (INVITTOX
ProtocolNo.120)Pearson's
correlation
coefficients forin vitro-in vivo
data T0
Mean FL20(mg/ml) w/o SD
or SEM T4
from both labssummarised inin vitro- in vivo
correlationgraphsFluorescein Leakage Assay Background Review Document

Study nameEvaluation of a Human
Corneal Epithelial Cell Line
as an In Vitro Model for
Predicting Ocular Irritation(Kruszewski et al.,1997)

EvaluationofthePrevalidation Process: The
Fluorescein Leakage Assay(Phase II) (Southee, 1998)

Evaluation of thePrevalidation Process: The
Fluorescein Leakage Assay(Phase III) (Southee, 1998)
Assessment of Initial
Damage and Recovery
Following Exposure of
MDCK Cells to an Irritant(Clothier et al., 1999)

Study
management

Chemicalselection,
chemicalcoding?

Process of
datacollection
andstatisticalanalysis

In-house

In-house. No
chemicalcodingIndependent

Independent.
No chemicalcodingIndependent

In-house

Independent.
ChemicalcodingIn-house. No
chemicalcodingAvail. ofstandard
prediction
model

Quality of data(in vivo and invitro GLP
compliant)Avail. ofstandard
protocol*In-house; nostatisticalanalysis

N

In vitro: non-GLP,
In vivo: GLP notstatedN

Independent-analyses
performed by
BIBRA.

Y- (basedon Brantom
et al.,(1997))

Independent-analyses
performed by
BIBRA.

In-house10

Y- (basedon Brantom
et al.,(1997))

In vitro: non-GLP
In vitro: non-GLP;
In vivo: GLP
unknown(historical datafrom Company #1 and Gautheron
et al., (1994))

N

In vitro: non-GLP;
In vivo: GLP(historical datafrom ECETOC,(1992))

Data format in
publication

N

Mean FR85(%)
T0
Raw FL20(mg/ml) andmean FL20(mg/ml) T0, T4
summarised ingraphs.

N (INVITTOX
ProtocolNo.120)

Raw FL20(mg/ml) andmean FL20(mg/ml) T0, T4
summarised ingraphs.

N- combined
FL/AB assay

Mean FL15(mg/ml) ± SD;
concentration
response
curves T0

Fluorescein Leakage Assay Background Review Document

Study name

Ocular Irritancy Assessmentof Cosmetics FormulationsandIngredients:
Fluorescein Leakage Test.
(Zanvit et al., 1999)
Comparative Evaluation ofFive In Vitro Tests forAssessing the Eye Irritation
PotentialofHair-care
Products (Jones et al.,
2001)
The Prediction of Human
Skin Responses by using
the Combined In Vitro
Fluorescein
Leakage/Alamar
Blue(Resazurin)
Assay.
(Clothier et al., 2002)

Study
management

Chemicalselection,
chemicalcoding?

Process of
datacollection
andstatisticalanalysis

Independent

Independent.
ChemicalcodingIndependent

In-house

In-house.
Formulations
coded inpublication.

In-house; nostatisticalanalysis

In-house

Independent(provided byvariouscompanies).
ChemicalcodingIn-house

11

Avail. ofstandard
prediction
model

Quality of data(in vivo and invitro GLP
compliant)Avail. ofstandardised
protocol*Data formatinpublication

N (INVITTOX
ProtocolNo.120)

Correlationgraphs for invitro and invivo
classifications.
Mean FL20(mg/ml) values
w/o SD or
SEM for 9
chemicals
only. T0 or T4

Y

In vitro: non-GLP;
In vivo: GLP

N

In vitro: non-GLP;
In vivo: GLP notstatedN

Mean FL%
w/o SD or
SEM T0, T72

Y

In vitro: non-GLP;
In vivo: human
occluded patch
test 1h, 24h

N -combined
FL/AB assay

FL%
summarised
in graphs T0

Fluorescein Leakage Assay Background Review Document

FRAME In-house data

Studymanagement

Study nameCompany # 4 (FRAME,
circa 1991)
FRAMEReportonComparison of 40 Cosmetic
and Domestic Formulations
Supplied by Company # 8
and Evaluated by 3 In Vitro
Cytotoxicity Tests (FRAME,
1992)
Company
1992)

#

5

Company
1993)

#

5

Process of datacollection andstatisticalanalysis

In-house

Independent.
ChemicalcodingIn-house; nostatistical analysis

In-house

Independent.
ChemicalcodingIn-house

Independent.
ChemicalcodingIn-house

Independent.
ChemicalcodingIn-house; nostatistical analysis

In-house

Independent.
ChemicalcodingIn-house; nostatistical analysis(FRAME,

Final Report on Testing of12
Mild
Surfactants
supplied by Company # 5
for Cytotoxicity Testing at
the FAL (FRAME, 1992)

Chemicalselection,
chemicalcoding?

(FRAME,

Avail. ofstandard
prediction
model

Quality of data(in vivo and invitro GLP
compliant)Avail. ofstandardised
protocol*Data formatinpublication
Mean FL20(mg/ml) ±SEM T0, T4,
T24, T48,
T72

N

In vitro: nonGLP

N

In-house; nostatistical analysis

N

In vitro: nonGLP

INVITTOX
Protocol No.71

In-house; nostatistical analysis

N

In vitro: nonGLP

INVITTOX
Protocol No. 71

Raw dataFL20, FL50
(mg/ml), T0
Raw dataFL20, FL50
(mg/ml), T0,
T72

12

N

In vitro: nonGLP

INVITTOX
Protocol No. 82

Raw data FL20 (mMl)
T0, T4, T24,
T48, T72

N

In vitro: nonGLP

INVITTOX
Protocol No. 82

Raw data FL% T0, T4,
T24, T72

Fluorescein Leakage Assay Background Review Document

Study nameFive Company # 5 Baby
Products (FRAME, 1994)
Results from the Fixed
Dose Fluorescein Leakage
In Vitro Cytotoxicity Test on
4 Company # 5 Test
Sample Formulations(FRAME, 1994)
Sainsburys, Effects of 6
Coded Chemicals on MDCK
Cells (FRAME, 1998)

Study
management

Chemicalselection,
chemicalcoding?

Process of datacollection andstatisticalanalysis

Avail. ofstandard
prediction
model

Quality of data(in vivo and invitro GLP
compliant)Avail. ofstandardised
protocol*In-house

Independent.
ChemicalcodingIn-house; nostatistical analysis

N

In vitro: nonGLP

INVITTOX
Protocol No. 82

Data formatinpublication
Raw data FL%, T0, T4,
T24, T48,
T72

In-house

Independent.
ChemicalcodingIn-house; nostatistical analysis

INVITTOX
Protocol No. 82

Raw data FL% T0, T4,
T24, T48,
T72

In-house

Independent.
ChemicalcodingIn-house; nostatistical analysisN

Raw data FL20 (mg/ml)
T0, T72

N

In vitro: nonGLPN

In vitro: nonGLP*. INVITTOX Protocols detailed in parentheses were assigned based on the authors of this BRDs knowledge of the protocol. Many FL
assay protocols described in the literature were accepted as INVITTOX Protocols at a later date. GLP= good laboratory practice; SD=
standard deviation; SEM= standard error of the mean; T= time-point; w/o= without

13

Fluorescein Leakage Assay Background Review Document

2. Test Definition (Module 1)
2.1. Rationale for the proposed test method
2.1.1.

Intended uses/ purpose

The FL assay was designed to measure the chemical-induced loss of trans-membrane
impermeability of a confluent epithelial monolayer. Damage to inter-cellular adhesion
molecules is an important event in chemical-induced eye irritation, and can be modelledusing the FL assay. The FL assay was specifically developed to detect potentially mildand moderate irritants to the human eye, which are often cosmetic products.
The FL assay is performed on a confluent monolayer of non-replicating cells which aregrown on permeable inserts. Madin-Darby Canine Kidney (MDCK) renal tubularepithelial cells are typically used as they form tight junctions and desmosomes similar to
those found on the apical side of conjunctivae and corneal epithelia (INVITTOX Protocol
No. 71). Test materials are applied to the cells growing on the apical side of the insert
for a short exposure, e.g. one minute or 15 minutes. The test material is then removed
and fluorescein dye added to the insert; the amount of dye that passes through themonolayer and insert within a given time is recorded. Due to the short exposure
periods, the FL assay generally measures the effects of relatively high chemical
concentrations. FL assay data differs from many other in vitro cytotoxicity assays whichmeasure the effects of relatively low chemical concentrations and longer exposures oncell viability and replication rates. An advantage of the short incubation period used in
the FL assay is that water-based ingredients and formulations can be tested neat if they
can be easily removed after the short exposure period. This allows more directcomparisons of the FL assay results with the chemical effects in vivo, which is theendpoint assessed by regulatory authorities. As the FL assay can be repeated on thesame cells for up to 72h, delayed effects and recovery can be measured in addition to
immediate acute effects. Recovery is an important part of a chemical’s toxicity profile
which is also assessed by the in vivo ocular irritation test.
Throughout Europe and the USA, the FL assay is used in industry, as a screening step
to detect potential eye irritants, in the early developmental phase of product ingredientsand formulations. Within a test battery, the FL assay is particularly useful for comparing
and distinguishing mild chemicals/formulations with very similar eye irritation potencies(INVITTOX Protocol No. 86). A FL assay protocol is used routinely by Company # 3, forpredicting the eye irritation potential of surfactant-based cosmetic products. Surfactants
are surface-active agents which exert immediate damage at the site of contact, including
damage to cell junctions, which is measured by the FL assay. The data generated using
the FL assay are used by the company for in-house assessments and do not constituteany basis for regulatory testing.
There are four principal FL protocols which have been accepted as INVITTOX
Protocols; ‘The Fluorescein Leakage Test’ (INVITTOX Protocol No. 71), ‘Fixed Dose
Procedure for the Fluorescein Leakage Test’ (INVITTOX Protocol No. 82), ‘Transepithelial Permeability Assay’ (INVITTOX Protocol No. 86), ‘The Fluorescein Leakage
Test -SOP of Company # 4 (INVITTOX Protocol No. 120) (Annex I). With the exceptionof INVITTOX Protocol No. 82, these protocols have featured, or provided the basis for
similar protocols, in large-scale international validation and/or evaluation studies; The
EC/HO International validation study on alternatives to the Draize eye irritation test
(Balls et al., 1995), CTFA Evaluation of alternatives program: an evaluation of in vitro
14

Fluorescein Leakage Assay Background Review Document

alternatives to the Draize primary eye irritation test (Gettings et al., 1996), the European
Cosmetic Toiletry and Perfumery Association (COLIPA) study on alternatives to the
Draize rabbit eye irritation test (Brantom et al., 1997), the Interagency Regulatory
Alternatives Group (IRAG) Working Group 3: Cell-function based assays (IRAG)(Botham et al., 1997). These studies assessed many in vitro assays, including the FL
assay, to determine their abilities and applicabilities for replacing the current in vivo
method for predicting chemical-induced in vivo eye irritation potential in humans. The
studies evaluated in vitro assays which measured acute effects, rather than chronic
effects or recovery.
2.1.2.

Regulatory rationale and applicability

No FL assay protocol has been validated as a regulatory test for predicting in vivo ocular
irritation. Scientific consensus is that no single in vitro cell-based test can replace thecurrent in vivo test for eye irritation testing. The FL assay is recommended for use
within a test battery of other in vitro tests for eye irritation, to enable the range ofpotencies and mechanisms of chemicals in vivo to be covered.
The validity of the FL assay for predicting chemical-induced in vivo eye irritation
potential, led to it undergoing prevalidation (Southee, 1998). The prevalidation process
took place in three phases; Phase I for protocol refinement, Phase II for protocol
transfer, and Phase III for protocol predictivity. Phase I was successful in determining asingle and appropriate FL assay protocol for testing in the later phases. A number ofproblems prevented the chosen FL assay protocol entering validation although it was
concluded that the assay showed some relationship to the in vivo MMAS data with goodinter-laboratory variability. On the basis of this study, the tested protocol was accepted
as INVITTOX Protocol No. 120.
2.1.3. Scientific basis for the test (mechanistic)
The FL assay was developed by Tchao (1988) as a model for detecting materials thatare potentially irritating to the eye. In vivo, the tight junctions and desmosomes of thecorneal epithelium prevent solutes and foreign materials moving into the cornea.
Solutes in the cornea can induce water to move by osmosis into the cornea, thus
causing oedema. Loss of trans-epithelial impermeability, due to damaged tight junctions
and desmosomes, is one of the early events in chemical-induced ocular irritation.
A
confluent layer of MDCK cells consists of inter-cellular tight junctions anddesomosomes. The confluent monolayer used in the FL assay is non-proliferating
which models the non-proliferating state of the in vivo corneal epithelium. Whilst
desmosomes maintain cell to cell adhesion, tight junctions form between adjacent cells
and form a permeability barrier that can prevent the movement of molecules as small as
350MW. Tight junctions are found at the apical surfaces of conjunctiva, corneal andskin epithelia. It is assumed that a significant part of ocular irritation is related to thestate and ability of the corneal epithelium to act as a barrier against foreign andpotentially irritant materials (Botham et al., 1997). Increasing the permeability of thecorneal epithelium in vivo has been found to accompany the inflammation and surface
damage observed when eye irritation develops (Igarashi, et al., 1989; Ward et al.,1997b).

15

Fluorescein Leakage Assay Background Review Document

General FL assay method outline
A confluent layer of MDCK cells is grown on the semi-permeable membrane of an insert;
the inserts are placed into the wells of 96 or 24 well plates (figure 2.1.3.1.).

Figure 2.1.3.1. Diagram of MDCK cells grown on an insert membrane for the FL assay(from Wilkinson, 2006).
MDCK cells grown on porous membranes orientate correctly with prominent apicalmicrovilli and cellular adhesion molecules including tight junctions and desmosomal
junctions. As the MDCK monolayer forms tight junctions similar to those found in thecorneal epithelium, the FL assay is considered an appropriate model for detecting
damage to the tight junctions of the cornea. The cells are exposed to the test material
for a short period (~one minute), which can be sufficient time for damage to occur to thetight junctions and desmosomes of the monolayer. The test material is then removed
and the non-toxic, highly fluorescent sodium-fluorescein dye (Chan and Hayes, 1994) is
added to the apical side of the monolayer for a defined period (~30-60 minutes).
Sodium-fluorescein has a molecular weight of 376.3, which is too large to pass throughthe tight junctions that prevent the passage of molecules with molecular weights ≥350.
Chemicals that disrupt the tight junctions of the monolayer allow sodium-fluorescein dye
to ‘leak’ through the monolayer and the porous membrane into the well. The amount of
sodium-fluorescein that leaks to the basal side of the monolayer, within a specified time
period, is measured. Sodium-fluorescein dye can be measured spectrophotometricallyat 490nm or spectrofluorometrically at excitation and emission respective wavelengthsof 485/530nm.

16Fluorescein Leakage Assay Background Review Document

FL is calculated in reference to readings from a blank control where a confluent
monolayer is not treated and a maximum leakage control where the FL through an insert
without cells is measured (figure 2.1.3.2.).
FLD = [(A-B)/(C-B)] x (MC - MB ) + MB
D = % inhibition.
A = % damage (20% or 50% fluorescein leakage)
B = % fluorescein leakage < A
C = % fluorescein leakage > A
MC = Concentration (mg/ml) of C
MB = Concentration (mg/ml) of B
Figure 2.1.3.2. Equation for the calculation of the chemical/formulation concentration
causing 20 percent FL (FL20) (from INVITTOX Protocol No. 71).
FL20 is the endpoint typically used in the various FL assay protocols. Specific damage
to the cellular adhesion molecules occurs before damage to cell membranes and intracellular contents. Thus, the chemical concentrations inducing damage predominately tothe tight junctions are possibly more accurately identified by FL20 rather than FL50, which
is more likely to include non-specific cell damage. The amount of FL is proportional tothe chemical-induced damage to the tight junctions, desmosomes and cell membranes.
The damage to the tight junctions and desmosomes, induced by a test material gives anindication of its eye irritation potential in vivo. The tight junctions of MDCK cells cansometimes be restored following initial damage, if returned to fresh culture medium(Clothier et al., 1994). As the sodium-fluorescein dye is non-toxic at the concentration
used, the cells can be re-cultured after the FL assay has been performed. The FL assay
can then be repeated at later time-points in order to assess recovery following the initial
exposure to the test material. The speed at which FL reduces following the chemical
exposure, relates to the rate at which the monolayer re-gains trans-epithelial
impermeability. Recovery is an important and relevant endpoint to assess as rapid
recovery of trans-epithelial impermeability in vivo prevents further exposures andsecondary insults.
Like other cell based assays, the FL assay is reported not to have a good predictive
capacity for test materials that have high acidity or alkalinity, fixative properties andreactivity with medium contents. The ocular irritation potential of materials that arewater soluble and/or where the toxic effect is not affected by dilution are generally
predicted accurately using the FL assay. In comparison to other in vitro tests, a concern
unique to the FL assay is that damage to the insert membranes can occur due to themultiple rinsing steps which feature in most protocols. Routine observations of the cellson the insert membranes throughout the exposure and the recovery phases should
detect damaged confluent layers and/or insert membranes and prevent misinterpretation of results.

17

Fluorescein Leakage Assay Background Review Document

Table 2.1.3. Summary of the physicochemical properties that the FL assay is (in)capableof modelling.

Physicochemical Property
AlcoholFixative
Extreme pH
Gases
Liquids
Solid materials
Emulsions
Granular materials
Suspensions
Coloured materials
Toxicity affected by dilution
Highly viscous materials
Volatile materials
Reactive chemistries
Hydrophobic/lipophilic chemicals
Neat concentrations of chemicals
MW > 350

FL assay capable of testingmaterials with this physicochemical
property?
YNYN
Y (if aqueous soluble)
Y (if aqueous soluble, but cannot be
tested in its solid form)Y
Y (if forms stable emulsion)
Impaired***Impaired**N*
Impaired
ImpairedN
NYN* the FL assay is unable to measure the toxicity of chemicals that have basic toxic
mechanisms which are affected by dilution.
** the FL assay is able to measure coloured materials which can be fully removed fromthe insert following the chemical exposure and therefore do not interfere with OD
readings.
*** solid materials suspended in liquid have the propensity to precipitate out and the final
concentration exposed to cells can be difficult to determine.
The FL assay has predominately been used to test surfactant and surfactant-basedmaterials. The TEP assay (INVITTOX Protocol No. 86) is used in-house at Company #3 for testing surfactants and surfactant-based formulations only. The FL assay protocolfeatured in the COLIPA study was used to test only surfactants and surfactant-based
formulations as a prediction model (PM) was only available for surfactant-basedmaterials (Zanvit et al., 1999). The TEP assay was evaluated in the CTFA study Phase
III, where surfactant-based formulations were tested (Gettings et al., 1996). Surfactants
are found in the majority of cosmetic and house-hold products and therefore constitute awide-ranging and relevant chemical class for ocular irritation testing. Surfactants are
generally mild materials, as determined by Draize test scores, which cause immediate
damage at the site of contact, including damage to the expression of adhesion
molecules. There is limited data regarding the predictive capacity of the FL assay forother chemical classes. Shaw et al., (1990, 1991) reported a good correlation between
FL20 results and in vivo ocular irritation data for 22 chemicals, which included 13
chemicals also tested in the EC pilot study (anon, 1991). Clothier et al., (1994) statedthat the EU risk phrase classifications for the 21 chemicals tested in the EC pilot study
were accurately predicted using the Fixed Dose FL assay (INVITTOX Protocol No. 82).
18

Fluorescein Leakage Assay Background Review Document

As the cells used in the FL assay are representative of corneal epithelial cells only, theassay is limited in the range of it’s predictive capacity. Although the FL assay does notmodel all types of possible corneal ocular irritation measured by the in vivo test, i.e.
damage to the corneal stroma, it can measure effects that impact on chemical-induced
stromal damage. For example, sodium dodecyl sulphate (SDS) is hypothesised tocause corneal opacity by permeating across the corneal epithelium to the stroma(Tchao, 1988).
In summary, the FL assay has many features rendering it a suitable as an in vitro model
for predicting in vivo ocular irritation, e.g., relevant, cell types, chemical concentrations,
exposures, and endpoint. The FL assay is particularly advantageous in that it allows
effects to be measured which occur prior to, or even independent of cell death. Often,cell death does not occur in the cases of mild irritation and therefore a sensitive assay
like the FL assay is essential. The assay is particularly useful as it allows recovery to bemeasured up to 72h following an initial exposure.
2.1.4. Similarities and differences of modes of action in the test method and thereference species
Most regulatory authorities require eye irritation data from the in vivo ocular irritation testdeveloped by Draize et al., (1944), e.g. OECD TG 405 (2002), EU Commission Directive
2004/73/EC (2004). Generally, the albino rabbit is the reference animal for in vivo eye
irritation testing; healthy young adult male and female albino rabbits are used. The
albino rabbit is used due to its relatively large eyes which make adverse effects easier toobserve. Rabbits are used despite their ocular physiology known to be considerably
different from that of humans.
The Draize test for ocular irritation (Draize et al., 1944) subjectively scores the chemicalinduced effects, specific to the cornea, conjunctiva and iris (figure 2.1.4.). The corneal
epithelium is responsible for excluding foreign material from the surrounding
environment and is therefore an important factor in protecting the eye from irritantresponses (Curren and Harbell, 1998). An in vitro system which models the barrier
function of the corneal epithelium, such as the FL assay is required if the Draize test is
to be replaced. In the Draize scoring system, the cornea has the greatest weighting
ascribed to the scores, indicating that these effects are the most important in terms of
the resulting overall chemical-induced eye irritation; the number of days for the effects toclear from all the different tissues is also scored (figure 2.1.4.).
The conjunctiva is the external layer of the eye which is most vulnerable to the external
environment. It is a non-keratinised squamous epithelium with secreting cells which
covers the exposed surface of the eyeball and the inner surface of the eyelids (Atkinson,
1993; Curren and Harbell, 1998). It also has glands which provide moisture and secrete
the components of the tear film. Foreign materials in the conjunctiva can induce
inflammation. Inflammation is noted when the network of blood vessels in the
conjunctiva dilate and produce an appearance of increased redness. Oedema can thenoccur due to changes in the capillary permeability of the blood vessels which allows
fluids to leak into the interstitial spaces. Severe oedema can occur to an extent which
impairs closure of the eye lid. Mild irritants can cause conjunctivitis without coupleddamage to the cornea (Curren and Harbell, 1998). Despite the different degrees ofconjunctival damage that can occur, chemical-induced effects have less weighting than

19

Fluorescein Leakage Assay Background Review Document

the cornea and iris, in the Draize scoring system. This is primarily due to the reversibilityof chemical-induced effects to the conjunctiva.
The iris is situated under the cornea within aqueous humour. It is a vascular structure
comprised of loose connective tissue, muscle and pigmented cells (Atkinson, 1993).
Through muscular control of pupil dilation it regulates the amount of light that enters theeye and reaches the retina. Irritation can cause the vessels to dilate and leak vascular
fluid which can cause oedema. Protein leakage from the vessels can cause aqueous
flare which alters the refractive index of the aqueous humour (Atkinson, 1993). Severe
damage can cause iris tissue destruction, resulting in the iris being unresponsive to light(Curren and Harbell, 1998).

20

Fluorescein Leakage Assay Background Review Document

I. Cornea
A.Opacity-Degree of Density (area which is most dense istaken for reading)
No opacity
Scattered or diffuse area-details of iris clearly visible
Easily discernible translucent areas, details of iris slightly obscured
Opalescent areas, no details of iris visible, size of pupil barely discernible
Opaque, iris invisible
B.Area of Cornea Involved
One quarter (or less) but not zero
Greater than one quarter, less than one-halfGreater than one half, less than three quarters
Greater than three quarters, up to whole area

0
1
2
3
4
1
2
3
4

Score equals A x B x 5 Total maximum = 80
II. Iris
A.Values
NormalFolds above normal, congestion, swelling, circumcorneal injection(any one or all of these or combination of any thereof), iris still reacting
to light (sluggish reaction is positive)
No reaction to light, haemorrhage; gross destruction (any one or allof these)
Score equals A x 5

01
2

Total maximum = 10

III. Conjunctivae
A.Redness (refers to palpebral conjunctivae only)
Vessels normal
0
Vessels definitely injected above normal
1More diffuse, deeper crimson red, individual vessels not easily discernible 2
Diffuse beefy red
3B.Chemosis
No swelling0
Any swelling above normal (includes nictating membrane)
1
Obvious swelling with partial eversion of the lids
2
Swelling with lids about half closed
3
Swelling with lids about half closed to completely closed
4
C.Discharge
No discharge
0
Any amount different from normal (does not include small amount observedin inner canthus of normal animals)
1
Discharge with moistening of the lids and hairs just adjacent to the lids
2
Discharge with moistening of the lids and considerable area around the eye3
Score (A + B + C) x 2

Total maximum = 20

Figure 2.1.4. Weighted scoring system for ocular lesions (from Draize et al., 1944).

21

Fluorescein Leakage Assay Background Review Document

Although cell-based in vitro models are unable to attain the complexity of the in vivo
situation, a number of cell lines that are not ocular in origin have been found to produce
cytotoxic data, that can be used to predict the in vivo eye irritation potential of test
materials (Borenfreund and Borrero, 1984).
At an ECVAM Expert Meeting (Scott et al., (under preparation)) four different types of
eye irritation processes were proposed as; membrane lysis, coagulation, saponification,and reactive chemistries. A list is given of the events that occur in ocular irritation which
also incorporates the eye irritation processes proposed (Scott et al., (under preparation))(in bold font); the latter effects are the most severe (table 2.1.4.1.). Loss of transepithelial permeability is an important step in the eye irritation process, and it occurs
before those mechanisms that were highlighted at the ECVAM Expert Meeting (Scott etal., (under preparation)).
The extent and duration of loss of trans-epithelial
impermeability impact on the mechanisms defined by Scott et al., (under preparation).

22

Fluorescein Leakage Assay Background Review Document

Table 2.1.4.1. Summary of the events involved in chemical-induced eye irritation in vivo
which are (not) modelled by the FL assay.
Modelled by
Event involved in chemical-induced eye irritation
the FL assay
Chemical interaction with tear film (Klyce and Beuerman,
1988; Hackett and McDonald, 1994)
N
Chemical binding to the conjunctival epithelium (Hackett
and McDonald, 1994; Hogan and Zimmerman, 1962)
N
Adhesion molecules compromised (Farquhar and Palade,
1963; van Meer et al., 1992; Katahira et al., 1997)
Y
Corneal epithelium damage (Dua et al., 1994)
Y* inhibition of receptor-mediated membranetransport (Dearman et al., 2003)
Y* compromise of cell membrane integrity of upper
corneal epithelium (Dua et al., 1994, Hackett and
McDonald, 1994: Maurer and Parker, 1996)Y* cell membrane lysis of all corneal epithelium
layers (Hackett and McDonald, 1994)
Y
Hydration of corneal stroma (Hackett and McDonald,1994).
N
Cross-linking of proteins in corneal stroma (Butler andHammond, 1980; Eurell et al., 1991; Chan and Hayes,
1994)
N
Erosion of corneal stroma (Baldwin et al., 1973; Hackett
and McDonald, 1994; Maurer et al., 1997)
N
Cell damage to corneal epithelium and limbus (Jacobs
and Martens, 1990; Wilhelmus, 2001)
N
Dilation and increased lymphatic leakage from scleral
vasculature (oedema and erythema) (Hackett and
McDonald, 1994)
N
Stimulation of nerve endings, i.e. enhanced blinking,tearing (Chan and Hayes, 1994)
N
Erosion of nerve endings in cornea and sclera (Butler andHammond 1980; Klyce and Beuerman, 1988; Araki et al.,
1994)
N
Duration of response, i.e. length of time cell responses
deteriorate. Duration of response covers the effects ofreactive chemicals which can cause coagulation,
saponification, that are effects which develop andincrease over time. (Hubert, 1992; Maurer and Parker,
1996)
Y
Recovery from response, i.e. length of time for cell
responses to return to control levels (Hubert, 1992)
Y
Text in italics indicates irreversible responses.
Based upon these defined effects (table 2.1.4.1.) the FL assay is capable of detecting
chemicals that induce the third and fourth elements of irritation described. Due tofundamental differences between the in vitro and in vivo situation, e.g., in vitro the cells
are grown as a monolayer, and in vivo the cornea consists of 5-7 epithelial layers and23

Fluorescein Leakage Assay Background Review Document

the conjunctiva 2-3 epithelial layers, care must be taken when interpreting the in vitro
data. In vitro cell-based assays cannot model chemical-induced effects to the tear film.
In vivo, chemical interaction with the tear film modifies the chemical concentration andexposure duration to the cornea. As the cornea is the tissue with the greatest weighting
for effects in the Draize scoring system, the effect of the tear film has an important effecton chemical induced eye-irritation. Similar to all in vitro methods, systemic effects suchas hormones, blood flow, and the immune system on chemical-induced toxicity cannot
be replicated.
In addition to the immediate chemical-induced effects measured by the FL assay, it isalso capable of measuring reversibility, delayed effects and recovery (Clothier et al.,1994). In vivo, the corneal epithelium has demonstrated recovery following exposure to
mild irritants (Clothier and Sansom, 1996). Recovery is an important aspect of the eyeirritation process that does not always relate to the degree of initial insult (Clothier et al.,1994). Use of the non-toxic (at the concentrations used) sodium-fluorescein dye allows
recovery of the impermeable monolayer to be assessed. In vitro, recovery has also
been observed in the FL assay over a 72h period following initial chemical exposure.
Following the initial chemical insult, recovery is assessed by the reduction of FL overtime. The ability of the FL assay to measure delayed effects and recovery is a uniqueproperty of this cell-based in vitro model. Recovery measured by the FL assay allows
comparisons to the in vivo Draize test which measures ‘days to clear.’A proposal for categorising the principal mechanisms of common classes of test
chemicals into the four categories of mechanisms that can cause ocular irritation wasmade during an ECVAM Expert Meeting for testing strategies (Scott et al., (underpreparation)) (table 2.1.4.2.).
Table 2.1.4.2. Examples of chemical classes and their effects through the different
mechanisms categorised (Scott et al., (under preparation)).
MembraneLysissurfactants (all)organic solvents
ketonesalcohols
volatile liquids
etherspolyethers
estersaromatic amines

Coagulation
acids-concentrated-diluted-derivatives
cationic
surfactantsorganic solvents

Saponification
alkaline materials-concentrated-diluted-derivatives

Reactive
Chemistries
peroxides
mustards
alkyl halides
epoxidesbleaches(oxidisers)

The majority of chemical classes exert membrane lysis (table 2.1.4.2.). In some cases,
a chemical class can have more than one mechanism of action, i.e. cationic surfactants
can cause cell damage via coagulation and membrane lysis (table 2.1.4.2.). Thus, theFL assay, which specifically detects membrane damage, should be capable of detecting
such eye irritants regardless of the different chemical classes, but dependent on
concentration. The chemical types for which the FL assay, and most cell-based in vitro
assays, cannot measure potential eye irritation are generally those that are strong,24

Fluorescein Leakage Assay Background Review Document

acids, alkalis, fixatives, and viscous. These chemicals have mechanisms that are notmeasured by the FL assay, e.g. extensive coagulation, saponification or specific reactive
chemistries. Chemicals known to have a high ocular irritation potential, through testingfor skin irritation, are normally assumed to be strong eye irritants, without additional invivo testing specifically for eye irritation. The FL assay, designed to detect sub-lethal
damage to cell membranes and tight junctions, is an appropriate in vitro test for
detecting mild ocular irritants. Assuming that the FL assay is able to measuremembrane damage induced by all the chemical classes which cause membrane lysis(as defined by Scott et al., (under preparation)), the assay will have a large and relevant
applicability domain. Overall, the FL assay allows for the testing of eye irritation induced
by a wide range of chemicals.
The sodium-fluorescein dye used in the FL assay is non-toxic at the concentrations usedand has also been used in humans to assess damage to the corneal epithelium(INVITTOX Protocol No. 71). In vivo, chemicals can adversely affect cell adhesion
molecules, which then allow fluorescein to penetrate into the stroma. Fluorescein
detected in the stroma indicates damaged areas of the epithelium (Igarashi, 1986; Chanand Hayes, 1994). FL not only occurs due to damaged adhesion molecules but also
due to membrane damage. In this context, the FL assay does measure one of themechanisms of eye irritation defined at the ECVAM Expert Meeting (Scott et al., (underpreparation)) that cannot be distinguished from the FL due to impaired adhesion
molecules. In order to determine the type of cell damage that has led to trans-epithelial
permeability, results from the FL assay have been compared with results from the
Neutral Red Release (NRR) assay which specifically measures membrane damage(Hubbard et al., 1994).
The effects of certain physiological features of the eye such as the tearing system, on
resulting toxicity, are difficult to identify. Inherently variable lachrymation rates affect theconcentration and duration of chemical exposures in the eye. It is difficult to identify
actual in vivo exposure durations and concentrations, and replicate them for in vitro
testing. It is estimated that following an accidental exposure, the foreign material islikely to remain in the eye for approximately 30 seconds before it is removed by blinking,tear formation and rinsing. In comparison to humans, rabbits have a slower blink rate
and a less efficient tearing system (from Europeans for Medical Progress,www.curedisease.com: Problems with the Draize Test, accessed 23.08.05), which
results in longer exposure durations in the rabbit eye in comparison to humans. The
exposure period used in the FL assay is a compromise between reproducing theprobable length of time of an in vivo accidental chemical exposure, whilst also allowing
sufficient time for experimental procedures to be performed reproducibly. In comparison
to most other cytotoxicity assays for eye irritation, the short exposure period allows theeffects of high chemical concentrations to be measured, thus replicating the in vivo test
situation in the rabbit. Unlike the in vivo situation, solids cannot be tested in the FL
assay unless suspended in a liquid vehicle.
A problem associated with the short exposure period of the FL assay is the difficulty ofefficiently removing the test materials after the short exposure period. This isparticularly true for viscous materials, such as gels and creams which are the type ofmaterials often tested using the FL assay. Due to the short exposure period, the mild
materials often need to be tested neat in order to produce a response which can bemeasured. Therefore the problems associated with viscous materials cannot be
reduced by dilution. Test materials can also bind to the insert membrane, thus making
25

Fluorescein Leakage Assay Background Review Document

their removal very difficult. Chemical binding to the insert membrane, is more common
for cationic materials, such as benzalkonium chloride, which are attracted to thepositively charged membrane (Balls et al., 1995). Increased washing steps to remove
the test material from the insert can also lead to insert membrane damage and thus
erroneous results. Alternatively, if test materials are not removed fully and/or efficiently,
they can potentially physically block the passage of the sodium-fluorescein through theinsert, which would cause chemical effects to be under-estimated. In general, additional
uncontrolled exposure time is a greater proportion of the short exposure period of the FL
assay, than with assays with longer exposures. This leads to greater variability inresults, and low assay reproducibility. As the FL assay can be repeated at multiple timepoints, erroneous results due to ineffective removal of the test material would be more
likely detected in comparison to cell viability assays which use single time-points. The
efficient removal of test agents from the eye is also a concern of the in vivo test.

26

Fluorescein Leakage Assay Background Review Document

2.2. Test method protocols
The major components of the test method protocol(s) featured in the literature and inhouse studies are shown in chronological order and/or associated studies (table 2.2.).
Full protocol information is provided in Appendix II
Table 2.2. Test method protocols
INVITTOX Protocols

Fluorescein
Leakage
Test-INVITTOX
Protocol No. 71

Fixed
Dose
FLT
-INVITTOX Protocol No.
82

Trans-epithelial
permeability (TEP)
Assay -INVITTOX
Protocol No. 86

Cell Type/Strain

Based on Tchao (1988)
MDCK CB997 (ECACC:
84121903)

Based on FLT INVITTOX
Protocol No. 71
MDCK CB997 (ECACC:
84121903)

Validatedin-house
Company # 3
MDCK NBL-2 (ATCC:
CCL 34)

Seeding(cells/ml)

4x105(400µl/insert)

4x105(400µl/insert)

N –but cells have beenused upto passage 40

5x105
cells/ml(200µl/insert)
Cells used after threepassages post-thawing;
no upper limit stated96
Millicell-HA
12mm(0.45µm
pore
size;
Anocell 10 inserts canalso be used)

96

48

AnoporeTM 10; 0.2µm or
0.02µm pore size

Costar 6.5mm Transwell
tissue culture inserts;
0.45µm pore size

Duration of Exposure(mins)

1 or 15

1

15

Exposure at RT?

Y

N -incubator

Study/Company/
Organisation

Protocol Used/Basis

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)andcells/mlN

cells/ml

Type(s) of Materials
tested?

N/A

not stated
Chemicals from Comm.
of European Commun.
collaborative study on theevaluation of alternative
methods to the eyeirritation test (anon, 1991)

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

0.1mg/ml Na-fluorescein
in HBSS (0.01% (w/v))(0.02% Na-fluorescein in
HBSS for Anocell inserts)

0.02% Na-fluorescein in
HBSS

0.02% Na-fluorescein in
HBSS

Time
allowedfor
Fluorescein Leakage(mins)

30 RT

60 RT

Maximum
Leakage
Control(insertwithout cells) (Y/N)

Y

Y

30 RT
N
-materialconcentrations attain aplateauof
maximumleakage

Negative Control

HBSS

HBSS

HBSS

Positive Control

100mg/ml Brij 35

130mg/ml glacial acetic
acid

None -to be selected byCompany # 3

27

In-house
testingsurfactant-basedmaterialsofFluorescein Leakage Assay Background Review Document

Endpoint (e.g. FL10,
FL20, %FL)

FL20, FL50 (mg/ml) T0; if
FL20 is not reached themax FL% should bequotedalongside
concentration
causingleakage

FL% T0Recovery
Timepoint(s)Following
Initial Exposure (h)N4, 24, 48 and 72 (underconsideration)

Prediction ModelN

Y -50mg/ml differentiatedirritants from non-irritants

28

EC50 % T0

N
EC50 <1.8% fail; EC50
≥2.2% pass. EC50 1.8%
-2.2% =borderline.

Fluorescein Leakage Assay Background Review Document

Study/Company/
Organisation

Protocol Used/Basis
Cell Type/Strain
Seeding(cells/ml)

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)andFLT -SOP Company # 4
INVITTOX Protocol No.
120(developedasconsequence of ECVAM
Prevalidation
Study,
1998)
Based
on
FLT
INVITTOX Protocol No.
71
MDCK NBL-2 (ATCC:
CCL34)
4x105
cells/ml(500µl/insert=
2x105
cells/insert)
3-30.
96
Millicell-HA
12mm(0.45µm pore size)

Duration of Exposure(mins)

15

Exposure at RT?

Y

Type(s) of Materials
tested?

surfactants
surfactant-based
formulations

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

0.01mg/ml Na-fluorescein
in HBSS (0.001% (w/v/)

Timeallowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

and

30 RT
Y

Negative Control

HBSS

Positive Control

Endpoint (e.g. FL10,
FL20, %FL)

0.16mg/ml SDS in HBSS
FL10, FL20 (mg/ml) T0,
T4.
Even if FL10 isattained,maxFL%should be quoted withconcentration
causingFL.

Recovery
Timepoint(s)Following
Initial Exposure (h)429Fluorescein Leakage Assay Background Review Document

Prediction Model

PM from COLIPA study
for surfactants only; FL20T4>100mg/ml =nonirritant/slight, MMAS < 15;
20-100mg/ml = moderate,
MMAS 15-30; <20mg/ml=
irritant/severe,
>
30
MMAS

30

Fluorescein Leakage Assay Background Review Document

Studies Reported

Study/Company/
Organisation

Trans-epithelial
PermeabilityofFluorescein In Vitro as
an Assay to Determine
Eye Irritants (Tchao,1988) Poster

Protocol Used/BasisN

Cell Type/Strain
Seeding(cells/ml)

Insert Type(s)Pore Size(s)andDuration of Exposure(mins)
Exposure at RT?

Predicting
OcularIrritancy and Recovery
from
Injuryusing
Madin-Darby
CanineKidney Cells (Shaw etal., 1991)

MDCK

MDCK
CB997
(ECACC: 84121903)

MDCK

1.5x10 cells/insert

105 cells/insert (in 400µl
medium)

Cells
grown
confluenceNnot stated

not stated96

96

Anocell 10

Anotec 10mm porous
tissue culture insert

1

1
not stated16
chemicalswithvarious mechanisms andpotencies.

Density

PassageRange
defined?
Timetoattainconfluency? (h)LossofTransepithelial
Impermeability of a
Confluent Layer ofMadin-Darby
CanineKidney (MDCK) Cells
as a Determinant ofOcularIrritancy
Potential (Shaw et al.,
1990)

5

72
Millicellwith(surfactant-free)membrane,diameter

-

to

HATF
12mm

15
o

Type(s) of Materials
tested?

surfactants

not stated
Chemicals with range of
mechanistic
activities
and potencies; 13 takenfrom EC pilot study

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

0.02% Na-fluorescein in
HBSS

0.02% (w/v) fluorescein
in HBSS

0.02% (w/v) fluorescein
in HBSS30, 24oC

60 RT

60 RT

Not statedY

Y

Negative Control

HBSS

distilled water

distilled water

Positive Control

N/A

not stated

not statedEndpoint (e.g. FL10,
FL20, %FL)

Preliminary experiments-increasesinFL
observed

FL20 and FL50 (mg/ml)
T0

FL20 (mg/ml) T0

Recovery
Timepoint(s)Following
Initial Exposure (h)

N

N

72

Prediction Model

N

N

N

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control (insert without
cells) (Y/N)

24 C

31

Fluorescein Leakage Assay Background Review Document

Company # 4 Inhouse
Fluorescein
Leakage Test SOP(1992)

Human Corneal
Epithelial Primary
Cultures and Cell Lines
with Extended Life
Span: In Vitro Model for
Ocular Studies (Kahn
et al., 1993)
Stated measurements
were according to
Tchao (1988)
Human corneal
epithelial cell line

Study/Company/
Organisation

Company # 4 (FRAME,
circa 1991)

Protocol Used/Basis
Cell Type/Strain

Shaw et al., (1990)
MDCK CB997 (ECACC:
84121903)

Seeding(cells/ml)

1x106 cells/cm2 insert

Company # 4 (1992)
MDCK
CB997
(ECACC: 84121903)
cells/ml
4x105(500µl/insert=
2x105
cells/insert)

Shaw et al., (1990)

3-30.

1-5

72-96

96

Not statedAnotec 10

Millicell-HA
12mm,
0.45µm pore size

Duration of Exposure(mins)

1

15

Cellagen disks CD24
N/A –assay used todetermine theimpermeability of layer

Exposure at RT?

Shaw et al., (1990)

not stated

Not stated

Type(s) of Materials
tested?

4 surfactants and 3
Company # 4 products

N/A

N/A

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

200mg/L

10µg/ml Na-fluorescein
in HBSS

0.02% Na-fluorescein
in PBS

60 RT

30 RT

30

Y

Y

Not stated

not statedDensity

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)

and

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)
Negative Control

2.4x105 cells/cm2

Positive Control

acetic acid

HBSS
0.16mg/ml
HBSS

Endpoint (e.g. FL10,
FL20, %FL)

FL20, FL50 (mg/ml)

FL20 (units not stated)
T0, T4

FR%

Recovery
Timepoint(s)Following
Initial Exposure (h)724, 24, 48, 72

N/A

Prediction Model

N

N

N

32

N/A
SDS

in
N/A

Fluorescein Leakage Assay Background Review Document

DevelopmentofaFixed Dose Approachfor The Fluorescein
Leakage Test. (Clothieret al., 1994)

Study/Company/
Organisation

Cell Type/Strain

Method according to
Shaw et al., (1991)
MDCK(methodaccording to Shaw et al.,(1991))

Seeding(cells/ml)

Density

Method according
Shaw et al., (1991)

Passagedefined?

Range

Protocol Used/Basisto

Investigations of the
MDCK
Permeability
Assay as an In Vitro
Test of Ocular Irritancy(Gautheron et al., 1994)

Use
of
In
Vitro
Methodology to Predict
Irritancy Potential of
Surfactants (Hubbard
et al., 1994)

Tchao (1988)

Shaw et al., (1990)

MDCK
Laboratories)(Flow
Shaw et al., (1990)

1.8-2 x105 cells/insert

Shaw et al., (1990)

not stated

Shaw et al., (1990)

Time
toattainconfluency? (h)Method according to
Shaw et al., (1991)‘based on exposure of aconfluent layer of MDCK
cells’

96

Shaw et al., (1990)

Insert Type(s)Pore Size(s)andMethod according
Shaw et al., (1991)

toMillipore HA

Shaw et al., (1990)

Duration of Exposure(mins)

to15

1

Exposure at RT?

Method according
Shaw et al., (1991)
Method according
Shaw et al., (1991)

Y

Type(s) of Materials
tested?

21 chemicals from the
EC/HO study

42 chemicals with rangeof chemical structuresand irritancy potential

Shaw et al., (1990)
6 commercially available
surfactants,
11
formulations (4 baby
shampoos and 7 bathproducts)

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

Method according
Shaw et al., (1991)

0.2mg/ml Na-fluorescein
in HBSS (pH adjusted to
pH 7.4)

Shaw et al., (1990)

30 RT

Shaw et al., (1990)

Y

Shaw et al., (1990)

HBSS pH adjusted to 7.4

Shaw et al., (1990)

Not stated

Shaw et al., (1990)

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

to

to

60 RT
to

Negative Control

Method according
Shaw et al., (1991)
Method according
Shaw et al., (1991)
Method according
Shaw et al., (1991)

toPositive Control
Endpoint (e.g. FL10,
FL20, %FL)

FL% T0FL20, FL50 (mg/ml) T0

FL50 (mg/ml) T0; FL%
T0

Recovery
Timepoint(s)Following
Initial Exposure (h)

72

N/A

24, 48

to33

Fluorescein Leakage Assay Background Review Document

Prediction Model

50mg/ml hypothesised tobe the cut-off point todistinguish
R36/R41
chemicals from NI if
FL20% was taken to
indicatesignificant
toxicity.

N

34

N

Fluorescein Leakage Assay Background Review Document

The
EvaluationofPesticide
Ingredientsand Formulations In
Vitro and Correlations
with In Vivo Data
(Clothier et al., 1995)

Study/Company/
Organisation

The EC/HO International
Validation Study on
Alternatives
totheDraize Eye Irritation
Test (Balls et al., 1995)

Protocol Used/Basis

According(1988)

Cell Type/Strain

MDCK (strain not stated)

INVITTOX Protocol No.
71
MDCK(INVITTOX
Protocol No. 71)

Seeding(cells/ml)

not stated

1x107 cells/ml

to

Tchao

Density

PassageRange
defined?
Timetoattainconfluency? (h)Effects of Surfactant
Re-treatment In Vitro:
A Method to Evaluate
Changesin
Cell
Junctions and Cell
Viability.
(Clothierand Sansom, 1996)
INVITTOX Protocol No.
71 (combined with AB
assay)MDCK CB997
60µl of 2x105 cells/ml
added to 7 or 8 wells ofeach strip

not stated

INVITTOX Protocol No.
71
INVITTOX Protocol No.
71

Millipore HA

Anopore 10

Anoporemembrane,
0.2µm pore size1
not stated
60 chemicals ranging inmechanisms and potency
for which historical in vivo
data was available- dataprimarily from ECETOC
database (1992)

1
INVITTOX Protocol No.
71

1
INVITTOX Protocol No.
71 and No. 80

Pesticideformulations,vehicles w/o pesticide
ingredients, and pure
pesticides.

CAPB

not stated

200mg/l Na-fluorescein in
PBS

0.01% Na-fluoresceinin a 1:10 dilution of AB
in HBSSnot stated

60 (RT unknown)

60 (incubator)

Y

Y

Y

Negative Control

not stated

not stated

Positive Control

not statedacetic acid

HBSS
INVITTOX Protocol No.
71 and No. 80

Endpoint (e.g. FL10,
FL20, %FL)

FL20 (mg/ml)

FL20 (mg/ml) T0, T72

FL% T0 AB% T072(initial), then 1, 24, 72
after 1st and 2ndtreatments

Insert Type(s)Pore Size(s)not statedandDuration of Exposure(mins)
Exposure at RT?

Type(s) of Materials
tested?
Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)
Timeallowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

Recovery
Timepoint(s)Following
Initial Exposure (h)

N/A

35

passage range 13<24
used
48-72

Fluorescein Leakage Assay Background Review Document

Prediction Model(not applied to publishedresults) FL20 values:<100mg/ml= R36 or R41;>750mg/ml= NI; 100750mg/ml= R41 if norecovery after 72h, R36 if
recovery after 72h

N

36

N

Fluorescein Leakage Assay Background Review Document

CTFA Evaluation of
Alternatives Program:
An Evaluation of In
Vitro Alternatives tothe Draize Primary Eye
Irritation Test: Phase III(Gettings et al., 1996)

Evaluation of a Human
Corneal Epithelial Cell
Line as an In Vitro
Model for Predicting
OcularIrritation(Kruszewski
etal.,1997)

EvaluationofChemically
InducedToxicity Using an In
Vitro Human Corneal
Epithelium (Ward et al.,1997b)

none

none

Cell Type/Strain

Based on Tchao (1988)
MDCK NBL-2(ATCC: CCL 34)

HCE-T

HCE-T

Seeding(cells/ml)

1.5x105 cells/insert

not stated

not statedN

1.5-2x105 cells/insert
not stated but cell line
only attains approx 20
passages48
Costar 6.5mm Transwell
tissue culture inserts

168
Cellagen,
14mm
collagen-membrane

168
Cellagen,
14mm
collagen-membrane

15

5

5

Study/Company/
Organisation

Protocol Used/Basis

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s) andPore Size(s)Duration of Exposure(mins)
Exposure at RT?

not stated

37 C

37oC

Type(s) of Materials
tested?

23
surfactant-based
formulations

Surfactant formulations
and twenty chemicals
with various properties

Benzalkonium
SDS,
isopropanol

0.02% Na-fluorescein in
HBSS

0.02%
(w/v)
Nafluoresceininhighcalcium KGM medium
w/o growth supplements

0.02%
(w/v)
fluorescein

30 RT

30 Incubator

30 Incubator

YY

Negative Control

not stated
HBSS -used as nontreated control

high calcium KGM

high calcium KGM

Positive Control

Not stated

Not stated

Not statedEndpoint (e.g. FL10,
FL20, %FL)

EC50 (%) T0

TEP assay relative FR85(%)

TEP assay relative FR85
(%)

24

48

N

N

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)
Timeallowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

Recovery
Timepoint(s)Following
Initial Exposure (h)Prediction ModelN
Regression modelling ofin vitro endpoint and invivo data to enableprediction of MAS values
for any formulationo37

chloride,ethanol,Na-Fluorescein Leakage Assay Background Review Document

IRAG Working Group
3: Cell Function-based
Assays (Botham et al.,1997) LAB A

IRAG Working Group
3: Cell Function-based
Assays (Botham et al.,1997) LAB B

Evaluation of Tissue
CultureInsertMembraneCompatibility in the
Fluorescein
Leakage
Assay (Ward et al.,1997a)

Cell Type/Strain

Based on Tchao (1988)
MDCK(ECACC: 84121903)

Based on Martin andStott (1992)
MDCK(ECACC: 84121903)

Method modified from
Shaw et al., (1990)
MDCK
CB997
(ECACC: 84121903)

Seeding(cells/ml)

not stated

not stated

2x105 cells/insert

not stated

not stated

Method modified from
Shaw et al., (1990)

48-96

48-96

Millicell-HA

Costar Transwell tissue
culture insert

72
Anopore with aluminium
oxide membrane (Nunc),
0.2µm, 0.02µm poresizes; Millicell-CM insert
with
PTFE
Bioporemembrane coated withhuman placental type IV
collagen,0.4µm poresize; Millicell-HA insert
withmembranecomposedof
mixedcellulose esters 0.45µm
pore size (Millipore)

Duration of Exposure(mins)

15

15

1

Exposure at RT?

not stated

RT

Type(s) of Materials
tested?

42 (14 surfactants, 16
alcohols,
12
otherchemicals MAS range,
0-98)

not stated25
surfactant-based
personal care products(MAS 0-40) performed inmodified
Draize(anaesthetic) test and 28
shampooswithundefined modification to
DraizeFluorescein
Concentrationdeliveredtomonolayer (mg/ml)

0.02%
(w/v)
Nafluorescein in HBSS

0.02%
(w/v)
Nafluorescein in HBSS

0.01% Na-fluorescein in
HBSS

30 RT

30 RT

30 RT

not stated

not stated

Y

Negative Control

not stated

not stated

HBSS

Positive Control

not stated

not stated

Method according
Shaw et al., (1990)

Study/Company/
Organisation

Protocol Used/Basis

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)

and

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control (insert without
cells) (Y/N)

38

6 known surfactantstoFluorescein Leakage Assay Background Review Document

Endpoint (e.g. FL10,
FL20, %FL)

FL20 and FL50 (mg/ml)
T0

FL50 (mg/ml) T0

FL20 (mg/ml) T0

Recovery
Timepoint(s)Following
Initial Exposure (h)

N

N

72

Prediction Modelnot stated

not statedN

39

Fluorescein Leakage Assay Background Review Document

Study/Company/
Organisation

A Summary Report ofthe
COLIPA
International Validation
Study on Alternatives
to the Draize Rabbit
Eye
Irritation
Test(Brantom et al., 1997)

OcularIrritancy
Assessmentof
Cosmetics
FormulationsandIngredients:
Fluorescein
Leakage
Test.
(Zanvit et al.,1999) DATA FROMCOLIPA

Protocol Used/Basis

Protocol of Cottin et al.,(1992)

Company # 4
Protocol (1992)

Cell Type/Strain

MDCK NBL-2

MDCK NBL-2

MDCK NBL-2

Seeding(cells/ml)

Densitysee entry for Zanvit etal., (1999)

4x105 cells/ml (500µl per
insert= 2x105 cells/insert)

PassageRange
defined?
Timetoattainconfluency? (h)see entry for Zanvit etal., (1999)
see entry for Zanvit etal., (1999)

2x105 cells/(not written
but assume) insert
N -presence of tightjunctions verified using
immunofluorescencewith ZO-1 antibody.

Insert Type(s)Pore Size(s)andDuration of Exposure(mins)

see entry for Zanvit etal., (1999)

96
Millipore
HA
cellulose
membranes

Test

FLT- SOP Company # 4(SOP used in the
ECVAM Prevalidation
Study 1996-1998) –submitted by Company#4
Based on FLT Protocol
INVITTOX Protocol No.
71

not stated
4

mixed
ester

Millicell-HA
0.4µM pore size

Exposure at RT?

15
see entry for Zanvit etal., (1999)

Type(s) of Materials
tested?

4
surfactants(diffconcentrations) and 23
surfactant-based
formulations soluble in
HBSS;
30materials
tested by FRAME, 33
materials by Company #4 -differed per laboratory

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

see entry for Zanvit etal., (1999)

10µg/ml Na-fluorescein

0.01mg/ml
in
HBSS(0.001% (w/v/) Solution
Na-fluorescein

see entry for Zanvit etal., (1999)

30 Incubator

30

Y

yes

not stated

HBSS

not stated

0.16mg/ml SDS

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)
Negative Control
Positive Control

see entry for Zanvit etal., (1999)
see entry for Zanvit etal., (1999)
see entry for Zanvit etal., (1999)

15

15

not stated4
surfactants(diffconcentrations) and 23
surfactant-based
formulations soluble in
HBSS;
30materials
tested by FRAME, 33
materials by Company #4-differedperlaboratory

Y

12mm,

40

surfactants

Fluorescein Leakage Assay Background Review Document

Endpoint (e.g. FL10,
FL20, %FL)
Recovery
Timepoint(s)Following
Initial Exposure (h)Prediction Model

FL20 (mg/ml) T4

FL20 (mg/ml) at T0 or T4
calculated as specified in
INVITTOX Protocol No.
71. If FL20 not reached,maximum FL% attained
is noted along with thetest concentration

FL10, FL20 (mg/ml) after
30mins and 4h sincecontact of test material.
Even if FL10 is attained,
max% FL should bequoted along with mg/ml
conc. causing the FL

4
PM for FL20 T4 values
only.
Classificationbased PM for surfactantbased materials only,
developed by Company# 4 (historical data of 43
surfactant
ingredientsand formulations forwhich in vivo data wasavailable). FL20 (mg/ml)>100, non irritant/slight,< 15 MMAS; 20-100,
moderate, 15-30 MMAS;<20, irritant/severe, >20
MMAS

4
PM for FL20 T4 values
only.
Classificationbased PM for surfactantbased materials only,
developed by Company# 4 (historical data of 43
surfactant
ingredientsand formulations forwhich in vivo data wasavailable). FL20 (mg/ml)>100, non irritant/slight,< 15 MMAS; 20-100,
moderate, 15-30 MMAS;<20, irritant/severe, >20
MMAS

4H

41

PM as used in the
COLIPA
study;>100mg/ml=nonirritant/slight, MMAS <15;
20-100mg/ml=
moderate, MMAS 15-30;<20mg/ml=
irritant/severe,
>
30
MMAS

Fluorescein Leakage Assay Background Review Document

Study/Company/
Organisation
Protocol Used/Basis
Cell Type/Strain

Fluorescein
LeakageTest: a Useful Tool in
OcularSafetyAssessment (Cottin andZanvit, 1997)
N/A
MDCK(ATCC: CCL 34)

Fluorescein
LeakageTest: a useful tool in
ocularsafetyassessment Toxicology
in Vitro 11(1997)3 99405 M. COTTIN–
submitted by Company#4
FLT- SOP Company # 4
MDCK NBL-2

2x10 cells/insert

4x105 cells/ml (500µl per
insert= 2x105 cells/insert)N

between 3rd and 30th
after thawing

96
Millicell-HA, 0.45µm poresize

4Millicell-HA
12mm,
0.45µM pore size

Duration of Exposure(mins)

15

15

Exposure at RT?
Type(s) of Materials
tested?

Y
Surfactant and surfactantbased formulations

Y

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

10µg/ml Na-fluorescein in
HBSS

0.01mg/ml
in
HBSS(0.001% (w/v/) Solution
Na-fluorescein

30 RT

30 RT

Y

yes

Negative Control

HBSS

HBSS

Positive Control

not statedEndpoint (e.g. FL10,
FL20, %FL)

FL10, FL20 (mg/ml) T0,
T4

0.16mg/ml SDS
FL20(mg/ml)after
30mins, 4h and 72 h
since contact of test
material.

Recovery
Timepoint(s)Following
Initial Exposure (h)4, 24, 48, 72

72H

Prediction Model

FL20 <20mg/ml =irritant;
FL20≥20mg/ml
and<100mg/ml =moderately
irritant; FL20 ≥100mg/ml= slightly irritantNo

Seeding(cells/ml)

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s) andPore Size(s)Timeallowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

5

surfactants

42

Fluorescein Leakage Assay Background Review Document

Assessment of Initial
DamageandRecovery Following
Exposure of MDCK
Cells to an Irritant(Clothier et al., 1999)
Based on INVITTOX
ProtocolNo.
71(combined with AB
assay–INVITTOX
Protocol No. 80).

EvaluationofthePrevalidation Process:
The
Fluorescein
Leakage Assay (Phase
II) (Southee, 1998)

EvaluationofthePrevalidation Process:
The
Fluorescein
Leakage Assay (Phase
III) (Southee, 1998)

Cell Type/Strain

Prevalidation of the FL
Assay
based
on
INVITTOX Protocol No.
71
MDCK NBL-2 (ECACC:
85011435)

Prevalidation of the FL
Assay
based
on
INVITTOX Protocol No.
71
MDCK
NBL-2
(ECACC:85011435)

MDCK

Seeding(cells/ml)

2x105 cells/ml

2x105 cells/ml

8x104 cells/insert

3<30

3<30

96
Millicell-HA
12mm
diameter, 0.45µm pore
size,

96
Millicel-HA
12mm
diameter, 0.45µm pore
size,

<100
according to INVITTOX
Protocol No. 71

Duration of Exposure(mins)

15

15

5

Exposure at RT?

Y

not stated

Type(s) of Materials
tested?

5% triton x-100, CTAB,
Company # 3 Baby
Shampoo,glycerol,ammonium nitrate

Y
Mild surfactants relevant
to cosmetic testing with invivo data available fromBIBRA after obtaining
from Company # 1 andSigma.

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

0.01% (w/v) fluorescein in
HBSS with Ca++ and
Mg++

0.01%
(w/v)
Nafluorescein in HBSS with
Ca++ and Mg++

0.01% Na-fluorescein in
HBSS

30±2 Incubator

30±2 Incubator

60 IncubatorYY

not stated

Negative Control

HBSS

HBSS

HBSS

Positive Control

0.16mg/ml SDS in HBSS

0.16mg/ml SDS in HBSS

Endpoint (e.g. FL10,
FL20, %FL)

FL20 (mg/ml) T0, T4

FL20 (mg/ml) T0, T4

not stated
FL15 (mg/ml) (in order
to compare results withhuman corneal model
endpoint of fluorescein
retention 85%) T1.5

Recovery
Timepoint(s)Following
Initial Exposure (h)4424, 48, 72, 96

Study/Company/
Organisation

Protocol Used/Basis

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)

and

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

43

Anoporemembrane,
0.2µm pore size

3 known irritants

Fluorescein Leakage Assay Background Review Document

Prediction Model

Based upon COLIPA PM
using FL20 T4 results:>
100mg/ml=
nonirritant/slight;
20100mg/ml = moderate;
<20
mg/ml
=
irritating/severe.

Based upon COLIPA PM
using FL20 T4 results:>
100mg/ml=nonirritant/slight;
20100mg/ml=moderate;<20 mg/ml=irritating/severe

44

N/A

Fluorescein Leakage Assay Background Review Document

Comparative Evaluationof Five In Vitro Tests forAssessingtheEye
Irritation Potential of
Hair-care
Products(Jones et al., 2001)

The
Predictionof
Human Skin Responses
by using the Combined
In Vitro Fluorescein
Leakage/Alamar
Blue(Resazurin)
Assay.
(Clothier et al., 2002)

Cell Type/Strain

Modification of Tchao(1988)MDCK (obtained from R
Tchao)

Adaptation(1988)
MDCK
CB997

Seeding(cells/ml)

cells/insert
2x105(500µl/insert)

5x105(200µl/insert)

2-30.

1<30

not stated (poss. 7 days)

Study/Company/
Organisation

Protocol Used/Basis

Density

PassageRange
defined?
Timetoattainconfluency? (h)

of

Tchao

cells/ml,

andMillicell HA, 0.45µm poresize

72Millicell-HA
12mm,
0.45µmpore
size;
Anoporemembranes
0.2µm pore size

Duration of Exposure(mins)

10secsshampoos,
30secs conditioners

1

Exposure at RT?

Y

Type(s) of Materials
tested?

Shampoosconditioners

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

0.01%
Na-fluorescein(w/v) in HBSS with Ca2+and Mg2+

0.01% Na-fluorescein in
HBSS

30 RT

60 (55) Incubator

YY

Negative Control

not stated

Positive Control

not stated

HBSS
1mg/mlCAPB(nonpreserved 50% activity)
or 0.16mg/ml SDS

Endpoint (e.g. FL10,
FL20, %FL)

FL% T0, T72

FL%, AB% T0

Recovery
Timepoint(s)Following
Initial Exposure (h)24, 48, 72

4, 24, 48, 72

Insert Type(s)Pore Size(s)Timeallowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

andnot statedhandwash formulations,laundry
detergent
formulations
andmoisturisers

45

Fluorescein Leakage Assay Background Review Document

Prediction Model

Results compared to the
FL result for benchmark
shampoo and conditioner.
Material acceptable ifsame as benchmark;'furtherinvestigation'
defined by ±20% frombenchmark value

Generatedfromtheresults for the handwash
products, and evaluatedwith results from laundry
powdered cleaners andthe moisturisers. Criteria
given for FL and AB
results
inducedbyvarious concentrations at
different time-points foreach level of in vivo score
0-4 in 0.5 increments.

46

Fluorescein Leakage Assay Background Review Document

Frame In-house Studies

Study/Company/
Organisation

Company # 4 (FRAME,
circa 1991)
INVITTOX Protocol No.
71.

Protocol Used/Basis
Cell Type/Strain
Seeding(cells/ml)

Density

PassageRange
defined?
Timetoattainconfluency? (h)INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

and

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

Duration of Exposure(mins)INVITTOX Protocol No.
71.

Exposure at RT?

INVITTOX Protocol No.
71.

Insert Type(s)Pore Size(s)Type(s) of Materials
tested?
Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)
Timeallowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

4 surfactants and 3
finished products
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

FRAMEReportonComparisonof40
Cosmetic and Domestic
Formulations Suppliedby Company # 8 andEvaluated by 3 In Vitro
Cytotoxicity
Tests(FRAME, 1992)

Company
Chemicals
1992)

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

40
cosmetics
anddetergent formulations

5 cosmetic ingredientsand final product

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

INVITTOX Protocol No.
71.

#
5(FRAME,

Positive Control

INVITTOX Protocol No.
71.
INVITTOX Protocol No.
71.

Endpoint (e.g. FL10,
FL20, %FL)

FL20. FL50 (mg/ml)

FL20, FL50 (mg/ml)

FL20, FL50 (mg/ml)

Recovery
Timepoint(s)Following
Initial Exposure (h)4, 24, 48, 72

N/A

72

Prediction Model

N

N

N

Negative Control

47

Fluorescein Leakage Assay Background Review Document

Study/Company/
Organisation

Final Report on Testingof 12 Mild Surfactants
supplied by Company #5
forCytotoxicity
Testing at the FAL(FRAME, 1992)

Protocol Used/Basis

Shaw et al., (1990, 1991)/
INVITTOX Protocol No. 82

Cell Type/Strain

MDCK (assume CB997)

Seeding(cells/ml)

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)54x10(400µl/insert)

cells/insert

Exposure at RT?

Five Company # 5
Products(FRAME,1994)

INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.

Shaw
1991)

et

al.,

(1990,INVITTOX Protocol No.
82.

4x105
cells/insert(400µl/insert)

MDCK (assume CB997)

96

INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.

Anocell, 0.2µm pore size

INVITTOX Protocol No.
82.

Anocell, 0.2µm pore size

1
INVITTOX82.

INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.

1
INVITTOX Protocol No.
82.

not statedandDuration of Exposure(mins)

Company # 5 (FRAME,
1993)

Protocol

No.

15-20
96

Type(s) of Materials
tested?

12 mild surfactants

Chemicals remain coded

5 baby care productswater in oil and oil inwater emulsions

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

not stated

INVITTOX Protocol No.
82.

not stated30

INVITTOX Protocol No.
82.

INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.
130mg/ml acetic acidTime
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)
Negative Control

Y
INVITTOX
82.

Positive Control

130mg/ml acetic acid

INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.
INVITTOX Protocol No.
82.

Endpoint (e.g. FL10,
FL20, %FL)

FL%

FL%

FL%

Recovery
Timepoint(s)Following
Initial Exposure (h)4, 24, 48, 72

24, 48, 72

24, 48Prediction Model

N/A

N

N/A

ProtocolNo.

48

Fluorescein Leakage Assay Background Review Document

Study/Company/
Organisation

Results
from
the
Fixed
Dose
fluorescein leakage In
Vitro Cytotoxicity teston 4 test Sample
Formulations
fromCompany#
5(FRAME, 1994)

Company # 6, Effectsof 6 Coded Chemicals
on
MDCK
Cells
(FRAME, 1998)

Protocol Used/Basis

Shaw et1991)

not stated

Cell Type/Strain

MDCK CB997

Seeding(cells/ml)

Density

PassageRange
defined?
Timetoattainconfluency? (h)Insert Type(s)Pore Size(s)

and

al.,

(1990,MDCK NBL-2 (ECACC)

5cells/insert
4x10(400µl/insert)

2x105
cells/insert(400µl/insert)N

not stated96

96

Anocell,
size

0.2µm

pore
Anopore or Millipore

Duration of Exposure(mins)

2

1 or 15

Exposure at RT?

not stated

not stated

Type(s) of Materials
tested?

Company # 5 samples

Company # 6 codedsamples

Fluorescein
Concentrationdeliveredtomonolayer (mg/ml)

not stated

0.01% (w/v) fluorescein
solution

60 RT

60 RT

Y

Y

Negative Control

Y

HBSS

Positive Control

130mg/ml acetic acid130mg/ml acetic acid

Endpoint (e.g. FL10,
FL20, %FL)

FL%

FL20 (mg/ml)

Recovery
Timepoint(s)Following
Initial Exposure (h)4, 24, 48, 72

4, 72

Prediction Model

N/A

N/A

Time
allowedfor
Fluorescein Leakage(mins)
Maximum
Leakage
Control(insertwithout cells) (Y/N)

49

Fluorescein Leakage Assay Background Review Document

Red font indicates protocol information received from companies directly. CAPB=
cocamidopropylbetaine; CTAB =cetyl trimethyl ammonium bromide; ECACC =European
Collection of Cell Cultures; FLT =fluorescein leakage test; GLP= good laboratory
practice; HCE-T =human corneal epithelium-transfected; NI= non-irritant; RT =room
temperature; SD =standard deviation; SEM =standard error of the mean; T=time-point;
WP =well plate(s).

50Fluorescein Leakage Assay Background Review Document

2.2.1. Description of protocol components and rationale for differences, if
available
The following sections detail the various protocol components of the FL assay andprovide rationales for the protocol differences. Details of the ECVAM Prevalidation
study (Southee, 1998) which compared various protocol elements are given whereappropriate.
i. Cell type
Two MDCK cell-strains exist and feature equally in the FL assay INVITTOX protocols;
INVITTOX Protocols No. 71 and No. 82 employ the MDCK cell strain CB997, whilst
INVITTOX Protocols No. 86 and No. 120 use the NBL-2 cell strain. The NBL-2 cells
were more frequently cited in the USA publications whilst the CB997 cells werecommonly used in Europe-based studies.
INVITTOX Protocol No. 120 was developed following the ECVAM sponsored FL assay
Prevalidation study (Southee, 1998). Phase I of the ECVAM Prevalidation study
undertook a number of experiments for protocol refinement. The protocol variables
investigated were; MDCK cell strain, growth medium and insert membrane. The aim of
the study was to determine which combination produced maximum barrier function (0%leakage).
As NBL-2 cells were reported to form a better barrier than CB997 cells, the optimum
growth medium for this cell strain was investigated. Both MDCK cell strains were grownin the following medium combinations:-

Minimum Essential Medium (MEM) + 10% Foetal Bovine Serum (FBS) + 1% Lglutamine
Dulbecco’s Modified Eagles Medium (DMEM)/F12 (1:1) with 15mM Hepes + 10%
heat inactivated FBS + 1% L-glutamine.

MEM supplemented with 10% FBS and 1% glutamine was found to be the optimum
growth medium for the NBL-2 cell strain; the different types of media used in the various
INVITTOX protocols supports this finding. INVITTOX Protocol No. 71 and No. 82
culture MDCK CB997 cells in DMEM/Nutrient Mix F12 (1x concentrate with L-glutamineand 15mM Hepes), supplied by Gibco (UK). INVITTOX Protocol No. 86 cultures MDCK
NBL-2 cells in Eagle’s MEM made up in Earle’s BSS (supplier not stated). In INVITTOX
Protocol No. 120, MDCK NBL-2 cells are cultured in modified Eagle’s medium with
Earle’s salts (2x concentrate with L-glutamine and without phenol red) supplied by Gibco(USA). Use of different media according to the cell strain cultured was supported by theliterature (Clothier et al., 1994; Shaw et al., 1990; 1991; Zanvit et al., 1999). All
protocols supplement the medium with 10% FCS/FBS.
The FL Assay Prevalidation study (Southee, 1998) tested the growth of both MDCK cellstrains on Millipore-HA, Costar Transwell and Nunc Anopore inserts to determine which
promoted optimum growth and barrier function. Cells were treated with 0.16mg/ml SDS
and the amount of FL(%) at T0 and T4 was recorded. The combination giving the mostconsistent and complete barrier function was MDCK NBL-2 cells grown on Millipore-HA
inserts and cultured in MEM supplemented medium. Both cell strains grown on
51

Fluorescein Leakage Assay Background Review Document

Anopore membranes and treated with SDS had a tendency to lift and peel away fromthe membrane.
The TEP assay has been performed using a human corneal epithelial cell line
developed by Gillette Medical Evaluation Laboratories (Kruszewski et al., 1997; Ward etal., 1997b). Human corneal epithelium primary cultures transfected with the SV40 large
T antigen plasmid pRSV-T (Ward et al., 1997b) produced a cell line (HCE-T) that
expresses human corneal epithelia features for approximately 24 passages (Kruszewski
et al., 1997). The advantages of using the HCE-T cell line, for predicting damage to thehuman corneal epithelium, is that it forms a similar number of layers as found in the invivo situation and the cells are of human origin. A human corneal epithelial cell line isalso used at the FAL. In comparison to the HCE-T cell line, the J-HCET cell line used at
FAL expresses similar properties to normal human corneal epithelial cells up to
approximately 100 passages (Araki-Sasaki et al., 1995).
Bridging Study:
In order to replicate the in vivo situation more closely, the SV40 transfected Japanesehuman corneal epithelial cell line (J-HCET) has been used at the FAL. These
immortalised human corneal epithelial cells stratify 4-6 layers when grown on collagenmembranes at air-liquid interfaces. In comparison to MDCK cells, J-HCET cells grewless satisfactorily on the inserts and generally took a longer time to form confluent layers(Cheah; BMedSci, 2000).
The ‘J-HCET protocol’ was used to predict the effects of repeated exposures to low
doses of surfactants on the corneal epithelium barrier. The J-HCET cell model which
consisted of stratified cell layers was less sensitive than the MDCK cell model which
formed a monolayer (Clothier et al., 1999). Various protocol modifications were made toobtain reproducible results with the J-HCET cell line; J-HCET cells were grown on
0.4µm Nunc polycarbonate membrane inserts whereas MDCK cells were grown on
0.2µm Anopore inserts. Fluorescein Isothiocyanate Dextran (FD) with molecularweights of 4.4kD (FD-4) and 9.5kD (FD-9) were used in conjunction with J-HCET cells
as sodium-fluorescein gave variable results and a greater rate of FL than with MDCK
cells, suggesting that the intra-cellular spaces of the tight junctions were larger in the JHCET cell line. The FL assay in the J-HCET model ranked the four surfactants inagreement with in vivo rankings (Cheah; BMedSci, 2000). Further work was deemednecessary in order to determine the use of this cell model for predicting in vivo ocular
irritation (Cheah; BMedSci, 2000).
More recent studies have shown that in the correct culture conditions the J-HCET cells
can generate an equally tight junction complex and para-cellular cleft as found with
MDCK cells, i.e. a 6% maximum of the no cells control can be applied as for the MDCK
cells (Moore et al., 2005; Wilkinson and Clothier, 2005).
ii. Cell seeding
Seeding density is important to ensure that the cells attain a confluent monolayer before
the FL assay is performed. A confluent layer is needed to enable tight junctions to form
between the cells and produce an impermeable layer to the sodium-fluorescein dye.
iii. Cell passage number
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Fluorescein Leakage Assay Background Review Document

The cell passage number is important to ensure that the cells used for the FL assay
function similarly. INVITTOX Protocol No. 120 reports that the MDCK cells should bewithin the passage range 3-30 from thawing. Many other publications regarding the FL
assay have also stated that experiments were performed with cells within this passage
range. Cells within this passage range have similar functionality which aids assay
results to be reproducible.
iv. Chemical solvent
All protocols use HBSS or deionised water rather than medium as the chemical solvent.
Some protocols stipulate that the HBSS used is without phenol red. Phenol red could
potentially remain in the wells and interfere with the sodium-fluorescein optical density(OD) readings by the spectrofluorimeter. HBSS is used rather than medium to reduce
the effects of the medium components interfering with the test material, and also to
reduce the possibility of the medium components binding to, or disrupting the insert
membrane. Using HBSS as the solvent reduces the likelihood of variation due tounpredictable properties and effects of the medium. Mineral oil is used for insoluble
materials.
INVITTOX Protocol No. 120 states that the HBSS should also contain calcium. Calcium
is important to maintain the tight junctions. INVITTOX Protocol No. 71 does not
stipulate that HBSS should contain calcium as it was not so commonly available at thetime the protocol was developed. The author of INVITTOX Protocol No. 71 now statesthat HBSS preferably should contain calcium, although as the exposure period for
INVITTOX Protocol No. 71 is shorter than INVITTOX Protocol No. 120, the absence of
calcium from HBSS would have a lesser adverse effect. Also, some residue of cell
culture medium which contains calcium is likely to remain in the well during the chemical
exposure.
v. Washing steps
In the literature, washing steps are often performed before the test chemical is added tothe cells. Of the INVITTOX protocols, only INVITTOX Protocol No. 86 uses a washingprocedure before adding the test chemical. With increased experience with the FL
assay it became apparent that a washing step before the chemical exposure increased
assay reproducibility by reducing the variable effect of test chemical interactions withmedium components (personal comm. R Clothier). HBSS is commonly used as thewashing solution although distilled water features in some protocols. The number oftimes the washing is repeated varies between protocols from 1 to 5. The advantage ofmultiple washing steps to increase method reproducibility, is offset by the greater
chance of damage to the cell monolayer and/or insert membrane.
Washing steps after the test material is removed and before sodium-fluorescein isadded to the cell monolayer feature consistently in the INVITTOX Protocols. HBSS iscommonly used as the washing solution for one or two rinses, and PBS or distilled waterless so. INVITTOX Protocol No. 86 features ten rinses. Efficient washing is necessary
to ensure the test material is fully removed and that uncontrolled prolonged exposures
do not occur, which could impact on the reproducibility and predictivity of the assay. If
the test chemical remains in the insert, it has the potential to physically block thesodium-fluorescein dye from leaking through the cell monolayer and insert membrane,
53

Fluorescein Leakage Assay Background Review Document

thus leading to an under-estimation of the chemical’s toxicity. Also, if the chemicalremains in the well when sodium-fluorescein is added, unknown prolonged exposuresand uncontrolled interactions between the chemical and the sodium-fluorescein dye canoccur that can impact on the reproducibility and predictivity of the assay, e.g. binding of
the two solutions or binding between solutions and the membrane.
vi. Duration of chemical exposure
The duration of the test material exposure is usually one minute or 15 minutes.
INVITTOX Protocol No. 71 uses both exposures according to the potency of material
being tested; although no guidance is given for determining which material exposure
period to use, the one minute exposure is commonly reported in the literature for thisprotocol. INVITTOX Protocol No. 82 uses a one minute chemical exposure period. This
protocol differs from others as it determines the amount of FL (%) caused by exposure
to a fixed chemical concentration. INVITTOX Protocol No. 86 and INVITTOX Protocol
No. 120 use a 15 minute chemical exposure period as the NBL-2 cell strain is known toform a greater number of tight junctions, and therefore form a more impermeablemonolayer than the CB997 cell strain used in INVITTOX Protocols No. 71 and No. 82.
vii. Sodium-fluorescein dye concentration
The sodium-fluorescein dye is used by all INVITTOX protocols and with the exception ofstudies conducted using human corneal cells, features consistently in the literature. The
concentration of the sodium-fluorescein dye varies according to each protocol;
INVITTOX Protocol No. 71 and No. 120 use 0.01% sodium-fluorescein whereas
INVITTOX Protocol No. 82 and No. 86 use 0.02% sodium-fluorescein. INVITTOX
Protocol No. 71 states that 0.02% sodium-fluorescein maybe used when Anocell inserts
are used instead of Millicell-HA inserts. Anocell inserts have a smaller pore size thanMillicell-HA inserts, and therefore a higher concentration of the sodium-fluorescein dye
is necessary in order to obtain sufficient ODs to distinguish different levels of damage tothe monolayer. All protocols use Millicell-HA inserts with the exception of INVITTOX
Protocol No. 86 which uses Costar Transwell inserts.
viii. Insert typeThe various insert types used for the FL assay were known to alter the results
considerably (Ward et al., 1997a). Inserts principally vary in the following parameters;
number of pores, pore size, formation of the o-ring, membrane surface charge, presence
and type of coating (e.g. Laminin Type I or Collagen IV). These differences can affectcell attachment, formation of tight junctions, and impact on the sensitivity of cell
responses to various chemical exposures. The main properties of four different inserts
commonly used in the FL assay protocols are shown (table 2.2.1.1.)

54

Fluorescein Leakage Assay Background Review Document

Table 2.2.1.1. Insert properties (modified from Ward et al., 1997a)
InsertName
AnoporeAnoporeMillicell-HA
Millicell-CM

Material
Aluminium oxide
Aluminium oxide
Mixed cellulose esters
Stretched polytetrafluorethylene(Biopore) (coated with human placental type IV
Collagen)Thickness
(µm)
45
45
80-150

Pore Size
(µm)
0.2
0.02
0.45

50 (insert
alone)

0.4

The insert membranes promote differentiation of the cells. The epithelial monolayergrown on the membranes acts as a barrier to apical and basal media. Anopore
membranes (Anocell inserts) possess a very flat surface to which cells attach via small
cytoplasmic extensions. The Millicell-HA membranes have an uneven, matted surface towhich the cells project downwards (Zanvit et al., 1999). Cells grown on the Anopore
inserts form a greater number of tight junctions and can be more sensitive to toxic
effects, than cells grown on the Millipore-HA inserts (Atkinson, (1995), unpublishedobservations). This difference is probably related to cell attachment to the insert
membranes as MDCK cells have greater attachment to Millicell-HA membranes incomparison to Anopore membranes. The various sensitivities of the cells grown on
different membranes is overcome by using a 15 minute toxicant exposure for cells
cultured on the Millicell-HA insert membranes (Zanvit et al., 1999) which contrasts to theone minute exposure used for cells grown on the Anopore membrane. The original FL
assay developed by Tchao (1988) used a 0.45µm pore-sized insert membrane and a 15
minute exposure. Shaw et al., (1990) modified this protocol to use 0.02µm pore-sized
insert membranes with a one minute exposure. The one minute exposure period
models the approximate length of time a material is likely to remain in the eye in vivo,
following an accidental exposure in humans.
The two inserts most commonly cited in the FL assay publications were the Millicell-HA
insert and the Anocell insert. Ward et al., (1997a) performed a bridging study todetermine the effects of insert membranes (featured in table 2.2.1.1.) on cell growth and
FL assay sensitivity. Only the Millicell-CM membrane required a coating solution which
consisted of human placental type IV collagen. This insert was not cited in any of the
INVITTOX Protocols. Cell attachment to the membranes was good, with FL through themonolayer less than 10% for all the inserts tested. The following surfactants were
tested in each insert; SDS, tween 20, triton x-100, benzalkonium chloride, cetrimide,
CAPB. The majority of test materials caused different results according to the insert
used. The cationic surfactants benzalkonium chloride and cetrimide produced similar
results regardless of insert type, e.g., 1mg/ml of both test chemicals induced an amountof FL which remained the same or decreased at higher test concentrations. The amountof FL induced by the threshold concentration of 1mg/ml varied between inserts (Ward etal., 1997a). The Millicell-HA insert produced a wide range of FL20 results in comparison
to the other inserts tested (Ward et al., 1997a). In the absence of a monolayer, thecationic surfactants bind to insert membranes and/or blocked pores to different degrees.
As FL cannot be determined to be solely due to the insert or cell monolayer,
experiments using a no cell control help to interpret the effects of these chemicals on thecells.

55

Fluorescein Leakage Assay Background Review Document

The FL assay data from the EC/HO study (Balls et al., 1995) which used Millicell-HA
inserts, was compared to data for the same chemicals tested on Anopore membranes(Ward et al., 1997a). The results from the Anopore membranes were found to give a
better correlation to Draize MMAS scores than the Millipore membranes. The regulatory
bodies generally required MAS scores.
In comparison to the Millipore membrane, an advantage of the Anopore membrane isthat it becomes opaque when wet. This allows damage to the membrane and/or cellmonolayer, which may occur due to the multiple washing steps, to be easily observed.
The FL Prevalidation study tested CB997 and NBL-2 MDCK cell strains in combination
with Millipore (0.45µm pore size), Anopore (0.2µm pore size), and Costar inserts (0.4µm
pore size) (Southee, 1998). The Anopore membrane was more robust than the MillicellHA insert (Southee, 1998). Where damage to the membrane is visible, results from theassay are discarded. Despite the advantages of Anopore membranes, the FL Assay
Prevalidation study observed that both MDCK cell strains had a tendency to peel-off the
Anopore membrane and cause barrier permeability.
Test materials can impede FL through the insert membrane via a number of interactions,e.g. attraction of charges between the test chemical and insert membranes or cells, or aphysical blockage due to the molecular weight of a chemical. Chemical binding to themembrane, is more common for cationics, e.g. benzalkonium chloride, which are
attracted to the positively charged membrane (Balls et al., 1995). Negatively charged
cell surface proteins can also attract positively charged surfactants. Chemical binding to
the insert membrane or cell surface, increases the chemical exposure period but canalso physically reduce FL, since the sodium-fluorescein dye can bind to the cationicsurfactant bound to the insert membrane. This can be readily monitored by exposing themembrane to the top concentration of the chemical tested and then adding sodiumfluorescein dye at the normal concentration for the standard time. If binding of thesodium-fluorescein dye occurs, the insert membrane appears yellow after the test
material has been washed-off. It is essential to know the binding properties of the test
materials in order not to over-estimate the toxic potential of those materials that remain
exposed to cells longer than the defined exposure period. In contrast, materials thatblock the pores and impede FL will cause toxicity to be under-estimated. Viscousmaterials are difficult to remove from the inserts, which pose the problem of continued
exposure and/or physically blocking the membrane pores and preventing FL; both
effects will produce erroneous results. The different properties of the insert membranes
determine which test materials they will interact with, e.g. acetic acid reacts with someMillicell-HA membranes but not the Anopore membrane (INVITTOX Protocol No. 120).
ix. Duration for FL
Following test chemical/formulation exposure and removal, the subsequent time given
for FL to occur varies according to each protocol. A 30 minute period at room
temperature (INVITTOX Protocol No. 71, INVITTOX Protocol No. 86, INVITTOX
Protocol No. 120) is commonly used. Other protocols employ a 60 minute period, which
can be either at room temperature (INVITTOX Protocol No. 82) or incubated at 36oC
(Clothier et al., 1997; Clothier et al., 2002). The incubator is used with a FL period of 60
minutes since it is possible that a prolonged exposure to room temperature could
damage the cells, and impact on FL due to the stress of external conditions. The
incubator is also used where the laboratories are interested in combining the FL assay
with a cell activity assay e.g., Resazurin/Alamar Blue (AB) assay. There is no evidence
56

Fluorescein Leakage Assay Background Review Documentthat a 30 minute exposure at room temperature damages the cells. An advantage ofusing a 60 minute period to allow for FL is that higher amounts of leakage occur. Thus,
a 60 minute period for FL will potentially allow for a greater degree of discrimination
between FL assay results, than the 30 minute period. A greater range of values isparticularly important for the Fixed Dose FL assay (INVITTOX Protocol No. 82) where
only one concentration is tested. This will allow the toxicity of a number of test materials
at a set concentration to be compared. The advantage of the 30 minute period for FL isthat the assay is quicker to perform. Both 30 minute and 60 minute periods are
sufficient to produce reproducible FL assay results.
x. Time-point FL is measured
The time-points used to measure FL following the chemical exposure varies between
protocols. The most common time-point for measuring acute effects is immediately
following the chemical exposure. As some protocols allow 30 minutes or 60 minutes for
FL to occur, the time of measuring the amount of FL varies by 30 minutes. The othercommon time-point for measuring acute effects is at 4h (INVITTOX Protocol No. 120).
Additionally, the cell monolayers have a greater tendency to ‘peel-off’ the membrane
when the FL assay is performed immediately following the exposure, which is less likely
to occur when the assay is performed at 4h. Subsequently, the 4h time-point isgenerally used when the results are entered into a PM to predict human eye irritation(Southee, 1998; Zanvit et al., 1999). Zanvit et al., (1999) also stated that the FL
measured 4h following the chemical exposure produced results with a better predictive
capacity for surfactants.
Additional time-points used to assess recovery and delayed effects are 24h, 48h, 72h(INVITTOX Protocol No. 86 and No. 120). Measurements can be made at 24h intervals
over a total of 72h; the 72h time-point features frequently in the literature (Clothier et al.,
1994; Clothier et al., 1999; Clothier et al., 2002; Cottin and Zanvit, 1997). Variability canoccur at 72h and this can be due to the effects of repeated FL assays or the differential
time for recovery between replicates. A measurement for recovery at the 72h time-point
was incorporated into the PM used by the Fixed Dose FL assay (INVITTOX Protocol No.
82) (Clothier et al., 1994). The EC/HO study also proposed a PM that included 72h
results which could be used to distinguish R36 and R41 classified ocular irritants.
xi. Positive Controls
The various FL Assay INVITTOX Protocols use different acceptance criteria and positive
controls. INVITTOX Protocol No. 71 uses 100mg/ml Brij 35 which should cause
approximately 30% FL, although results are accepted if the positive control produces
20% to 40% FL. INVITTOX Protocol No. 120 uses a positive control of 130mg/ml glacialacetic acid, which should produce approximately 50% damage to the cell layer; results
are also accepted if the positive control produces FL results in the range of 30-70%(Southee, 1998). INVITTOX Protocol No. 82, uses 0.16mg/ml SDS which must induce
less than 50% leakage in order for the results to be accepted. In the literature, SDS is
the most common chemical used as a positive control but the range of FL which it must
induce for results to be accepted varies between the publications. INVITTOX Protocol
No. 86 does not state which chemical is to be used as a positive control. However, thisprotocol featured in the CTFA study Phase III where a Company # 3 baby shampoo wasused as a positive control (Gettings et al., 1996). Results were only accepted if the test
concentrations of Company # 3 baby shampoo, reached a plateau of 100% FL over57

Fluorescein Leakage Assay Background Review Document

more than one concentration. This acceptance criteria controls only for the correct
concentration range being tested and does not aid test reproducibility.
With the exception of INVITTOX Protocol No. 120, all INVITTOX protocols state a range
for which the positive controls must induce a certain amount of FL. Assuming thatsuitable chemicals have been chosen as positive controls, INVITTOX Protocol No. 71,
with the smallest range for the positive control, would be expected to produce the most
reproducible results for the test chemicals.
xii. FL endpoint measured
The endpoint to be measured as stated in the various INVITTOX Protocols differs, but
are typically; FL10 (INVITTOX Protocol No. 120), FL20 (INVITTOX Protocol No. 71, No.
82, No. 120), FL50 (INVITTOX Protocol No. 71), EC50 (INVITTOX Protocol No. 86) and
FL% (INVITTOX Protocol No. 71). The FL assay has also been performed on a humancorneal cell line where the chemical concentration giving rise to 85% fluorescein
retention (FR85) was recorded which equates to FL15 (Clothier et al., 1999).
FL20 features most predominately in the literature. FL20 is used in order to determine theconcentrations that might cause damage to the tight junctions rather than non-specificcellular damage which is more likely to be measured at the FL50 endpoint. The highchemical concentrations required to induce FL50 values within a short exposure could
possibly cause severe damage to the cells and even cell loss.
Prediction Models
Prediction models (PMs) are necessary to interpret the FL assay results in terms ofpotential ocular irritancy to humans. In various publications, PMs have been defined for
all four INVITTOX Protocols; only INVITTOX Protocols No. 86 and No. 120 actually
feature PMs. A PM for INVITTOX Protocol No. 71 data was devised following theresults of the EC/HO study (Balls et al., 1995); the PM does not appear in INVITTOX
Protocol No. 71 (table 2.2.1.2.).
Table 2.2.1.2. PM for data generated using INVITTOX Protocol No. 71 as presented in
the EC/HO Study publication (Balls et al., 1995).
FL20 (mg/ml)>750mg/ml>100 ≤750mg/ml and recovery
after 72h>100 ≤750mg/ml and further
deterioration after 72h

EUClassification
Not Classified
R36
R41

There was no definitive PM reported for the method in INVITTOX Protocol No. 82 (table
2.2.1.3.).

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Fluorescein Leakage Assay Background Review Document

Table 2.2.1.3. The PM for INVITTOX Protocol No. 82 (taken from the publication of
Clothier et al., (1994)).
Chemical tested at 50mg/ml

causes <FL20 (%)
causes ≥FL20 (%) withconsideration of recovery or
further deterioration at 72h

EU Classification
Not Classified

R36/R41

NB. Clothier et al., (1994) state that the fixed concentration tested can differ according
to prior knowledge of the types of test materials.
INVITTOX Protocol No. 120 contains the following PM (table 2.2.1.4.).
Table 2.2.1.4. PM from INVITTOX Protocol No. 120
FL20 (mg/ml)>100 mg/ml
20 -100 mg/ml<20 mg/ml

EU Classification
non-irritant/slightmoderate
irritant/severe

Table 2.2.1.5. shows the PM which features in INVITTOX Protocol No. 86.
Table 2.2.1.5 PM from INVITTOX Protocol No. 86 (TEP assay)
EC50 (%)≤1.8%>1.8% <2.2%≥ 2.2%TEP Assay Rating
failborderline/undeterminedpass

Briefly, the PMs used with the INVITTOX Protocols vary in the following ways:- number of classifications- correlation with standard classification system (i.e. EU risk phrases)- size of the range of values used to distinguish different irritancy classes- use of recovery data- types of materials PM is applicable for** although not explicitly stated in INVITTOX Protocol No. 120, the PM was devised
based on the results of surfactant-based formulations and only applied to these types oftest materials in the studies reported in the literature (Zanvit et al., 1999).
Both INVITTOX Protocol No. 71 and No. 82 have PMs reported in the literature which
predicted EU classifications. INVITTOX Protocol No. 120 is comprised of three classes
which distinguish two levels of irritancy, but not according to a standard classification
system. INVITTOX Protocol No. 86 features a PM which is only able to distinguishirritants and non-irritants but not according to a standard classification system. The
different PMs have been challenged to various levels according to the types of materials
tested to generate the data which were entered into the PMs.

59

Fluorescein Leakage Assay Background Review Document

2.2.2.

Proposed critical components of the protocol that impact on
reproducibility and/or predictive capacity of the assay

Of the protocol steps listed in the previous section, the following elements wereconsidered ‘critical’ in terms of the reproducibility and predictive capacity of the FL assay
protocols (figure 2.2.2.1.):•

Cell type, cell strain -in medium with a sufficient calcium concentration to ensure
tight junction formation and integrity.

•

Cell seeding -to ensure confluence of the cells at the times of exposure.

•

Confluence and tight junction integrity checked for each insert before FL assay–fluorescein leakage must be below 6%.

•

Passage number range to ensure even and reproducible tight junction formation.

•

Insert and membrane type:-affects cell growth-chemical binding can be membrane type specific•

Test chemicals should be prepared in calcium-containing HBSS or medium
without serum to avoid serum protein binding.

•

Test chemical exposure period duration –differs according to cell type and cellstrain but should enable immediate chemical effects to be measured•

Ambient temperature of exposure is room temperature (23-25oC) or at 37oC in theCO2 incubator. The lower room temperature has been found to have little effecton the rate of FL, but the temperature can be of relevance when longer than 1-5
minute chemical exposures are employed.

•

Time given for FL to occur to ensure fluorescence OD values are within a range
to produce a dose-response curve•

Endpoint FL20, FL50, FL%, calculated as % of non-cell control inserts treated with
top concentration of test chemical set at 100%.

•

Time-point FL is measured, i.e. immediately, or at 4h, then 24h, and/or 48h
and/or 72h for recovery.

•

Prediction model

Figure 2.2.2.1. Critical elements of FL assay protocols
The rationales for these critical protocol steps were stated in the previous section(section 2.2.1):

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Fluorescein Leakage Assay Background Review Document

2.2.3. List of studies with similar protocols (no protocol differences, or no impact
of protocol differences on predictive capacity)
Various FL assay protocols have been grouped according to the number of features thatthey have in common.
Group 1 INVITTOX Protocol No. 71: These studies used the same cell strain, test
material exposure duration, and FL duration:• Loss of Trans-epithelial Impermeability of a confluent layer of Madin-Darby Canine
Kidney (MDCK) Cells as a Determinant of Ocular Irritancy Potential (Shaw et al.,1990)
• Predicting Ocular Irritancy and Recovery from Injury using Madin-Darby Canine
Kidney Cells (Shaw et al., 1991)• Development of a Fixed Dose Approach for The Fluorescein Leakage Test.
(Clothier et al., 1994) (INVITTOX Protocol No. 82)• Evaluation of Tissue Culture Insert Membrane Compatibility in the Fluorescein
Leakage Assay (Ward et al., 1997a)• The Evaluation of Pesticide Ingredients and Formulations In Vitro and Correlations
with In Vivo Data (Clothier et al., 1995)• The EC/HO International Validation Study on Alternatives to the Draize Eye
Irritation Test (Balls et al., 1995)• FAL In-house studies
The study of Tchao et al., (1988) could not be included in this group because it was not
documented which cell strain was used in the protocol.
The study of Jones et al., (1988) could not be included in this group because the cells
used were obtained from Tchao and the cell strain was not documented. In addition, thechemical exposure duration and the endpoint measured using this protocol also differedto those listed in this section.
The inserts used in the two studies performed by Shaw et al., (1990; 1991) were thesame, although marketed under different names; Anocell 10 (Shaw et al., 1990) andAnotec 10mm (Shaw et al.,1991). The endpoints differed between the Shaw et al.,(1990; 1991) studies and the Fixed Dose FL assay of Clothier et al., (1994). The Fixed
Dose FL assay tested a set concentration of 50mg/ml for each test material. Materials
that attained FL20 (%) or above at this concentration were classified as potential irritants;
those that induced less-than FL20 were classed as non-irritants. No PM was used in thestudies by Shaw et al., (1990; 1991), although a good correlation between the ranking of
the FL20 results with in vivo EU risk phrase classifications were reported. The predictive
capacity of the protocols for in vivo classifications were similar as comparisons of theresults from Shaw et al., (1990) with the Fixed Dose FL assay data (Clothier et al., 1994)
generally indicated the same chemicals to be either irritants or non-irritants. For
example, 50mg/ml DMSO did not cause any FL in the Fixed Dose FL assay and was
ranked as one of the least toxic chemicals tested in the study of Shaw et al., (1990).
Group 2- INVITTOX Protocol No. 120: These studies had the same protocol elements
in terms of cell strain, test material exposure duration, insert type, duration for FL:• A Summary Report of the COLIPA International Validation Study on Alternatives tothe Draize Rabbit Eye Irritation Test (Brantom et al., 1997)
61

Fluorescein Leakage Assay Background Review Document• Evaluation of the Prevalidation Process: The Fluorescein Leakage Assay (Southee,1998)• Ocular Irritancy Assessment of Cosmetics Formulations and Ingredients:
Fluorescein Leakage Test. (Zanvit et al., 1999)
The protocols featured in the following publications could not be grouped with INVITTOX
Protocol No. 120 and similar protocols’ as the FL assay was performed immediately
after the chemical exposure:••

Gautheron et al., (1994a.) Investigations of the MDCK Permeability Assay as anin vitro test of Ocular Irritancy
Fluorescein Leakage Test: a Useful Tool in Ocular Safety Assessment (Cottin
and Zanvit, 1997)

Group 3- INVITTOX Protocol No. 86: Data from the CTFA study Phase III was also
submitted to the IRAG study, thus identical protocols are featured.
• CTFA Evaluation of Alternatives Program: An Evaluation of In Vitro Alternatives
to the Draize Primary Eye Irritation Test, Phase III (also submitted to IRAG
Working Group 3: Cell Function-based Assays (Botham et al., 1997) Lab B)• Company # 3
Group 4- INVITTOX Protocol No. 71 combined with the Alamar Blue assay•••

Effects of Surfactant Re-treatment In Vitro: A Method to Evaluate Changes in Cell
Junctions and Cell Viability (Clothier and Sansom, 1996)
The Prediction of Human Skin Responses by using the Combined In Vitro
Fluorescein Leakage/Alamar Blue (Resazurin) Assay. (Clothier et al., 2002)
Assessment of Initial Damage and Recovery Following Exposure of MDCK Cells
to an Irritant (Clothier et al., 1999)

The protocols featured in the following publications could not be grouped with other
INVITTOX Protocols due to many differences but most significantly, different cell types:•••

Human Corneal Epithelial Primary Cultures and Cell Lines with Extended Life
Span: In Vitro Model for Ocular Studies (Kahn et al., 1993)
Evaluation of a Human Corneal Epithelial Cell Line as an In Vitro Model for
Predicting Ocular Irritation (Kruszewski et al., 1997)
Evaluation of Chemically Induced Toxicity Using an In Vitro Human Corneal
Epithelium (Ward et al., 1997b)

62

Fluorescein Leakage Assay Background Review Document

2.2.4. Known applicability and limitations of the assay (including ranges of
irritancy, types of substances, technical limitations)
The FL assay was designed to measure chemical-induced permeability of a monolayer
in order to model loss of impermeability of the corneal and conjunctival epithelia. The
FL assay was specifically developed to detect potentially mild and moderate irritants tothe human eye, which are often cosmetic products. The technical limitations of theassay and the applicability domain are discussed below.
There are certain technical limitations specific to MDCK cell culture. The tight junctions
that block the passage of the sodium-fluorescein dye through the monolayer are
increasingly compromised with increasing cell passage number. Incomplete formationof the tight junctions results in increased FL in the non-treated control. Therefore, adefined permissible maximal leakage in the non-treated controls is important. Heat
inactivated-FCS can be difficult to obtain commercially, and the process of heatinactivating serum can cause protein precipitations to form that require filtration; thisprocess in-house can lead to inter-laboratory and intra-laboratory variations in thequality of the serum batch to batch. Protocols do not generally give details on themethod for heat-inactivating serum which could reduce variability and improve tightjunction competence. As with all in vitro assays there is the potential for the cells to
become transformed over time, thus it is vital that passage number ranges for theassays are stated. However, the risk is that cell stocks are depleted quickly, as with theGillette human corneal cells that require a low passage number.
An advantageous feature of the FL assay is the short exposure which is pertinent to thein vivo scenario, where an accidental exposure is likely to be blinked and washed out by
tearing in approximately one minute. Due to the short exposure period, highconcentrations of test materials can be tested which are relevant to human accidental
exposures. High chemical concentrations can react with the insert membranes, and isparticularly noticeable for corrosive compounds. However, one can assume that any
chemical causing membrane corrosivity would also have a significant ocular irritancy
potential. There are some examples of chemicals at high concentration that react with
the insert membrane and compromise fluorescein leakage through the membrane. Thiscan be monitored by exposing an insert with no cells to the highest chemical
concentration tested.
A problem associated with some test materials is the difficulty in efficiently removing
them from the insert after a short incubation period; the incubation periods featured in
the literature range from ten seconds to 15 minutes. This is particularly applicable forviscous materials, such as gels and creams that are commonly formulated to have low
irritation potentials and for which the FL assay was designed to measure. A ten second
exposure is less likely to produce reproducible results than a 15 minute exposure
because if the test material is not removed fully or efficiently, the impact is a greater
proportion of a ten second period in comparison to a 15 minute period. Thus, thereproducibility of the assay is reflected by the ability to control the real/effective
exposure period.
The majority of FL assay protocols featured in the literature tested materials solubilisedin either HBSS or distilled water. Chemicals that are not soluble in HBSS or distilledwater can only be used if they form a stable suspension or emulsion. However,
emulsions and suspensions will not be as homogeneous as a solubilised material and63

Fluorescein Leakage Assay Background Review Document

different areas of the cell monolayer will receive different concentrations of the test
material which will cause variable results. It is not easy to establish that an emulsion orsuspension is uniform or stable, although the short exposure duration provides less time
for the emulsion or the suspension to degrade. Therefore it is important to specify the
total time between emulsion or suspension preparation and end of the exposure time.
Materials that are not soluble or do not form stable aqueous emulsions can besolubilised or suspended in mineral oil.
Highly volatile materials such asacetylformaldehyde need to be solubilised in mineral oil to reduce evaporation andensure that they remain in contact with the cells for the specified exposure period.
INVITTOX Protocols No. 71 and No.120 state that products can be tested neat.
Formulations that are tested neat are typically creams and gels that are already diluted
in a vehicle for direct human exposure, and have low potency. These can be difficult toremove from the insert after the short exposure period. Solids cannot be reliably tested
as the concentration in contact with the cells cannot be assumed to be equal to thatplaced on the cell monolayer, nor uniformly distributed. Also, solids cannot be easily
removed from the cell surface following the short exposure period (Balls et al., 1995).
Like other cell based assays, the FL assay does not have a good predictive capacity fortest materials that have fixative properties and/or reactivity with medium contents. The
FL assay is useful for testing only mild to moderate irritant chemicals within a defined
range where it can measure mechanistic damage caused to the adhesion molecules.
Testing surfactants requires the ionic properties of the chemicals to be taken into
consideration when interpreting results. For example, the pH of cell culture medium canaffect whether an amphoteric surfactant will display anionic or cationic properties(Cheah; BMedSci, 2000). Materials that are water soluble and/or the toxic effect is notaffected by dilution are generally predicted accurately using the FL assay. It is importantthat the basic toxic mechanism is not affected by dilution as the formation of micelles by
surfactants can unpredictably alter cellular responses over a concentration range; this
impacts on the predictive capacity of in vitro assays. Micelles are surfactant molecules
grouped into spheres which reduce the surfactant surface area available for exposure to
the cells. At a set concentration, referred to as the ‘critical micelle concentration’ (CMC),
surfactants form micelles. Thus, it is possible that a higher surfactant concentration
produces less irritancy than a lower concentration in vitro and in vivo, as micelles haveformed at the higher concentration. As the FL assay can test neat or high test material
concentrations, the unpredictable effects of dilution on the CMC could remain
undiscovered. The effect of CMC is a particular problem for the Fixed Dose FL assay,
as only one concentration is measured and the effects of micelle formation on irritancy
potential will not be easily detected. In most of the Fixed Dose FL assay studies
conducted at FAL, a series of fixed doses are tested in order to discriminate between
micellar and non-micellar effects.
The sodium-fluorescein dye is solubilised in HBSS as phenol red and bovine serum
found in the medium could potentially interfere with the OD readings. The humancorneal model uses high calcium KGM medium. As KGM medium is without serum, thismedium type is less likely to interfere with test results than a full serum-medium used for
MDCK cells. However, the KGM medium does have a buffering capacity greater than
the salt solutions used in other FL assays that can compromise the effects of acids or
alkalis.
The inserts used for the FL assay were not designed specifically for the FL assay but asfilters. Insert manufacturers have issued warnings regarding potential membrane64

Fluorescein Leakage Assay Background Review Document

incompatibilities with test chemicals being filtered. It is important to know of any
interaction between the insert membrane and test material in order to be able toconfidently interpret FL assay results. For example, test materials can bind to the insert
membrane, thus making their removal very difficult. Chemical binding to the insert
membrane, is more common for cationic materials, such as benzalkonium chloride,
which are attracted to the positively charged membrane (Balls et al., 1995). The
different inserts available are known to affect the results as acetic acid is incompatible
with the Millicell-HA insert but often used as a positive control for the Anopore
membrane (Balls et al., 1995). Negatively charged cell surface proteins could also
attract positively charged surfactants. Increasing the number of washing steps toremove the test material from the insert membrane can also lead to insert membrane
damage and therefore erroneous results. Routine observations of the monolayer on theinserts throughout the exposure and the recovery phases should detect damaged
monolayers and/or insert membranes and prevent erroneous results being accepted.
Anopore membranes are opaque when wet and so damage to the monolayer and themembrane can be easily observed and the results from the assay discarded.
Alternatively, if test materials with high molecular weights are not removed fully and/or
efficiently, they can physically block the passage of the sodium-fluorescein dye throughthe insert, which could cause chemical effects to be under-estimated. In general,
additional uncontrolled exposure time is a greater proportion for the FL assay with ashort exposure period (~one minute), than with the assays with longer exposures (~15minutes). This leads to greater variability in results, and low assay reproducibility. As
the FL assay can be repeated at multiple time-points, erroneous results due toineffective removal of the test material would more likely be detected in comparison tocell viability assays which use single time-points. The efficient removal of test agentsfrom the eye is also a concern of the in vivo test.
Like many in vitro assays for eye irritancy, there are a number of endpoints that aremeasured in vivo that presently cannot be measured or replicated using the FL assay.
The effect of lachrymation which reduces toxic effects by diluting and removing the test
material are difficult to quantify and replicate in vitro. However, the results gained using
the Draize test in rabbits eyes are not wholly representative of the human situation due
to the species variation in lachrymation rates. Pathological conditions such as chemosis
which are scored in the Draize test cannot be measured using an in vitro test.
In contrast to most cell-based in vitro assays for eye irritation, reversibility and recovery
of effects can be measured using the FL assay for up to 72h following the initial
exposure. For assessing recovery, the inserts are removed to new wells containing
fresh medium following the chemical exposure. Transferring the filter inserts can cause
bubbles to form under the basal side of the insert membrane which can impede contact
between the cells and the medium. Bubbles can also reduce the passage of fluoresceinfrom the apical side of the insert membrane into the solution below the insert membraneand distort results (Balls et al., 1995). Repeated assays on the same population of cells
are more likely to cause damage to the monolayer.
Attempts to study the effects of repeated chemical exposures have proved unsuccessful
following the second exposure due to cumulative cell damage which destroys themonolayer (Clothier and Sansom, 1996).
A combined FL/AB assay has been developed to try to distinguish the specific damage
caused to the tight junctions from cell membrane damage. A disadvantage specific to65

Fluorescein Leakage Assay Background Review Document

the combined FL/AB assay is that the AB assay results can be erroneous due to thelength of time taken to perform the protocol steps. AB reduction is affected bytemperature (Clothier, personal communication). The combined assay is a lengthy
procedure which involves temperature changes of the cell culture. In addition, high celldensity and prolonged culture times allow reversal of the AB reduction process which
can lead to an over-estimation of toxicity (Larson et al., 1997). Therefore the AB assay
results may not correctly represent the chemical-induced damage to cell viability. This
would cause FL results to be read incorrectly leading to over-estimations or underestimations of chemical effects on tight junctions.
In conclusion, the FL assay is better suited to measure high concentrations of test
materials that have low to mid-range toxicity and are soluble in water or HBSS. Test
materials that are difficult to remove from the inserts due to viscosity or binding to themembrane are not accurately measured. Materials that have their basic toxic
mechanism affected by dilution are not accurately measured by the FL assay.

66

Fluorescein Leakage Assay Background Review Document

2.2.5. Othersi. Combined assays
The FL assay has been used in combination with other assays, principally cell viability
assays. These assay combinations allow the specific damage caused to the tightjunctions to be distinguished from general cell loss and non-specific cell damage
affecting viability and/or membrane functions.
A combined FL/AB assay was used to predict human skin responses to a set of test
materials comprised of surfactant-based handwashes, laundry detergents andmoisturisers (Clothier et al., 2002). The AB assay measures the reduction of resazurin
to resorufin by a number of enzymatic pathways, and the rate of reduction can be used
as a measure of cell viability (Page et al., 1993; O’Brien et al., 2000). Using a combined
FL/AB assay, chemical effects on cell viability and barrier integrity can be measured. A
significant loss of resorufin production and an increase in FL can indicate loss of cell
viability leading to cell death, whilst FL with only small decreases in resorufin production
indicates reversible barrier damage to the cell monolayer. Details of this study are given
in brief.
Following a one minute exposure, a combined solution of sodium-fluorescein dye andAB was placed onto the MDCK monolayer, and incubated for 60 minutes. AB was also
placed into the well to allow apical and basal uptake. Following incubation, the solution
in the wells was measured for FL. The solution from the insert was then added to that in
the corresponding well and measured for resorufin production. Repeat assays withfluorescence readings were performed 4h, 24h, 48h and 72h after the initial test material
exposure. The results from the two assays were considerably different at the various
time-points. For example, 24h after a 25mg/ml exposure to ‘Kids’ antibacterial
moisturising handwash’ cellular activity measured by the AB assay was restored. The
epithelium impermeability, as measured by the FL assay, was not restored until the 48htime-point.
A PM was generated using FL/AB data from the hand-wash formulations. FL/AB data
were compared to human patch test data from experiments which were performed
during the same time-period as the FL assay. The FL/AB data for the laundry
detergents and moisturisers were entered into the PM to predict the human patch data.
The PM was under-predictive for two of ten handwashes and over-predictive for two of
six moisturisers and eight out of ten laundry powders. The in vivo patch test data towhich the in vitro data was compared was also notably variable (Clothier et al., 2002).
The combined FL/AB assay was also used to study recovery following repeat exposures
to surfactants (Clothier and Sansom, 1996). The effect of repeated surfactant
exposures were of interest as many surfactant-based products, such as cosmetics, aredesigned for repeated use. Adverse reactions are potentially more likely to be observed
following multiple exposures rather than a single acute exposure. Therefore, the abilityof the FL/AB assay to measure the effects of repeated exposures to the surfactant
CAPB was assessed. MDCK cells were grown to confluence and the FL/AB assay
performed prior to test material exposure. The cells were exposed to CAPB for one
minute and the FL/AB assay carried out immediately and then at 24h and 72h following
exposure. After the 72h FL/AB assay, the cells were re-treated with one of four different
surfactants for one minute. The FL/AB assay was then performed at 1h, 24h and 72h67Fluorescein Leakage Assay Background Review Document

following the initial exposure. After the second exposure, cells exposed to a pretreatment of CAPB did not recover as well as those that only received HBSS. In
general, the lower concentrations of the second surfactant treatment were found topromote recovery as determined by decreased FL and increased AB reduction (Clothierand Sansom, 1996).
The combined FL/AB assay was used with the J-HCET cell line (Araki-Sasaki et al.,1995) to predict the effects of repeated exposures to low doses of surfactants on thecorneal epithelium barrier. The J-HCET cells were grown in low concentrations ofsurfactant containing medium prior to toxicant exposure (Cheah; BMedSci, 2000). Pretreatment of a low dose surfactant was hypothesised to reduce the sensitivity of the cells
to the following acute dose as measured by the FL assay. These experimental
conditions reflected the in vivo scenario of wearing eye drops or being repeatedly
exposed to contact lens solutions. Cells were treated with low doses of either CAPB or
benzalkonium chloride and then treated with either SDS, tween 20, CAPB or
benzalkonium chloride (Cheah; BMedSci, 2000). A five minute chemical exposure wasused in combination with a FL15 endpoint (Cheah; BMedSci, 2000). FL/AB assays
following this treatment and prior to the acute exposure showed that the cell junctions
were not affected by these low doses. FD was used in conjunction with J-HCET cells as
sodium-fluorescein dye produced variable results and a greater rate of FL than with
MDCK cells. For this study, FDs with molecular weights of 4.4kD (FD-4) and 9.5kD (FD9) were used to assess the size of the tight junctions. FD-4 at a concentration of 200µM
showed the smallest variation and optimum FL OD values.
Throughout theexperiments, AB values did not differ considerably, indicating that measured FL was due
to damaged tight junctions rather than cell damage. The FL/AB assay was repeated at
24h and 48h after the second treatment, and recovery as indicated by reduced FL, wasobserved by 48h for all treated cells. In agreement with in vivo data, the FL assay
ranked the four surfactants accordingly, with the cationic being the most toxic followedby the anionic, amphoteric and non-ionic surfactants (Cheah; BMedSci, 2000). Further
work was deemed necessary in order to determine the use of this cell model for
predicting the in vivo situation (Cheah; BMedSci, 1997).
The FL assay has also been used in combination with the Neutral Red Uptake (NRU)
assay which was used to distinguish tight junction damage from loss of MDCK cell
viability caused by ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA) (Carter;
BMedSci, 1994). EGTA was tested as it was hypothesised to be a more appropriate
positive control than the acetic acid which was then routinely used. EGTA chelates
extracellular calcium which is required for tight junction function (Carter; BmedSci,
1994). A combined NR dye/ sodium-fluorescein dye solution was added to the inserts
and the protocols modified to allow a 30 minute incubation with the sodium-fluorescein
dye and a 60 minute incubation with the NR dye. Following a one hour incubation, thedyes were removed, cells washed, and a fixative added to each well. OD values were
not significantly different for the FL assay performed alone or in combination with the
NRU assay. The addition of the fixative for the NRU assay prevented repeated FL
assays being performed on the same cells so recovery could not be assessed. EGTA
was found to require more than a one minute exposure to cause damage to the tightjunctions and was therefore concluded not to be an appropriate positive control (Carter;
BMedSci, 1994).

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Fluorescein Leakage Assay Background Review Document

ii. Storage of seeded MDCK cells
The FL assay has featured in a number of large-scale validation studies (Balls et al.,
1995; Gettings et al., 1996; Zanvit et al., 1999). This had led to investigations to study
the feasibility of storing pre-prepared plates seeded with MDCK cells. The blood
substitute HypoThermasolTM was successfully used to store human epidermal cells
which were viable and retained functional integrity at 4oC. The ability of MDCK cells toremain viable and form tight junctions following 60 hours of storage in HypoThermasolTM
was investigated. Five surfactants representing a range of ionics and cytotoxic
potencies were tested using INVITTOX Protocol No. 71 and INVITTOX Protocol No. 82.
A 60h exposure of MDCK cells to HypoThermasolTM at 4oC did not affect MDCK cell
growth rate nor their ability to form tight junctions. In comparison, cells stored in
medium for 60h at 4oC detached from the membrane when returned to 37oC. FL rates
were comparable for both cold-stored and normally plated MDCK cells following
treatments with the five surfactants for both acute exposure and recovery time points.
Annex I

Standard FL assay protocols: a. The Fluorescein Leakage Test, INVITTOX
Protocol No. 71; b. Fluorescein Leakage Test - SOP of Company # 4,
INVITTOX Protocol No. 120; c. Trans-epithelial Permeability (TEP) Assay,
INVITTOX Protocol No. 86; d. Fixed Dose Procedure for The Fluorescein
Leakage Test, INVITTOX Protocol No. 82 ; e. Company # 3 Robotic TransEpithelial Permeability (TEP) Assay ; f. ECVAM Prevalidation Study Phase
II Protocol; g. ECVAM Prevalidation Study Phase III Protocol

Appendix II Complete list of protocol steps

69

Fluorescein Leakage Assay Background Review Document

3. Within-laboratory reproducibility (Module 2)
Fourteen FL assay data sets were appropriate for within-laboratory reproducibility
analyses. The mean, SD and CV were calculated for all the chemicals and formulations
tested in these data sets (refer Annex II). The relevant information for these studies isgiven in table 3.1.

70

Fluorescein Leakage Assay Background Review Document

3.1

Table presenting the results and relevant information for each study

INVITTOX Protocol No. 71

Study

Variability
Reported

No. ofTest
SubstancesType of Test
Substances

No. ofLaborat-oriesNo. ofOperators

No. ofExperiments

No. ofreplicates (wells)

Data
FormatReport on Comparison of 40
Cosmetic and Domestic
Formulations Supplied byCompany # 8 and Evaluated by 3
In Vitro Cytotoxicity Tests(FRAME, 1992)

No

40

surfactant-based
formulations

1

3

4

3

FL20, FL50
(mg/ml) T0

3

FL20 (mM)
T0, T24, T48,
T72

3

FL20, FL50
(mg/ml) T0
and T72

Final Report on Testing of 12 Mild
Surfactants supplied by Company# 5 for Cytotoxicity Testing at the
FAL (FRAME, 1992)
Use of In Vitro Methodology to
Predict Irritancy Potential of
Surfactants (Hubbard et al., 1994)
Use of In Vitro Methodology to
Predict Irritancy Potential of
Surfactants (Hubbard et al., 1994)–re-runs based on activity
The Evaluation of Pesticide
Ingredients and Formulations In
Vitro and Correlations with In Vivo
Data (Clothier et al., 1995)

n/a

No

12

10

mild surfactants

1

1

surfactants

Not stated

3

n≤ 4

n≤ 5

No

10

surfactants

1

3

n≤ 5

3

FL20 FL50(mg/ml) T0
and T72No4

pesticides pure
ingredients

1

1

4

3

FL20 (mg/ml)
T0 and T72

71

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 120

No. ofTest
SubstancesType of Test
Substances

No. ofLaborat-oriesNo. of Operators

No.
of
Exp
eriments

30

1

Not stated4

3

FL20 (mg/ml)
T0 and T72

ECVAM Prevalidation Study-Phase II (Southee, 1998)

No
A hierarchical
ANOVA
noted
laboratory
variation(results notreported).

Surfactants andsurfactant-based
formulations
soluble in HBSS

5

4 chemicals and 1
formulation

4

n≤5

3

FL20 (mg/ml)
T0 and T4

ECVAM Prevalidation Study-Positive control data (Southee,1998) Phase IINo

1

0.16mg/ml SDS

4

FRAME =1,
ECVAM =2,
Company # 3=1,
Company #7=not
statedFRAME =1 (2occasions 1 otherperson), ECVAM=2, Company #3=1,
Company # 7=not
statedn≤2
33FL20 (%) T0
and T4

3

FRAME =1 (2occasions 1 otherperson), ECVAM=2
Company # 3 =1n≤53FL20 (mg/ml)
T0 and T4

3

FRAME =1,
ECVAM =unknown,
Company # 3 =1

n≤1
9

3

FL20 (%) T0
and T4Study
A Summary Report of the COLIPAInternational Validation Study on
Alternatives to the Draize Rabbit
Eye Irritation Test (Brantom et al.,1997) FRAME

ECVAM Prevalidation Study-Phase III (Southee, 1998)

ECVAM Prevalidation Study-Positive control data (Southee,1998) Phase III

Variability
Reported

No

No

10

1

surfactants

0.16mg/ml SDS

72

No. ofreplicates
Data(wells) Format

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 86

Study

Variability
Reported

No. ofTest
Substances

CTFA Phase III TEP assay data(submitted by CTFA)

No

23

TEP assay Formulation data(submitted by Company # 3)No41

surfactant-based
formulations
formulations;some surfactantbased

Variability
Reported

No. ofTest
SubstancesType of Test
Substances

1

Not stated

n≤ 4

3

1

Not stated

n≤ 4

3

Type of Test
Substances

No. ofLaborat-oriesNo. ofOperators

No. ofExperiments

No. ofreplicates (wells)

Data
Format1

Not statedn≤4

Not stated

EC50 (%) T0

1

Not statedn≤3

Not stated

EC50 (%) T0

No. ofLaborat-oriesNo. ofOperators

No. ofExperiments

No. ofreplicates (wells)

Data
FormatINVITTOX Protocol No. 82

StudyCompany # 5 test concentration X(FRAME, 1993)No

12

surfactants forcosmetics

Company # 5 test concentration XI(FRAME, 1993)No

12

surfactants forcosmetics73

FL (%)T0,
T24, T48,
T72
FL (%)T0,
T24, T48,
T72

Fluorescein Leakage Assay Background Review Document

3.2.

Compilation of Results

3.2.1. Statistical approach(es) used: description & rationale for the approach used
Data were analysed according to ECVAM guidance. The SD and the CV were plottedagainst mean FL assay values for the different endpoints, FL (mg/ml), FL (%) (figure
3.2.1.a-b). The aim was to determine which measure of variability (i.e. S.D or CV)
produced the most consistent level of variability over the entire range of mean FL assay
values. The most consistent measure of variability would then be used in further
analyses to assess protocol within-laboratory variability.

200

Variability

150

100

50

0
0.1

1.0

10.0

Mean FL20 (mg/ml)

a.

74

100.0

1000.0Fluorescein Leakage Assay Background Review Document

100000

Variability

1000

10

0

0
0.01

0.10

1.00
M e an FL(%)

10.00

100.00b.

Variability

100

50

0
0.1

c.

1.0

10.0

100.0

Mean FL20 (mM)

Figure 3.2.1. ◊ SD and ♦ CV values plotted against the mean FL assay results; a. FL20
(mg/ml) ; b. FL (%) (n≥2); c. FL20 (mM) . Materials which produced greater-than or lessthan values in any experimental run were not plotted. Data for these graphs were takenfrom the studies listed in table 3.1. The raw data are visible in the Annex file titled‘Intralaboratory II’ and are arranged according to the units of measurement, e.g. %, mM,
mg/ml.
75

Fluorescein Leakage Assay Background Review Document

Figure 3.2.1.a. predominately contains data produced by measuring FL immediately
following a one minute chemical exposure, and to a lesser extent from FL assays
measured 72h and then 4h, 24h and 48h following the initial test material exposure. The
mean FL20 (mg/ml) values covered a larger range than the mean FL (%) values.
Figure 3.2.1.b. consisted predominately of data produced by measuring FL immediately
following a one minute exposure and 4h exposure, and to a lesser but equal extent fromFL assays measured at 24h, 48h and 72h following the initial test material exposure.
For FL (%) data, the range of values covered by the SD and CV values were similaralthough the CV values were higher values in comparison to the SD values.
Figure 3.2.1.c. consisted of data produced from a single study where only mild
surfactants were tested. Data are shown for FL measured immediately following a one
minute exposure and then at 4h, 24h, 48h and 72h following the initial test ma
terial exposure. The CV values were higher in comparison to the SD values andcovered a wider range.
For the larger data sets, the SD values increased as the mean FL20 (mg/ml) and FL%
values increased. The CV measurement, which exhibited random distribution acrossthe range of FL20 (mg/ml) values and FL% values, was selected for analyses.
3.2.2. Results and discussion
Table 3.2.2. shows the summary results for each study featured in table 3.1.. For eachtest material, greater-than values were not included in the calculations of mean, SD and
CV. In some cases a single value was used to determine the mean as the greater-thanvalues generated by some experimental repeats were not included in the calculation.
For each study, the overall mean, overall median, mean SD, median SD, mean CV andmedian CV were calculated from the mean, SD and CV for each test material. For test
materials that only produced greater-than values, no mean, SD or CV was calculated forthat test material. For studies that contained some test materials with no mean, mean
SD or mean CV values, the overall mean, overall median, mean SD, median SD, mean
CV and median CV values could not be calculated. Annex II shows the raw data used
to produce the summary values in table 3.2.2., and the mean, mean SD and mean CV
for each test material where calculated.

76

Fluorescein Leakage Assay Background Review Document

Table 3.2.2. Summary table of the overall reproducibility per study. There were 25 data sets (compared to 14 in table 3.1) as somestudies contained multiple data sets (e.g. ECVAM Prevalidation study (Southee, 1998)).
INVITTOX Protocol No. 71

Study
Report on Comparison of 40
Cosmetic and Domestic
Formulations Supplied byCompany # 8 and Evaluated by
3 In Vitro Cytotoxicity Tests(FRAME, 1992)

Company #5 Chemicals(FRAME, 1992)
Final Report on Testing of 12
Mild Surfactants supplied byCompany # 5 for Cytotoxicity
Testing at the FAL (FRAME,
1992)
Use of In Vitro Methodology to
Predict Irritancy Potential of
Surfactants (Hubbard et al.,1994)
Use of In Vitro Methodology to
Predict Irritancy Potential of
Surfactants (Hubbard et al.,1994) –re-runs based on
activity

No. ofNo. of Test ExperiSubstances ments

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV (%)*

40

4

FL20
(mg/ml)
T0

24.8

-

-

5.2

-

-

n≤ 5

FL20
(mg/ml)T0 and
T72

-

-

-

-

-

-

n≤ 4

FL20
(mM) T0,
T4, T24,
T48

-

-

-

-

-

-

n≤ 5

FL20
(mg/ml)T0 and
T72

5.1

2.8

63.2

5.5

2.6

58.6

n≤ 5

FL20
(mg/ml)T0 and
T72

16.2

9.1

56.5

11.2

5.9

56.8

6

12

10

10

77

Fluorescein Leakage Assay Background Review Document

Study
The Evaluation of Pesticide
Ingredients and Formulations In
vitro and Correlations with In
Vivo Data (Clothier et al., 1995)

No. ofNo. of Test ExperiSubstances ments

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV(%)*

4

FL20(mg/ml)T0 and
T72

-

-

-

-

-

-

No. ofNo. of Test ExperiSubstances ments

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV(%)*12n≤ 4FL(%) T0,
T24, T48,
T72

6.1

6.57

94.9

0.3

0.3

98

n≤ 4

FL(%) T0,
T24, T48,
T72

26.2

-

-

16.8

-

-

4

INVITTOX Protocol No. 82

StudyCompany # 5 test concentration
X (FRAME, 1993)

Company # 5 test concentration
XI (FRAME, 1993)

1278

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 120

Study
A Summary Report of the
COLIPA International Validation
Study on Alternatives to the
Draize Rabbit Eye Irritation Test
(Brantom et al., 1997) FRAME
ECVAM Prevalidation Study(Southee, 1998) Phase II–FRAME Laboratory
ECVAM Prevalidation Study(Southee, 1998) Phase II–Company # 7 Laboratory
ECVAM Prevalidation Study(Southee, 1998) Phase II–ECVAM Laboratory
ECVAM Prevalidation Study(Southee, 1998) Phase II– Company # 3 Laboratory

No. ofNo. of Test ExperiSubstances ments

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV(%)*

30

4

FL20(mg/ml)
T4

-

-

-

-

-

-

n≤5

FL20
(mg/ml)T0 and T4

129.9

35.6

36.9

19.7

7.9

42.2

n≤5

FL20
(mg/ml)T0 and T4

250.3

-

-

24.1

-

-

n≤5

FL20
(mg/ml)T0 and T4

149.5

-

-

13.9

-

-

n≤5

FL20
(mg/ml)T0 and T4

-

-

-

-

-

-

5

5

5

5

79

Fluorescein Leakage Assay Background Review Document

Study

No. ofNo. of Test ExperiSubstances ments

ECVAM Prevalidation Study(Southee, 1998) Phase II–FRAME Laboratory

1 -Positive

ECVAM Prevalidation Study(Southee, 1998) Phase II– Company # 7 Laboratory

1 -Positive

ECVAM Prevalidation Study(Southee, 1998) Phase II–ECVAM Laboratory

1 -Positive

ECVAM Prevalidation Study(Southee, 1998) Phase II– Company # 3 Laboratory

1 -Positive

ECVAM Prevalidation Study(Southee, 1998) Phase III–FRAME Laboratory

1 -Positive

ECVAM Prevalidation Study(Southee, 1998) Phase III–ECVAM Laboratory

1 -Positive

ECVAM Prevalidation Study(Southee, 1998) Phase III– Company # 3 Laboratory

1 -Positive

Control Data

Control Data

Control Data

Control Data

Control Data

Control Data

Control Data

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV(%)*n=3

FL (%) T0
and T4

79.7

13.9

17.4

79.7

13.9

17.4

n=6

FL (%) T0
and T4

11.4

4.9

73.6

11.4

4.9

73.6

n=23

FL (%) T0
and T4

12.8

7.7

67.3

12.8

7.7

67.3

n=5

FL (%) T0
and T4

17.1

5.7

35.3

17.1

5.7

35.3

n≤19

FL (%) T0
and T4

4.3

2.7

128.0

4.3

2.7

128.0

n≤15

FL (%) T0
and T4

9.3

1.9

35.4

9.3

1.9

35.4

n≤5

FL (%) T0
and T4

10.5

3.8

603.0

10.5

3.8

603.0

80

Fluorescein Leakage Assay Background Review Document

Study
ECVAM Prevalidation Study(Southee, 1998) Phase III–FRAME Laboratory
ECVAM Prevalidation Study(Southee, 1998) Phase III–ECVAM Laboratory
ECVAM Prevalidation Study(Southee, 1998) Phase III– Company # 3 Laboratory

No. ofNo. of Test ExperiSubstances ments

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV (%)*

10

n≤3

FL20
(mg/ml)T0 and T4

50.6

6.7

28.5

1.9

0.6

16.4

n≤5

FL20
(mg/ml)T0 and T4

28.0

-

-

7.6

-

-

n=2

FL20
(mg/ml)T0 and T4

-

-

-

-

-

-

10

10

81

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 86

Study

No. ofNo. of Test ExperiSubstances ments

Data
FormatOverall
Mean**

Mean
SD*

Mean
CV
(%)*

OverallMedian**MedianSD*

MedianCV (%)*

CTFA Phase III TEP assay
data (submitted by CTFA)

23

EC50 (%)
T0

3.7

0.4

13.5

1.9

0.2

14.3

2.8

0.2

8.1

2.1

0.1

7.3

n≤4

TEP assay Formulation data
EC50 (%)(submitted by Company # 3) 41n≤3T0* calculated from the SD and CV calculated for each test material** calculated from the mean value for each test material82

Fluorescein Leakage Assay Background Review Documenti. Protocols
The FL20 (mg/ml) data were predominately produced by the FAL using INVITTOX
Protocol No. 71 or a similar protocol with slight modifications. INVITTOX Protocol No.
71 was based on the protocol of Tchao (1988). Data from the Hubbard et al., (1994)
paper used the protocol of Shaw et al., (1990). This was one of the first FL assay
protocols published and was very similar to the first FL assay protocol developed by
Tchao (1988). An advantage of the data provided by the FAL is that it contained
information regarding the time and date of the individual experiments. This information
enabled analyses of operator and time variability to be carried-out (Section 3.2.3.).
FL20 (mg/ml) data were also produced using INVITTOX Protocol No. 120, or a slightly
modified protocol, which was performed by a number of different laboratories as part of
the ECVAM Prevalidation study (Southee, 1998). Phase II of the ECVAM Prevalidation
study used a slightly different protocol to the Phase III protocol that was later adopted as
INVITTOX Protocol No. 120. Raw positive control data for 0.16mg/ml SDS were alsoavailable from the ECVAM Prevalidation study Phase II and Phase III for which theendpoint was FL%.
There were fewer data for INVITTOX Protocol No. 86 which were produced by Company# 3. There were even fewer data for a protocol similar to INVITTOX Protocol No. 82;
results were reported for 12 surfactants that were tested at two different concentrations.
A summary of all the differences between the protocols discussed in this section,
including those not known to be significant, are given in table 3.2.2.1.

83

Fluorescein Leakage Assay Background Review Document

Table 3.2.2.1. The main protocol differences between the FL assay protocols for which data were available to assess intra-laboratory
variability.

Protocol Step

Evaluation of thePrevalidation
Process: The
Fluorescein
Leakage Assay(Phase II)(Southee, 1998)
MDCK NBL-2(ECACC:
85011435)

Evaluation of thePrevalidation
Process: The
Fluorescein
Leakage Assay(Phase III)
(Southee, 1998)
MDCK NBL-2(ECACC:
85011435)

FLT- SOP
Company # 4
INVITTOX
Protocol No. 120(developed asconsequence of
ECVAM
Prevalidation
Study, 1998)

Loss of Transepithelial
Impermeability ofa Confluent Layer
of Madin-Darby
Canine Kidney(MDCK) Cells asa Determinant ofOcular Irritancy
Potential (Shawet al., 1990)
MDCK CB997(ECACC:84121903)

Fluorescein
Leakage Test.
INVITTOX
Protocol No. 71
MDCK CB997(ECACC:84121903)

Trans-epithelial
Permeability of
Fluorescein In
Vitro as an Assayto Determine Eye
Irritants (Tchao,
1988)

Seeding Density

2x10 cells/ml

2x10 cells/ml

MDCK NBL-2(ATCC: CCL34)
4x105 cells/ml(500µl/insert=
2x105 cells/insert)

Medium Type

MEM

MEM

MEM w/o phenol
redMillicel-HA, 12mm
diameter (0.45µm
pore size)
Insert washed with
500µl HBSS with
Ca++, Mg ++ in
insert and well.
Prior to treatment,
medium from underinsert is removed,
replaced with HBSS
Ca++, Mg ++Millicel-HA, 12mm
diameter (0.45µm
pore size)
Insert washed with
500µl HBSS with
Ca++, Mg ++ in
insert and well.
Prior to treatment,
medium from underinsert is removed,
replaced with HBSS
w/o Ca++ and Mg++

Millicel-HA, 12mm
diameter (0.45µm
pore size)

Anocell 104x105 cells/ml(400µl/insert)
DMEM x1 (Lglutamine and15mM HEPES)/Ham's F12 (1:1)
Millicel-HA, 12mm
diameter (0.45µm
pore size), Anocell
10 inserts can also
be usedN

500µl distilled
water

N

HBSS

15

15

15

1

1 or 15

15

Cell Type/Strain

Insert Type's, Pore
Size's, coating

Rinsing before
Material Incubation(solution andvolume)Material Exposure(mins)

5

5

84

105 cells/insert (in400µl medium)
DMEM/Ham's F12
(1:1)

MDCK
1.5x105
cells/insert

MEM
Millicell with HATF
(surfactant-free)membraneFluorescein Leakage Assay Background Review Document

Protocol Step
Material Volume(µl)

Solvent

Fluorescein
Addition Outline
Fluorescein
Volume added (µl)
Fluorescein
Concentrationdelivered tomonolayer (mg/ml)

FLT- SOP
Company # 4
INVITTOX
Protocol No. 120(developed asconsequence of
ECVAM
Prevalidation
Study, 1998)

Loss of Transepithelial
Impermeability ofa Confluent Layer
of Madin-Darby
Canine Kidney(MDCK) Cells asa Determinant ofOcular Irritancy
Potential (Shawet al., 1990)

Fluorescein
Leakage Test.
INVITTOX
Protocol No. 71

Trans-epithelial
Permeability of
Fluorescein In
Vitro as an Assayto Determine Eye
Irritants (Tchao,
1988)

100µl

200µl

500µl

HBSS or mineral
oil

HBSS

Inserts checked fordamage andmoved to 24WP
with 400µl HBSS;
400µl 0.01% Nafluorescein in
HBSS added toinsertFluorescein added
to each insert
which is placed in
fresh 24WP
containing 500µl
buffer

400µl

500µl

Evaluation of thePrevalidation
Process: The
Fluorescein
Leakage Assay(Phase II)(Southee, 1998)

Evaluation of thePrevalidation
Process: The
Fluorescein
Leakage Assay(Phase III)
(Southee, 1998)

200µl
HBSS containing
Ca++ and Mg++ orlight mineral oil.
Prepared just before
use in case ofemulsion and/or
suspensions

200µl
HBSS with Ca++and Mg++, or lightmineral oil.
Prepared just before
use in case ofemulsion and/or
suspensions

500µl

Treated inserts
transferred to new
24WP containing
500µl HBSS with
Ca++, Mg ++/well.
200µl 0.01%
fluorescein added toeach insert.

Treated inserts
transferred to new
24WP containing
500µl HBSS with
Ca++, Mg ++/well.
200µl 0.01%
fluorescein added toeach insert.

Place washedinserts into new
24WP with 500 µl
warm HBSS, 500µl
0.01% Nafluorescein in
HBSS put into
each insert

tested neat or in
distilled water
Following washing
step, inserts
removed to freshwells containing
500µl warm
Dulbecco's Ca2+
PBS. 500µl 0.02%
w/v fluorescein in
Ca2+ PBS added
and plateincubated at RT

200µl

200µl

500µl

500µl

0.01% (w/v) Nafluorescein in HBSS
with Ca2+ andMg++

0.01% (w/v) Nafluorescein in HBSS
with Ca++ andMg++

0.01mg/ml Nafluorescein in
HBSS (0.001%(w/v/)

HBSS

85

0.02% (w/v) Nafluorescein in
HBSS

0.1mg/ml Nafluorescein HBSS
(0.01% (w/v)) (0.02%
Na-fluorescein HBSS
for Anocell inserts)

0.02% Nafluorescein in
HBSSFluorescein Leakage Assay Background Review Document

Protocol Step

Evaluation of thePrevalidation
Process: The
Fluorescein
Leakage Assay(Phase II)(Southee, 1998)

Evaluation of thePrevalidation
Process: The
Fluorescein
Leakage Assay(Phase III)
(Southee, 1998)

Time allowed for
Fluorescein
Leakage (mins)
Vehicle Control

30±2 Incubator
HBSS

30±2 Incubator
HBSS

Endpoint

FL20 (mg/ml) T0, T4

FL20 (mg/ml) T0, T4

Plate gently shaken for
1min, then 200µl
samples taken from theplate and transferred to
96WP 485-

Plate gently shaken for
1min then 200µl samples
taken from the plate andtransferred to 96WP

490nm/650nm(excitation/emission)

485-490nm/650nm(excitation/emission)

Optical Density
Wavelengths(excitation/emission)

FLT- SOP
Company # 4
INVITTOX
Protocol No. 120(developed asconsequence of
ECVAM
Prevalidation
Study, 1998)

Loss of Transepithelial
Impermeability ofa Confluent Layer
of Madin-Darby
Canine Kidney(MDCK) Cells asa Determinant ofOcular Irritancy
Potential (Shawet al., 1990)

Fluorescein
Leakage Test.
INVITTOX
Protocol No. 71

30 RT
HBSS
FL20, FL10
(mg/ml) T0, T4

60 RT
medium
FL20, FL50
(mg/ml) T0

30 RT
HBSS
FL20, FL50
(mg/ml) T0485nm/530nm(excitation/
emission)

492nm/620nm(excitation
emission)

485nm/530nm(excitation/
emission)

86

Trans-epithelial
Permeability of
Fluorescein In
Vitro as an Assayto Determine Eye
Irritants (Tchao,
1988)
30 24oC
HBSS
EC50 (%) T0

490nm

Fluorescein Leakage Assay Background Review Document

No study has been reported which compared the within-laboratory variability for
INVITTOX Protocol No. 71 or for INVITTOX Protocol No. 120. From the FL assay data
available to this BRD, the CV values were plotted against the mean values for
INVITTOX Protocol No. 71 and INVITTOX Protocol No. 120, as an approach to compare
their relative reproducibilities. This analysis has limitations as different types ofmaterials have been tested in the two protocols. Data from INVITTOX Protocol No. 71
and from similar protocols (i.e. Shaw et al., (1990) were plotted; data from INVITTOX
Protocol No. 120 and from similar protocols (i.e. ECVAM Prevalidation Study) were alsoplotted (figure 3.2.2.1.).

200

Variability (CV)

150

100

50

0
0.1

1.0

10.0

100.0

1000.0M e an FL20 (mg/ml)

Figure 3.2.2.1. The CV values for INVITTOX Protocol No. 71 ♦, and INVITTOX Protocol
No. 120 ◊ plotted against FL assay results for all available raw data. Materials that had
greater-than or less-than FL20 (mg/ml) values were not plotted (n≥2).
There were a similar number of data points for both INVITTOX Protocol No. 71 and
INVITTOX Protocol No. 120. INVITTOX Protocol No. 71 was employed in a greaternumber of smaller studies (i.e., fewer test materials) in comparison to INVITTOX
Protocol No. 120 which was carried out in fewer but larger-scale studies where many
more materials were tested, i.e. ECVAM Prevalidation study (Southee, 1998) and the
COLIPA Eye Irritation International Study (Brantom et al., 1997). In addition, data from
INVITTOX Protocol No. 71 was produced exclusively from the FAL, whilst INVITTOX
Protocol No. 120 was carried out by a number of different laboratories. The data plottedin figure 3.2.2.2. show that a wider range of FL20 (mg/ml) values were produced by thematerials tested using INVITTOX Protocol No. 120 and that intra-laboratory variability
was comparable for INVITTOX Protocol No. 71 and INVITTOX Protocol No. 120 overthe same range of FL20 (mg/ml) values.
TEP assay data submitted by Company # 3, and from the CTFA study Phase III wereplotted (figure 3.2.2.2.). The CV was plotted against the mean EC50 values for the test
formulations. The data were from an in-house protocol of a robotic TEP assay. The
87

Fluorescein Leakage Assay Background Review Document

assay was essentially the same as INVITTOX Protocol No. 86 which was developed byCompany # 3. The CTFA Phase III compositions were known, whilst only an outline of
the compositions for 17 of the 41 formulations tested in-house by Company # 3 wasprovided.

Variability (CV)

30

20

10

0
0.1

1.0

10.0

100.0

Mean EC50 (%)

Figure 3.2.2.2. The CV values for TEP assay data plotted against the mean TEP assay
values (n=2) for Company # 3 formulations ▲, and CTFA Phase III formulations ∆.
The core range of mean EC50 values was relatively small in comparison to the data setsfor INVITTOX Protocol No. 71 and INVITTOX Protocol No. 120. Additionally, the CV
values produced by the data set were smaller in comparison to INVITTOX Protocol No.
71 and INVITTOX Protocol No. 120. This could be due to the use of EC50 values rather
than FL20 values. FL20 values are likely to produce more variable results than EC50
values as the concentrations which cause 20% effects, tend to be more variable as they
are at the start of a dose response curve, whilst EC50 concentrations are more likely to
be in the linear part of the dose-response curve. It is difficult to directly compare thelevel of variability produced by the various FL assay protocols due to the different formatof the data, although the CV values did seem generally lower for the TEP assay. Thisfinding is indicative of an established system that is in routine use.
Positive Control Data over time, from the ECVAM Prevalidation study (Southee, 1998)
The ECVAM Prevalidation study (Phase II and Phase III) (Southee, 1998) was one offew studies featuring a FL assay protocol to have well documented positive control data.
The test protocol was similar to INVITTOX Protocol No. 120. The level of FL (%) that
resulted from the positive control (0.16mg/ml SDS) was plotted against time, todetermine variability (figure 3.2.2.3. a-b). The number of data points varied perlaboratory, as they differed in their use of the positive control. The FAL and ECVAM
performed a positive control per chemical/plate, whilst Company # 7 and Company # 3
used the same positive control data for all chemicals performed within that run.

88

Fluorescein Leakage Assay Background Review Document

100
90
80
70
FL%

60
50
40
30
20
10
0
1

Days 10100

1Days 10

100

a.
110
100
90
80
FL%

70
60
50
40
30
20
10
0

b.
Figure 3.2.2.3. The effect of time on the ECVAM Prevalidation Phase II SLS positive
control FL% values for the four participating laboratories at a.) T0; b.) T4; □, FAL;■,Company # 7; ▲, Company # 3 ; ∆, ECVAM.

- - - - - - - - - Pre-defined range inwhich the positive control values should fall for the FL assay results from the test
chemicals to be accepted. A version of INVITTOX Protocol No. 120 was used.
For all laboratories at both time-points, the FL positive control results did not vary withtime (day) (figure 3.2.2.3.). Where two or more positive control values were generated
for a single day, (FAL, ECVAM) the degree of variation between the two points showed
no increase or decrease in relation to time. Although the FAL performed theexperiments within a shorter period of time than the other laboratories, the range of FL
assay values was greater for the FAL at T0 and T4. In this case, variation between
89

Fluorescein Leakage Assay Background Review Document

experimental runs did not increase in proportion to the time taken to perform theexperiments. In comparison to T0 data, the range of FL% values for the FAL at T4
increased slightly whilst the range of FL% values decreased slightly for the other threelaboratories.
Similar analyses were performed using the ECVAM Prevalidation study Phase III
positive control data (results not shown). The range of data was similar to that observedin figure 3.2.2.3.b. FAL produced positive control data within a range similar to the otherlaboratories, but all results were outside the defined acceptable values. Despite slight
protocol modifications and an increase in the range of acceptability to 15-30%, there
was only a slight improvement in the number of assays from all laboratories withacceptable positive control results. Although many of the FL values were outside theacceptance range, three out of four laboratories produced data within the range of 030% whilst the data from the FAL ranged from 50-100%.
ii. Test Materials
The number and types of materials tested in the protocols varied considerably. The
number of materials and the range of in vivo irritancy levels for each of the studies areshown for those studies reporting in vivo data (table 3.2.2.2.). The table provides
information regarding the range of in vivo ocular irritation covered by the studies but
does not include the majority of data analysed in this section as they were without
corresponding in vivo data.
Table 3.2.2.2. Relevant in vitro and in vivo information for the types of test materials andpotencies.

Study
The Evaluation of Pesticide
Ingredients and Formulations In
Vitro and Correlations with In
Vivo Data (Clothier et al., 1995)
Evaluation of the Prevalidation
Process: The Fluorescein
Leakage Assay (Phase II)(Southee, 1998)
Evaluation of the Prevalidation
Process: The Fluorescein
Leakage Assay (Phase III)
(Southee, 1998)
CTFA Evaluation of Alternatives
Program: Phase III, TEP assay(Gettings et al., 1996)
Ocular Irritancy Assessment of
Cosmetics Formulations andIngredients: Fluorescein
Leakage Test. (Zanvit et al.,1999) COLIPA DATA

Types ofmaterials tested

Range ofFL20 values

2 pesticide
chemicals and 2
formulations

Range
of in
vivo
scores1>250mg/ml

Type of invivo data
Draize (MASor MMAS)scores upto
96h

0-57

4 cosmetic
ingredients and 1
formulation

0.006mg/ml
-neat

MAS

0-44

10 mild surfactants

0.3470.5mg/ml

MMAS

0-37

0.24 – 30.3
(EC50(%))

MAS

2.3-430.2>1072mg/ml

MMAS

0.67-108

25 surfactantbased formulations
11 cosmetic
surfactants and
23 (Company # 4)
20 (FAL)surfactant-based
formulations

INVITTOX Protocol No. 71 raw data were predominately available for surfactants andsurfactant-based formulations; there were data for only two pure chemicals which were90

Fluorescein Leakage Assay Background Review Document

pesticides. These data represent the fact that the FL assay has been predominately
used by the cosmetics industry for product development.
Raw INVITTOX Protocol No. 120 data were available for fewer different types ofmaterials; the majority were surfactants. INVITTOX Protocol No. 120 was tested in the
ECVAM Prevalidation study Phase III; ten mild surfactants were tested in four different
laboratories. The CVs for the surfactants were plotted against the mean FL20 (mg/ml)
data T4 (figure 3.2.2.4.).
160
140
120

Variability (CV)

100
80
60
40
20
0
0

1

10

100

1000

Mean FL20 (mg/ml)

Figure 3.2.2.4. The CV values plotted against mean FL20 (mg/ml) T4 values for the ten
surfactants tested in three laboratories; □, FAL; ▲, Company # 3; ∆, ECVAM (n≥2).
Greater-than values were not included in the calculation of the means and were notplotted.
The Company # 4 SOP (1992), which formed the basis for INVITTOX Protocol No. 120,
was tested in the COLIPA Eye irritation international validation program (Phase I)(Brantom et al., 1997); only surfactants and surfactant-based solutions soluble in HBSS
were tested. The compositions of these test materials are provided (Formulation Annex
B). The data from the COLIPA study were included in the analyses for INVITTOX
Protocol No. 120. The test materials were tested by Company # 4 and the FAL but data
were only available from the FAL. The CVs for the test materials were plotted against
the mean FL20 (mg/ml) data (figure 3.2.2.5.).

91Fluorescein Leakage Assay Background Review Document

120

Variability (CV)

100
80
60
40
20
0
0

1

10

100

1000

Mean FL20 (mg/ml)

Figure 3.2.2.5. The CVs plotted against the mean T4 FL20 (mg/ml) values for surfactants
and surfactant-based formulations tested by the FAL as part of the COLIPA study (n≥2).
Greater-than values were not included in the calculation of the mean and were notplotted. The Company # 4 SOP (1992), which formed the basis for INVITTOX Protocol
No. 120, was used. .
The compositions of the formulations for the TEP assay data submitted by Company #3 were only provided for 17 formulations (Formulation Annex C). The in vivo Draize
data for these 17 formulations indicated that the products were mostly ‘mild’ and‘moderate’ irritants and relatively few were ‘severe’ irritants. The formulations used inPhase III of the CTFA study were generic surfactant-based formulations representativeof those found in the cosmetics industry. The compositions of the CTFA Phase III
formulations were available (Formulation Annex A), and they were known to be
predominately non-irritants and severe irritants. The CVs were plotted against the mean
TEP assay results for formulations tested by Company # 3 (figure 3.2.2.6.a.) and theCTFA study Phase III formulations (figure 3.2.2.6.b.).

92

Fluorescein Leakage Assay Background Review Document

30

Variability (CV)

20

10

0
0.1

1.0

10.0

100.0

10.0

100.0

M ean EC50 (%)

a.

Variability (CV)

30

20

10

0
0.1

1.0
Mean EC50 (%)

b.
Figure 3.2.2.6. The CVs plotted against the mean EC50 (%) values for a. formulations
tested by Company # 3 (n≥2); b. CTFA Phase III data (n≥3). INVITTOX Protocol No. 86
was used to generate both data sets.
In comparison to the data sets for INVITTOX Protocol No.71 and INVITTOX Protocol
No.120, the data range for INVITTOX Protocol No. 86 was limited. This was probably
due to the data supplied here was from Company # 3 which routinely use the TEP assay
to test surfactants and surfactant-based formulations only. The core range of mean
EC50 values was relatively small (1-10%) in comparison to the data sets for INVITTOX
Protocol No. 71 and INVITTOX Protocol No. 120 (1-100mg/ml). Additionally, the CV
values produced by the data set were relatively small in comparison to the otherprotocols.
93

Fluorescein Leakage Assay Background Review Documentiii. Classifications
The FL assay can produce FL20 values that cover up to six orders of magnitude (table
3.2.2.2.). A potential advantage of such a wide range of FL20 values is that large rangesof values can be used to assign the same classification if there is equal correlation with
FL20 values and in vivo scores across the entire range of FL20 values. This entails thatassay reproducibility does not need to be high in order to obtain reproducible predictedclassifications for in vivo ocular irritation.
FL assay PMs featured in the CTFA study Phase III (Gettings et al., 1996), COLIPA
study (Brantom et al., 1997), and the ECVAM Prevalidation study (Southee, 1998). The
PMs were comprised of ranges of FL assay values that were correlated to in vivo scores
and/or classifications of irritancy. None of the classification PMs that were applied to
the FL assay study data were for the EU Risk Phrase Classification system, the GHS
classification system, or the EPA classification system. The predicted classifications
were assigned to the raw data (i.e. each experimental run) to determine to what extent
variable FL assay values affected the resulting predicted classifications (Annex II). The
classification systems as featured in the literature were used (rather than the EU, GHS
and EPA classification systems).
The PM used in the COLIPA study (Brantom et al., 1997) was defined using historical
data for 43 test materials, prior to the testing of COLIPA test materials. The COLIPA PM(table 3.2.2.3.) was also used in the ECVAM Prevalidation study (Southee, 1998).
Table 3.2.2.3. PM from the COLIPA study for a version of INVITTOX Protocol No. 120.
FL20 (mg/ml) T4>100
20-100<20
T4= Four hour time-point

Classification
Non-irritant/slightModerate
Irritant/severe

Draize MMAS
<15
15-30
>30

There were 30 COLIPA test materials for which raw data were available from the FAL.
Of these materials, three had mixed classifications based on the results from the various
experimental repeats (n≥2). All three materials with mixed classifications were classified
as having both ‘moderate’ and ‘irritant/severe’ levels of ocular irritancy. Importantly, theassay and PM appeared able to distinguish irritants from non-irritants. However, theseresults were for only surfactants and surfactant-based materials that were soluble in
HBSS.
The same PM as featured in the COLIPA study was applied to the ECVAM Prevalidation
Study Phase III FL20 (mg/ml) T4 data (Southee, 1998) as the test protocols in each study
were very similar. The number of mixed classifications from the three different
laboratories are presented (table 3.2.2.4.).

94

Fluorescein Leakage Assay Background Review Document

Table 3.2.2.4. Variation in classifications assigned according to the COLIPA PM for thethree laboratories that participated in the ECVAM Prevalidation study Phase III (n ≥2).

Laboratory
FAL
ECVAM
COMPANY#3

No. ofmaterials
tested910

No. and combination of mixed
classifications
1x moderate and irritant/severe
1x moderate and irritant/severe
1x non-irritant/slight andmoderate100

Due to the very few materials which produce data leading to mixed classifications, theresults suggested that the protocol was reproducible in terms of classifications. The
different laboratories showed very similar levels of reproducibility, i.e. the number of
mixed classifications for any given test material ranged from 0-1. It is known to bedifficult for in vitro tests to distinguish different levels of irritancy so it is not unusual thatthe 2/3 of the mixed classifications were comprised of moderate and irritant/severeclassifications. Importantly, the FL assay protocol distinguished irritants and nonirritants. Those materials that were classified as irritants according to the in vivo dataand not detected as irritants according to the FL assay results would be of greater
concern.
In the CTFA study Phase III publication (Gettings et al., 1996), a post-hoc thresholdvalue of ≤2.60 (%) was assigned to the TEP assay data, to try to classify irritants andnon-irritants according to the FHSA classification system (Gettings et al., 1996). Basedupon this threshold value, the FHSA classifications were assigned to the raw data to
determine if for a given test material the various TEP assay experimental runs led todifferent irritancy classifications (Annex II). Of 23 surfactant-based formulations tested,
six produced TEP assay results which led to mixed classifications (n≥3).
No in vitro classification system was available for the Company # 8 test formulations thatwere tested using INVITTOX Protocol No. 71 (data included in figure 3.2.2.1.). As thecompositions of the test formulations were unknown it was difficult to confidently apply
another PM to this data. In addition, much of the raw Company # 8 data (Annex II) were
produced by using different exposure periods, which reduced the number ofexperimental repeats per test material with the same exposure period.
In general, all of the analyses from the different studies were slightly skewed as forsome formulations there were more experimental repeats than others. A greaternumber of experimental repeats provides greater opportunity for more diverse
classifications to be assigned to the same test material. However, a high number ofexperimental repeats generally only occur in the case of low reproducibility in the priorexperimental runs.

95

Fluorescein Leakage Assay Background Review Document

3.2.3. Additional analyses of operator and time variability
Ideally, operator and time variability would have been assessed using results from a
positive control chemical. However, there were a limited number of available sets ofresults that had positive control data. Subsequently, the approach taken was based on
identifying outlier experimental runs.
The mean, SD and CV were calculated for each test material. As the CV does not take
into account the size of the numbers analysed, it is more difficult to attain a low CV value
with numbers of lower values than with numbers of higher values. Therefore thefollowing rules were applied for data handling:-if the mean FL20 was ≤20mg/ml or 20mM, outlier experimental runs were
removed in order to attain a CV ≤ 60%
if the mean FL20 was ≥20mg/ml or 20mM, outlier experimental runs were
removed in order to attain a CV ≤40%

The experimental run(s) producing the value that caused a CV greater than 40% or 60%
were identified. For this type of analysis, there were a number of criteria that needed to
be fulfilled:--only FL20 mg/ml or FL20 mM data were used
the number of experimental runs per test material had to be greater than three(data sets that had only three experimental repeats for some test materials werealso included if the majority of test materials in the data set had more than threeexperimental runs).
for test materials that did not have three experimental repeats, all experimental
runs for that test material were removed from the data set and were not included
in any analyses.
the number of experimental runs remaining, after removal of those causing the
CV to be ≥ 40%, had to be at least three per test material‘greater-than’ values were changed to actual values, i.e. ‘>500’ became ‘500.’

For determining ‘operator variability’ the name of the operator(s) who produced theoutlier value(s) was recorded. For determining ‘time variability,’ the date that produced
the outlier value(s) was recorded. The number of times that the various operators anddates were identified as causing a CV greater than either 40% (or 60% depending on
the mean FL20 value) were recorded and presented as a proportion of the number ofexperiments they had performed for the entire data set (table 3.2.3.1.). If the removal ofdata points did not attain a CV below 40% or 60%, the ‘removed’ data points were notcounted as outliers, but were counted in the total number of experiments that had been
performed in the data set. This allowed one to observe if a single operator, or
combination of operators, were consistently responsible for test materials that produced
CVs above the set threshold.
For test materials that did not attain an acceptable CV value, it was assumed that thetest material had inherently variable properties that prevented reproducible results frombeing attained. In some cases the test material was known to have variable properties,
but often the test material was coded so inherent variation could only be assumed by thevariable results. Inclusion of this data enabled the robustness of the various FL assay
protocols to be fully tested.
96

Fluorescein Leakage Assay Background Review Document

Table 3.2.3.1. Results and relevant information for each study

Study
A Summary Reportof the COLIPAInternational
Validation Study on
Alternatives to the
Draize Rabbit Eye
Irritation Test
(Brantom et al.,1997) FRAME
A Summary Reportof the COLIPAInternational
Validation Study on
Alternatives to the
Draize Rabbit Eye
Irritation Test
(Brantom et al.,1997) FRAME
The Evaluation of
Pesticide Ingredientsand Formulations In
vitro andCorrelations with In
Vivo Data (Clothieret al., 1995)
The Evaluation of
Pesticide Ingredientsand Formulations In
vitro andCorrelations with In
Vivo Data (Clothieret al., 1995)

Operator
Variability

Time Variability

Operator
No.

n/a18.02.94. 1/3
21.02.94. 1/5
28.02.94. 0/5
04.03.94. 0/5

notstatedn/a

18.02.94. 1/4
21.02.94. 0/5
28.02.94. 0/5
04.03.94. 0/5

notstatedn/a

13.07.92.
17.07.92.
20.07.92.
20.07.92.

0/4
0/4
0/4
0/4

n/a

16.07.92.
17.07.92.
23.07.92.
30.07.92.

2/4
0/4
0/4
0/4

97

1

1

Exp.

No.

4

4

4

4

Well
ReplicateNo.

No. of Test
Substancesa., b., c.,

Data
Format

3

a. 5
b. 0
c. 0

FL20
(mg/ml)
T0

3

a. 5
b. 0
c. 0

FL20
(mg/ml)T72

3

a. 4 pesticide
formulations
b. 0
c. 0

FL20
(mg/ml)T0

3

a. 4 pesticide
formulations
b. 0
c. 1

FL20
(mg/ml)T72

Fluorescein Leakage Assay Background Review Document

Study
Use of In Vitro
Methodology to
Predict Irritancy
Potential of
Surfactants(Hubbard et al.,1994)

Use of In Vitro
Methodology to
Predict Irritancy
Potential of
Surfactants(Hubbard et al.,1994) –re-runs
based on activity

Use of In Vitro
Methodology to
Predict Irritancy
Potential of
Surfactants(Hubbard et al.,1994) –re-runs
based on activity

Report on
Comparison of 40
Cosmetic andDomestic
Formulations
Supplied byCompany # 8 andEvaluated by 3 In
Vitro Cytotoxicity
Tests (FRAME,
1992)

Operator
Variability

n/a

C 3/28D 3/8
F 1/1

C 1/28
D 0/8
F 0/1

A 1/5
B 0/9
F 4/64

Time Variability
21.09.92.

0/6

28.09.92.

0/4

08.10.92.

0/6

09.10.92.

0/4

13.10.92.

1/6

11.01.93.

0/6

15.11.92.

2/10

27.01.93

1/4

01.02.93

2/6

02.02.93

0/4

08.02.93

0/8

05.02.93

0/2

15.02.93

0/1

19.02.93

0/5

19.04.93

0/4

29.03.93

1/2

20.04.93

2/4

OperatorNo.

notstated3

27.01.93

0/4

01.02.93

1/6

02.02.93

0/4

08.02.93

0/8

05.02.93

0/2

15.02.93

0/1

19.02.93

0/5

19.04.93

0/4

29.03.93

1/2

20.04.93

2/4

03.04.92 0/2
10.04.92 0/2
01.05.92. 0/2
03.07.92. 0/1
05.05.92. 0/1
06.03.92. 0/2
07.01.92. 0/2
09.03.92. 0/2
11.05.92. 2/6
12.05.96 0/6
13.03.92. 0/3
15.05.92. 0/2
15.06.92 0/2
17.03.92. 0/4
19.06.92 0/1
21.05.92. 0/4
22.05.92.1/20
23.03.92. 2/4
23.06.92. 0/2
24.04.92. 0/2
27.01.92. 0/1
29.04.92 0/1
29.06.92 0/2
31.03.92. 0/4

3

3

98

Exp.
No.

n≤ 5

n≤ 5

n≤ 5

4

Well
ReplicateNo.

No. of Test
Substancesa., b., c.,

Data
Format3

a. 10
surfactants
b. 0
c. 1

FL20
(mg/ml)
T0

3

a. 10
surfactants
b. 0
c. 0

FL20
(mg/ml)
T0

3

a. 10
surfactants
b. 0
c. 4

FL20
(mg/ml)
T72

3

a. 40
b. 12
c. 8
d. 6

FL20
(mg/ml)T0

Fluorescein Leakage Assay Background Review Document

Study

Company # 5 test
concentration X

Company # 5 test
concentration XI
Final Report on
Testing of 12 Mild
Surfactants suppliedby Company # 5 for
Cytotoxicity Testingat the FAL (FRAME,
1992)

Operator
Variability

Time
Variability

n/a09.02.93. 0/9
16.02.93. 0/11
08.02.93. 0/4
03.02.93. 2/10
06.02.93. 0/9

n/a

09.02.93. 2/9
16.02.93. 0/10
08.02.93. 0/3
03.02.93. 0/10
06.02.93. 0/9

n/an/a –all CVs
were below
thresholdvaluesOperator No.

1

Exp. No.

n≤ 4

1

n≤ 4

2

n≤ 4 forthe 5materials
analysed

Well
Replicate No.

No. of Test
Substancesa., b., c.,

Data
Format

3

a. 12
b. 2
c. 6

FL20
(mg/ml)
T24

3

a. 12
b. 2
c. 3

FL20
(mg/ml)
T24

a. 5
b. 0
c.0

FL20,
(mM)
T0,
T24,
T48

3

a. total number of materials in the data set; b. number of test materials that had two or
fewer experimental repeats, thus all test material data were removed from the analyses;
therefore the final number of test materials analysed is ‘No. of substances in data set’minus ‘number of test materials that had ≤2 experimental repeats’; c. number of test
materials which did not have a CV below the threshold values. Outliers for these test
materials were not included in the analyses for operator and time variability. However,this information was included in the counts of total number of runs per date and total
number of runs per operator, which enables one to observe if a particular operator
and/or dates were consistently responsible for the high variation.

99

Fluorescein Leakage Assay Background Review Document

i. Operator Variability
In order to evaluate operator variability, the number of outlier experimental runs per
operator was calculated from all the appropriate data sets featured in table 3.2.3.1.
(table 3.2.3.2).
Table 3.2.3.2. Operator variability for all the data sets featured in table 3.2.3.1
Data set

A

B

C

OperatorD

Use of In Vitro Methodology to
Predict Irritancy Potential of
Surfactants (Hubbard et al., 1994).
T0
Use of In Vitro Methodology to
Predict Irritancy Potential of
Surfactants (Hubbard et al., 1994).
T72
Report on Comparison of 40
Cosmetic and Domestic
Formulations Supplied by Company# 8 and Evaluated by 3 In Vitro
Cytotoxicity Tests (FRAME, 1992)

1/5

0/9

4/64

Total
%
Data sets

1/5
20%
1

0/9
0%
1

8/120
6.7%
3

E

F

3/28

3/8

1/1

1/28

0/8

0/1

3/16
18.8%
1

0/0
n/a
n/a

1/2
50%
2

For each operator, the total number of outlier experimental runs was shown in relation to
the total number of runs recorded (table 3.2.3.2). To allow operator variability to becompared, the number of outliers produced by each operator was calculated andpresented as a percentage of the total number of runs performed. The results show that
operator variability ranged from 0% to 50%. It was difficult to confidently assess therelationship between the number of experiments performed and the level of operator
variability due to the limited number of data sets and differing number of experiments ineach. However, the data did not indicate a relationship between operator variability andthe number of experiments performed, i.e. operator A had a variability of 20% based on
5 experimental runs whilst operator D had a variability of 18.8% based on 16
experimental runs.
The number of studies was also recorded to observe if data from a greater number ofstudies increased the overall operator variability calculated. There was no correlation
between operator variability and number of studies, i.e. variability for operator A and B
was calculated using data from one study and were 20% and 0% respectively.
From this limited data, the results showed that operators do vary in their abilities toattain reproducible FL assay results for this protocol. With the exception of operator F(only two experiments performed), the reproducibility ranged from 0-20%. This indicatedthat this FL assay protocol was reproducible as the maximum rate of outliers produced
was approximately 20%. As different data sets were used to assess operator variability,
it should be noted that some of the data sets would have contained test materials withproperties causing them to be difficult to test using the FL assay, thus leading to variable
results impacting on the assessment of operator variability. Where the same sets of100

Fluorescein Leakage Assay Background Review Document

materials were tested by different operators, it did not follow that each operator tested
exactly the same set of test materials.
ii. Time Variability
The effect of length of time between experimental runs
As the experimental runs were arranged in chronological order for each chemical, aquick observation of the data indicated that no particular run (i.e. the first or last) was
consistently the outlier run (data not shown). Subsequently, one could state thatvariation in experimental runs was not due to the time they were conducted, i.e. it could
have been surmised that over time, more experience with the protocol would be
developed so this could have resulted in increased reproducibility over time. The data
did not indicate this to be the case, for example, three experimental repeats performed
one week or three weeks apart were just as likely to produce outlier results.
The effect of failed experimental runs
It was clear that some experimental runs ‘fail’ for the majority of chemicals tested on thatparticular date. The data sets with more than 10 test materials were analysed todetermine the number of experimental runs that produced 50% or more outlier results
for the materials tested on that particular date (table 3.2.3.3.).
Table 3.2.3.3. Impact of failed experimental runs on overall reproducibility.

Study

No. of dates
which
produced
outliers

No of dates
with ≥50%
outlier
results

Did the dates with morethan 50% outlier results
account for more than
50% of the total numberof outliers within thedata set?

Use of In Vitro Methodology to Predict
Irritancy Potential of Surfactants(Hubbard et al., 1994). T0
Use of In Vitro Methodology to Predict
Irritancy Potential of Surfactants(Hubbard et al., 1994). Re-runs basedon activity T0

2

0

n/a

4

1 (only 2
chemicals
tested)No- account for 1 outliers
out of a total of 6

Use of In Vitro Methodology to Predict
Irritancy Potential of Surfactants(Hubbard et al., 1994). Re-runs basedon activity T72

1

0

n/a

3

1 (only 4
chemicals
tested)No- account for 2 outliers
out of a total of 5

Report on Comparison of 40 Cosmetic
and Domestic Formulations Supplied byCompany # 8 and Evaluated by 3 In
Vitro Cytotoxicity Tests (FRAME, 1992)

a. Did the dates with more than 50% outlier results account for more than 50% of thetotal outlier dates within the data set?
The analysis of the effect of failed runs was hampered by the lack of large available data
sets that were required in order for numerous materials to have been tested per
experimental run. There were only two experimental runs that produced ≥50% outliers
but only four materials were tested in these runs. In general, the number of materials
tested in an experimental run was low due to the complexity and time required to carryout the FL assay protocol.
101

Fluorescein Leakage Assay Background Review Document

Overall it appeared that ‘failed’ experimental runs were not responsible for a significant
proportion of the total number of outliers within a data set. The majority of outliers were
produced for one or two materials within an experimental run containing a high numberof chemicals. In conclusion, this analysis indicated that this FL assay protocol was
generally reproducible as there were not a great proportion of outliers.
The effect of potential inherent chemical variation
To determine if specific materials were responsible for the majority of outliers withineach data set, the number of materials for which there were two or more outliers wasrecorded (table 3.2.3.4.). For this analysis, materials had to have five or moreexperimental repeats in order to have sufficient occasions for two outliers to occur,
therefore only a few materials from each study qualified for analysis.
Table 3.2.3.4. The number of materials for which two or more outliers were identified inrelation to the total number of materials within the data set

Study
The Evaluation of Pesticide Ingredientsand Formulations In Vitro andCorrelations with In Vivo Data (Clothier etal., 1995) T0
The Evaluation of Pesticide Ingredientsand Formulations In Vitro andCorrelations with In Vivo Data (Clothier etal., 1995) T72
Use of In Vitro Methodology to Predict
Irritancy Potential of Surfactants(Hubbard et al., 1994). T0
Use of In Vitro Methodology to Predict
Irritancy Potential of Surfactants(Hubbard et al., 1994). Re-runs based on
activity T0
Use of In Vitro Methodology to Predict
Irritancy Potential of Surfactants(Hubbard et al., 1994). Re-runs based on
activity T72
Final Report on Testing of 12 Mild
Surfactants supplied by Company # 5 for
Cytotoxicity Testing at the FAL (FRAME,
1992)

No. ofchemicals withat least one
outlierNo. ofchemicals with
two of more
outliers

No. ofchemicals with
2 outliers as aproportion ofchemicals withoutliers

0

0

0%

2

0

0%

3

0

0%

4

2

50%

1

0

0%

0

0

0%

T=time point
Data for 10 materials were analysed and only two test materials produced two or more
outliers. More materials require testing, but this data indicated that outliers tended to berandom and were not due to potential inherent variation of the materials being tested.
In conclusion, the findings suggested that the effect of time on the reproducibility of the
FL assay was random. More data is required to conclusively determine if and how time102

Fluorescein Leakage Assay Background Review Document

could affect FL assay reproducibility. There are a number of inter-related factors that
could be responsible for the random distribution of outliers identified in these analyses.

103

Fluorescein Leakage Assay Background Review Document

3.3.

Additional studies where raw data are not available: attempt to combine the data using weight-of-evidence approaches

Table 3.3. Presents the results and relevant information for each study containing non-raw data.

Study

Variability
Reported

No. of Test
SubstancesType of Test
Substances

No. ofLaboratories

No. of Operators

No. ofExperiments

No. ofreplicate(wells)Data
Format

N/A

-

-

-

-

-

-

-

-

104

Fluorescein Leakage Assay Background Review Document

The weight-of-evidence approach applied was developed by the authors of this BRD.
I. Protocols
A substantial amount of data was available to allow within-laboratory reproducibility for
INVITTOX Protocol No. 71 and INVITTOX Protocol No. 120 to be evaluated. Fewerdata were available for materials tested using INVITTOX Protocol No. 82 and INVITTOX
Protocol No. 86. There were only provisional data for INVITTOX Protocol No. 82;
INVITTOX Protocol No. 71 had been used to test formulations at two fixed
concentrations only.
Overall, more data were available for materials that were tested using INVITTOX
Protocol No. 71, but INVITTOX Protocol No. 120 had been used in more laboratories.
Data analysed for both protocols covered similar FL20 (mg/ml) ranges and similar CV
ranges. Applying a weight-of-evidence approach, there was greater weighting for
INVITTOX Protocol No. 120 as data had been collected from two multi-laboratory
studies that used this protocol; the COLIPA study (Brantom et al., 1997) and the
ECVAM Prevalidation study (Southee, 1998). A PM was devised before the start of boththese studies; both studies also undertook independent statistical analyses. Statisticalanalyses of the COLIPA study found that reproducibility was good as the predictedclassifications for in vivo ocular irritation were similar from both laboratories. In
comparison, much of the data evaluated here, generated by INVITTOX Protocol No. 71
were produced in-house and did not undergo extensive nor independent statisticalanalyses.
ii. Test materials
Similar numbers of test materials have been tested using INVITTOX Protocol No. 71
and INVITTOX Protocol No. 120. The variety of test materials used to assess intralaboratory variability differed according to each INVITTOX Protocol. Chemicals werepredominately tested using INVITTOX Protocol No. 120 whilst more formulations were
tested using INVITTOX Protocol No. 71. This was because INVITTOX Protocol No. 120
featured in a number of large-scale validation/evaluation studies whilst INVITTOX
Protocol No. 71 had been predominately used by the FAL to test formulations provided
by industry.
There was some INVITTOX Protocol No. 86/ TEP assay data forformulations that had been tested in the CTFA study Phase III (Gettings et al., 1996)
and used in-house by Company # 3.
The ICCVAM/NICEATM BRDs for organotypic models for ocular irritation requested
data from pure chemicals. The chemicals also had to cover the potency range andmechanisms that the organotypic model was designed to test. Applying the same
approach here would cause the data for formulations to have less weighting than thedata for chemicals, although it is acknowledged that it is important to determine thepredictive capacity of the FL assay for formulations. Using the ICCVAM/NICEATM
approach for determing the predictive capcity of organotypic models for ocular irritation,
the results from the ECVAM Prevalidation study Phase III (Southee, 1998) would
receive greater weighting because pure chemicals were tested. As industry is likely to
use the FL assay to test formulations rather than pure chemicals it is also important toknow that the FL assay is capable of testing both formulations and chemicals.
INVITTOX Protocol No. 120 also featured in the COLIPA study where it was used to test
33 test materials in two different laboratories. Surfactants and surfactant-based
formulations that were soluble in HBSS were tested. The range of potency of the test
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Fluorescein Leakage Assay Background Review Document

materials (according to the in vivo data) was predominately from non-irritants tomoderate irritants. Overall, greater weighting was given to INVITTOX Protocol No. 120
data which were generated by testing both surfactants and surfactant-based
formulations (Formulation Annex B) and had been used in a number of different
laboratories.
iii. Classifications
From the collected literature, classification systems were present for the CTFA study
Phase III (Gettings et al., 1996), COLIPA study (Brantom et al., 1997) and the ECVAM
Prevalidation study Phase III (Southee, 1998). INVITTOX Protocol No. 120 featured in
the COLIPA study and the ECVAM Prevalidation study whilst INVITTOX Protocol No. 86
featured in the CTFA study Phase III.
The weight-of-evidence approach to the classifications can be analysed using different
criteria. The COLIPA and ECVAM Prevalidation study (Southee, 1998) receive goodweighting as the classification PM was defined prior to the testing of the materials.
However, the in vivo data and in vivo classification system used to define the in vitro
classification system were not transparent as it was only written that the PM wasdeveloped using
Company # 4 data for 43 surfactants and surfactant-based
formulations. In comparison, the CTFA Study established post-hoc threshold values for
the TEP assay data, which produced the most accurate classifications possible inrespect to the FHSA irritancy classifications. Thus, the CTFA classification model,
based on a recognised in vivo classification scheme, would receive greater weighting
than the PM used in the ECVAM Prevalidation study and the COLIPA study. A greaternumber and a wider range of test materials were tested using the protocol and PM
featured in the COLIPA study and the ECVAM Prevalidation study (INVITTOX Protocol
No. 120) in comparison to the number tested in the CTFA study Phase III (INVITTOX
Protocol No. 86). The COLIPA PM was also applied to data from a number of different
laboratories which contrasts to the one laboratory featured in the CTFA study Phase III.
In conclusion, the data analysed in this section provide greater weighting for the
reproducibility of INVITTOX Protocol No. 120 in comparison to the other FL assay
INVITTOX Protocols. When variability data (CV values) were compiled for all of the
INVITTOX Protocols, variability for INVITTOX Protocol No. 120 and INVITTOX Protocol
No. 71 were comparable despite the data for INVITTOX Protocol No. 120 originatingfrom a number of different laboratories. The within-laboratory variability was lower for
INVITTOX Protocol No. 86 due to the data originating only from one laboratory and thesingle type of materials tested.
Annex II

Raw data used in the statistical analyses for intra-laboratory variability

Annex A

CTFA Study Phase III formulation ingredients (from draft HET-CAM
BRD: Appendix C2 (ICCVAM/NICEATM, 2004)

Annex B

COLIPA Study test chemicals and formulations compositions (fromCOLIPA)

Annex C

Formulation compositions from Company # 3.

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Fluorescein Leakage Assay Background Review Document

4. Transferability (Module 3)
4.1. Brief description of study results on transferability and availability ofStandardised Operating Procedures (SOPs)
According to ‘A modular approach to the ECVAM principles on test validity’ (Hartung etal., 2004), the aim of assessing transferability is to determine how much training isnecessary to be able to establish the test in a naïve laboratory and reproduce assay
predictions. The Evaluation of the Prevalidation Process: The Fluorescein Leakage
Assay (Southee, 1998) is the only study which was known to have investigated protocol
transfer of a FL assay protocol (see section 5.2.). Phase II of this study specificallyinvestigated protocol transferability by testing a single protocol in four different
laboratories with various levels of experience with the chosen protocol, and the FL
assay in general. The Company # 7 laboratory was responsible for refining the test
protocol and also participated in testing the materials. The FAL participated in the study
and represented a laboratory experienced with the assay in general but not the specifictest protocol. Company # 3 also participated in the study; they were familiar with asimilar FL assay but their participation also allowed assay transferability to the USA to
be determined. ECVAM participated in the study as a naïve laboratory was required to
fully assess the completeness of the protocol. Five materials which covered a range of
irritancy were tested. Variability was quantified by calculating the CV for the 0% and100% leakage controls and the positive control (0.16mg/ml SLS) data. The CVs varied
considerably between laboratories with the ECVAM laboratory generally producing themost variable results. The laboratories also differed as to which control produced thehighest CV. All laboratories produced very few results that had control data within thevarious pre-defined acceptance ranges of the different control values. The results
indicated that there were different levels of intra-laboratory variability in the laboratories
and that the protocol did not transfer well. Further refinements to the protocol weremade for Phase III of the study which investigated protocol performance, i.e. increasing
the ranges of results for the 0% leakage control and the positive control, increased
number of washing steps, clarification of protocol steps
For Phase III, ten mild surfactants were tested twice in each laboratory. Analyses
included data that did not satisfy the acceptance criteria as all laboratories experienced
problems to consistently fulfil the acceptance requirements. Inter-laboratory variability
was lowest when the results from ECVAM and Company # 3 (Pearson’s correlation
coefficient 0.98) were compared and highest when the results from the FAL andCompany # 3 (Pearson’s correlation coefficient 0.82) were compared. Following theresults of this study, this protocol was accepted as INVITTOX Protocol No. 120.
Different FL assay protocols have been tested in large-scale validation and/or evaluation
studies. Some studies tested a single protocol in a number of independent laboratories
which enabled inter-laboratory variability to be assessed. In the absence of specificstudies for protocol transferability, inter-laboratory variability indicates protocol
transferability to some extent. None of the studies used any of the standardised
INVITTOX protocols, but as a result of these studies, some protocols were later adopted
as INVITTOX protocols.

‘The EC/HO International validation study on alternatives to the Draize eye irritation test
(Balls et al., 1995),’ investigated a number of in vitro assays for their ability to predict in107

Fluorescein Leakage Assay Background Review Document

vivo MMAS scores. It was stated in the publication of Balls et al., (1995) that the FL
assay protocol evaluated, was based on the method of Tchao (1988); the protocolfeatured in this study was later accepted as INVITTOX Protocol No. 71. This protocol
was tested in four laboratories; sixty chemicals of high purity were tested. Pearson’s
correlation coefficients were calculated to compare the results from the different
laboratories and values ranged from 0.167 to 0.778 (Balls et al., 1995). All the low
correlation co-efficients were obtained when comparisons were made with one particularlaboratory which indicated that this laboratory did not follow the protocol in a similarmanner to the other three. A general concern of this study was that the protocol was notequally adhered to by all participating laboratories (R Clothier, personal communication),and thus inter-laboratory variation was reflected in the Pearson correlation co-efficients.
Certain participating laboratories had used a FL assay method before, and since thiswas one of the first large multi-laboratory studies it was not fully realised that protocol
adherence should be checked during the generation of the data and not at the end of
the study. The format of the data submitted by some laboratories showed clearly thatthe protocol had not been followed. Thus, whilst modern validation studies agree upon
the test protocols and undergo training before the study is embarked upon, in the
EC/HO study (Balls et al., 1995) the protocol was not tested via initial training or via a
training set of chemicals; these are now required for a study to be considered as eligible
for a validation study.
‘The COLIPA Eye irritation international validation program (Phase I)’ (Brantom et al.,
1997; Zanvit et al., 1999) was the only other published study known to have tested asingle protocol in more than one laboratory. The FL assay protocol’s predictive capacity
for in vivo MMAS values was evaluated. The protocol was performed according to
Cottin et al., (1992). Only two laboratories (FAL and Company # 4) tested the protocol
and therefore it was difficult to draw definitive conclusions regarding between-laboratory
variability. Additionally, both laboratories were experienced with the FL assay protocol
used or a similar version which entails that transferability to a naïve laboratory was notassessed. Classifications based on the in vivo data were compared to the FL assay
predicted classifications using Kappa (k) analysis where a value of one indicatedcomplete agreement between values. Although no direct analyses for inter-laboratory
variability were performed, both laboratories were similar in the number of correctlypredicted in vivo classifications; FRAME K= 0.65±0.3, Company # 4 K= 0.78±0.2(Zanvit et al., 1999). These results indicated that the protocol was performed similarly inboth laboratories although it is known that some information is lost when qualitativeclassifications are considered rather than quantitative values. From these results, one
could conclude that this protocol was successfully transferred to the different
laboratories. However, it should be noted that of a range of test materials available fortesting in the COLIPA study, the FL assay protocol was only used to test mild
surfactants and surfactant-based formulations that were soluble in HBSS.
Subsequently, the type of material tested was the same for which the FL assay was
specifically designed to test and therefore the assay and PM would be expected toperform well. If other types of materials had been tested, the results from the different
laboratories may have varied to greater extents and affected the conclusions regarding
protocol transferability. A modified version of this protocol was accepted as INVITTOX
Protocol No. 120 following the results of the ECVAM Prevalidation Study (Southee,1998).

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4.2.

Facilities and major fixed equipment needed

The fixed equipment required to carry out the FL assay are generally those required forcell culture (table 4.2.)
Table 4.2. The fixed equipment required to carry out the FL assay
Equipment-laminar flow hoodUse.

cell culture only, as the FL assay does not need to be
performed using sterile conditions-incubator (37oC, 5% CO2 ,90% humidity)
cell culture and Na-fluorescein dye incubation- cell counter or counting slide
cell culture and cell seeding into wells-inverted phase microscope
cell counting and/or checking monolayer confluency- spectrofluorimeter (485nm, 530nm filters) reading Na-fluorescein dye OD-balance
weighing test materials for solubilisation
warming cell culture materials and solubilising test
-water bathmaterials-fridgestoring cell culture media-freezer, liquid nitrogen containercell storage

4.3.

Required level of training, expertise, and demonstrated proficiency needed

The availability of standardised FL assay INVITTOX Protocols No. 71, No. 82, No. 86,
No.120, make the FL assay straight-forward to perform as instructions are clear andprecise. However, the INVITTOX protocols are rarely cited in the literature, unless by
the research groups that devised them.
In general the FL assay is relatively easy to perform for anyone with basic cell culture
experience. Both strains of the MDCK cells grow well in culture and on the inserts. In
comparison to other in vitro assays, the principal difficulty of the FL assay protocol isthat special care is needed when dealing with the monolayer grown on the insert
membranes to ensure that neither are damaged. Rinsing steps following test material
exposure and prior to adding the sodium-fluorescein dye are incorporated into all the
INVITTOX Protocols. The rinsing steps are required to ensure that the test material
does not remain in the insert and produce erroneous results.
The FL assay can be used to measure recovery after an initial exposure which entails
that the cells can remain cultured on the inserts for up to 96 hours. During this time,
multiple FL assays are performed on the same population of cells, thus providing many
opportunities for damage to the monolayer and insert to occur. After the FL assay iscompleted, the remaining sodium-fluorescein dye is removed from the insert and theinserts placed into new 24 well plates containing fresh medium; fresh medium is thenalso added to the inserts. Of the four different INVITTOX Protocols, recovery is only
featured in INVITTOX Protocol No. 82, although other INVITTOX Protocols have been
adapted to measure recovery.
An understanding of solubility is important to ensure that the correct solvent is used tosolubilise the test materials and that concentrations are uniform; this ensures exposures
are comparable for subsequent experimental repeats. Studies using coded materials
should provide adequate solubility instructions. Knowledge of the properties of test
materials is required to ensure that they are compatible for testing with the FL assay, as109

Fluorescein Leakage Assay Background Review Document

some chemicals can interact with the sodium-fluorescein dye. Sufficient controls, i.e.
with and without cells should highlight these effects.
The FL assay was designed to measure the effects of the damage caused by test
materials to a cell monolayer in order to be able to predict damaging effects to thecorneal epithelium in vivo. The assay has been predominately used to measure theeffects caused by materials relevant to the cosmetics industry, such as surfactants,
which are likely to enter the eye through accidental exposures. Surfactants are often
supplied as stock concentrations ≥ 30% that are viscous gels, creams, or formulations
that can be applied neat or diluted using volume/volume aqueous solvents. Viscousmaterials are not always easily removed and special care is required to ensure that themonolayers and/or insert membranes are not damaged during the removal process.
Some experience in efficiently applying and removing viscous test materials after a shortincubation are needed to enable experiments to be replicated and to ensure exposures
are not longer than defined by the protocol. This problem is a greater concern for those
protocols using one minute exposures, as any variation in exposure period will be agreater proportion of one minute in comparison to the five minutes used by otherprotocols. It is especially important to remove all the test material in the case of the FL
assay, as any remaining in the insert could cause the membrane pores to be blocked
and physically prevent FL. Dyes which alter the colour of the sodium-fluorescein dye
could potentially distort results if left in the well, although a no-cell control exposed to thesame dye concentration can indicate this as a problem. In general, careful observationof the insert and monolayer is required at all stages of the FL assay to ensure that themonolayer is not damaged and that the test material has been fully removed.
Training is also required to ensure that the operator is able to perform all the necessary
steps quickly following the short test material exposure, i.e. aspiration, washings andfixation/desorbing of the remaining dye. This should be undertaken with the positive
control that can be used to confirm reproducibility of results for the operator and ensure
they are comparable to published data for the chosen protocol.
A level of specific technical instruction/assistance is normally required for setting up thefixed laboratory equipment e.g. the incubator and laminar flow hood, and for operating
the plate reader.

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Fluorescein Leakage Assay Background Review Document

5. Between-laboratory reproducibility (Module 4)
Various FL assay protocols have featured in a number of large-scale studies whichassessed the ability of the protocols for predicting the effects of chemical-induced in vivo
ocular irritation. Normally, the FL assay was tested in two or more laboratories which
also allowed the results to be used to assess between-laboratory variability. Table 5.1.
shows the studies where a single FL assay protocol was tested in more than one
laboratory and raw in vitro data were available.

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5.1.

Table presenting the relevant results and information for studies where raw in vitro data are available.

Study
Evaluation ofthe
Prevalidation
Process: The
Fluorescein
Leakage Assay(Phase II)(Southee,1998)

Evaluation ofthe
Prevalidation
Process: The
Fluorescein
Leakage Assay(Phase III)
(Southee,1998)

No. ofChemicals

410No. ofProducts

1

0

Code

Results(e.g., ANOVA SD,
CV, Range, sim ofclassif.)

Not
state
d

A
hierarchical
ANOVA performed
for the Southee
report (1998) noted
inter-well,interplate,interexperiment, interday
andinterlaboratory variation.

Y

ANOVA
analysisfoundstatisticaldifference in results
from
the
three
laboratories for fourchemicals

Lab.
No.

4

3

Exp.
No.

3-8
Proto
col
speci
fied 2(but n=
2-3
FRAM
E;
n=2-3
ECVA
M; n=2
COMP
ANY #
3)

Replicat
e No.

Data
Format(raw,
summary)

ChemicalClasses3

Raw FL20(mg/ml)T0,
T4values per
chemicalperlaboratory

triton X-100 (5%),
CTAB, Johnson's
baby shampoo,
glycerol,ammonium
nitrateRaw FL20(mg/ml)T0,
T4values per
chemicalperlaboratory

Mild surfactants
relevanttocosmetic testingwith in vivo datareadily available
from
BIBRA
(originally
from
AVON
and
Sigma).

3

T=time point, MMAS= modified maximal average score, MW= molecular weight

112

Rangesof
Toxicity

Physicochemical
properties

MMAS
Range:
0-44

Viscousliquids,hydrophilic
emulsifiers,
liquids, MW
80.04-364.45

MMAS
Range:
0-34

Viscousliquids, liquids,
powders, MW:
288.37687414.6

Fluorescein Leakage Assay Background Review Document

5.2. Discussions from the literature
The following comments were taken from the ECVAM Prevalidation Report (Southee,1998) and the accompanying independent statistical analysis report prepared by BIBRAInternational (Lovell, 1998).
‘The Evaluation of the Prevalidation Process: The Fluorescein Leakage Assay’ allowed
between-laboratory variability in Phase II and Phase III of the study to be investigated(Southee, 1998). Phase II specifically investigated protocol transferability althoughprotocol modifications (i.e. the acceptance criteria) were possibly based upon initial
results. In Phase I of the prevalidation process, Company # 7 developed a FL assay
protocol that was then transferred to the FAL laboratory. The FAL was experienced inperforming a slightly different FL assay protocol. In addition to Company # 7 and the
FAL, ECVAM participated in Phase II and had no experience of performing any FL
assay protocol. The research laboratory of Company # 3 also participated in Phase II;
this laboratory had devised the TEP assay (INVITTOX Protocol No. 86) and their
participation in this study allowed problems associated with setting up the assay in the
USA to be highlighted. Data generated by these three laboratories allowed many
aspects of protocol transferability to be tested and between-laboratory variability to beassessed.
The five materials tested in Phase II were, 5% triton X-100, CTAB, glycerol, ammonium
nitrate and a Company # 3 baby shampoo. All chemicals were distributed to the othertest laboratories by Company # 7. It was understood that the Company # 3 baby
shampoo formulation was obtained by the laboratories independently as it was reported
that Company # 3 baby shampoo only was distributed by Company # 7 to Laboratory 4(Company # 3) as the formulation was unique to the UK.’ Under statistical guidance, thefive test materials were tested as follows:-

3 chemicals on 5 separate occasions
2 chemicals on 2 separate occasions

The FL20 (mg/ml) values and the following control values were recorded immediately
following the exposure (T0) and four hours later (T4); FL% for 0.16mg/ml SLS, FL% for
the untreated monolayer (0% leakage control), FL% for the insert only (100% leakage
control). For FL20 (mg/ml) values to be accepted, the control values had to be within
pre-defined ranges.
The materials were tested at different periods by the various laboratories. However, alllaboratories conducted the testing within two month periods. The results were collectedby Company # 7 and sent to BIBRA International (UK) for independent statisticalanalyses. Variability was quantified by calculating the CVs for the 0% and 100% FL
controls, and the positive control (0.16mg/ml SLS). The CVs for these controls varied
considerably between laboratories with the ECVAM laboratory generally producing themost variable results. The laboratories also varied as to which control produced thegreatest mean CV value. All laboratories produced only a few results that had control
data within the acceptance ranges. The positive control values from FAL wereconsistently below 15% and outside of the specified acceptance range of 15.1-28.3%.
Some of the positive control results submitted by ECVAM were also outside of theacceptance range. Both laboratories felt confident that their FL20 results were valid
despite failing positive control results. The Company # 3 laboratory had more control
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Fluorescein Leakage Assay Background Review Document

data within the acceptance range but some runs did fail which resulted in additional runs
being performed until the acceptance criteria were fulfilled. As all laboratories had someproblems to fulfil the acceptance criteria of the positive control, it was undecided by thestudy organisers whether it was a poor choice for a positive control and subsequently notest chemical data were excluded as protocol modifications were allowed at this stage ofthe study. These results suggested that there were disparate levels of intra-laboratory
variability in the different laboratories, and that the protocol did not transfer well. It was
concluded that there was inter-laboratory variation which was either due to protocol
ambiguity or the protocol not being fully adhered to.
One possible source of inter-laboratory variation was the different concentration ranges
used by the various laboratories to determine the FL20 value. In addition, somelaboratories changed the range of concentrations used for the different experimental
runs to a greater extent than others.
Some laboratories performed a greater number of experimental repeats than was
originally decided. It also appeared that some laboratories included the results fromrange-finding experiments, although these were not adequately distinguished from theresults of confirmatory assays. The inclusion of range-finding experiments from somelaboratories and not others was reported to be a major cause of between-laboratory
variation. Some laboratories submitted greater-than or less-than values which wereconsidered to be redundant in the statistical analyses and furthermore were ‘not allowed’by the protocol (Lovell, 1998).
Whilst some laboratories performed control experiments on a per chemical basis, otherperformed positive controls per plate or per day. This impacted on the likelihood of FL20
values being accepted, e.g., if a control value failed it could cause one chemical run to
be excluded in one laboratory, a plate of results to be excluded in another, and anumber of plates to be excluded in the other.
A hierarchical ANOVA performed for the ECVAM Prevalidation study report noted interwell, inter-plate, inter-experiment, inter-day and inter-laboratory variation for the Phase II
data (Southee, 1998).
Further refinements to the protocol were made before Phase III testing began, i.e.
increasing the ranges of FL% results for the untreated monolayer (0% leakage) control
and the positive control.
The aim of Phase III was to determine protocol predictivity; as the protocol was tested inthree different laboratories, inter-laboratory variability could also be tested. Ten mild
surfactants, coded by BIBRA International, were tested twice in each laboratory. Mildsurfactants were tested as results from the COLIPA study indicated that the FL assay
was particularly useful for predicting lower levels of ocular irritation and showed better
predictivity for surfactants (Zanvit et al., 1999).
Raw and log-transformed FL20 (mg/ml) T4 ECVAM Prevalidation study Phase III data
were analysed for inter-laboratory variability by ANOVA analyses. Significant betweenlaboratory variation was reported for four of the ten surfactants when both types of data
were analysed. The between-laboratory variability for each chemical was quantified bydefining the value below which the difference between test results from different

114

Fluorescein Leakage Assay Background Review Document

laboratories would be expected to lie with 95% probability. The lowest level of betweenlaboratory was recorded for deoxycholic acid, and the highest level for polysorbate-60.
Kappa analysis was performed to determine the agreement of predicted classifications
between the laboratories. Linear kappa values ranged from 0.44-0.82 according towhich laboratory data sets were compared.
Following this study, comments from the participating laboratories were submitted toCompany # 7. Those factors which could have impacted on between-laboratory
variability are given below.
The FAL highlighted the problem of viscous solutions that can be difficult to pipetteaccurately and to wash off at the end of the exposure. Materials which are solubilised inmineral oil can precipitate out in a heterogeneous manner and lead to variable results.
ECVAM suggested that as SLS, (positive control) is hygroscopic it needed to be handled
carefully. It was also stated that it was unclear whether the positive control was to bemade up fresh every time it was required. Although the results for the test materials
were not discarded on the basis of the control results, it was clear from the ECVAM
comments that some ambiguity in the protocol existed and that the SLS results could bea source of variation in the future. It was also stated that the solubilisation instructions
for the test materials were sometimes unclear. CTAB was also reported to bind to theinsert membrane at concentrations above 1mg/ml.
The Company # 3 laboratory suggested that SLS may not be a suitable positive control.
They reported that when assays ‘failed’ due to the positive control, the test samples
results were in agreement with other runs that had passed. They also stated that fiveconcentrations were too few to determine an adequate dose-response curve.
All laboratories highlighted the disadvantage of using Millicell-HA filters as themonolayer cannot be observed. Damage caused to the monolayer due to the many
protocol procedures can go undetected and is therefore a potential source of interlaboratory variability.
5.3.

Compilation of results

5.3.1. Statistical approach(es) used: description & rationale for the approach used
to determine between-laboratory reproducibility
For each data set, the mean, SD and CV were calculated per test material and for theentire data sets.
For each phase of the ECVAM Prevalidation study, one-way ANOVAs were performed
using the mean results for each test material to determine if the variation between the
results from the different laboratories was equal. Mean results were used as thenumber of experimental repeats varied according to test material and laboratory.
Pearson correlation coefficients were calculated for the data sets generated by thevarious laboratories. This allowed all the data sets produced by the different
laboratories featured in each study to be compared.

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Fluorescein Leakage Assay Background Review Document

For this BRD, analyses were performed to determine the agreement of predictedclassifications of ocular irritation between the participating laboratories for thePrevalidation study Phase III. The PM used to transform the FL assay values into
predicted classifications for ocular irritation was the same as featured in thePrevalidation report (Southee, 1998).
5.3.2. Relevant results and information for each study
Table 5.3.2.1.a. Summarised ECVAM Prevalidation Phase II FL20 (mg/ml) data (All raw
data for Phase II, for each laboratory provided in Annex III on CD)i) T0chemical
10% CTAB
Company # 3 baby
shampooGlycerol
Triton X-100
Ammonium Nitrate

FRAME
1.59

COMPANY
#7

2.12

ECVAM
0.73

8.50
387.9019.97
156.0714.31
952.7715.90
243.63

11.91
532.22
15.60
147.22

FRAME
1.41

COMPANY
#7
1.30

ECVAM
0.57

9.83
376.5719.47
327.9332.32
978.877.14
353.8312.80
764.61
11.91
242.95

COMPANY
#3
0.57

Mean
1.25

11.61
750.0022.27
155.68
Mean

SD
0.73

CV (%)
58.28

11.58
655.7218.43
175.65172.532.38
247.73
3.24
45.51
59.92

20.58
37.78
17.57
25.91
32.02

COMPANY
#3
0.42

Mean
0.92

SD
0.50

CV (%)
54.19

8.34
750.006.56
330.29Mean

15.82
717.51
11.27
313.75211.8611.15
250.22
5.97
48.63
63.29

70.49
34.87
52.95
15.50
45.60

ii) T4chemical
10% CTAB
Company # 3 baby
shampooGlycerol
Triton X-100
Ammonium Nitrate

Table 5.3.2.1.b. Summarised ECVAM Prevalidation Phase III FL20 (mg/ml) data (All raw
data for Phase III, for each laboratory provided in Annex III on CD)i) T0chemicalBenzalkonium Chloride (1%)
Brij-35
Cetyl stearyl alcohol (50% in corn oil)
Deoxycholic Acid
Lauryl Sulfebetaine
N-lauroyl sarcosine sodium salt
Polysorbate-60
Sodium C14-C16 olesulfonate
Sodium Laureth Sulfate Pres 35%
Triton X-155

FRAME
26.38
7.12
0.53
2.96
0.485
436.860.4
2.595
1.693333

ECVAM
18.97633
200.0418.35567
1.028267
2.743167
0.773333
57.6685
0.376333
8.41
7.9386

116

COMPANY#3
17.5035
199.82
10.9405
0.1975
0.953
0.569
50
0.329
2.028
1.2285
Mean

Mean
20.95
199.93
12.14
0.59
2.22
0.61
181.51
0.37
4.34
3.62
42.63

SD
4.76
0.16
5.71
0.42
1.10
0.15
221.17
0.04
3.53
3.75
24.08

CV (%)
22.70
0.08
47.06
71.44
49.65
24.35
121.85
9.81
81.31
103.51
53.18

Fluorescein Leakage Assay Background Review Documentii) T4chemicalBenzalkonium Chloride (1%)
Brij-35
Cetyl stearyl alcohol (50% in corn oil)
Deoxycholic Acid
Lauryl Sulfebetaine
N-lauroyl sarcosine sodium salt
Polysorbate-60
Sodium C14-C16 olesulfonate
Sodium Laureth Sulfate Pres 35%
Triton X-155

FRAME
20.415
2.17
0.48
1.65
1.19
399.310.43
5.1
0.933333

ECVAM
15.65133
121.9911.84217
2.02112.695967
1.345675.32067
0.415317
5.116367
7.278467

COMPANY#3
26.516
200
2.629
0.468
1.84650.628537.5450.58
4.46850.9665
Mean

Mean
20.86
161.00
5.55
0.99
2.06
1.05
170.73
0.48
4.89
3.06
37.07

SD
5.45
55.16
5.46
0.89
0.56
0.38
198.86
0.09
0.37
3.65
27.09

CV (%)
26.11
34.26
98.37
90.25
26.93
35.76
116.4819.18
7.55
119.4357.43

ECVAM Prevalidation study Phase II (Southee, 1998)
There was no statistical difference between the data sets for the five materials tested n
the four different laboratories when T0 or T4 data were analysed (one-way ANOVA).
The Pearson correlation coefficients for T0 data ranged 0.98-1 and for T4 data ranged
0.87-1.
ECVAM Prevalidation study Phase III (Southee, 1998)
Ten mild surfactants were coded and distributed by BIBRA. They were tested blindly atleast twice in the three participating laboratories. There was no statistical difference
between the data sets produced by the three participating laboratories when either T0 or
T4 data were analysed (one-way ANOVA). Pearson correlation coefficients ranged
0.94-1 for T0 data, and 0.82-0.98 for T4 data.
ECVAM Prevalidation Study –Positive control data
Positive control data were available for Phase II and Phase III of the ECVAM
Prevalidation study, and were used here to assess between-laboratory variation (figure
5.3.2.1. and figure 5.3.2.2.).

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Fluorescein Leakage Assay Background Review Document

120
100

Mean FL (%)

80
60
40
20
0

Company # 7
Laboratory 1

FRAME
Laboratory 2

ECVAM
Laboratory 3

Company # 3
Laboratory 4

Laboratory

Figure 5.3.2.1. Mean FL% ±SD values for Phase II positive control data (0.16mg/ml
SLS) from each laboratory at T0 (pattern) and T4 (blank); n≥ 5. A version of INVITTOX
Protocol No. 120 was used.
The T0 and T4 positive control data from the four test laboratories were statisticallydifferent (one-way ANOVA, p< 0.0001). This finding was based on different numbers ofexperimental runs from each laboratory. The mean values for the positive control data
were similar for three of the four test laboratories at T0 and T4 (figure 5.3.2.1.). In
comparison to the other laboratories, the FAL (FRAME Alternatives Laboratory) reported
a greater amount of FL. At T0, the FAL had the smallest CV equal to 16%, whilst CV
values for other laboratories ranged from 23% to 46%.
All laboratories had greater intra-laboratory variation for T4 data in comparison to T0
data, with CVs ranging from 19% (FAL) to 131% (Company # 7). One could expect
greater variation at T4 because the relatively short chemical exposure would cause
differing levels of cell damage. At T4 the monolayer begins to repair itself and the rateof the recovery would depend on the number of cells initially damaged and the extent of
the damage, thus leading to high levels of variation in the amount of FL measured.
Using positive control data, the between-laboratory variation for the three laboratories
that participated in Phase III was investigated; ECVAM, FRAME and Company # 3(figure 5.3.2.2.). The main protocol differences between Phase II and Phase III were theacceptance criteria ranges for the control values which were increased slightly (0%leakage control, positive control) or the boundaries shifted (100% leakage control).

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Fluorescein Leakage Assay Background Review Document

30

Mean FL (%)

25
20
15
10
5
0
FRAME Laboratory 2

ECVAM Laboratory 3
Laboratory

Company # 3
Laboratory 4

Figure 5.3.2.2. Mean FL% ±SD values for Phase III positive control data (0.16mg/ml
SLS) from each laboratory at T0 (pattern) and T4 (blank); n≥ 5. A version of INVITTOX
Protocol No. 120 was used.
In comparison to Phase II data, all the laboratories showed relatively similar mean T0 FL
assay values in Phase III. This finding was based on different amounts of data fromeach laboratory. There was no statistical difference between the positive control datafrom the different laboratories when either T0 or T4 data were compared. This indicatedthat protocol clarifications made after Phase II were effective as the protocol produced
comparable Phase III results for the positive control between the laboratories. The SDs
were similar for all T0 and T4 data with the exception of data from the Company # 3laboratory at T4. For all laboratories, the difference between the mean values for T0
and T4 increased in each test laboratory in comparison to Phase II results.
Despite increased correlation between the Phase III T0 results from the different
laboratories, only the Company # 3 laboratory had a mean value that fell well within theacceptance range of 15-30%. ECVAM had a mean value of 15% which was just within
the range of acceptance, whilst the FAL had a mean value of 7%. All T4 mean values
from the different laboratories failed to fall within the acceptance range. This result
indicated that a different concentration of SLS may be required for it to be used as asuitable positive control, or that a different chemical would be more appropriate.
Phase III data were analysed to determine the proportion of identical predictedclassifications from the three different test laboratories. The ten test surfactants covereda wide range of MMAS values. The PM applied to the data was taken from the COLIPA
study (Brantom et al., 1997) (table 5.3.2.2.).

119Fluorescein Leakage Assay Background Review Document

Table 5.3.2.2. PM applied to ECVAM Prevalidation study Phase III data
FL20 (mg/ml) T4>100
20-100<20Classification
Non irritant/ SlightModerate
Irritant/ Severe

Draize MMAS
<15
15-30
>30

Table 5.3.2.3. Mean FL20 (mg/ml) T4 data and classification for each test material ineach laboratory.
Company
Chemical
FRAMEClassific. ECVAM Classific. # 3
Classific.
Benzalkonium
Chloride (1%)
20.42 Moderate
15.65
Irritant
26.52 Moderate
Non
Non
Not
Irritant/
Irritant/
Not
Brij-35
TestedTested121.99Slight200
SlightCetyl stearyl
alcohol (50%
Irritant
11.84
Irritant
2.63
Irritant
in corn oil)
2.17
Deoxycholic
Irritant
2.02
Irritant
0.47
Irritant
Acid
0.48
LaurylSulfebetaine
1.65
Irritant
2.70
Irritant
1.85
IrritantN-lauroylsarcosine
sodium salt
1.19
Irritant
1.35
Irritant
0.63
Irritant
Non
PolysorbateIrritant/60
399.31Slight75.32 Moderate
37.5 Moderate
Sodium C14C16
olesulfonate
0.43
Irritant
0.42
Irritant
0.58
Irritant
SodiumLaurethSulfate Pres35%
5.1
Irritant
5.12
Irritant
4.47
Irritant
Triton X-155
0.93
Irritant
7.28
Irritant
0.97
Irritant
NB. N=3 for ECVAM laboratory and FAL, n=2 for the Company # 3 laboratory.

120

Fluorescein Leakage Assay Background Review Document

Table 5.3.2.4. Summary table of proportion of identical predicted classifications from each laboratory

ReportClassif.
Scheme

No.
of
Lab.s

ECVAM
Prevalidation(Southee, 1998)

Refer totable
5.3.2.2.

3

Materials with
100%
agreement
betweenlaboratories

Materials
with atleast 1
divergentclassif.

Materials
with 3different
classif.s

All materials with classificationdifferences compared *ECVA
MFALFALCompa
ECVAM
Company
ny # 3classif.
# 3 classif. classif.
comparis compariso compar
on
n
ison

77.8%
(7/9)

22.2%
(2/9)

0%
(0/9)

77.8%
(7/9)

88.9%
(8/9)

90%
(9/10)

Only materials which differed by 2
classifications compared**
FALECVAM
classif.
comparison

FALCompan
y#3
classif.
comparison

ECVAMCompan
y#3
classif.
comparis
on

100%
(9/9)

100%
(9/9)

100%
(10/10)

Values in brackets are the numbers used to determine the percentage of identical predicted classifications from the total number ofclassifications.
* materials that differed by one or more classifications when comparing the different laboratories were counted**only materials that produced classifications that differed by two classes were counted (i.e. non-irritant and irritant)
NB. Analyses which included data from the FAL were for nine test chemicals only as no value was produced for Brij-35
The results showed that the proportion of identical predicted classifications was relatively high and that predicted classifications for thesurfactants only ever differed by one classification. One would expect the proportion of identical predicted classifications to be high as
only surfactants were tested and the test materials had similar potencies, towards the mild end of the ocular irritation scale. In
addition, the PM used to convert the FL assay results into the predicted classifications for ocular irritation was specifically developed
for surfactants and surfactant-based formulations only. Therefore a high proportion of identical predicted classifications from thevarious laboratories would be expected as the protocol was not thoroughly challenged by a wide range of chemical classes andpotencies.

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Fluorescein Leakage Assay Background Review Document

5.4.1. Additional studies where raw data are not available: attempt to combine the data using weight-of-evidence approaches
Table 5.4.1. Relevant results and information for studies where raw data were not available

Study
The EC/HOInternational
Validation
Study on
Alternatives tothe Draize Eye
Irritation Test
(Balls et al.,
1995)
A Summary
Report of the
COLIPA
International
Validation
Study on
Alternatives tothe Draize
Rabbit Eye
Irritation Test
(Brantom etal., 1997)

No. ofChemicals

60

11No. ofProducts

018(commonto bothFRAME
andCompany# 4 lab.)

Coded?

Results

Y

Pearsoncorrelation
coefficients forinter-laboratory
variability ofFL20 values
ranged from0.214-0.841.

Y

Kappa statistics
for correlation ofin vivoclassifications
and predictedclassification;
FAL K= 0.65±
0.3; Company #4 K= 0.78± 0.2

Lab.
No.

4

2

Exp.
No.

3

4

122

ReplicateNo.

Data
Format(raw,
summary)

RangesPhysicochemical
properties

3

Mean FL20(mg/ml) T0
values per
chemicalperlaboratory

ChemicalClasses
60 chemicals
ranging inmechanisms andpotency for which
historical in vivo
data wereavailable- dataprimarily fromECETOC
database

MMAS
Range:
0-108

Liquids,
crystals,
powdersMW:
39.99707-1228

3

Mean FL20(mg/ml) T4
valuessurfactants andsurfactant-basedmaterials soluble
in HBSS

MMAS
Range:
0-110

Solids, liquids
MW 39.997071228

Fluorescein Leakage Assay Background Review Document

5.4.2. Discussions from the literature
Possible sources of variation acknowledged by the literature reporting these studies
were discussed.
The EC/HO International validation study on alternatives to the Draize eye irritation test
(Balls et al., 1995)
The EC/HO International validation study on alternatives to the Draize eye irritation test
(Balls et al., 1995) investigated a number of in vitro assays for their ability to predict invivo MMAS scores. The FL assay test protocol was based on the method of Tchao(1988) and was later accepted as INVITTOX Protocol No. 71. The FL assay was
conducted in the following laboratories, FAL; Company # 4, State University of New
York, the Philadelphia College of Pharmacy & Science. Inter-laboratory variability wasassessed as part of this study.
Sixty chemicals of high purity were tested. The test chemicals were supplied by BIBRAInternational (UK), and when available came from the same source as those used forthe previously conducted in vivo tests. Therefore, one can assume that where possible,
the chemicals supplied to the different laboratories were from the same suppliers andthat variation caused by different chemical batches was minimal. Balls et al., (1995)
stated that a wide range of chemicals with different mechanisms and potencies wereused in order to fully challenge the various alternative methods tested. However, mostalternative methods are developed and used in industry to test a specific chemical classor similar toxicity mechanisms, and thus predictive capacity would be expected to be low
for a wide range of chemicals.
Pearson’s correlation coefficients were calculated to compare the results from thedifferent laboratories and ranged 0.214-0.841. (Balls et al., 1995). All the low correlationco-efficients were obtained when comparisons were made with one particular laboratory.
Although this finding could indicate that the protocol was not sufficiently detailed,therefore preventing all participating laboratories from following it correctly, as FAL
participated in this study it was known that not all laboratories followed the FL assay test
protocol equally (R Clothier, personal communication).
Various factors were stated in the Balls et al., (1995) publication regarding the limitationsof the FL assay method. These are discussed in detail in section 2. of this BRD. As
these limitations would also impact on between-laboratory variability, they are also listed
here briefly:-viscous non-toxic materials can be difficult to remove after the one minute exposure-binding of test chemicals to the insert membrane, (e.g. cationic surfactants) can occur-transfer of the inserts to new wells can cause bubbles to form underneath themembranes and impede FL-damage to the insert membrane can occur during the many handling procedures of the
protocol-some materials are incompatible with the O-ring of the membrane-highly volatile materials need to be sealed with mineral oil to ensure continued contact
with the cell monolayer-materials need to be tested as, either an aqueous solution, mineral oil solution,suspension, or as a microemulsion formulation

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Fluorescein Leakage Assay Background Review Document

The COLIPA Eye irritation international validation program (Phase I) (Brantom et al.,
1997; Zanvit et al., 1999)
The COLIPA Eye irritation international validation program (Phase I) (Brantom et al.,
1997; Zanvit et al., 1999) evaluated a FL assay protocol, for its predictivity of in vivo
MMAS scores and inter-laboratory variability. The protocol was performed according to
Cottin et al., (1992), which was the basis for INVITTOX Protocol No. 120. The protocol
was tested in only two laboratories (FAL and Company # 4) which made it difficult todraw definitive conclusions regarding inter-laboratory variability. Classifications basedon the in vivo data were compared to the FL assay predicted classifications using kappa(k) analysis where a value of one indicated complete agreement between the values.
Although no direct analyses for inter-laboratory variability were reported in the
publications, both laboratories reported a similar number of correctly predicted in vivo
classifications; FRAME K =0.65±0.3, Company # 4 K =0.78±0.2 (Zanvit et al., 1999).
Four of the test materials tested by Company # 4 had their irritancy potential overpredicted which compared to the six over-estimated by FAL; four were common to bothlaboratories. None of the materials tested in Company # 4 laboratory had their irritancy
potential under-estimated whilst the hand cleanser formulation was under-predicted
when tested in the FAL. It was stated that this test sample contained a mineral spiritthat was not very stable in the formulation; this could have caused the different results inthe two laboratories.
Zanvit et al., (1999) stated that there were too few test materials with mid-range irritancy
potentials to enable the predictive capacity of the protocol to be fully tested. It was also
stated that the protocol was not designed to measure materials with severe irritancy
potentials (MMAS 50 to 110) and that another in vitro test should be performed before
the FL assay, to detect severe materials which cannot be accurately measured using the
FL assay. Forty materials were tested in this study although the results for only ~30
which were surfactants and surfactant-based formulations considered to be soluble in
HBSS were accepted. The two laboratories differed as to which materials they
considered to be soluble in HBSS.
It should be noted that of a range of test materials available for testing in the COLIPA
study, the FL assay was only used to test surfactants and surfactant-based formulations
that were soluble in HBSS. Subsequently, the range of materials tested was limited tothe type which the FL assay was designed to test and therefore would be expected toperform well. If other types of materials had been tested, the results from the different
laboratories may have varied to greater extents and indicated that the protocol was notvery transferable. However, as the only available PM was specific for surfactants,
comparisons of the predictive capacity of the protocol was limited to this chemical class.

124

Fluorescein Leakage Assay Background Review Document

5.4.3.

Compilation of results

5.4.3.1.

Statistical approach(es) used: description & rationale for theapproach used to determine between-laboratory reproducibility

ANOVA analyses were not performed because there were no individual data for eachexperimental run per test material from each laboratory. Hence the results of an ANOVA
analysis could be artifactual.
For each study, Pearson’s correlation coefficients were calculated using the various data
sets generated by the different laboratories. This allowed all the data sets produced by
the laboratories featured in each study to be compared.
For the results from the COLIPA study, the mean CVs were calculated for the entire
data set, and for ‘formulations only’ and for ‘chemicals only.’ The CVs were calculated
for each laboratory.
For Prevalidation study Phase III data, analyses were performed to determine theagreement between the participating laboratories for predicted classifications of ocular
irritation. The PM used to transform the FL assay values into predicted classifications of
irritancy was the same as that featured in the COLIPA study.

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Fluorescein Leakage Assay Background Review Document

5.4.3.2 Compilation of results
Table 5.4.3.2.1. Between-laboratory FL20 (mg/ml) data for COLIPA study test materials:i) chemicals
ChemicalLab 24

Lab 25

Triton X-100 1%

58.98

51.05

55.02

5.61

10.19

SLS 3%

9.28

6.7

7.99

1.82

22.83

Triton X-100 5%

13.27

13.1

13.19

0.12

0.91

Benzalkonium chloride 1%

10.88

6.8

8.84

2.88

32.64

SLS 15%

4.36

4.3

4.33

0.04

0.98

SLS 30%

0.88

0.42

0.65

0.33

50.04

Triton X-100 10%

5.16

7.4

6.28

1.58

25.22

Benzalkonium chloride 5%
Benzalkonium chloride
10%
Cetylpyridinium bromide
6%
Cetylpyridinium bromide
10%

4.12

3.8

3.96

0.23

5.71

3.63

0.46

2.05

2.24

109.61

12.83

0.35

6.59

8.82

133.91

7.45

0.38

3.92

5

127.69

10.26

2.61

47.25

Mean

126

Mean

SD

CV (%)

Fluorescein Leakage Assay Background Review Documentii) formulations
Formulation

Lab 24

Perfumed skin lotion

930

Polishing scrub

Lab 25Mean

SD

CV (%)

>1062

n/a

n/a

n/a

649.85311

480.43

239.6

49.87

Shampoo #1 normal

5.29

5.9

5.6

0.43

7.71

Eye make-up remover

193.12

161.5

177.31

22.36

12.61

Hand cleaner

10.3

24

17.15

9.69

56.49

Hair dye base F#1992>872.5

n/a

n/a

n/a

Sunscreen lotion

979

>1009

n/a

n/a

n/a

Emulsion antiperspirant

1057>1062.5

n/a

n/a

n/a

Gel cleaner

35.38

17.75

Hand soap

4.86

Shampoo - baby

7.41

Sunscreen SPF 15

344.6

Liquid soap #1

5.82

6.15

5.99

0.23

3.9

Shampoo antidandruff

3.13

2.5

2.82

0.45

15.83

Shampoo 2-in-1

4.59

4.15

4.37

0.31

7.12

244.25

44.95

144.6

140.93

97.46

30.1

30.1

n/a

n/a

Mouthwash
Toothpaste
Cleansing foam III
Moisturiser withsunscreen

26.57

-

4.86
3.6

n/a

5.51

-

344.6

-

12.47

46.93
n/a

2.69
n/a

48.94
n/a

9.67

-

9.67

n/a

n/a

498.35-498.35n/an/aShower gel

7.16

6.2

6.68

0.68

10.16

Skin cleaner

5.92

3

4.46

2.06

46.29

Hair dye base form #2

981

>1001

n/a

n/a

n/a

Hair dye base form #3

974

>750

n/a

n/a

n/a

Mean

104.06

127

35.99

33.61

Fluorescein Leakage Assay Background Review Document

Table 5.4.3.2.2. Between-laboratory FL20 (mg/ml) data for EC/HO study test chemicals
Chemicalsodium hydroxide (10%)
benzalkonium chloride (10%)
trichloroacetic acid (30%)
cetylpyridinium bromide (10%)
cetylpyridinium bromide (6%)
benzalkonium chloride (5%)
captan 90 concentratechlorhexidine
cyclohexanol
quiniacrine
promethazine HClparafluoraniline
triton X-100 (10%)
acetonehexanol
1-naphthalene acetic acid, Nasalt
sodium oxalate
isobutanol
imidazole
sodium lauryl sulphate (15%)
2-ethyl-1-hexanol
4-carboxybenzaldehyde
methyl ethyl ketone
pyridine
1-naphthalene acetic acidbenzalkonium chloride (1%)
2,2-dimethylbutanoic acidgammabutyrolactone
thioureaoctanolmethyl acetate
L-aspartic acidbenzoyl-L-tartaric acidtriton X-100 (5%)
potassium cyanate
isopropanol
sodium perborate
dibenzyl phosphate
2,5-dimethylhexanediol
methyl cyanoacetate
sodium hydroxide (1%)
ethanol
2,6-dichlorobenzoyl chloride
ammonium nitrate

Lab
18

Lab
19

Lab
20

Lab
21

Mean SD

CV
(%)

6.3
19.0
120.0
27.0
93.0
18.0
>100.0>100.0473.0>750.0
12.0
55.0
90.0
523.0
270.0

13.3
>25.0
>250.0
>25.0
>25.0
>25.0
>250.0>250.0>938.0>250.053.0>1146.0
99.4
709.3
12.7

5.8
>1000.0>1000.0>960.0>810.0>1000.0>250.0>250.0741.0>250.065.1>1120.0339.0
678.0>750.0

10.0
>25.0
>25.0
>25.0
>100.0>25.0>100.0>100.0>1000.0>250.0
>25.0
665.0
839.0
>1000.0

8.9
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
298.4
687.3
n/a

3.5
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
270.2
129.8
n/a

39.6
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
90.6
18.9
n/a

171.0
>100.0
369.0
135.0
12.0
230.0
>100.0256.0
176.0>100.0
91.0
172.0
133.0
>100.0
198.5
361.0
16.0
49.5
167.0
>750.0
549.0
50.5
21.0
>750.0>1100.0132.0
516.0>1500.0551.0>250.0>250.0>250.0
86.3
8.9
78.4
>250.0273.3
315.8>250.0>100.0>250.0160.7>250.082.4
518.8>250.0>25.0
163.7>500.0715.0>250.0>250.0>750.0>1115.0
125.2
698.6>1448.0>750.0>500.0>750.0>770.0185.0
24.0>730.0>250.0235.0
371.0>250.0>960.0>810.0482.0>250.0>770.0>870.0>250.0>250.0625.0>500.0618.0>250.0>250.0>250.0>750.0127.0
590.0>1400.0>750.0245.0>250.0>1000.0
185.0
70.0>1000.0>100.0>1000.0989.0>250.0>250.0>100.0152.0>250.0>1000.0>1000.0>100.0>100.0715.0>750.0992.0>250.0>25.0>750.0>100.0133.0>1000.0>250.0>750.0

n/a
n/a
n/a
147.8
28.7
n/a
n/a
n/a
463.0
n/a
n/a
n/a
231.9
n/a
n/a
n/a
n/a
n/a
417.7
n/a
718.5
n/a
n/a
n/a
n/a
129.3
n/a
n/a
n/a

n/a
n/a
n/a
47.3
28.3
n/a
n/a
n/a
360.2
n/a
n/a
n/a
167.1
n/a
n/a
n/a
n/a
n/a
293.7
n/a
194.6
n/a
n/a
n/a
n/a
3.8
n/a
n/a
n/a

n/a
n/a
n/a
32.0
98.5
n/a
n/a
n/a
77.8
n/a
n/a
n/a
72.1
n/a
n/a
n/a
n/a
n/a
70.3
n/a
27.1
n/a
n/a
n/a
n/a
2.9
n/a
n/a
n/a

128

Fluorescein Leakage Assay Background Review Document

Table 5.4.3.2.2. continued
Chemical
ethyl-2-methylacetoacetate
sodium lauryl sulfate (3 %)ethyl acetatemaneb
fomesafen
tetraaminopyrimidine sulphate
toluenebutyl acetate
trichloroacetic acid (3%)
methyl isobutyl ketone
tween 20ethyl trimethyl acetate
methylcyclopentane
cetylpyridinium bromide (0.1%)
glycerolpolyethylene glycol 400

Lab
18

Lab
19

Lab
20

Lab
21

Mean

SD

CV
(%)

25.0
231.0
>100.0>100.0>100.0>860.0>940.0803.0>800.0653.0>850.0>750.0>900.0>1200.0>1200.0>990.014.7>887.0>250.0>250.0>250.0>800.0>874.0>1006.0>792.0>1101.0>844.0>741.0>984.0>1254.0>1110.0

369.0
73.0
746.0
>750.0>750.0>250.0>830.0>860.0>1000.0>770.0>750.0>800.0>670.0>960.0>1000.0>1100.0>1000.0317.0>1000.0>250.0>250.0>100.0>1000.0>100.0>250.0>1000.0>1000.0>1000.0>1000.0>1000.0>1000.0
>1.0

n/a
107.4
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a

n/a
142.0
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a

n/a
132.2
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a

Mean

294.4 149.1

60.2

NB. It was known that the various laboratories differed as to the highest test
concentration used per chemical. Therefore, the greater-than values from the different
laboratories were not indicative of the level of toxicity of the chemical but the testing
strategies adopted by the different laboratories. The overall mean, SD and CV
calculated for the EC/HO study was based only on the actual data and not greater-thanvalues. Subsequently, the overall mean, SD and CV values calculated are skewed as
there were very few chemicals for which actual data were available.
The EC/HO International validation study on alternatives to the Draize eye irritation test
(Balls et al., 1995)
An inter-laboratory correlation of the FL20 (mg/ml) data from each laboratory wasreported in the EC/HO study publication (Balls et al., 1995) and agreed with thePearson’s correlation coefficients performed for this BRD (table 5.4.3.2.3.).
Table 5.4.3.2.3. Inter-laboratory correlation of the FL20 (mg/ml) data from the four
laboratories (from Balls et al., 1995)
Laboratory
Lab
Lab
Lab
Lab
18
19
20
21
Lab 18 1
Lab 19 0.841 1
Lab 20 0.490 0.513 1
Lab 21 0.373 0.424 0.214 1
The proportion of identical predicted classifications for the EC/HO chemicals tested in
the four participating laboratories was calculated (table 5.4.3.2.5.). There was no PM
featured in the EC/HO study that could be used with the FL assay data generated as the
PM proposed in the publication required 72h FL assay recovery data to distinguish EU
R36 and R41 classifications. The PM was modified by the authors of this BRD and
129

Fluorescein Leakage Assay Background Review Document

applied to the EC/HO FL20 (mg/ml) data to distinguish EU irritants (R36/R41) and Not
Classified classifications only (table 5.4.3.2.4.).
Table 5.4.3.2.4. Modified PM from EC/HO study used to assign irritant and non-irritant
EU classifications.
EU
Classification
Not Classified
R36/R41

FL20 (mg/ml)>750mg/ml≤ 750mg/ml

The modified PM was applied to the mean results for each chemical from eachlaboratory. SD or SEM values were not available (table 5.4.3.2.5.).

130

Fluorescein Leakage Assay Background Review Document

Table 5.4.3.2.5. Predicted classifications for EC/HO study data generated using the PMfeatured in table 5.4.3.2.4.
Chemical concentrations (mg/ml) causing FL 20%
Lab 18 Classif.
Lab19 Classif.
Lab 20 Classif.

Lab 21

Classif.

>100

unknown>250

unknown>250

unknown>250

unknown

1-naphthalene acetic
acid, Na salt

171

R36/R41.

>250

unknown>500

unknown

245

R36/R41.

2,2-dimethylbutanoic
acid

172

R36/R41.

>250

unknown

810

NC

>100

unknown

2,5dimethylhexanediol

>750

NC.

>750

NC

>250

unknown

>750

NC

>1500
230

NC
R36/R41.

>1448
78.4

NC
R36/R41.

>1400
>730

NC
unknown

>250
>1000

unknown
NC

>100
523
551

unknown
R36/R41.
R36/R41.

>250
709
>750

unknown
R36/R41.
NC.

>250
678
>750

unknown
R36/R41.
NC

>100
839
>750

unknown
NC
NC

91

R36/R41.

>100

unknown

>960

NC.

>250

unknown

19

R36/R41.

>25

unknown

>1000

NC.

>25

unknown

18

R36/R41.

>25

unknown

>1000

NC.

>25

unknown

49.5>940

R36/R41.
NC.

>25
>874

unknown
NC.

>250
>860

unknown
NC.

>100
>100

unknown
unknown>100

unknown>250

unknown>250

unknown>100

unknown

>900

NC

>984

NC

>960

NC

>1000

NC

27

R36/R41>25

unknown>960

unknown>25

unknown

93
>100
473

R36/R41
unknown
R36/R41

>25
>250
>938

unknown
unknown
NC

>810
>250
741

NC
unknown
R36/R41

>100
>100
>1000

unknown
unknownNCdibenzyl phosphateethanol
ethyl acetate

21
516
231

R36/R41
R36/R41
R36/R41

>250
699
>887

unknown
R36/R41
NC

>250
590
746

unknown
R36/R41
R36/R41

>25
>1000
>1000

unknownNCNC

ethyl trimethyl acetate

>850

NC

>844

NC

>800

NC

>1000

NC

ethyl-2methylacetoacetate

*

N/A

>990

NC

369

R36/R41>1000

NC

Chemical Name
1-naphthalene acetic
acid

2,6-dichlorobenzoyl
chloride2-ethyl-1-hexanol
4carboxybenzaldehyde
acetoneammonium nitratebenzalkonium
chloride (1 %)benzalkonium
chloride (10%)
benzalkonium
chloride (5%)
benzoyl-L-tartaricacidbutyl acetate
captan 90
concentrate
cetylpyridinium
bromide (0.1%)
cetylpyridinium
bromide (10%)
cetylpyridinium
bromide (6%)
chlorhexidine
cyclohexanol131

Fluorescein Leakage Assay Background Review Document

Chemical Name
Fomesafen

Chemical concentrations (mg/ml) causing FL 20%
Lab 18 Classif.
Lab19 Classif.
Lab 20 Classif.
>100
unknown >250
unknown >750
NC

Lab 21>250Classif.
R36/R41.

gammabutyrolactone
glycerolhexanol
imidazoleisobutanol
isopropanol
L-aspartic acidmaneb
methyl acetate

133
>1200
270
135
369
549
16
>100
361

R36/R41
NC
R36/R41
R36/R41
R36/R41
R36/R41
R36/R41
unknown
R36/R41

161
>1254
12.7
86.3
>250
715
>250
>250
519

R36/R41
NC
R36/R41
R36/R41
unknown
R36/R41
unknown
unknown
R36/R41

482
>1000
>750
185
>770
618
>250
>750
>870

R36/R41
NC
NC
R36/R41
NC
R36/R41
unknown
NC
NC

152
>1000
>1000
185
>1000
992
>100
>250
>1000

R36/R41
NC
NC
R36/R41NCNC
unknown
unknownNC

methyl cyanoacetate

>1100

NC

>1115

NC

>750

NC

>100

unknown

methyl ethyl ketone
methyl isobutyl
ketone256

R36/R41273

R36/R41235

R36/R41

>1000

NC

>800

NC

>792

NC

>770

NC

>1000

NC

methylcyclopentane
octanolparafluoraniline
polyethylene glycol
400

>750
198.5
55

NC
R36/R41
R36/R41

>741
82.4
>1146

unknown
R36/R41
NC

>670
>770
>1120

unknown
NC
NC

>1000
>1000
*

NC
NC
N/A

>1200

NC

>1110

NC

>1100

NC

>1

unknown

potassium cyanate
promethazine HClpyridinequiniacrine
sodium hydroxide(1%)
sodium hydroxide(10%)
sodium lauryl sulfate(3 %)sodium laurylsulphate (15 %)sodium oxalate
sodium perborate
tetraaminopyrimidine
sulphatethiourea
toluenetrichloroacetic acid(3%)
trichloroacetic acid
(30%)

>750
12
176
>750

NC
R36/R41
R36/R41
NC

>500
53
316
>250

unknown
R36/R41
R36/R41
unknown

>500
65.1
371
>250

unknown
R36/R41
R36/R41
unknown

>750
>25
989
>250

NC
unknownNC
unknown

132

R36/R41125

R36/R41

127

R36/R41133

R36/R41

6.3

R36/R41

13.3

R36/R415.8

R36/R4110

R36/R4125

R36/R4114.7

R36/R4173

R36/R41317

R36/R41

12
>100
50.5

R36/R41
unknown
R36/R41

8.87
>250
>250

R36/R41
unknown
unknown

24
>750
>250

R36/R41NC
unknown

70
>250
>250

R36/R41
unknown
unknown

>100
>100
>860

unknown
unknown
NC

>250
>250
>800

unknown
unknown
NC

>250
>250
>830

unknown
unknown
NC

>100
>250
>1000

unknown
unknown
NC

803

NC

>1006

NC

>1000

NC

>250

unknown120

R36/R41>250

unknown

>1000

NC

>25

unknown

triton X-100 (10 %)triton X-100 (5 %)tween 20

90
167
653

R36/R41
R36/R41
R36/R41

99.4
164
>1101

R36/R41
R36/R41
NC

339
625
>750

R36/R41
R36/R41
NC

665
715
>1000

R36/R41
R36/R41NC* no results reported; NC =EU Not Classified, GHS No Category132

Fluorescein Leakage Assay Background Review Document

The proportion of identical predicted classifications from each laboratory (table
5.4.3.2.5.) was assessed and the results summarised in table 5.4.3.2.6. Only the results
for chemicals which had results leading to four classifications based on the prediction
model were used (i.e. the classifications for any chemical which had one or morelaboratories producing an unknown classification were not used). It was consideredinappropriate to utilise ‘unknown’ as a classification as it was known that the various test
laboratories used different critieria to determine which was the highest test concentration
used. Therefore the analyses were performed using the classifications for only 25
chemicals.

133

Fluorescein Leakage Assay Background Review Document

Table 5.4.3.2.6. Summary table of proportion of identical predicted classifications from each laboratory

All materials with classification differences compared

ReportClassif.
SchemeRefer
table
EC/HO Study(Balls et al., 1995) 5.4.3.2.4.

No.
of
Lab.’
s

Materials with
100%
agreement
betweenlaboratories

Materials
with atleast 1
divergentclassif.
from anylaboratory

4

52%(13/25)48%(12/25)Materials
with 3different
classif.s

Lab 18Lab 19classifcomparison

Lab 18Lab 20classifcomparison

Lab 18Lab 21classifcomparison

Lab 19Lab 20classifcomparison

Lab 19Lab 21classifcomparison

Lab 20Lab 21classifcomparis
on

N/A

84%
(21/25)80%(20/25)52%(13/25)80%
20/25)

68%(17/25)72%(18/25)

Similar analyses performed for other datasets (i.e., Prevalidation study Phase III, COLIPA study data) calculated the proportion ofchemicals which produced more than one class difference in the predicted classifications, i.e the number of chemicals that werepredicted to be Not Classified and R41 irritants. As the PM for the EC/HO dataset was unable to distinguish R36 and R41 irritants,
this analysis could not be performed for this dataset.
Only approximately 50% of the test materials produced the same classification in all four participating laboratories. The proportion ofidentical classifications from the laboratories was lowest when the classifications from all other laboratories were compared withlaboratory 21; excluding analyses for laboratory 21, the proportion of identical classifications for the other laboratories was consistently
above 80%.

134

Fluorescein Leakage Assay Background Review Document

The COLIPA Eye irritation international validation program (Phase I) (Brantom et al.,
1997; Zanvit et al., 1999)
A one-way ANOVA compared the FL20 values produced by Company # 4 laboratory andthe FAL for materials found to be compatible for testing with this assay (33 test materials
for Company # 4, 30 test materials for the FAL). Interestingly, the two participating
laboratories did not agree totally about which materials they found to be soluble.
Twenty-nine identical materials were tested in both laboratories. There was no
statistical difference between the data sets, subsequently the Pearson correlation
coefficient was also found to be high; 0.98. The mean CV for the ‘chemicals only’
analysis was calculated to be 43% whilst the mean CV for the ‘formulations only’
analysis was 25%. These results indicate relatively good inter-laboratory variability
although it is acknowledged that the test materials were those specifically suited fortesting with the FL assay.
The number of experimental repeats performed and the acceptance criteria were notstated.
Predicted classifications for the COLIPA data were generated using the PM as featured
in Brantom et al., (1997) (table 5.3.2.2.).
Table 5.4.3.2.7. shows the predictedclassifications for both laboratories. Data were provided by COLIPA.
Table 5.4.3.2.7. Predicted classifications for COLIPA Study data generated using the
PM featured in table 5.3.2.2.

Test Chemical

Chemical concentrations causing FL20%(mg/ml) T4
Lab 24Company
Lab 24
Lab 25
Lab 25#4Classif.
FRAME
Classif.

Benzalkonium
chloride 1%

10.9

Irritant/
Severe

6.8

Irritant/
SevereBenzalkonium
chloride 10%

3.6

Irritant/
Severe0.5

Irritant/
SevereBenzalkonium
chloride 5%

4.1

Irritant/
Severe

3.8

Irritant/
SevereCetylpyridinium
bromide 10%

7.5

Irritant/
Severe0.4

Irritant/
SevereCetylpyridinium
bromide 6%

12.8

Irritant/
Severe0.4

Irritant/
SevereCleansing foam III

9.7

Irritant/
Severe**Emulsion
antiperspirant

1057.0NI/Slight>1062.50

NI/Slight

Eye make-up
remover

193.1

NI/Slight

161.5

NI/Slight

Gel cleaner

35.4

Moderate

17.8

Irritant/
SevereHair dye base F#1

992.0

NI/Slight>872.5

NI/Slight

Hair dye base F#2

981.0

NI/Slight>1001.00

NI/Slight

135

Fluorescein Leakage Assay Background Review Document

Test Chemical

Chemical concentrations causing FL20%(mg/ml) T4
Lab 24
Lab 24Company Test
Company Test
#4
Chemical # 4
ChemicalHair dye base
F#3

974.0

NI/Slight>750.00

NI/Slight

Hand cleaner

10.3

Irritant/
Severe24.0

Moderate

Hand soap

4.9

Irritant/
Severe**Liquid soap #1

5.8

Irritant/
Severe6.2

Irritant/
SevereMoisturiser
with sunscreen

498.4

NI/Slight**

Mouthwash

244.3

NI/Slight

45.0

Moderate

Perfumed skinlotionPolishing
scrub

930.0

NI/Slight>1062

NI/Slight

649.9

NI/Slight

311.0

NI/Slight

Shampoo baby

7.4

Irritant/
Severe

3.6

Irritant/
SevereShampoo #1
normal5.3

Irritant/
Severe5.9

Irritant/
SevereShampoo 2-in1

4.6

Irritant/
Severe4.2

Irritant/
SevereShampooantidandruff

3.1

Irritant/
Severe2.5

Irritant/
SevereShower gel

7.2

Irritant/
Severe6.2

Irritant/
SevereSkin cleanser

5.9

Irritant/
Severe3.0

Irritant/
SevereSLS 15%

4.4

Irritant/
Severe4.3

Irritant/
SevereSLS 3%

9.3

Irritant/
Severe

6.7

Irritant/
SevereSLS 30%Sunscreen
lotion0.9

Irritant/
Severe0.4

Irritant/
Severe979.0

NI/Slight>1009.00

NI/Slight

344.6

NI/Slight

*

*

*

*

30.1

Moderate

59.0

Moderate

51.1

Moderate

Triton X-100
10%

5.2

Irritant/
Severe7.4

Irritant/
SevereTriton X-100
5%

13.3

Irritant/
Severe13.1

Irritant/
SevereSunscreen
SPF 15
Toothpaste
Triton X-100
1%* Material not tested by the laboratory; NI =non-irritant136

Fluorescein Leakage Assay Background Review Document

Analyses were performed to determine the proportion of identical predicted classifications from each laboratory (table 5.4.3.2.8.).
Table 5.4.3.2.8. Summary table of proportion of identical predicted classifications from each laboratory

ReportClassif.
SchemeRefer to
COLIPA (Brantom table
5.3.2.2.
et al., 1997)

No. ofLab.’sMaterials with 100%agreement between
laboratories

Materials with atleast 1 divergent
classification*

Only materials which
differed by 2
classifications
compared**2

89.7% (26/29)

10.3% (3/29)

0% (0/29)

Values in brackets are the numbers used to determine the percentage of proportion of identical classifications from the different
laboratories.
* materials that differed by one or more classifications when comparing the different laboratories were counted**only materials that produced classifications that differed by two classes were counted (i.e. non-irritant and irritant)
NB. The analyses were limited due to only two participating laboratories.
The proportion of identical predicted classification from the two laboratories was high. There were no test materials that caused theclassifications from the two laboratories to differ by two classes (i.e. non-irritant and severe irritant). This was probably due to thelaboratories only testing surfactants and surfactant-based formulations that they considered to be soluble in HBSS. Unknown
solubility of test materials is likely to account for some between-laboratory variability as laboratories are likely to use different solventsor obtain different solubilised concentrations.

137

Fluorescein Leakage Assay Background Review Document

There were nine test chemicals that featured both in the EC/HO study (Balls et al., 1995)
and the COLIPA study (Brantom et al., 1997). Different protocols were used in thesestudies. The FAL and Company # 4 participated in both studies. The results for thesenine test chemicals from both studies and for both laboratories are shown (table
5.4.3.2.9.) (data from Zanvit et al., 1999).
Table 5.4.3.2.9. FL20 (mg/ml) data from the EC/HO study and the COLIPA study for thenine common test chemicals (from Zanvit et al., 1999). A version of INVITTOX Protocol
No. 120 was used.
FL20 COLIPA *FL20 EC/HO **ChemicalCompany FRAME
Company FRAME#4#4
SLS 3%
9.28
6.7
14.7
25
Triton X-100 5%
13.27
13.10
163.72167
Benzalkonium chloride 1%
10.88
6.8
>100
91
SLS 15%
3.6
4.3
8.87
12
Triton X-100 10%
5.16
7.4
99.44
90
Benzalkonium chloride 5%
4.12
3.8
>25
18
Cetylpyridinium bromide 6%
9.49
0.35
>25
93
Cetylpyridinium bromide 10% 7.45
0.38
>25
27
Benzalkonium chloride 10%
3.63
0.46
>25
19
* The protocol tested in the COLIPA study used a 15 minute chemical exposure durationand measured FL 4h following the chemical exposure** The protocol tested in the EC/HO study used a 1 minute chemical exposure durationand measured FL immediately following the chemical exposure
As there was no PM for the EC/HO FL20 (mg/ml) study data which converted the values
into classifications of ocular irritation it is difficult to compare the predictive capacity ofthe two protocols for ocular irritation. As the protocols differed greatly, it was notconsidered appropriate to apply the PM from the COLIPA study to the EC/HO study
data. Zanvit et al., (1999) reported Pearson’s and Spearman’s correlation coefficientsfor these data respective to in vivo MMAS. There was higher correlation with the in vivo
MMAS for the COLIPA data for both laboratories in comparison to the EC/HO study data
produced by the same laboratories (Zanvit et al., 1999).
5.4.3.3. Attempt to combine the data using weight-of-evidence approaches
If COLIPA study and ECVAM Prevalidation study data are considered representative of
INVITTOX Protocol No. 120, there were more data available to enable the interlaboratory variability of INVITTOX Protocol No. 120 to be evaluated in comparison to theother INVITTOX Protocols. There were no inter-laboratory data available for INVITTOX
Protocols No. 82 and No. 86. A protocol similar to INVITTOX protocol No 71 was tested
in the EC/HO study (Balls et al., 1995). This was a multi-laboratory study where 60 purechemicals were tested in four laboratories. In comparison, INVITTOX Protocol No. 120
was tested in the COLIPA study where 29 surfactants and surfactant-based formulations
were tested in two laboratories, and also in the ECVAM Prevalidation study; fourchemicals and one formulation were tested in four laboratories for Phase II and ten
surfactants were tested in three laboratories for Phase III. In addition to the test material
data available for this protocol there was also a considerable amount of positive control(0.16mg/ml SDS) data. Subsequently, in terms of the quantity of data, INVITTOX
Protocol No. 120 had greater weighting than INVITTOX Protocol No. 71.
138

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 71 was used to test a wide range of chemicals (EC/HO study)
and solubility was uncertain for some. In terms of the ranges of chemical classes,
INVITTOX Protocol No. 71 would receive greater weighting than INVITTOX Protocol No.
120. Data for both INVITTOX Protocol No. 120 and INVITTOX Protocol No. 71 covered
the same range of in vivo irritancy scores and therefore both protocols would receive
equal weighting for this criterion.
It was known that not all of the laboratories that participated in the EC/HO study fully
adhered to the FL assay protocol, therefore there was some doubt as to whether all the
results were representative of the same protocol (R Clothier, personal communication).
This fact was supported by the Pearson’s correlation coefficient calculations in the
publication and also the statistical analyses performed for this BRD; both found that
there were significant differences between the data sets from each laboratory.
Unfortunately, there was no other study reported where INVITTOX Protocol No. 71 hadbeen tested in more than one laboratory, which would have enabled one to determine if
the EC/HO study FL assay results for inter-laboratory variability were typical for thisprotocol. There was no statistical difference between the data sets submitted from thevarious laboratories that participated in the COLIPA study and also the ECVAM
Prevalidation Study Phase II and Phase III. Therefore, there was greater evidence insupport of the reproducibility INVITTOX Protocol No. 120 rather than irreproducibility of
INVITTOX Protocol No. 71.
Annex III

Between-laboratory FL assay data for a. COLIPA test materials:i) chemicals ii) formulations b. EC/HO study chemicals; c. ECVAM
Prevalidation Phase II; d. ECVAM Prevalidation Phase III –provided on CD

Annex A

CTFA study Phase III formulation ingredients (from draft HET-CAM
BRD: Appendix C2 (ICCVAM/NICEATM, 2004)

Annex B

COLIPA Study test chemicals and formulations compositions (fromCOLIPA)

Annex C

Formulation compositions from Company # 3

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6. Predictive Capacity (Module 5)
The majority of the literature that featured a FL assay protocol assessed the assay’s
ability to predict chemical-induced in vivo eye irritation, or less commonly skin irritation.
Most of the chemicals/materials tested using the FL assay have been cosmetic
ingredients and formulations, i.e. those that humans are likely to be routinely exposed toand are likely to gain entry to the eye, rather than exposure to industrial chemicals in theworkplace. In the literature, the in vivo data used to assess the predictive ability of the
FL assay were often Draize MAS or MMAS. In some cases, the in vivo ocular irritation
data were not provided in the publications but a statement made regarding thecorrelation of the FL assay ranking of materials with the in vivo ranking for toxicity,
based on the chemical structures and properties, i.e. Hubbard et al., (1994). EU risk
phrases featured in only one study (Clothier et al., 1994) and were assigned based on
the historical in vivo data available. No study featured classifications according to the
GHS or the EPA classification systems for ocular irritation.
It is to be noted that summarised in vivo data were often presented in the literature andthe variability of this data was rarely discussed. There is no consensus for how thevariability of in vivo data should be addressed. The impact of in vivo data variability
differs according to the EU, GHS and EPA classification schemes which use the raw invivo data in different ways, to determine the various classifications. ECVAM is currently
evaluating the effect of in vivo variability in these different classification systems.
Table 6.1.1. Provides information regarding the CTFA study Phase III (Gettings et al.,1996) which tested the TEP assay/ INVITTOX Protocol No. 86. This was the only data
set for which raw in vitro and in vivo data were available.

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6.1. Studies with available raw data
Table 6.1.1. Table presenting the relevant information for each study where raw in vitro and in vivo data were available
INVITTOX Protocol No. 86

Study
CTFA Evaluation of
Alternatives Program: An
Evaluation of In Vitro
Alternatives to the Draize
Primary Eye Irritation Test:
Phase III (Gettings et al.,1996) IRAG LAB B (Botham
et al., 1997)

No. ofNo. ofNo. ofLaboratories Chemicals Products

10

23 (data for2formulations
were notreported)

Coded
(Y/N)

ChemicalClasses

Ranges of
Toxicity
TestedPhysico-chemical
properties

independent
codingrepresentative
surfactant-based
personal careformulations

MAS Range:
2.3-43liquid formulations-compositions known

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6.1.2. In vivo reference data used to assess the performance of the FL assay for studies where raw in vitro and in vivo data
were available
Table 6.1.2. In vivo reference data used to assess the performance of the FL assay for studies where raw in vitro and in vivo data
were available
INVITTOX Protocol No. 86

Study
CTFA Evaluation of
Alternatives Program: An
Evaluation of In Vitro
Alternatives to the Draize
Primary Eye Irritation Test:
Phase III (Gettings et al.,1996) IRAG LAB B (Botham etal., 1997)

Species andProtocols used
as ref. Data

Draize test
–albino rabbits

Sources of Information
In vivo and in vitro
experiments conducted inparallel. Random block
design resulted inmaterials tested x6 on
different days in (3M, 3F)
albino rabbits, diluted
formulations tested on M
rabbits only

142

No. ofLabs

1

No. ofexperiments

6

Quality ofData (GLP)

Data Format

GLP

Raw animal data
available. In
publication,summarised MAS
values provided
for various ocular
tissues and total
MAS scores

Fluorescein Leakage Assay Background Review Document

6.1.3. Brief description of the studies with available raw in vitro and in vivo dataa. Statistical approaches detailed in the CTFA publications (Gettings et al., 1996)
The ‘response profile’ of the TEP assay (INVITTOX Protocol No. 86) data from a single
test laboratory was compared to MAS values for each formulation, and the agreement
between the data sets assessed. A post-hoc threshold value was assigned to the TEP
assay data, which produced the least number of false positive and false negativeclassifications of irritancy according to the FHSA classifications based on the in vivo
data. The ability of the TEP assay to separate pairs of test materials with significantly
different MAS values and to determine which was the least irritant according to the MAS
values was evaluated. This analysis was termed ‘concordance analysis’ and usedbecause it did not require subjective interpretation of the in vitro data. A disadvantage ofthis type of analysis was that when TEP assay results separated test materials that were
not separated by the in vivo data, it was considered to be a discordance. However, theseemingly greater sensitivity of the in vitro test was questionable in the absence of
supporting human data.
A Monte Carlo computer re-sampling procedure was used to give estimates of thevariability of the discordance rates for test materials that had different Draize MAS. This
analysis was performed to determine which of the numerous test assays had similar or
dissimilar discordance rates. The re-sampling was performed 100 times; the SDs of thesimulated values were used as estimates of the SDs for the discordance rates based on
observed MAS. A separation index was produced which indicated the extent ofagreement between the TEP assay data and the Draize data in their abilities to separate
the irritancy potential of pairs of test materials. The assays were ranked in order toselect the assay(s) that had discordance rate(s) similar to the discordance rate for the invivo data. The ranking was also performed using separation index values as thisreflected discordance rates when Draize data did and did not separate the pairs of test
materials.
Regression analyses were performed to describe the predictive capacity of the TEP
assay for MAS. Base-10 logarithm TEP assay data were plotted against in vivo MAS,and regression models fitted using weighted least squares; weights were inversely
proportional to the sum of the variability of the in vivo data and TEP assay data. The
regression models were fitted in order to smooth and interpolate the in vitro response
within the range of the observed in vivo data. 95% prediction bounds were calculated forthe range of logarithmic in vitro scores.
b. Principal results reported in the literature
Twenty-three of the 25 surfactant-based formulations were tested/reported for the TEP
assay from one laboratory only. A post-hoc threshold value enabled 14 of the FHSA
irritants and all of the non-irritants to be correctly identified. The specifity of the TEP
assay and PM was greater than the sensitivity, 100% and 78% respectively. Base-10
logarithm transformed data were used for the statistical comparisons with thequantitative in vivo data. Analyses of the discordance rates when the Draize test data
did and did not separate test materials and the separation index led to the TEP assay
being selected amongst 13 other assays for having greater concordance with the in vivo
data than the other assays evaluated.

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Table 6.1.3. The findings reported in the literature for those studies which used PMs to assess the predictive capacity of the TEP
assay.

Study
CTFA Evaluation of
Alternatives Program:
An Evaluation of In Vitro
Alternatives to the
Draize Primary Eye
Irritation Test: Phase III(Gettings et al., 1996)

Timepoint(s)used for PM

ProtocolPrediction/
Classification ModelTEP assay(INVITTOX
Protocol No.
86)

A post-hoc threshold value (≤2.6) was
assigned to achieve the least number offalse positives and false negatives
according to the FHSA classification
system15min

No of test
materials

Type of Test
materials

No. ofmaterials
underpredicted*

23

Surfactantbased
formulations

4* based on the threshold value in featured in Getttings et al., (1996)
NB. The assay featured in the CTFA publication (Gettings et al., 1996) was later accepted as INVITTOX Protocol No. 86.

144

No. ofmaterials
overpredicted*

0Fluorescein Leakage Assay Background Review Document

6.1.4. Compilation of data on predictive capacity of the test method from studies
with raw data available
Refer to Annex Vai -Compilation of raw TEP assay data and EU, GHS and EPA
classifications generated by entering raw in vivo data into the ECVAM template v6.
6.1.4.1. Description & rationale for the PM applied and statistical approaches used
a. Classification systems
The aim of these analyses was to determine the predictive capacity of the TEP assay for
the EU, GHS and EPA classification systems. The CTFA study did not report in vivo
eye irritation classifications for the EU, GHS nor the EPA classification systems.
Therefore, raw in vivo data (supplied by Company # 1) were entered into the ECVAM
template v6 which allowed raw Draize data to be converted into EU, GHS and EPA
classifications. These in vivo based classifications were used for comparisons with thepredicted classifications based on the TEP assay results.
b. Prediction models (PMs)
INVITTOX Protocol No. 86 includes a PM (table 6.1.4.1.1.). The EC50 (%) values
produced by the test materials are used to determine if the irritancy levels ‘pass’ or ‘fail.’
The PM is only capable of distinguishing potential irritants from non-irritants and notcapable of distinguishing different levels of irritancy (i.e. R36 and R41 irritants). The PMfeatured in INVITTOX Protocol No. 86 does not correlate the predicted classifications
with any classification schemes used for regulatory proposes. The PM contains a rangeof TEP assay values where it was not possible to determine conclusively if the materials
were irritants or non-irritants, these materials were classified as ‘borderline’.
Table 6.1.4.1.1. PM from INVITTOX Protocol No. 86
EC50 (%)≤1.8>1.8 < 2.2≥ 2.2

TEP Rating
failborderline/undeterminedpass

The PM from INVITTOX Protocol No. 86 had been applied to TEP assay data forsurfactant-based formulations tested in-house at Company # 3. Therefore it wasconsidered suitable to apply this PM to the data generated from the CTFA study Phase
III (Gettings et al., 1996). A modified version of the PM featured in INVITTOX Protocol
No. 86 was submitted to ECVAM; the same ranges of values were used, but correlated
to EU, GHS and EPA classifications (table 6.1.4.1.2.).

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Table 6.1.4.1.2. Modified TEP assay PM for predicting EU, GHS and EPA
classifications.
EC50 (%)≤1.8>1.8 < 2.2≥ 2.2

EU Classification
R36 or R41
Borderline/Undetermined
Not Classified

GHS Classification
Cat 1, Cat 2A or Cat 2B
Borderline/Undetermined
No Category

EPA Classification
Category I/ II
Borderline/Undetermined
Category III/ IV

The predicted EU, GHS and EPA classifications based on the TEP assay data werecompared to the actual classifications obtained by entering raw in vivo data into the
ECVAM template v6. The same ranges of TEP assay values were used to distinguishthe three principal classes used in the EU and GHS classification systems. The EPA
classification system has four principal classes, but the PM was not able to distinguishCategory IV and Category III test materials. The PM was not capable of distinguishingsub-categories, i.e. Category 2A and Category 2B GHS classifications. All Category IV
and Category III materials were treated as non-irritants for analyses in this BRD. The
conclusions formed regarding the predictive capacity of the TEP assay would be thesame for all classification systems unless the in vivo classifications did not correlate, i.e.
a test material with an R36 EU classification would be expected to have a Category 2
GHS classification and a Category II EPA classification.
Company # 3 also submitted to this BRD, TEP assay/INVITTOX Protocol No. 86 data forformulations, and corresponding raw in vivo data. The data set supplied by Company #3 could not be analysed to assess the protocol’s predictive capacity for ocular irritation,
due to the format of the in vivo data; greater-than values were predominately reported
which prevented EU, GHS and EPA classifications from being generated by the ECVAM
template v6. The raw in vitro and in vivo data from Company # 3 were provided in
Annex Vc of this BRD.
6.1.4.2. Description of the performance compared to the reference and eventually
the human situation for each study
Concordance analyses were performed according to ECVAM guidance. Contingency
tables (tables 6.1.4.2.1.a-c.) show the concordance of predicted and actualclassifications for the EU, GHS and EPA classification systems.
TEP assay data from the CTFA study Phase III were analysed (Gettings et al., 1996).
Of 25 test formulations, TEP assay data for only 23 formulations were reported; no data
were reported for the polishing scrub (HZT) and the facial cleanser (HZZ). It was notstated in the publication (Gettings et al., 1996) whether these two formulations were nottested or whether the results were not accepted or submitted.

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Table 6.1.4.2.1.a. Contingency tables for CTFA Phase III data generated using
INVITTOX Protocol No. 86 and the PM featured in table 6.1.4.1.2. -EU classification
systemEU Classification
Test
Prediction
NC
Borderline
R36/R41
Total

NC
8
2
3
13

R36
0
0
0
0

R41
2
0
8
10

23

The results showed that the TEP assay and PM had a tendency to over-predict theirritancy potential of the surfactant-based formulations. Nearly 40% of the EU Not
Classified formulations were classified as irritants or borderline materials. All but two of
the R41 formulations were correctly classified; the facial cleansing foam and gel
cleanser were misclassified as Not Classifieds. This would seem to indicate that either
the TEP assay was not capable of detecting the irritancy potential of all types of
surfactant-based formulations or that the ranges of values of the PM needed someadjustment. As a version of this assay is routinely used at Company # 3 for testing
surfactants and surfactant-based formulations, it is hypothesised that the PM required
some refinement rather than the assay being incompatible with the test formulations.
Table 6.1.4.2.1.b. Contingency tables for CTFA Phase III data generated using
INVITTOX Protocol No. 86 and the PM featured in table 6.1.4.1.2. -GHS classification
systemGHS Classification
Test
Prediction
NC
Borderline
Cat 1/ Cat 2
Total

NC
10
1
3
14

Cat 2
0
1
0
1

Cat 1
0
0
8
8

23

The results for the GHS classifications show that all the Category 1 irritants werecorrectly predicted. This contrasts to the results for the EU and EPA classification
system where two severe irritants were predicted to be non-irritants. One Category 2
irritant and one No Category formulation were considered ‘borderline’ and did notreceive any classification.

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Table 6.1.4.2.1.c. Contingency tables for CTFA Phase III data generated using
INVITTOX Protocol No. 86 and the PM featured in table 6.1.4.1.2. -EPA classification
systemEPA Classification
Test
Prediction
Cat III/IV
Borderline
Cat I/ Cat II
Total

Cat IV20
02

Cat III
6
2
3
11

Cat II
0
0
0
0

Cat I
2
0
8
10

23

The predictive capacity of INVITTOX Protocol No. 86 was similar for the EU, GHS andEPA classification systems due to the general concordance for the classifications basedon the in vivo data, i.e. a R36 (EU) irritants were generally also labelled Category 2(GHS) and Category II (EPA) irritants.
The predictive capacity for the three different classification systems indicated that further
work was required to increase the predictive capacity of the protocol and/or PM. If
INVITTOX Protocol No. 86 was to be used for regulatory purposes, work should also
focus on determining a range of values that could be used to distinguish the mild irritantsfrom the severe irritants.
Summary tables were compiled in order to compare the predictive capacity of the TEP
assay for the different classification schemes (tables 6.1.4.2.2.a-c). Under ECVAM
guidance the predictive capacity of the assay was ascertained by determining thepredictive capacity for the ‘non-irritants versus the rest’ and the ‘severe irritants versus
the rest.’ It was determined by ECVAM that if there were fewer than 10% mild irritants inthe data set, only the ‘non-irritants versus the rest’ analysis should be performed, as the‘severe irritants versus the rest’ analysis would only produce similar results in theabsence of data for mild irritants. For the data sets featured in tables 6.1.4.2.2.a-c,
there were less than 10% of the formulations classed as mild irritants in all theclassification systems, therefore only the ‘non-irritants versus the rest’ analysis was
performed for these data.
For interpretation of the ‘non-irritant versus the rest’ analyses below, the following
should be noted:-Concordance referred to the number of correctly predicted non-irritants and irritants by
the FL assay; irritants that were identified but predicted to have the wrong classification
were still counted as correctly identified.
-Sensitivity referred to the number of correctly predicted irritants by the FL assay as aproportion of the number of actual irritants. This analysis only determined that irritants
were predicted to be irritants by the FL assay but did not distinguish between the
different irritancy classifications predicted (e.g., R36 and R41). ‘Borderline’ formulations
were not counted as an irritancy classification.

148

Fluorescein Leakage Assay Background Review Document-Specificity referred to the number of correctly predicted non-irritants by the FL assay as
a proportion of the total number of actual non-irritants.
-Positive Predictivity referred to the number of correctly predicted irritants as aproportion of the total number of predicted irritants i.e. this analysis only determined that
irritants were predicted to be irritants by the FL assay but did not distinguish between
the different irritancy classifications predicted (e.g., R36 and R41).
-Negative Predictivity referred to the number of correctly predicted non-irritants as aproportion of the total number of predicted non-irritants-False Positive Rate referred to the number of non-irritants predicted to be irritants as aproportion of the total number of non-irritants-False Negative Rate referred to the number of irritants predicted to be non-irritants as aproportion of the total number of irritants
These descriptions are relevant for all ‘non-irritant versus the rest’ analyses throughout
Section 6 of this BRD.

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Fluorescein Leakage Assay Background Review Document

Table 6.1.4.2.2.a. Evaluation of the performance of INVITTOX Protocol No. 86 for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest
Data Source

Anal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

CTFA Phase III(Gettings et al.,
1996)

Refer
to
Table
6.1.4.1
.2.

23

69.6

80

61.5

72.7

80

27.3

20

16/23

8/10

8/13

8/11

8/10

3/11

2/10

Table 6.1.4.2.2.b. Evaluation of the performance of INVITTOX Protocol No. 86 for predicting ocular irritation according to the
GHS classification system –Non-irritants versus the rest
Data Source

Anal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

CTFA Phase III(Gettings et al.,
1996)

Refer
to
Table
6.1.4.1
.2.

23

78.3

88.9

71.4

72.7

100

23.1

0

18/23

8/9

10/14

8/11

10/10

3/13

0/8

Table 6.1.4.2.2.c. Evaluation of the performance of INVITTOX Protocol No. 86 for predicting ocular irritation according to the
EPA classification system –Non-irritants versus the rest
Data Source

Anal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

CTFA Phase III(Gettings et al.,
1996)

Refer
to
Table
6.1.4.1
.2.

23

69.6

80

61.5

72.7

80

27.3

20

16/23

8/10

8/13

8/11

8/10

3/11

2/10

NB. As the PM was not able to distinguish EPA Category III and Category IV materials, materials with these classifications wereconsidered in the analyses concerning non-irritants. Refer to Annex Vai for the origins of the in vitro data and in vivo classifications.

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Fluorescein Leakage Assay Background Review Document

6.1.4.3. DiscussionsaDescriptions of limitations of the test method (i.e. applicability domain
based on the results of the compiled data)
Analyses to determine the applicability domain of the TEP assay/INVITTOX Protocol No.
86 were limited to analyses of the data for the surfactant-based formulations tested in
the CTFA study Phase III (Gettings et al., 1996). As only formulations were tested, thepredictive capacity of this protocol and PM for chemicals could not be established.
The PM was not capable of distinguishing different levels of irritancy, and there was alsoa range of values within which the protocol and PM could not distinguish materials as
either irritants or non irritants, but which were labelled as ‘borderline.’ Two of the 23
formulations could not have their EU, GHS, EPA classifications predicted because theresults fell within the range of ‘borderline’. If it was known that the assay had not beenable to test the two materials for which data was not submitted, in addition to the two
formulations classed as ‘borderline’ materials, the PM was not able to predict theirritancy for 16% (4/25) of the test materials. As there were only data for 21 test
formulations, the various results summarised in tables 6.1.4.2.2.a-c. were greatly
affected by each individual result.
The concordance rates for the ‘non-irritants versus the rest’ for the three classification
systems ranged 70-78%. The protocol and PM had a greater predictive capacity for the
GHS classifications rather than the EU and EPA classifications. Concordance rates andsensitivity were higher, and false positive and false negative rates were lower for the
GHS classifications in comparison to the EU and EPA classifications. The negative
predictivity was the same for the EU and EPA classifications but higher for the GHS
classifications.
There were more formulations classified as non-irritants than irritants in each of thethree classification systems, and the data indicated that the protocol and PM had ahigher sensitivity rather than specificity for all three classification systems. This finding
suggested that further work was required to modify the protocol and/or PM to increase
the specificity of the assay and PM.
The physical properties of the test formulations were evaluated to determine if certain
features affected the predictive capacity of INVITTOX Protocol No. 86. As the predictive
capacity of INVITTOX Protocol No. 86 for the EU, GHS and the EPA classifications weresimilar, comparisons were made with EU classifications only. All of the test formulations
were liquids so it could not be determined if the assay had a good predictive capacity forsolids which are known to be difficult to measure in many in vitro assays. Two of thethree EU Not Classified formulations that were misclassified as being EU irritants andboth the EU R41 irritants misclassified as Not Classified formulations were coloured
materials. As 17 of 23 formulations were coloured, the data did not conclusively indicatethat colour did or did not affect the predictive capacity of the TEP assay. There were 13
viscous formulations (12 viscous plus one gel) in the data set, ten of these viscous
formulations were also coloured. Only one of the five materials with their irritancy
potential over-predicted was a viscous liquid. One of the two coloured formulations that
had their irritancy potential under-predicted was also viscous. One Not Classified
formulation that was neither coloured nor viscous was also misclassified as a R36/R41
irritant. As 13 of the 23 test materials were viscous solutions, the data did not indicate
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Fluorescein Leakage Assay Background Review Documentthat the assay was unable to measure materials with viscosity and that the predictive
capacity of the assay was adversely affected. As many test formulations were bothcoloured and viscous it was difficult to distinguish the effects of these two physicalproperties on the predictive capacity of the protocol. Overall, there was greater
evidence in support of colour rather than viscosity reducing the predictive capacity of the
protocol and PM.
In consideration that the TEP assay had only been used to measure the effects ofmaterials which it was designed to test, the overall predictive capacity of the assay
based on a limited data set was not promising. In addition, the assay was only
evaluated for its ability to distinguish irritants from non-irritants as the PM was not able
to distinguish different levels of irritancy. As there were few formulations labelled as‘mid-range’ irritants according to the in vivo data, it was difficult to surmise from therange of data whether the assay would be capable of distinguishing mid-range irritants if
the PM was modified. Further work could investigate if there are different ranges ofvalues that could be used to distinguish mild and severe irritants, and this would
probably involve removing the range of (‘borderline’) values within which the PM isunable to distinguish materials as either irritants or non-irritants.
b. Possible rationale for the differences observed
As only one type of material was tested using the TEP assay/INVITTOX Protocol No. 86
in a single laboratory, possible rationale for differences observed was limited to thevariation between the predictive capacity of the protocol and PM for the various
classification systems (EU, GHS, EPA). As there were few test materials in the data set,the concordance values etc., calculated were greatly affected by the prediction of every
single test formulation. For example, sensitivity was calculated as 80% for the EUclassification system and the EPA classification based on the correct prediction of 8/10
irritants, whilst sensitivity was calculated as 88.9% for the GHS classification system
based on the correct prediction of 8/9 irritants. Therefore, although the data indicatethat the predictive capacity of the protocol varies considerably according to the different
classification systems, further testing is required to give significance to the resultspresented.

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Table 6.2.1. Table presenting the relevant information for each study where raw data were not available
INVITTOX Protocol No. 71 (and similar)

Study
Loss of Trans-epithelial
Impermeability of a Confluent
Layer of Madin-Darby Canine
Kidney (MDCK) Cells as a
Determinant of Ocular
Irritancy Potential (Shaw etal., 1990)
The EC/HO International
Validation Study on
Alternatives to the Draize Eye
Irritation Test (Balls et al.,
1995)

No. ofNo. ofNo. of
Coded
Laboratories Chemicals Products (Y/N)

ChemicalClasses

Ranges of
Toxicity
Tested1Nmetals,surfactants,
alcohols, solvents

non-irritants,
R41 irritants;
one R36 irritantcompounds only, solids
and liquids.
MW: 32.042-1228
liquids, crystals,
powders.
MW: 39.99707-1228
Cypermethrin -palebrown, viscous
liquid/semi-solid, MW
416.31; permethrin Seige II- gel, lanasolliquid, MW 391.28

MW: 288.37687-1228,
liquids

22

0

4

60

0

Y

pure chemicals
ranging inmechanisms andpotency

The Evaluation of Pesticide
Ingredients and Formulations
In Vitro and Correlations withIn Vivo Data (Clothier et al.,
1995)

1

2

4

Y

pesticides andvehicles

MMAS Range:
0-108
permethrin,
severe irritant(K-C) andcypermethrin,
minimal irritant(K-C)

Evaluation of Tissue Culture
Insert Membrane
Compatibility in the
Fluorescein Leakage Assay.
(Ward et al., 1997a)

1

6

0

N

surfactants (nonionics and ionics)

MMAS Range:
0-108

153

Physico-chemical
properties

Fluorescein Leakage Assay Background Review Document

FRAME In-house (non-published) -INVITTOX Protocol No. 71

Study

Report on Comparison of 40
Cosmetic and Domestic
Formulations Supplied byCompany # 8 and Evaluatedby 3 In Vitro Cytotoxicity Tests(FRAME, 1992)

No. ofNo. ofNo. of
Coded
Laboratories Chemicals Products (Y/N)

ChemicalClasses

Ranges of
Toxicity
Tested1

variety ofcosmetics andindustrial
cleaners
representing
different levels of
toxicity, mainly
surfactant-based
formulations

24h LVETMMAS Range:
0-44.7

formulations, liquids,
powders, creams.
MW: 149.1894-1228 (2compounds).

No. ofNo. ofNo. of
Coded
Laboratories Chemicals Products (Y/N)

ChemicalClasses

Ranges of
Toxicity
TestedPhysico-chemical
properties

1

mixture ofindustrial
chemicals
representing
those found inindustry

non-irritants andR41 chemicals;
only 2 R36
chemicals

liquids, solids
acetyldehyde volatile.
MW: 39.997 – 376.275

Surfactant-based
formulations-shampoos andconditioners

mild, moderateand substantial
irritants relative
to the toxicitiesof these producttypes

formulation gels

0

40

Y

Physico-chemical
properties

INVITTOX Protocol No. 82

Study

Development of a Fixed DoseApproach for The Fluorescein
Leakage Test. (Clothier et al.,
1994)
Comparative Evaluation ofFive In Vitro Tests forAssessing the Eye Irritation
Potential of Hair-care
Products (Jones et al., 2001)

1

21

0

0

17

Y

Y

154

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 86
Refer to Section 6.1.
INVITTOX Protocol No. 120 (and similar)

Study

No. ofNo. ofLaboratories Chemicals

No. of
Coded
Products (Y/N)

ChemicalClasses

Fluorescein Leakage Test: a
Useful Tool in Ocular Safety
Assessment (Cottin andZanvit, 1997)

1

20

23

Surfactantbased
formulations
only

Evaluation of thePrevalidation Process: The
Fluorescein Leakage Assay(Phase II) (Southee, 1998)

4

4

1

Not statednon-ionic,
anionic, cationicsurfactants andsurfactant-based
products
glycerol, triton X100, CTAB,
ammonium
nitrate,Company # 3
Baby shampoo

3

10

0

Y

mild surfactantsYsurfactants andsurfactant-based
formulations
soluble in HBSS

Evaluation of thePrevalidation Process: The
Fluorescein Leakage Assay(Phase III) (Southee, 1998)
Ocular Irritancy Assessmentof Cosmetics Formulations
and Ingredients: Fluorescein
Leakage Test. (Zanvit et al.,1999) DATA FROM COLIPA

2

11

23

155

Ranges of
Toxicity
TestedPhysico-chemical
properties

MAS Range:
0.3-50.3

limited information, MW
228.4172-1310. lotions,gels for formulations

MMAS Range:
0-33.8

MW: 80.04-364.45,
liquids

MMAS Range:
0-37

MW: 288.37687-414.6,
liquids, powders, flakes

MMAS Range:
0-108

For chemicals only:
liquids, viscous liquids,
crystals, flakes, MW:
250.3802-384.4419

Fluorescein Leakage Assay Background Review Document

OthersStudy
Investigations of the MDCKPermeability Assay as an In
Vitro Test of Ocular Irritancy(Gautheron et al., 1994) IRAGLAB A (Botham et al., 1997)
Evaluation of a Human
Corneal Epithelial Cell Line asan In Vitro Model for
Predicting Ocular Irritation(Kruszewski et al.,1997)
Assessment of Initial Damage
and Recovery Following
Exposure of MDCK Cells toan Irritant (Clothier et al.,1999)

No. ofNo. ofNo. of
Coded
Laboratories Chemicals Products (Y/N)

ChemicalClasses

Ranges of
Toxicity
Tested

1

32

0

N

surfactants,
alcohols,
miscellaneous

MAS Range:
0-98

1

20

25

N

Chemicals, many
surfactants

MMAS Range:
1.33-64.75

liquids, solids,
MW: 41.0524-448.0873
MW 39.99707384.4419, liquids,
crystals, solids, 25
surfactants from CTFA
Phase III study

1

3

0

N

known irritants(surfactants)

MMAS Range:
4- 56

Tween 20, isopropanol,
benzalkonium chloride

Blue font indicates that raw in vivo data were available.

156

Physico-chemical
properties

Fluorescein Leakage Assay Background Review Document

6.2.2. In vivo reference data used to assess the performance of the FL assay
Table 6.2.2. In vivo reference data used to assess the performance of the FL assay
INVITTOX Protocol No. 71 (and similar)

Study

Loss of Trans-epithelial
Impermeability of a Confluent
Layer of Madin-Darby Canine
Kidney (MDCK) Cells as a
Determinant of Ocular Irritancy
Potential (Shaw et al., 1990)
The EC/HO International
Validation Study on
Alternatives to the Draize Eye
Irritation Test (Balls et al.,
1995)

The Evaluation of Pesticide
Ingredients and Formulations
In Vitro and Correlations withIn Vivo Data (Clothier et al.,1995)
Evaluation of Tissue Culture
Insert Membrane Compatibility
in the Fluorescein Leakage
Assay. (Ward et al., 1997a)

Species andProtocols used asref. Data

Draize test
Draize test
-ECETOC (1992)
normally NZ White
rabbits
OECD Guidelines for
Testing of Chemicals(1987) No, 45 "Acute
Eye Irritation/
Corrosion" using NZWhite Rabbit-statedin confidential report
only

Draize test -normally
NZ White rabbits

Sources ofInformation
Botham et al., (1989)
Acute and topical
toxicity profiles for
substances involvedin the in vitro
validation of the invivo ocular irritation
model. Nottingham,
FRAME
Historical data from
Bagley et al., (1992),
ECETOC, (1992)

No. of Labs

No. ofexperim
ents

1

1-6
(accordin
g to
Bothamet al.,(1989)).

unknown

3-6.

Rat oral LD50 (mg/ml)
from either Purchaseet al. (1993) and/or
RTECS (1985).
Summarised Draizescores for individual
tissues

1

OECD
Guideline
405

Draize MMAS datafrom Balls et al.,(1995) and ECETOC(1992)

unknown

3-6.

157

Quality of
Data
(GLP)

Data Format

N

Data summarised
into NC, R36, R41
categories andocular irritation
categories (OIC)

GLP

Summarised
MMAS forindividual
chemicalsOECD
Guideline
405

In vivo Draizescores provided
by manufacturernot stated if MAS
or MMAS

GLP

Mean MAS ±SD

Fluorescein Leakage Assay Background Review Document

FRAME In-house (non-published) -INVITTOX Protocol No. 71

StudySpecies andProtocols used
as ref. Data

Sources ofInformation

No. of Labs

No. ofexperiments

Quality of
Data
(GLP)

Data Format

Report on Comparison of 40
Cosmetic and Domestic
Formulations Supplied byCompany # 8 and Evaluatedby 3 In Vitro Cytotoxicity Tests(FRAME, 1992)

24h LVET

24h LVET MAS from
Company # 8

1

unknown

not statedMean LVET 24h
MASSpecies andProtocols used asref. Data

Sources ofInformation

No. of Labs

No. ofexperiments

Quality ofData (GLP)

Data Format

Draize test

Historical Draize test
data interpreted andreclassified from
Botham et al., (1989)

many

1-6
(according to
Botham etal., (1989)).

N

Materials
categorised as
R41, R36 or NC

not stated

No in vivo scores
given. Test
materials ranked
according tobenchmarkvalues.

INVITTOX Protocol No. 82

Study

Development of a Fixed
Dose Approach for The
Fluorescein Leakage Test.
(Clothier et al., 1994)
Comparative Evaluation ofFive In Vitro Tests forAssessing the Eye Irritation
PotentialofHair-care
Products (Jones et al., 2001)

Draize test
–species not stated

Historical MAS
scores and market
research

158

1

N/A

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 120 (and similar)

Study

Fluorescein Leakage Test: a
Useful Tool in Ocular Safety
Assessment (Cottin andZanvit, 1997)
Evaluation of thePrevalidation Process: The
Fluorescein Leakage Assay(Phase II) (Southee, 1998)

Evaluation of thePrevalidation Process: The
Fluorescein Leakage Assay(Phase III) (Southee, 1998)
Ocular Irritancy Assessmentof Cosmetics Formulations
and Ingredients: Fluorescein
Leakage Test. Zanvit et al.,(1999)

Species andProtocols used asref. Data

Draize test

Sources ofInformation

No. ofLabs

MAS data

unknown

Draize test

MMAS data -assume
historical but source
not statedunknown
Historical MMAS data
supplied by Company# 1 and Gautheron etal., (1994b) which
met requirements set
by BIBRA
unknown

Draize test (as for
COLIPA)

Draize MMAS data
provided by BIBRAInternational from the
COLIPA study(Botham et al., 1997) unknown

Draize test

159

No. ofexperiments

Quality ofData (GLP)
Method of
the Officiel
de la
Republique
Français,(24/10/198
4).

MAS and mean
Draize scores forevery chemical at
1h, 24h, 48h, 72h,
96h, 7 days

unknown

MMAS scores
given forindividual
chemicals

unknown

unknown

Mean MMAS ±SD scores given
for individual
chemicals

3

OECDGuideline
405

Raw animal data
available (fromIIVS); MMAS in
publication

3

unknown

Data Format

Fluorescein Leakage Assay Background Review Document

OthersSpecies andProtocols used asref. Data

Evaluation of a Human
Corneal Epithelial Cell Line asan In Vitro Model for
Predicting Ocular Irritation(Kruszewski et al.,1997)

Draize test -normally
NZ White rabbits

Sources ofInformation
24h total Draizescores from EEC
study (Gautheron
et al., 1994b) and
Kennah et al.,(1989)
Data fromECETOC Eye
Irritation Reference
Chemicals Data
Bank (ECETOC
1992) and from
CTFA Phase III(Gettings et al.,
1996)

Assessment of Initial Damage
andRecovery
Following
Exposure of MDCK Cells to an
Irritant (Clothier et al., 1999)

Draize test
(ECETOC-normally
NZ White rabbits)

Historical MMAS
data fromECETOC (1992)

Study
Investigations of the MDCKPermeability Assay as an In
Vitro Test of Ocular Irritancy(Gautheron et al., 1994a)
IRAG LAB A (Botham et al.,
1997)

Draize test

160

No. ofLabs

1

unknown1

No. ofexperiments

Quality ofData (GLP)

3data fulfils
IRAG
criteria, but
GLP status
not stated

Data Format
17 chemicals with
24h total Draizescores, cornea,
conjunctivalscores, and days
to clear

3-5
dependingon material
and assay

GLP

Mean Draize MAS
w/o SD or SEM

n≥3

unknown

Summarised
MMAS

Fluorescein Leakage Assay Background Review Document

6.2.3. Brief description of the studies without raw data available
a. Statistical Approaches
The statistical approaches used for analysing the predictive capacity of the FL assay
protocols featured in table 6.2.1. were outlined in brief below. Studies featured in table
6.2.1. and not discussed below did not statistically analyse the data. In these cases, thepredicted classifications or rankings of the in vitro and the in vivo data were usuallyreported.
INVITTOX Protocol No. 71 (and similar)
The EC/HO International Validation Study on Alternatives to the Draize Eye Irritation
Test (Balls et al., 1995).
The statistics featured in the EC/HO study were employed to evaluate inter-laboratory
variability and the relationship between the in vitro data and the in vivo data (MMAS).
The in vitro data were plotted against MMAS data from the ECETOC Technical Reporton Eye Irritation (ECETOC, 1992) and additional in vivo data for 14 other test chemicals.
Greater-than and less-than values were also plotted and were included as the maximalor minimal concentration tested in calculations for; linear regression, 95% confidence
intervals and correlation coefficients.
Evaluation of Tissue Culture Insert Membrane Compatibility in the Fluorescein Leakage
Assay (Ward et al., 1997a).
A MMAS value of less than 15 was used to indicate surfactants with mild or no irritancy.
A cut-off value of 50mg/ml was used to discriminate irritants from non-irritants according
to FL20 (mg/ml) values measured immediately following the chemical exposure.
INVITTOX Protocol No. 82 (and similar)
Comparative Evaluation of Five In Vitro Tests for Assessing the Eye Irritation Potentialof Hair-care Products (Jones et al., 2001)
The amount of FL (%) measured immediately after and 72 hours following the chemical
exposure were compared to the respective FL (%) results from a benchmark shampoo
and conditioner. If the FL (%) value was lower than the benchmark, the test material
was considered to have an ‘acceptable‘ level of irritancy; if the FL (%) value was higher
than the benchmark, the test material was considered to have an ‘unacceptable’ level ofirritancy. FL (%) values that were similar to the benchmark, i.e. ±20% were considered
to require ‘further investigation’ before concluding if the test material had acceptable orunacceptable levels of ocular irritancy.
INVITTOX Protocol No. 120 (and similar)
A Summary Report of the COLIPA International Validation Study on Alternatives to the
Draize Rabbit Eye Irritation Test (Brantom et al.,1997)
Data submitted to the COLIPA study underwent an independent quality check by BIBRAInternational (UK). The publication stated that the protocol used was according to Cottin
161

Fluorescein Leakage Assay Background Review Document

et al., (1992) but a very similar version of this protocol was later accepted as INVITTOX
Protocol No. 120.
The FL assay PM was based on data from 43 surfactants and surfactant-based
formulations that were dissolved or suspended in HBSS. The PM used log10 FL assay
results taken 4h after the 15 minute chemical exposure. Cut-off values for the in vitro
data indicated that three classes of observed in vivo ocular irritation could be
distinguished; ‘non-irritant/slight’, ‘moderate’, and ‘irritant/severe’.
Log10 FL assay 4h results were plotted against observed MMAS values. Cut-off values
were applied to the in vitro and in vivo data. The classifications based on the in vitro
results showed good correlation to the in vivo classifications.
The lead laboratories provided a 95% prediction interval for the PMs. The 95%
prediction interval refers to the range in which 95% of the predicted values were likely tofall within. The 95% prediction/confidence intervals (CIs) were produced for Altman and
Bland comparisons which were used as they allow false positives and false negatives tobe considered. The difference between the average observed MMAS values and theaverage predicted MMAS values was plotted as a continuous line. The ±2SD from themean was plotted on the graph which represented the 95% CI. Wide intervals indicateda reduced predictive capacity of the in vitro assay for in vivo ocular irritation. A meandifference greater than zero indicated that the assay had the tendency to under-predictin vivo eye irritation; a mean difference less than zero indicated that the assay had thetendency to over-predict in vivo eye irritation. The mean difference of the FL assay wasplotted as zero. Values that were plotted above the CI represented materials with theirin vivo ocular irritancy potential under-predicted. Values that were plotted below the CI
indicated materials that had their in vivo ocular irritancy potential over-predicted.
A classification PM was also produced and analysed by kappa statistics using equal
weightings, linear weightings and quadratic weightings. Linear weightings give a greater
weight to predicted values that differ by two (rather than one) classification groups inrelation to the in vivo classification. Quadratic weightings give an even greater
weighting than the linear analyses, to results that differ by two classifications in respect
to the in vivo classifications. The 95% CIs were included in all of the analyses. Altman
Bland comparisons were carried out on the classifications.
Fluorescein Leakage Test: A Useful Tool in Ocular Safety Assessment (Cottin andZanvit, 1997).
Pearson (linear) and Spearman (rank) correlations were computed for the FL assay dataand corresponding historical MAS data at 0h, 1h, 24h and day 7 for the 43 test samples.
NB. Greater-than 1000mg/ml data were plotted as ‘1000mg/ml’ data. Weighted kappa
statistics were used to measure the agreement between classifications based on in vivo
and in vitro data.
Evaluation of the Prevalidation Process: The Fluorescein Leakage Assay (Phase III)
(Southee, 1998).
The data were independently analysed by BIBRA International. FL20 (mg/ml) results
measured 4h after the 15 minute chemical exposure were used for comparisons with invivo MMAS data. The results were classified into various categories of ocular irritation.
162

Fluorescein Leakage Assay Background Review Document

Kappa statistics were used to compare inter-laboratory reproducibility of predicted invivo classifications, and to assess the capacity of the FL assay protocol for predicting invivo classes of ocular irritation. Linear and quadratic kappa statistics were used.
Quadratic weightings give an even greater weighting, than the linear analyses, to resultsthat differ by two classifications in respect to the in vivo classifications.
OthersInvestigations of the MDCK Permeability Assay as an In Vitro Test of Ocular Irritancy(Gautheron et al., 1994).
The FL50 (mg/ml) results were compared to the in vivo data (Gautheron et al., (1994b)(Kennah et al., 1989)) using the Pearson correlation test. It was indicated that due tothe low amount of variation in the in vivo data the Pearson’s correlation test could only
be performed for comparative purposes, thus the correlation was given little weighting.
Evaluation of a Human Corneal Epithelial Cell Line as an In Vitro Model for Predicting
Ocular Irritation (Kruszewski et al.,1997).
FR85 (%) values were used which measure the concentration of the test material causing
fluorescein retention to decrease to 85% relative to the negative control. FR85 (%)
values were compared to MMAS values, corneal scores, corneal opacity scores, corneal
area scores, iris scores, conjunctival redness scores, conjunctival discharge scores, and‘days to clear’ scores for the 25 CTFA study Phase III surfactant-based formulations.

163

Fluorescein Leakage Assay Background Review Documentb. Main results
Table 6.2.3. The findings reported in the literature for those studies which used PMs to assess the predictive capacity of the various
FL assay protocols.
INVITTOX Protocol No. 71 (and similar)

Study

No of test
materials

Type of Test
materials

No. ofmaterials
underpredicted$

1min

60

Chemicalswith various
mechanisms

N/A**

N/A**

1min

12

surfactants00Timepoint(s)used for PM

Prediction/
Classification Model

The EC/HO International
Validation Study on
Alternatives to the
Draize Eye Irritation
Test (Balls et al., 1995)

ProtocolAccording to
Tchao (1988)(i.e.,INVITTOX
Protocol No.
71)

FL20 (mg/ml) values: <100mg/ml =R36
or R41; >750mg/ml =NI; 100-750mg/ml=R41 if no recovery after 72h, R36 if
recovery after 72h

Evaluation of Tissue
Culture Insert
Membrane Compatibility
In the Fluorescein
Leakage Assay (Ward etal., 1997a)

According to
Shaw et al.,(1990)–Anopore
insert used

FL20 (mg/ml) values: <50mg/ml=irritants; ≥50mg/ml =non-irritants

164

No. ofmaterials
overpredicted$

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 82 (and similar)

Study
Development of a Fixed
Dose Approach for The
Fluorescein Leakage
Test. (Clothier et al.,
1994)
Comparative Evaluationof Five In Vitro Tests forAssessing the Eye
Irritation Potential of Haircare Products (Jones etal., 2001)

ProtocolPrediction/
Classification Model

INVITTOX
Protocol No.
82 (Fixed
Dose)
Essentially aFixed Dose
method using
INVITTOX
Protocol No.
71*

50mg/ml hypothesised to be the cut-off
point to distinguish R36/R41 chemicals
from NC if ≥ FL20 (%) was taken to
indicate significant toxicity.
Results compared to the FL assay
values for benchmark shampoo andconditioner. Material acceptable if
lower than benchmark; 'further
investigation' defined by ±20% frombenchmark value

INVITTOX Protocol No. 86 (and similar)
See Section 6.1.

165

Timepoint(s)used for PM
1min andconsiderationof 72hrecoveryrates
1min (andassume withconsiderationof 72hvalues)No of test
materials

Type of Test
materials

No. ofmaterials
underpredicted$

No. ofmaterials
overpredicted$

22

Chemicalswith various
mechanisms

1

1

17

Shampooandconditioner
formulations

0 (2 nonclassified)

10 (2 nonclassified)Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 120 (and similar)

Study
A Summary Report of
the COLIPAInternational Validation
Study on Alternatives tothe Draize Rabbit Eye
Irritation Test (Brantom
et al., 1997) –FRAME
data
A Summary Report of
the COLIPAInternational Validation
Study on Alternatives tothe Draize Rabbit Eye
Irritation Test (Brantom
et al., 1997) –Company# 4 data
Fluorescein
LeakageTest: A Useful Tool in
OcularSafetyAssessment (Cottin andZanvit, 1997)
Evaluation of thePrevalidation Process:
The Fluorescein
Leakage Assay (Phase
III) (Southee, 1998)

Protocol

Cottin et al.,(1992) (i.e.,
INVITTOX
Protocol No.
120)

Timepoint(s)used for PM

Prediction/
Classification Model

For FL20 (mg/ml) value at T4:>100mg/ml =non irritant/slight, MMAS
<15; 20-100mg/ml =moderate, MMAS
15-30; <20mg/ml =irritant/severe,
MMAS> 304h

No of test
materials

Type of Test
materials

No. ofmaterials
underpredicted$

30

Surfactants
andsurfactantbased
formulations
soluble in
HBSS

1

6

0

4

2***

1***
6 (basedon meanclassif.
from threelabs)

Cottin et al.,(1992) (i.e.,
INVITTOX
Protocol No.
120)

For FL20 (mg/ml) value at T4:>100mg/ml =non irritant/slight, MMAS <15; 20-100mg/ml =moderate, MMAS
15-30; <20mg/ml =irritant/severe,
MMAS> 304h

33

Not stated(similar to
INVITTOX
Protocol No.
120)

For FL20 (mg/ml) value at T0:≥100mg/ml =slightly irritant,≥20,100mg/ml =moderate irritant<20mg/ml =irritant

0h43

INVITTOX
Protocol No.
120

Based upon COLIPA PM using FL20
(mg/ml) at T4: results: >100mg/ml=non-irritant/slight; 20-100mg/ml=moderate; <20 mg/ml =irritating/severe

4h10Surfactants
andsurfactantbased
formulations
soluble in
HBSS
Surfactants
andsurfactantbased
formulations
soluble in
HBSS

Mild
surfactants

0 (based onmean classif.
from threelabs)

No. ofmaterials
overpredicted$* The protocol used different incubation periods; 10 seconds for the shampoos and 30 seconds for the conditioners. ** PM reported in
the publication was not applicable to the data reported. *** results were taken directly from graphical representation of concordanceof classifications (Cottin and Zanvit et al., 1997). $ the results were based on the classification systems featured in the publications.

166

Fluorescein Leakage Assay Background Review Document

Additional discussions and comparisons of the conclusions for the studies featured intable 6.2.3. are detailed below. There are many different elements to the studies
featured in table 6.2.3. that cause the conclusions regarding the predictive capacity ofthe various FL assays to differ from study to study. Examples include; use of different
FL assay protocols, types of test materials, format and quality of in vivo data (i.e., MAS,
MMAS, qualitative classification groups), PM used, level of statistical analyses. The
principal reasons for the various findings regarding the FL assays predictive capacities
in the different studies are outlined.
INVITTOX Protocol No. 71 (and similar)
The predictive capacity of INVITTOX Protocol No. 71 for ocular irritation has been
evaluated in a number of studies. The largest study was the EC/HO study where 60
pure chemicals were tested in four laboratories (Balls et al., 1995). The FL assay was
initially designed to detect immediate damage caused by test materials to theimpermeability of an epithelial monolayer. The chemicals tested in the EC/HO study
covered a wide range of chemical classes and encompassed many different physicalproperties. The FL20 (mg/ml) data were compared to Draize MMAS scores using
Pearson’s correlation coefficients and Spearman’s rank correlation coefficients. The
Pearson’s correlation coefficients from the four laboratories testing the FL assay ranged
from -0.151 to -0.565. This range of values was comparable to other correlation
coefficients produced by a number of in vitro tests evaluated as part of this study.
INVITTOX Protocol No. 71 also featured in the study of Shaw et al., (1991). Twenty-two
chemicals were tested which covered a range of potencies and mechanisms. The FL
assay results were ranked and the order of the chemicals showed good correlation to invivo classifications which ranged from non-irritants, mild irritants and severe irritants.
The conclusions from study of Shaw et al., (1991) differed to those from the EC/HO
study predominately because Shaw et al., (1991) compared the FL values to aqualitative classification scheme rather than quantitative MMAS scores. Additionally,
fewer chemicals were also tested which created less opportunity for the assay to bechallenged by different chemical mechanisms and potencies.
This protocol was also used within a test battery, comprised of the NRR assay and the
kenacid blue assay, to test two pesticides and their formulations (Clothier et al., 1995).
The formulation vehicles without the pesticide ingredients were found to be more toxic
than the pure pesticide ingredients in all three assays. Permethrin was considered to bea severe eye irritant and cypermethrin a minimal eye irritant according to the Kay andCalandra scoring system.
The protocol of Shaw et al., (1990) was used to test the effects of EC/Home Officevalidation trial surfactants (Balls et al., 1995) on four types of inserts which can be used
for the FL assay; 0.2 and 0.02 pore sized Anopore inserts with aluminium oxide
membranes, 0.4 pore sized Millicell-CM inserts with PTFE biopore membranes and 0.45
pore sized Millicell-HA inserts with a membrane comprised of mixed cellulose esters(Ward et al., 1997a). It was concluded that the Anopore membrane produced resultsthat correlated better with MMAS than those obtained using a Millicell-HA membrane(Ward et al., 1997a).

167

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 82 (and similar)
INVITTOX Protocol No. 82 was developed as a consequence of the findings of the
EC/HO study (Balls et al., 1995). A set concentration was used to test all chemicals andthe amount of induced FL was used to distinguish irritants and non-irritants according to
the EU classification system. The protocol was very similar to INVITTOX Protocol No.
71 with the following exceptions; only one chemical concentration was tested, the lengthof time for FL to occur and the endpoint measured (FL%). The authors reported that achemical concentration of 50mg/ml was effective at distinguishing irritants from nonirritants. This was the only reported study where this particular Fixed Dose FL assay
has been used. The test chemicals were identical to those tested by Shaw et al., (1991)
who also reported that the protocol was able to distinguish different levels of irritancy.
Jones et al., (2001) stated that a method similar to that of Tchao (1989) was used to test
hair care products although an evaluation of the protocol described showed greaterconcordance with INVITTOX Protocol No 82. However, the exposure durations and thetime allowed for FL were shorter than those featured in INVITTOX Protocol No. 82. A
total of 17 shampoos and conditioners were tested neat and the FL assay performed at
0h and 72h. The results were compared to benchmark shampoo and conditioner
formulations for which in vivo data were available. The amount of FL produced by thetest formulations had to be lower than that caused by the respective benchmarks inorder for the test formulations to be considered to have ‘acceptable’ levels of potential
ocular irritation. No formulation had its irritancy potential under-predicted but ten
materials had their irritancy potentials over-predicted. It is difficult to compare theresults of this study with others that also featured INVITTOX Protocol No. 82 as here
formulations were tested rather than pure chemicals. In addition, the endpoint used bythis protocol differed to the FL20 (mg/ml) endpoint generally used with INVITTOX
Protocol No. 82. The type of in vivo data used for this study was not as clearly defined
as in the other publications.
INVITTOX Protocol No. 120 (and similar)
This protocol was accepted as an INVITTOX Protocol following the promising resultsfrom the COLIPA study (Brantom et al., 1997) and the ECVAM Prevalidation study(Southee, 1998).
In the COLIPA study, only surfactants and surfactant-based
formulations that were soluble in HBSS were tested. This contrasted to the studies for
INVITTOX Protocol No. 71 where numerous types of chemicals with various levels ofsolubility were tested. A classification PM was defined prior to the start of the COLIPA
study. Four test materials were over-predicted by Company # 4 and six materials wereover-predicted by the FAL; only one formulation was under-predicted by the FAL.
INVITTOX Protocol No. 120 was concluded to be a promising in vitro test based upon
the COLIPA results (Zanvit et al., 1999). This finding contrasted to the results presented
for INVITTOX Protocol No. 71 tested in the EC/HO study. However, the types ofmaterials tested in the COLIPA study were limited to surfactants soluble in HBSS.
There was also approximately half the number of materials tested in the COLIPA study
in comparison to the EC/HO study. However, the COLIPA study tested both chemicals
and formulations that enabled the predictive capacity of INVITTOX Protocol No. 120 for
surfactants and surfactant-based formulations to be ascertained.
Results for the nine materials common to the COLIPA study and the EC/HO study werecompared to the in vivo MMAS data using Pearson’s correlation and Spearman’s rank
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coefficients. INVITTOX Protocol No. 120 (COLIPA study data) was found to show agreater correlation to the in vivo data in comparison to INVITTOX Protocol No. 71(EC/HO study data) (Zanvit et al., 1999).
Cottin and Zanvit (1997) used the assay to measure the effects of surfactants andsurfactant-based products for 43 samples. The authors reported that different recovery
rates were observed for anionic (greater recovery) and cationic surfactants (lessrecovery) despite similar amounts of initial damage. The FL assay data were concluded
to correlate well with the historical Draize in vivo data according to Spearman’s
correlation test results.
A protocol similar to INVITTOX Protocol No. 120 was also tested as part of the ECVAM
Prevalidation study (Southee, 1998). The predictive capacity of the assay was tested inPhase III. Ten mild surfactants were tested in three independent laboratories. The
mean results (compiled from the three laboratories) were entered into the same PM thatfeatured in the COLIPA study. Six materials had their irritancy over-predicted and none
had their irritancy under-predicted. These results supported those from the COLIPA
study which indicated that the FL assay and PM had a tendency to over-predict rather
than under-predict ocular irritation potential. As the same PM was used in both studies
it was difficult to determine whether the protocol and/or the PM needed refinement to
reduce the incidence of over-predictions.
OthersGautheron et al., (1994a) tested a number of materials including surfactants andalcohols. The authors concluded that the FL assay data correlated well with 24h
historical in vivo data for surfactants rather than other types of test materials. Somesurfactants had their irritancy potential over-estimated. In vivo data were taken from two
different studies (Gautheron et al., 1994b; Kennah et al., 1989) that were judged to be‘not comparable’ according to the authors of the publication.
INVITTOX Protocol No. 71 was also used combined with the AB assay in the study ofClothier et al., (1999; Assessment of initial damage and recovery following exposure of
MDCK cells to an irritant). The FL15 (%) was measured to coincide with data fromanother type of FL assay performed with HCE-T cells. FL15 (%) data for three test
materials (tween 20, isopropanol, benzalkonium chloride (1%)) showed some correlation
to Draize MMAS.
A modified TEP assay, using HCE-T cells, was used to test 20 chemicals selected fromthe ECETOC database (1992) and the 25 formulations tested in CTFA study Phase III(Gettings et al., 1996) (Kruszewski et al., 1997). FR85 (%) data were correlated to
Draize MAS scores using Pearson Correlations which was r=0.71 for the chemicals and
r=0.86 for the CTFA study Phase III formulations. For both data sets, outlier chemicals
were as equally prevalent throughout the entire range of Draize MMAS scores. It wasreported that the most variable FR85 (%) results were for the CTFA formulations that had
low Draize scores leading the authors to conclude that the assay was not able todistinguish mildly irritating surfactant-based formulations (Kruszewski et al., 1997). The
data for the chemicals indicated that the assay was most effective for predicting theocular irritation potential of surfactants, alcohols and least effective for acids, bases andacetates (Kruszewski et al., 1997).

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6.2.4. Compilation of data on predictive capacity of the test method from studies
without raw data
Refer to Annex Vaii
6.2.4.1. Description & rationale for the Prediction Model(s) applied and statistical
approach(es) used
The aim of these analyses was to determine the abilities of the different FL assay
protocols to predict in vivo ocular irritation according to the EU, GHS and EPA
classification systems. However, only the publication reporting results for the Fixed
Dose FL assay assessed the protocol’s predictive capacity for EU classifications. No
studies featuring the FL assay assessed the assays predictive capacity for GHS or EPA
classifications. Subsequently there was limited relevant in vivo data available from theliterature to compare the predictive capacities of the various FL assay protocols.
ECVAM provided a template that allowed raw data from either three or six test animals
to be converted into EU, GHS and EPA classifications. ECVAM used this template toproduce EU, GHS and EPA classifications for the chemicals tested in the EC/HO study(Balls et al., 1995). The classifications were provided directly from ECVAM forconcordance analyses in this BRD. The chemicals tested in the COLIPA study werebased on those from the EC/HO study which enabled the same classifications based on
the in vivo data, provided by ECVAM for the EC/HO study, to be used.
Prediction models (PMs)In order to evaluate the predictive capacity of the various FL assays without bias, PMs
were submitted to ECVAM before all the FL assay data and the in vivo based EU, GHS,
and EPA classifications were compiled. The various PMs used to evaluate the datacollected for this BRD are detailed.
INVITTOX Protocol No. 71 with recovery data
The EC/HO study publication (Balls et al., 1995) proposed/devised a PM that could be
used for FL20 (mg/ml) data produced by INVITTOX Protocol No. 71, that also included
72h recovery data (refer to table 2.2.1.2.). This PM correlated FL20 (mg/ml) values with
the EU classification system. This PM was not used to evaluate the FL assay data
featured in the EC/HO study (Balls et al., 1995) as 72h data were not produced; the PM
was only stated as an ‘application’ for the results. Subsequently, for this BRD the PM
was only applied to the relatively few chemicals that also had 72h data which were takenfrom the publications of Shaw et al., (1991) and Ward et al., (1997a). Similar thresholdvalues as published in the PM for the EU classifications were also applied to therespective GHS and EPA classifications. The PM was modified to include clarifications
made by the authors of this BRD to specify recovery or deterioration; 30% increases ordecreases at the 72h FL measurement relative to the initial measurement were used(table 6.2.4.1.1.). In addition, the ranges of values used to determine R41 and R36
irritants were increased from >100 ≤ 750mg/ml to 0 ≤750mg/ml. The PM was not able todistinguish Category III and Category IV EPA classifications.

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Table 6.2.4.1.1. PM submitted to ECVAM for FL20 (mg/ml) values with recovery data,
based upon the PM featured in the EC/HO study (Balls et al., 1995).
FL20 (mg/ml)>750mg/ml≤750mg/ml and ≥30%
recovery at 72h≤750mg/ml and ≥30%
deterioration at 72h

EUClassification
Not Classified
R36

GHS
Classification
No Category
Cat 2A or Cat
2B

EPA
Classification
Category III/IV
Category II

R41

Cat 1

Category I

In order to evaluate the predictive capacity of this protocol and PM, the in vivo data weretaken from other sources. An explanation of the in vivo data sources and how EU, GHS
and EPA classes were assigned are given below.
ECVAM provided some raw in vivo data (originally from ICCVAM/NICEATM), whichwere to be entered into the ECVAM template v6 to generate the EU, GHS and EPA
classifications. The raw in vivo data for six out of 12 chemicals requiring classifications
could not be found in the TNO file (Prinsen and Koeter, 1993) due to chemical coding.
Other documents containing raw in vivo data also contained coded chemicals. As thepairing of in vitro and in vivo data was based purely on chemical name andconcentration due to a lack of chemical information, there was some doubt as to thesuitability of the pairings of in vitro and in vivo data. It was hypothesised that thevariability generated by possible differences between the ICCVAM/NICEATM andECVAM classifications would be minor in comparison to the variability generated bypairing the in vitro and in vivo data together based only on chemical name. Therefore it
was decided to take EU, GHS and EPA classifications directly from the
ICCVAM/NICEATM ‘Current Status of In Vitro Test Methods for Detecting OcularCorrosives and Severe Irritants Draft Background Review Documents (BRDs) (2004); in
vivo classifications from the Hen's Egg Test -Chorioallantoic Membrane (HET-CAM)
draft BRD (2004) were predominately used. Therefore, the in vivo data consisted ofthose classifications generated by entering data into the ECVAM template v6 and thoseclassifications taken directly from the ICCVAM/NICEATM publications. The authors of
this BRD acknowledge that the quality of the analyses performed with paired in vitro andin vivo data was considerably lower than those with raw in vivo data available.
Therefore the results and conclusions drawn from these analyses are considered to be
speculative and only potentially indicative of the predictive capacities of the different
protocols.
The criteria used by ICCVAM/NICEATM for assigning the EU, GHS and EPA
classifications to the various types of Draize data are outlined in brief. Data from one tosix animals were used. The criteria were:-

the test material had to be tested in at least three rabbits unless a severe effect(e.g. corrosion of cornea, lesion persistence) was observed in a single animal
upon which a classification could then be assigned
a volume of 0.1ml or 0.1g had to be tested. An exception was made for materials
that were tested in lower quantities but which produced a severe effect.
the minimum observations of the eye had to be made at 24h, 48h and 72h aftermaterial instillation if the lesions were not severe.
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For the GHS classification system (UN, 2003), classifications were assigned to eachindividual animal based on the average scores for corneal opacity, iritis, conjunctival
redness and/or conjunctival chemosis for the 24h, 48h and 72h time points. The
combinations of different GHS classifications given to the various animal results wereused to assign the irritancy classification to the test material. In order to use all the data
available, an additional rule was introduced into the classification scheme by ICCVAM/NICEATM (table 6.2.4.1.2.)
Table 6.2.4.1.2. ICCVAM/NICEATM criteria for assigning GHS classifications to Draize
data (from draft HET-CAM BRD (2004)).

Text in italics indicates the additional criteria added by ICCVAM/NICEATM.
The ICCVAM/NICEATM rules for applying the EU classifications (EU, 2001) to theavailable Draize data are described. Calculation of the EU classifications varied
according to the number of test animals. In the case of data from three animals,
average corneal opacity, iritis and conjunctival chemosis were scored for each animal
across the 24h, 48h and 72h time-points. When data were available for more than three
animals, the overall corneal opacity, iritis, conjunctival chemosis and redness werescored for all of the test animals across the 24h, 48h and 72h time-points. Using thesevalues, classifications were assigned based on the minimal positive average when data
were available for only three animals, and on the overall average when data wereavailable for more than three animals (table 6.2.4.1.3.). For test materials that produced
many different classifications for the group of test animals, no classification was
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Table 6.2.4.1.3. ICCVAM/NICEATM criteria for assigning EU classifications to Draize
data (from draft HET-CAM BRD (2004)).

The majority of data used by ICCVAM/NICEATM for assigning these classifications werebased on average score data, average animal data or irritancy classifications.
For EPA classifications to be assigned, normally at least three animals are tested (EPA,
1996). A positive response is classified as an opacity or iritis score of equal to or
greater-than one, or a redness or chemosis score of equal to or greater-than 2 (table
6.2.4.1.4.). The observed score can occur at any time up to 21 days following thematerial exposure. The most severe classification recorded for any of the test animals is
assigned to the test material.
Table 6.2.4.1.4. EPA classification system for ocular irritation (from draft HET-CAM BRD(2004)).

It is to be noted that the explanations for the in vivo data provided above were relevant
only for the in vitro data set which included recovery data. The remaining FL assay
protocols and PMs analysed were assessed according to classifications generated byentering in vivo data into the ECVAM template v6. Original individual animal data wereavailable for the CTFA study Phase III (Gettings et al., 1996), the EC/HO study (Balls etal., 1995) and the COLIPA study (Brantom et al., 1997).

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INVITTOX Protocol No. 71 (and similar)
The majority of data collected for this BRD were generated following a one minuteexposure and FL measured immediately afterwards. As no PM existed for this type of
data, a new PM was devised using the data from the EC/HO study (table 6.2.4.1.5.). In
devising the range of values for the three principal classes of EU, GHS and EPA
classification systems, greater efforts were made to reduce the number of false negative
predictions rather than false positive predictions. The PM was unable to distinguish
EPA Category III and Category IV irritancy classifications.
Table 6.2.4.1.5. PM for FL20 (mg/ml) values
FL20 (mg/ml)

EU
Classification

GHS
Classification

EPA
Classification>750mg/ml

Not Classified

Category III/IV>100 ≤ 750mg/ml

R36

No Category
Cat 2A or Cat
2B

Category II≤100mg/ml

R41

Cat 1

Category I

INVITTOX Protocol No. 120 (and similar)
A substantial amount of data was collected for materials that had been tested for a 15
minute exposure period. The majority of data were from the COLIPA study (Brantom etal., 1997), where the FL was measured 4h following the original chemical exposure. A
PM for this data type featured in the COLIPA study and distinguished three classes of
irritancy; non-irritant/slight, moderate, irritant/severe. The same ranges of FL20 (mg/ml)
values were used to distinguish the three principal classes featured in the EU, GHS andEPA classification systems (table 6.2.4.1.6.). The PM was not able to distinguish EPA
Category III and Category IV classifications.
Table 6.2.4.1.6. PM for FL20 (mg/ml) values produced 4h following a 15 minute chemical
exposure period (modified from Brantom et al., 1997).
FL20 (mg/ml)>100 mg/ml
20 -100 mg/ml<20 mg/ml

EU
Classification
Not Classified
R36
R41

GHS
Classification
No Category
Cat 2A or Cat 2B
Cat 1

EPA
Classification
Category III/IV
Category II
Category I

INVITTOX Protocol No. 82
The PM as featured in the publication of Clothier et al., (1994) was submitted for the
Fixed Dose FL assay. Clothier et al., (1994) proposed that 50mg/ml could be used to
distinguish non-irritants from R36 and/or R41 chemicals if FL20 (%) was taken to indicate
significant toxicity (table 6.2.4.1.7.). In the publication, consideration of recovery or
deterioration of effects at 72h following the chemical exposure, was used to distinguish
R36 and R41 irritants. The amount of recovery or deterioration required to beconsidered significant of an effect was not stated in the publication of Clothier et al.,(1994). Therefore, for analyses of this BRD, a 30% reduction in FL at 72h incomparison to the initial FL assay immediately following the exposure was taken to
indicate recovery. Less than 30% recovery or any further deterioration at 72h incomparison to the initial FL assay was taken to indicate R41 chemicals.

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Although the authors (Clothier et al., 1994) stated that further work was required tomodify this threshold value according to the type of materials being tested, the same
threshold value was used here as no additional data for this protocol were collected.
The EU classifications as stated in the publication were used to determine the predictive
capacity of the protocol and PM.
Table 6.2.4.1.7. Modified PM for INVITTOX Protocol No. 82 data (from Clothier et al.,(1994)).
Chemical tested at 50mg/ml

causes <FL20 (%)
causes ≥FL20 (%) with more than 30%
recovery at 72h
causes ≥FL20 (%) with less than 30%
recovery or further deterioration at 72h

EU Classification
Not Classified
R36
R41

INVITTOX Protocol No. 86
The only in vitro and in vivo data available for analyses of the TEP assay/ INVITTOX
Protocol No. 86 were analysed in section 6.1. of this BRD.
FL Assay with HCE-T cells
A modified FL assay was devised by Kruszewski et al., (1997). This protocol uses
human transfected corneal epithelium cells (HCE-T) in order to try and increase thepredictive capacity of the assay for human ocular irritation by using relevant cell types ofhuman origin. Data for this protocol were available for the CTFA study Phase III
surfactant-based formulations. The percentage of the test formulation which enabled
85% of the sodium-fluorescein dye to be retained was measured (FR85(%)). No PM wasavailable for this protocol so the CTFA study Phase III data were plotted against the EUclassifications generated by entering in vivo data into the ECVAM template v6 (figure
6.2.4.).

100

FR85(%)

10

1

0.1
0

NC 1

2
R36

3
R41

EU Classification

Figure 6.2.4. Correlation of FR85 (%) data with EU classifications.
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Fluorescein Leakage Assay Background Review Document

Both Not Classified formulations and R41 formulations were plotted within a similar
range of values (figure 6.2.4.). Based upon this limited data set, this protocol did notappear able to distinguish surfactant-based formulations with different potencies as
defined by EU classifications.
Comparisons of PMs
The range of values used in the PMs for the FL assay protocols, to distinguish thevarious categories featured in the EU, GHS and EPA classification systems, showedsome correlation when protocol differences were taken into consideration. The PM for
INVITTOX Protocol No. 71, uses the widest range of values to distinguish different
irritancy classifications in comparison to the other PMs. As only a one minute chemical
exposure is used, the concentration of the test material required to induce FL20 and labela test material as an irritant is higher in comparison to the PM for INVITTOX Protocol
No. 120. In comparison to INVITTOX Protocol No. 71, INVITTOX Protocol No. 120
uses, a different cell strain, a 15 minute chemical exposure and measures FL 4h after
the chemical exposure. The FL assay at 4h entails that some recovery or further
damage could occur before the assay is carried out. Due to these protocol differences,
INVITTOX Protocol No. 120 generally produces lower values than INVITTOX Protocol
No. 71 which is shown in the different ranges of values used for the PMs of these
protocols.
INVITTOX Protocol No. 82 uses a fixed concentration of the test material. The amountof FL produced immediately following the exposure and 72h later determines theclassification. The test concentration featured in the publication of Clothier et al., (1994)
was 50mg/ml although it was stated that this could be modified according to the type oftest material. As the Fixed Dose FL assay is very similar to INVITTOX Protocol No. 71,
the fixed concentration is within the range of values used to classify R41 irritants in the
PM for INVITTOX Protocol No. 71.
The PM reported in INVITTOX Protocol No. 86 uses relatively low values to distinguishirritants and non-irritants. This protocol shows many similarities to INVITTOX Protocol
No. 120 but the lower values reported for the PM of INVITTOX Protocol No. 86 wasprimarily due to the use of EC50 values rather than FL20 values.
6.2.4.2. Description of performance compared to reference and eventually, to thehuman situation for each study
The predictive capacities of the various FL assay protocols and their respective PMs for
the EU, GHS and EPA classifications were evaluated. The predicted EU, GHS and EPA
classifications were compared to the in vivo based EU, GHS and EPA classifications
obtained using either the ECVAM template v6 or from the ICCVAM/NICEATM BRDs fororganotypic models. As the predicted classifications used the same boundaries for all
three classification systems (EU, GHS, EPA), the conclusions formed regarding thepredictive capacity of the FL assay protocols were the same for all classification systems
unless the in vivo classifications did not correlate (i.e. a test material with an R36 EU
classification would be expected to have a Category 2 GHS classification and a
Category II EPA classification). None of the PMs were capable of distinguishing subcategories nor Category III or Category IV EPA classifications.

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Contingency tables were created to display the concordance of the predictedclassifications based on FL assay data with EU, GHS and EPA classifications based onin vivo data.
For all the different analyses performed in this section, tables of the in vitro and in vivo
data, including the sources for both types, were provided (Annex Vaii).
INVITTOX Protocol No. 71
Data for this protocol were generated in the EC/HO study (Balls et al., 1995). The
protocol was accepted as INVITTOX Protocol No. 71 following the results of this study.
The mean data reported for each of the four laboratories that participated in the EC/HO
study (Balls et al., 1995) were analysed individually as it was known that somelaboratories did not sufficiently adhere to the test protocol (personal communication, R
Clothier); to some extent this prevented the predictive capacity of the protocol frombeing significantly affected by the results of one or more laboratories that failed toadequately follow the protocol. The different testing practices of the various laboratories
was partially indicated by the different greater-than values that were reported by thedifferent laboratories; e.g. some laboratories tested the chemicals up to their maximum
level of solubility whilst others ceased testing at lower concentrations. Greater-thanvalues that did not allow an irritancy classification to be assigned or indicated two
possible classifications, were not reported in the contingency tables (6.2.4.2.1.a-c.) but
referred to in the text following the appropriate table.
INVITTOX Protocol No. 71 results from the four laboratories were produced for all of the
59 test chemicals except for parafluoraniline for which laboratory 21 failed to report a
result and ethyl-2-methylacetoacetate for which laboratory 18 failed to report a result.
No formulations were tested and therefore, the conclusions regarding the predictive
capacity of this protocol and PM were limited to chemicals only.
The EU, GHS and EPA classifications based on the in vivo data were provided by
ECVAM directly. The concordance of the predicted classifications generated byentering INVITTOX Protocol No. 71 data into the PM featured in table 6.2.4.1.5., with theEU, GHS and EPA classifications based on the in vivo data, was assessed (tables
6.2.4.2.1.a-c.).
Table 6.2.4.2.1.a. Contingency table for EC/HO study data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5. -EU classification system
EU classification
Test
Prediction
NC
R36
R41
Total

NC
41
12
4
57

R36
19
20
3
42

R41
12
13
21
46

145

Based on the in vivo data, the ‘study criteria were not met’ to be able to determine theocular irritation classifications for quiniacrine, potassium cyanate, L-aspartic acid,maneb, sodium oxalate, parafluoranile, triton X-100 (5%), formesafen, chlorhexidine and2,2-dimethylbutanoic acid. Thiourea was reported to have killed all the test animals.
Predictions for thiourea were not included in table 6.2.4.2.1.a. although all four177

Fluorescein Leakage Assay Background Review Document

laboratories produced results which according to the PM rendered the chemical aseither a NC or a R36 irritant.
There were nine incidences of NC and nine R36 classified chemicals according to the invivo data that could not be distinguished as either NC or R36 chemicals based on the
INVITTOX Protocol No. 71 data available. Dibenzyl phosphate was a R36 classified
chemical according to the in vivo data that could not be given any classificationaccording to the in vitro result from laboratory 21. There were ten incidences ofchemicals labelled as R41 according to the in vivo data that could not be assigned a
predicted classification because of the greater-than value being so low. There were 20
incidences of R41 labelled chemicals according to the in vivo data that produced in vitro
results which did not distinguish if they were NC or R36 irritants.
Based on the results presented in table 6.2.4.2.1.a., for which definitive prediction were
made, INVITTOX Protocol No. 71 had a greater predictive capacity for the NC chemicals(41/57 correctly predicted) rather than the R36 chemicals (20/42 correctly predicted) and
the R41 chemicals (21/46 correctly predicted).
There were slightly more GHS classifications based on the in vivo data, in comparison to
the number of available EU classifications. The concordance of predicted and actual
GHS classifications was shown (table 6.2.4.2.1.b.). Based on the in vivo data, the study
criteria were not met’ to be able to determine the GHS ocular irritation classifications for
2,2-dimethylbutanoic acid, fomesafen, L-aspartic acid, maneb, parafluoraniline,
potassium cyanate, and thiourea. Data for thiourea which killed the test animals were
not included in the table 6.2.4.2.1.b. This results for this chemical led to all chemicals
stating that either thiourea was a No Category or a Category 2 irritant; none of thelaboratories could distinguish between these two classifications.
Table 6.2.4.2.1.b. Contingency table for EC/HO study data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5. -GHS classification system

Test
Prediction
NC
Cat 2
Cat 1
Total

GHS classification
Cat
Cat
NC
2
1
36
24
14
4
32
13
3
4
21
43
60
48

151

In addition to the data presented in table 6.2.4.2.1.b. there were the following incidencesof chemicals that could not be given a definitive predicted classification; eight incidencesof No Category chemicals that were predicted to be No Category/Category 2, ten
incidences of Category 2 chemicals that were predicted to be No Category/Category 2
chemicals, 30 incidences of Category 1 chemicals that were predicted to be No
Category/ Category 2 chemicals. Also, there was one incidence of a No Category, one
incidence of a Category 2A, and ten incidences of Category 1 chemicals that could not
be given a predicted classification due to low greater-than values.
The predictive capacity of INVITTOX Protocol No. 71 for the GHS classifications wascomparable to the protocol’s predictive capacity for the EU classifications. There was a178

Fluorescein Leakage Assay Background Review Document

greater proportion of Category 2 irritants, based on the in vivo data, featured in the data
set for GHS classifications (60/151) in comparison to the number of R36 irritants(42/145). A similar proportion of GHS Category 2 irritants were correctly identified by
INVITTOX Protocol No. 71 in comparison to the number of R36 irritants correctly
identified; 32/60 and 20/42 respectively. More than half of the Category 1 irritants were
under-predicted to be Category 2 irritants or No Category chemicals.
The predictive capacity of INVITTOX Protocol No. 71 for the EPA classification system
was also investigated. The PM was not capable of distinguishing materials classified as
Category III and Category IV. Only glycerol and PEG 400 were classified as Category
IV irritants according to the in vivo data. There were no EPA classifications for thefollowing materials: cetylpyridinium bromide 6%, 2,2-dimethylbutanoic acid, pyridine,benzoyl-L-tartaric acid, L-aspartic acid, parafluoraniline, potassium cyanate,promethazine HCl, quiniacrine, chlorhexidine, imidazole and sodium oxalate as the‘study criteria were not met’.
Table 6.2.4.2.1.c. Contingency table for EC/HO study data generated using INVITTOX
Protocol No. 71 and the PM featured in table 6.2.4.1.5. -EPA classification system
EPA classification
Test
Prediction
Cat III/ IV
Cat II
Cat I
Total

Cat IV70
0
7

Cat III
29
23
3
65

Cat
II
16
15
6
37

Cat I
10
5
13
28

137

Data for thiourea which killed the test animals were not included in the table 6.2.4.2.1.c.
All laboratories produced data which could not distinguish the chemical as being either
Category II, Category III or Category IV.
In addition to the data presented in table 6.2.4.2.1.c. there were the following incidencesof chemicals that could not be given definitive predicted classification; fourteen
incidences of a Category III chemicals that were predicted to be Category II/III/IV, ten
incidences of Category II chemicals that were predicted to be Category II/III/IV
chemicals, 17 incidences of Category I chemicals that were predicted to be Category
II/III/IV chemicals. Also, there was one incidence of a Category IV, one incidence of a
Category II and seven incidences of Category I chemicals that could not be given a
predicted classification due to low greater-than values.
Based on the in vivo data, there were fewer in vivo EPA classifications in comparison to
the number of EU and GHS classifications. The predictive capacity of INVITTOX
Protocol No. 71 and PM for the EPA classifications for the non-irritants (Category IV)
and mild irritants (Category III) was lower than predictive capacity for the EU and GHS
non-irritants and mild irritants. A similar predictive capacity was calculated for thesevere irritants for all three classification systems.
The data featured in tables 6.2.4.2.1.a-c. were summarised in tables 6.2.4.2.2.a-c and6.2.4.2.3.a-c for analyses of ‘non-irritants versus the rest’ for all three classification
systems.

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Table 6.2.4.2.2.a Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5.

49

67.6

64.8

71.9

78.1

56.9

28.1

35.2

98/145

57/88

41/57

57/73

41/72

16/57

31/88

Table 6.2.4.2.2.b. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
GHS classification system –Non-irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5.

49

70.2

106/15
1

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

64.8

83.7

90.9

48.6

16.3

35.2

70/108

36/43

70/77

36/74

7/43

38/108

Table 6.2.4.2.2.c. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EPA classification system –Non-irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5.

49

62.0

60.0

63.9

60.0

63.9

36.1

40.0

85/137

39/65

46/72

39/65

46/72

26/72

26/65

NB. As the PM was not able to distinguish EPA Category III and Category IV materials, materials with these classifications wereconsidered in the analyses concerning non-irritants. Refer to Annex Vaii for the origins of the in vitro data and in vivo classifications.

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As more than 10% of the test materials were classed as mild irritants for the EU, GHS
and EPA data sets, ‘severe irritants versus the rest’ analyses was performed for theseclassification systems only (tables 6.2.4.2.3.a-c.).
For interpretation of the ‘severe irritants versus the rest’ analyses, the following should
be noted:-Concordance referred to the number of correctly predicted severe irritants by the FL
assay relative to the number of non-irritants and mild irritants; non-irritants and mild
irritants that were identified but predicted to have the wrong classification were still
counted as correctly identified.
-Sensitivity referred to the number of correctly predicted severe irritants by the FL assay
as a proportion of the total number of actual severe irritants.
-Specificity referred to the number of correctly predicted non-irritants and mild irritants by
the FL assay as a proportion of the total number of actual non-irritants and mild irritants.
-Positive Predictivity referred to the number of correctly predicted severe irritants as aproportion of the total number of predicted severe irritants.
-Negative Predictivity referred to the number of correctly predicted non-irritants and mild
irritants as a proportion of the total number of predicted non-irritants and mild irritants-False Positive Rate referred to the number of non-irritants and mild irritants predicted to
be severe irritants as a proportion of the total number of non-irritants and mild irritants-False Negative Rate referred to the number of severe irritants predicted to be nonirritants or and mild irritants as a proportion of the total number of severe irritants
These descriptions are relevant for all ‘severe irritant versus the rest’ analyses
throughout Section 6 of this BRD.

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Table 6.2.4.2.3.a Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EU classification system –Severe irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5.

49

77.9

113/14
5

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

45.7

92.9

75

78.6

7.1

54.3

21/46

92/99

21/28

92/117

7/99

25/46

Table 6.2.4.2.3.b. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
GHS classification system –Severe irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5.

49

77.5

117/15
1

Sensitivity%
No.

Specificity
%
No.

43.8

93.2

21/48

96/10
3

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

75.0

78.0

6.8

56.3

21/28

96/123

7/103

27/48

Table 6.2.4.2.3.c. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EPA classification system –Severe irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5.

49

82.5

113/13
7

Sensitivity%
No.

Specificity
%
No.

46.4

91.7

13/28

100/1
09

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

59.1

87.0

13/22

100/11
5

False
Positive Rate%
No.

False Negative
Rate
%
No.

8.3

53.6

9/109

15/28

NB. As the PM was not able to distinguish EPA Category III and Category IV materials, materials with these classifications wereconsidered in the analyses concerning non-irritants. Refer to Annex Vaii for the origins of the in vitro data and in vivo classification.

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INVITTOX Protocol No. 71 with recovery data
The predictive capacity of INVITTOX Protocol No. 71 which also produced recovery data
was analysed. Recovery data were only available for chemicals, therefore the predictive
capacity of the protocol and PM for formulations could not be ascertained. The in vitro
data were taken from the study of Shaw et al., (1991) and Ward et al., (1997a); Annex
Vaii provides the source of the in vitro data for each chemical. Chemicals that were only
tested once by Shaw et al., (1991) were not included in the analyses. It was alsodecided not to include Shaw et al., (1991) data for SDS as the test concentration was
not stated. FL assay data for SDS were available from the publication of Ward et al.,(1997a) where two different test concentrations were used.
There were no matching in vivo data for these studies so the in vitro and the in vivo data
were paired from different sources (i.e. classifications produced using the ECVAM
template v6 or taken directly from the ICCVAM/NICEATM BRDs) as described in section
6.2.4.1. Annex Vaii provides the source of the in vivo data for each chemical. It is
acknowledged that the in vivo and in vitro data which may have been generated by
testing slightly different forms of the test chemical could largely account for predictedmisclassifications. The results of this analysis are only to be used as an indication of the
protocol’s and PM’s predictive capacity
The PM used to analyse the data was taken from the EC/HO study (Balls et al., 1995)(table 6.2.4.1.1.). It was proposed that R36 and R41 irritants should be distinguishedaccording to whether there was recovery or deterioration at the 72h time-point incomparison to the amount of FL measured immediately after the chemical exposure(Balls et al., 1995). The degree of recovery or deterioration required in order to beclassed as significant was not stated in the publication and for the analyses of this BRD
was taken to be a 30% difference in relation to the first FL measurement.
Table 6.2.4.2.4.a. Contingency table for data generated using INVITTOX Protocol No.
71 with recovery time-point and the PM featured in table 6.2.4.1.1. -EU classification
systemEU classification
Test
Prediction
NC
R36
R41
Total

NC
1
1
1
3

R36
0
0
0
0

R41
0
3
7
10

13

In addition to the data presented in table 6.2.4.2.4.a., there were four chemicals thatwere Not Classifieds according to the in vivo data that were predicted to be either R36
or R41 irritants based on the FL assay results, these were; 1,2-propanediol, DMSO,
methanol and brij 35. Allyl alcohol was a R36 irritant and 1-butanol was a R41 irritantthat were predicted to be either a R36 or a R41 irritant. As the amount of recovery or
deterioration at the 72h time-point was not 30% different to the amount of FL at theoriginal measurement, the PM could not distinguish if the chemicals were R41 or R36
irritants. These results were not included in the calculations reported in table
6.2.4.2.5.a. For the definitive classifications featured in table 6.2.4.2.4.a., the protocol
and PM predicted all of the R41 irritants as irritants but only one of three Not Classified
chemicals were correctly predicted.
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There were fewer chemicals with GHS in vivo classifications in comparison to thenumber of chemicals with EU classifications; there were no GHS classifications for allyl
alcohol, benzethonium chloride, brij 35, DMSO, mercuric chloride and methanol. On
the contrary, triton X-100 (5%) which did not have an EU irritancy classification was
included in this data set.
Table 6.2.4.2.4.b. Contingency table for data generated using INVITTOX Protocol No.
71 with recovery time-point and the PM featured in table 6.2.4.1.1. -GHS classification
system.
Test
Prediction
NC
Cat 2
Cat 1
Total

GHS classification
Cat
Cat
NC
2
1
1
0
0
1
0
2
1
2
5
3
2
7

12

In addition to the data presented in table 6.2.4.2.4.b., there was also one No Category
chemicals according to the in vivo data, which was predicted to be either Category 1 orCategory 2 irritant; 1,2-propanediol. 1-Butanol is a Category 1 chemical that waspredicted to be a either a Category 1 or Category 2 irritant. The exact classifications
could not be determined as the recovery rate did not differ by 30% respective to theamount of FL first measured immediately after the chemical exposure. These result
were not included in table 6.2.4.2.4.b. and the calculations reported in tables 6.2.4.2.5.band 6.2.4.2.6. The predictive capacity of the FL assay and PM for the GHS
classification system was comparable to the predictive capacity for the EU classification
system.
There were fewer EPA classifications in comparison to the number of EU and GHS
classifications. Based on the in vivo data, the following chemicals did not have EPA
classifications; 1-butanol, allyl alcohol, benzethonium chloride, brij 35, cetylpyridinium
bromide 6%, DMSO, mercuric chloride and methanol.
Table 6.2.4.2.4.c. Contingency table for data generated using INVITTOX Protocol No.
71 with recovery time-point and the PM featured in table 6.2.4.1.1. -EPA classification
system.
EPA classification
Test
Prediction
Cat III/ IV
Cat II
Cat I
Total

Cat IV0000

Cat III
1
1
2
4

Cat
II
0
0
1
1

Cat I
0
2
4
6

11

In addition to the data presented in table 6.2.4.2.4.c., there was also one Category IV
chemicals (1,2-propanediol), according to the in vivo data, that was predicted to beeither a Category I or Category II irritant. This result was not included in the calculations
reported in table 6.2.4.2.5.c for analyses of the predictive capacity for ‘non-irritants
versus the rest.’

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Fluorescein Leakage Assay Background Review Document

The predictive capacity of INVITTOX Protocol No. 71 with recovery data, for the EPA
classifications was very similar to the predictive capacity for the EU and GHS
classification systems.

185

Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.5.a Evaluation of the performance of INVITTOX Protocol No. 71 with recovery data for predicting ocular irritationaccording to the EU classification system –Non-irritants versus the rest
Data
Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Shaw
et
al., (1991),
Ward et al.,(1997a)

Refer
to
table
6.2.4.1.1.

20

84.6

100

33.3

83.3

100

66.7

0

11/13

10/10

1/3

10/12

1/1

2/3

0/10

Table 6.2.4.2.5.b. Evaluation of the performance of INVITTOX Protocol No. 71 with recovery data for predicting ocular
irritation according to the GHS classification system –Non-irritants versus the rest
Data
Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Shaw
et
al., (1991),
Ward et al.,(1997a)

Refer
to
table
6.2.4.1.1.

20

83.3

100

33.3

81.8

100

66.7

0

10/12

9/9

1/3

9/11

1/4

2/3

0/9

Table 6.2.4.2.5.c. Evaluation of the performance of INVITTOX Protocol No. 71 with recovery data for predicting ocular
irritation according to the EPA classification system –Non-irritants versus the rest
Data
Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Shaw
et
al., (1991),
Ward et al.,(1997a)

Refer
to
table
6.2.4.1.1.

20

72.7

100

25

70

100

75

0

8/11

7/7

1/4

7/10

1/1

3/4

0/7

* refers to the total number of chemicals included in all the analyses (refer to Annex Vaii) NB. As the PM was not able to distinguish
EPA Category III and Category IV materials, materials with these classifications were considered in the analyses concerning nonirritants.
As more than 10% of the test materials were classified as mild irritants according to the GHS classifications based on the in vivo data,an analysis of ‘severe irritants versus the rest’ was performed for this classification system (table 6.2.4.2.6.).
186

Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.6. Evaluation of the performance of INVITTOX Protocol No. 71 with recovery data for predicting ocular irritationaccording to the GHS classification system –Severe irritants versus the rest
Data
Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Shaw
et
al., (1991),
Ward et al.,(1997a)

Refer
to
table
6.2.4.1.4

20

58.3

71.4

40

62.5

40

60

28.6

7/12

5/7

2/5

5/8

* refers to the total number of chemicals included in all the analyses (refer to Annex Vaii).

187

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3/5

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Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 120
The Company # 4 assay protocol was tested in the COLIPA study (Brantom et al.,1997). This protocol was slightly modified and later accepted as INVITTOX Protocol No.
120. In the COLIPA study the protocol was used to test only surfactants and surfactantbased formulations that were soluble in HBSS. The PM used results from the FL assay
performed 4h following the material exposure. The assay was tested in two laboratories(FAL and Company # 4) and the mean results from each of the laboratories were used
to determine the predictive capacity of this protocol. As the laboratories sometimes
differed in their judgement as to whether a certain material was soluble in HBSS, not allmaterials were tested in each laboratory; twenty-nine identical materials were tested inboth laboratories.
Raw in vivo data were entered into the ECVAM template v6 to generate the EU, GHS
and EPA classifications
Table 6.2.4.2.7.a. Contingency table for data generated using INVITTOX Protocol No.
120 and the PM featured in table 6.2.4.1.6. –EU classification system
EU classification
Test
Prediction
NC
R36
R41
Total

NC
19
5
6
30

R36
0
1
3
4

R41
0
0
27
27

61

Based on the in vivo data, there was no EU classification, for triton X-100 tested at 5%
INVITTOX Protocol No. 120 had a tendency to over-predict the irritancy potential of thetest materials. Nearly 40% of the Not Classified materials were misclassified as either
R36 or R41 irritants. More chemicals were over-predicted than formulations, as three of
11 different chemicals (including different chemical concentrations) had their irritancy
potential over-predicted in comparison to four of 22 formulations that had their irritancy
potential over-predicted.
INVITTOX Protocol No. 120 and the PM only classified one R36 material correctly whilst
three were misclassified as R41 irritants. All R36 test materials were chemicals with the
exception of the hand cleanser which was classified correctly as an R36 irritant by the
FAL but misclassified by Company # 4 as a R41 irritant. This was the only test material
which produced different classifications in the two laboratories.
The results in table 6.2.4.2.7.b show the predictive capacity of the protocol for GHS
classifications.

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Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.7.b. Contingency table for data generated using INVITTOX Protocol No.
120 and the PM featured in table 6.2.4.1.6. –GHS classification system

Test
Prediction
NC
Cat 2
Cat 1
Total

GHS classification
Cat
Cat
NC
2
1
17
2
0
5
1
0
3
8
27
25
11
27

63

The predictive capacity of the protocol for the GHS classifications was similar to that for
the EU classifications. The number of No Category materials misclassified as irritants,
and the number of Category 2 irritants misclassified as Category 1 irritants indicated thatthe assay and PM had a tendency to over-predict irritancy. There were no Category 1
test materials under-predicted which supported the finding that the assay over-predicted
rather than under-predicted potential irritancy. The Category 2 irritants were all
misclassified with the exception of the hand cleanser formulation tested at the FAL.
Table 6.2.4.2.7.c. Contingency table for data generated using INVITTOX Protocol No.
120 and the PM featured in table 6.2.4.1.6. –EPA classification system
EPA classification
Test
Prediction
Cat III/ IV
Cat II
Cat I
Total

Cat IV15
1
0
16

Cat III
4
4
5
13

Cat
II
0
1
8
9

Cat I
0
0
23
23

61

The concordance of the predicted and actual in vivo classifications were similar for all
three classification systems.
The data featured in tables 6.2.4.2.7.a-c. were summarised to determine the predictive
capacity of the protocol and PM for ‘non-irritants versus the rest’ (table 6.2.4.2.8.a-c.)and ‘severe irritants versus the rest’ (table 6.2.4.2.9.a-b.) for the EU, GHS and EPA
classification systems

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Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.8.a Evaluation of the performance of INVITTOX Protocol No. 120 for predicting ocular irritation according to the
EU classification system –Non-irritant versus the rest
Data Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Brantom et al.,(1997)

Refer
to
table
6.2.4.1
.6.

33

82.0

100

63.3

73.8

100

36.7

0

50/61

31/31

19/30

31/42

19/19

11/30

0/31

Table 6.2.4.2.8.b. Evaluation of the performance of INVITTOX Protocol No. 120 for predicting ocular irritation according to the
GHS classification system –Non-irritant versus the rest
Data Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Brantom et al.,(1997)

Refer
to
table
6.2.4.1
.6.

33

84.1

94.7

68

81.8

89.5

32

5.3

53/63

36/38

17/25

36/44

17/19

8/25

2/38

Table 6.2.4.2.8.c. Evaluation of the performance of INVITTOX Protocol No. 120 for predicting ocular irritation according to the
EPA classification system –Non-irritant versus the rest
Data Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Brantom et al.,(1997)

Refer
to
table
6.2.4.1
.6.

33

83.6

100

65.5

76.2

100

34.5

0

51/61

32/32

19/29

32/42

19/19

10/29

0/32

* 33 different materials were tested (33 by Company # 4 and 30 by FAL) NB. As the PM was not able to distinguish EPA Category III
and Category IV materials, materials with these classifications were considered in the analyses concerning non-irritants.
As more than 10% of the test materials were classified as mild GHS and EPA irritants according to the in vivo data, analyses of‘severe irritants versus the rest’ were performed for these classification systems (tables 6.2.4.2.9.a-b.).
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Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.9.a. Evaluation of the performance of INVITTOX Protocol No. 120 for predicting ocular irritation according to the
GHS classification system –Severe irritants versus the rest
Data Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

Brantom et al.,(1997)

Refer
to
table
6.2.4.1
.6.

33

82.5

100

69.4

71.1

69.4

52/63

27/27

25/36

27/38

25/3
6

False Positive
Rate
%
No.

False Negative
Rate
%
No.

30.6

0

11/36

0/27

Table 6.2.4.2.9.b. Evaluation of the performance of INVITTOX Protocol No. 120 for predicting ocular irritation according to the
EPA classification system –Severe irritants versus the rest
Data Source

Anal.

No.*

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

Brantom et al.,(1997)

Refer
to
table
6.2.4.1
.6.

33

78.7

100

65.8

63.9

65.8

48/61

23/23

25/38

23/36

25/3
8

False Positive
Rate
%
No.

False Negative
Rate
%
No.

34.2

0

13/38

0/23

* 33 different materials were tested (33 by Company # 4 and 30 by FAL) NB. As the PM was not able to distinguish EPA Category III
and Category IV materials, materials with these classifications were considered in the analyses concerning non-irritants.

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Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 82: Fixed Dose FL assay
For this data set, the percentage of FL (FL%) induced by 50mg/ml of the test chemical
was recorded. The PM (table 6.2.4.1.7.) as featured in the publication of Clothier et al.,(1994) was applied to distinguish irritants and non-irritants. It was stated that recovery
and deterioration should be taken into account when distinguishing R36 and R41
irritants. As the amount of recovery or deterioration that should take place in order to beconsidered as an effect was not stated in the publication, it was decided for the purposeof this BRD that a 30% difference in relation to the original FL assay data would be used
to indicate significant differences. In the Clothier et al., (1994) publication, theclassifications for the test chemicals were based on a literature search of historical
Draize data carried-out in the early 1990’s,. These classifications were used for the
analyses below as it was assumed that the correct in vivo data were matched to thechemical tested in vitro, i.e. the same chemical purity, concentration, CAS etc (Annex
Vaii)). Further analyses to determine the predictive capacity of the assay for the GHS
and EPA classifications were not performed as these classifications were not readily
available and the suitability of the paired in vitro and in vivo data would be questionable.
Table 6.2.4.2.10. Contingency table for data generated using INVITTOX Protocol No. 82
and the PM featured in table 6.2.4.1.7. –EU classification system
EU classification
Test
Prediction
NC
R36
R41
Total

NC
9
0
1
10

R36
0
0
2
2

R41
1
0
9
10

22

The protocol and PM performed well and allowed the majority of Not Classified
chemicals and irritant chemicals to be classified correctly. There were only two
chemicals classified as R36 irritants which entailed that the predictive capacity of thisprotocol and PM for mild irritants was not sufficiently tested; 2-methoxyethanol andchloroform were misclassified as R41 irritants.
The data in table 6.2.4.2.10. were analysed for the predictive capacity of ‘non-irritants
versus the rest,’ and the results presented in the summary table 6.2.4.2.11.

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Table 6.2.4.2.11. Evaluation of the performance of INVITTOX Protocol No. 82 for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest
Data Source

Anal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Clothier
etal., (1994)

Refer totable
6.2.4.1.7

22

90.9

91.7

90

91.7

90

10

8.3

20/22

11/12

9/10

11/12

9/10

1/10

1/12

As fewer than 10% of the test materials were classified as mild irritants according to the in vivo data, the analysis of ‘severe irritants
versus the rest’ was not carried-out for this data set.

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Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 71–Surfactants only
Data from the EC/HO study generated using INVITTOX Protocol No. 71 were analysed
to determine the predictive capacity of the assay and PM for testing surfactants only.
There were no INVITTOX Protocol No. 71 data available for surfactant-based
formulations. The chemicals that were tested as part of the EC/HO study were tested infour independent laboratories and therefore the results per chemical presented in tables
6.2.4.2.12.a-c. (excluding greater-than values and where results for chemicals were notreported) were in quadruplicate.
Table 6.2.4.2.12.a. Contingency table for data generated using INVITTOX Protocol No.
71 and the PM featured in table 6.2.4.1.5. –EU classification system
EU classification
Test
Prediction
NC
R36
R41
Total

NC
10
2
3
15

R36
0
0
0
0

R41
5
6
11
22

37

In addition to the results presented in table 6.2.4.2.12.a. there were three incidences of
R41 chemicals which received mixed Not Classified/R36 classifications and sevenincidences where no classification could be assigned due to the low greater-than value
reported.
The predictive capacity for ‘surfactants only’ did not differ greatly to the predictive
capacity for when all the EC/HO test chemicals were analysed. For each surfactant, theclassifications produced by each laboratory tended to agree and when there wasdiscordance they differed by only one classification. All the incidences of Not Classified
surfactants misclassified as R41 surfactants were for SDS tested at 3%; one laboratory
also misclassified SDS tested at 3% as an R36 irritant. There were no surfactants
classified as R36 irritants which prevented the predictive capacity of this protocol andPM for mildly irritating surfactants from being evaluated. All five incidences of R41
chemicals that were misclassified as Not Classified chemicals were reported byLaboratory 20. However, a total of three incidences of chemicals that were reported to
be Not Classified/R36 irritants and seven incidences of chemicals that could not begiven a predicted classification came from Laboratories 19 and 21. The FAL
participated in the EC/HO study and stated that the format of the data submitted
indicated that not all participating laboratories followed the protocol exactly.
Table 6.2.4.2.12.b. Contingency table for data generated using INVITTOX Protocol No.
71 and the PM featured in table 6.2.4.1.5. –GHS classification system
GHS classification
Test
Prediction
NC
Cat 2
Cat 1
Total

NC
10
2
3
15

Cat
2
0
4
0
4

194

Cat
1
5
2
11
18

37

Fluorescein Leakage Assay Background Review Document

In addition to the results presented in table 6.2.4.2.12.b. there was one No Category
chemical which was could not be given a classification based on the result reported by
one laboratory, three incidences of R41 chemicals that were classified as No
Category/Category 2, and seven incidences of R41 chemicals where no classification
could be assigned due to the low greater-than value reported.
The predictive capacity for the GHS classification system was very similar to the EUclassification system. The four incidences of Category 2 surfactants that were correctlyclassified by the protocol were all for triton X-100 tested at 5%. For the EUclassifications, the ‘study criteria were not met’ for triton X-100 tested at 5%.
Table 6.2.4.2.12.c. Contingency table for data generated using INVITTOX Protocol No.
71 and the PM featured in table 6.2.4.1.5. –EPA classification system
EPA classification
Test
Prediction
Cat III/ IV
Cat II
Cat I
Total

Cat IV30
03

Cat III
7
6
3
16

Cat
II
0
2
2
4

Cat I
4
0
8
12

35

For the EPA classification system, the ‘study criteria were not met’ for cetylpyridinium
bromide tested at 6%. Additionally, there was one Category IV chemical for which aclassification could not be predicted by Laboratory 21, six incidences of Category I
chemicals that could not be given a predicted classification and two incidences ofCategory I chemicals that were predicted to be either Category II, Category III or
Category IV chemicals (i.e. the classifications could not be distinguished)
The predictive capacity of the protocol and PM for the EPA classification system was
similar to that for the GHS classification system. The protocol and PM showed a similarrate of over-predicting the non-irritants and under-predicting the severe irritants for all
three classification systems. The only material classified as a Category II irritant,according to the in vivo data was triton X-100 tested at 10%. Triton X-100 (10%) wasclassified as a R41 and a Category 1 irritant in the EU and GHS classification systems
respectively.
The data in tables 6.2.4.2.12.a-c. were analysed and the results presented in thesummary tables 6.2.4.2.13.a-c. for analyses of ‘non-irritants versus the rest’. As fewer
than 10% of the test materials were classified as mild irritants according to the in vivo
data, the analysis of ‘severe irritants versus the rest’ was not carried-out for this data
set.

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Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.13.a Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest
Data Source

Anal.
Refer

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

37

73.0

77.3

66.7

77.3

66.7

33.3

22.7

to

Balls et al., table
1995
6.2.4.1.5

27/37

17/22

10/15

17/22

10/15

5/15

5/22

Table 6.2.4.2.13.b. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
GHS classification system –Non-irritants versus the rest
Data Source

Anal.
Refer

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

37

73.0

77.3

66.7

77.3

66.7

False Positive
Rate
%
No.

False Negative
Rate
%
No.

to

Balls et al., table
1995
6.2.4.1.5

27/37

17/22

10/15

17/22

10/15

33.3

5/15

22.7

5/22

Table 6.2.4.2.13.c. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EPA classification system –Non-irritants versus the rest
Data Source

Anal.
Refer

No.

Concordance%No.

Sensitivity%
No.

Specificity
%
No.

to

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive
Rate
%
No.

False Negative
Rate
%
No.

Balls et al., table
1995
6.2.4.1.5
37
62.9
22/35
75.0
12/16 52.6
10/19
57.1
12/21 71.4
10/14
47.4 9/19
25.0
4/16
NB. As the PM was not able to distinguish EPA Category III and Category IV materials, materials with these classifications wereconsidered in the analyses concerning non-irritants. Refer to Annex Vaii for the origins of the in vitro data and in vivo classifications.

196

Fluorescein Leakage Assay Background Review Document

INVITTOX Protocol No. 71–Alcohols only
All of the data included in this analysis were generated from the EC/HO study andtherefore results from four laboratories for each chemical were presented in table
6.2.4.2.14.a-c (excluding greater-than values and where results for chemicals were notreported). Data for the exact same chemicals were analysed for the predictive capacityof the protocol and PM for all three classification systems. Of all the data sets analysedin this section, this data set for alcohols contained the highest proportion of mild irritants.
Materials with this level of potency were known to be difficult to classify correctly.
Therefore one would aspect the concordance of predicted and actual classifications to
be lower for this data set in comparison to those containing few chemicals classified as
mild irritants.
Table 6.2.4.2.14.a. Contingency table for data generated using INVITTOX Protocol No.
71 and the PM featured in table 6.2.4.1.5. –EU classification system
EU classification
Test
Prediction
NC
R36
R41
Total

NC
7
7
1
15

R36
6
3
2
11

R41
5
2
0
7

33

In addition to the data presented in table 6.2.4.2.14.a. there was one incidence of a Not
Classified chemical which was classified as a No Category/R36, one incidence of a R36
chemical which was classified as a No Category/R36 and one incidence of a R41
chemical which was classified as a No Category/R36.
The results showed that this FL assay protocol had a poor predictive capacity foralcohols classified according to the EU classification system. More than 50% of the Not
Classified, R36 and R41 chemicals were classified incorrectly. In comparison to theanalyses for surfactants, there was a higher incidence of discordance between thepredicted classifications from each laboratory.
Table 6.2.4.2.14.b. Contingency table for data generated using INVITTOX Protocol No.
71 and the PM featured in table 6.2.4.1.5. –GHS classification system
GHS classification
Test
Prediction
NC
Cat 2
Cat 1
Total

NC
4
0
0
4

Cat
2
9
10
3
22

Cat
1
5
2
0
7

33

In addition to the data presented in table 6.2.4.2.14.b. there were two incidences ofCategory 2 chemicals which were classified as a No Category/Category 2 chemicals
and one incidence of a Category 1 chemical which was classified as a No
Category/Category 2 chemicials.
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Fluorescein Leakage Assay Background Review Document

The predictive capacity of INVITTOX Protocol No. 71 for the GHS classification system
was slightly better than for the EU classification system. Eight of the 16 Not Classified
alcohols in the EU system were classified as Category 2 irritants based the in vivo dataand subsequently were not misclassified by the assay and PM for GHS classifications.
The predictive capacity for the Category 1 irritants was comparable to the predictive
capacity for EU R41 irritants.
Table 6.2.4.2.14.c. Contingency table for data generated using INVITTOX Protocol No.
71 and the PM featured in table 6.2.4.1.5. –EPA classification system
EPA classification
Test
Prediction
Cat III/ IV
Cat II
Cat I
Total

Cat IV40
0
4Cat III
2
6
0
8

Cat
II
6
4
3
13

Cat I
5
2
0
7

32

In addition to the data presented in table 6.2.4.2.14.c. there were two incidences ofCategory II chemicals which were classified as a Category IV/Category III/Category II
chemicals and one incidence of a Category I chemical which was classified as a
Category IV/Category III/Category II.
The predictive capacity of the protocol and PM for the EPA classifications showed
greater similarity to the EU rather than the GHS classifications, i.e. a high number of
irritants (EPA Cat I) were misclassified as Category II or Category III/IV chemicals.
The data in tables 6.2.4.2.14.a-c. were analysed and the results presented in thesummary tables for ‘non-irritants versus the rest’ (6.2.4.2.15.a-c.) and ‘severe irritants
versus the rest’ (table 6.2.4.2.16.a-c.).

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Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.15.a Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EU classification system –Non-irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5

33

42.4

38.9

46.7

46.7

38.9

53.3

61.1

14/33

7/18

7/15

7/15

7/18

8/15

11/18

Table 6.2.4.2.15.b. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
GHS classification system –Non-irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5

33

57.6

51.7

100

100

22.2

0

48.3

19/33

15/29

4/4

15/15

4/18

0/4

14/29

Table 6.2.4.2.15.c. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EPA classification system –Non-irritants versus the rest
Data
SourceAnal.

Balls et al.,
1995

Refer
table
6.2.4.1.5

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity%
No.

False Positive
Rate
%
No.

False Negative
Rate
%
No.

33

46.9

45.0

50.
0

60

35.3

50.0

55.0

to
15/32

9/20

6/12

9/15

6/17

6/12

11/20

NB. As the PM was not able to distinguish EPA Category III and Category IV materials, materials with these classifications wereconsidered in the analyses concerning non-irritants. Refer to Annex Vaii for the origins of the in vitro data and in vivo classifications.

199

Fluorescein Leakage Assay Background Review Document

Table 6.2.4.2.16.a Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EU classification system –Severe irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5

33

69.7

0

88.5

0

76.7

100

23.3

23/33

0/7

23/26

0/3

23/30

3/3

7/30

Table 6.2.4.2.16.b. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
GHS classification system –Severe irritants versus the rest
Data
SourceAnal.

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

Balls et al.,
1995

Refer
to
table
6.2.4.1.5

33

69.7

0

88.5

0

76.7

100

23.3

23/33

0/7

23/26

0/3

23/30

3/3

7/30

Table 6.2.4.2.16.c. Evaluation of the performance of INVITTOX Protocol No. 71 for predicting ocular irritation according to the
EPA classification system –Severe irritants versus the rest
Data
SourceAnal.

Balls et al.,
1995

Refer
table
6.2.4.1.5

No.

Concordance%No.

Sensitivity%
No.

Specificity%
No.

Positive
Predictivity%
No.

Negative
Predictivity
%
No.

False
Positive Rate%
No.

False Negative
Rate
%
No.

33

68.8

0

88.
0

0

75.9

100

24.1

to
22/32

0/7

22/25

0/3

22/29

3/3

7/29

NB. As the PM was not able to distinguish EPA Category III and Category IV materials, materials with these classifications wereconsidered in the analyses concerning non-irritants. Refer to Annex Vaii for the origins of the in vitro data and in vivo classifications.

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Fluorescein Leakage Assay Background Review Document

6.2.4.3.

Discussions

a. Description of the limitations of the test method (e.g., applicability domain
based on results from compilation of data)
The results of the analyses conducted in section 6.2.4.2. were evaluated in an attempt
to determine and compare the predictive capacities of the various FL assay protocols
and PMs for which summarised in vitro and in vivo data were available.
The predictive capacities of the various FL assay protocols were evaluated in relation toin vivo data from regulated testing using rabbits’ eyes. Currently, no database
containing reliable data of chemical-induced human ocular irritation exists. Althoughrabbits’ eyes are known to have differences in comparison to the human eye that
potentially affect the results to a significant level, available human data tends to lackdetailed exposure information, i.e. test material concentration, formulation composition,
exposure duration. This information is required in order to accurately assess thepredictive capacity of any in vitro assay. In the case of products that have been tested
in humans’ eyes, the irritancy potentials of these materials were generally known to be
mild before testing. This tends to produce data sets which are biased towards the mildend on a scale of irritation. As the rabbits’ eye is generally acknowledged to be moresensitive to ocular irritation than humans’ eyes, one can state that the in vivo test using
the rabbit provides an error safety margin, i.e. a material that is predicted to be an irritantby the Draize test is likely to be less irritant in the human eye.
Efforts were made to compare the predictive capacities of the various FL assay
protocols whilst acknowledging the different amounts and quality of the in vitro and invivo data. The largest data set analysed in section 6.2.4.2. was for INVITTOX Protocol
No. 71 which featured in the EC/HO study (Balls et al., 1995). There was no PM
available for the type of data generated using this protocol, therefore a PM was createdby the authors of this BRD, based on the EC/HO FL assay data and the in vivo based
EU classifications provided by ECVAM. The PM was applicable for EU, GHS and EPA
classifications. The EU, GHS and EPA classifications were generated by entering theraw in vivo data into the ECVAM template v6. As the PM was developed post-hoc, one
could expect this protocol and PM to have a good predictive capacity, especially for the
EU classifications. The PM was only applied to the definitive predicted classifications(i.e. not the mixed classifications generated by the greater-than values reported by thetest laboratories). The data set contained information for a large number of chemicals
with different potencies, but no data for formulations. Therefore the conclusions for thepredictive capacity of INVITTOX Protocol No. 71 and the PM were limited to chemicals
only. When all the chemicals were analysed, the predictive capacity was relatively low
and concordance values ranged 62-70% for ‘non-irritants versus the rest’ analyses and
78-83% for ‘severe irritants versus the rest’ analyses according to the different
classification systems. Specificity was consistently higher than sensitivity for eachclassification system for analyses of ‘non-irritants versus the rest’ and for analyses of‘severe irritants versus the rest.’ Overall, these results indicated that the protocol had agreater predictive capacity for non-irritants for the EU, GHS and EPA classification
systems and mild irritants for the EU and GHS classification systems, rather than severe
irritants.
For the ‘alcohols only’ analyses, for analyses of ‘non-irritants versus the rest,’
concordance values were 58% and 47% for the GHS and EPA classifications
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Fluorescein Leakage Assay Background Review Document

respectively and lower for the EU classification system (42%). For the alcohols,
specificity was greater than sensitivity for all three classification systems for analyses of‘non-irritants versus the rest.’ For analyses of ‘severe irritants versus the rest’ theconcordance values were lower than those calculated for when all chemicals wereanalysed. For ‘alcohols only,’ sensitivity was 0% and specificity ≥88% for all threeclassification systems. These findings indicated that for alcohols, INVITTOX Protocol
No. 71 had a better predictive capacity for non-irritants and mild irritants rather thansevere irritants, as was the finding for the analysis for the entire data set.
For all three classification systems, the concordance values for ‘alcohols only’ werelower than those for ‘surfactants only’ and when the entire set of EC/HO chemicals wasevaluated. Approximately 25% of the EC/HO test chemicals were surfactants. In
relation to analyses for all test chemicals, when the predictive capacity of INVITTOX
Protocol No. 71 for ‘surfactants only’ was analysed, the concordance values showedslight increases for the EU and GHS classification systems and a very slight increase for
the EPA classification system for analyses of ‘non-irritants versus the rest.’ Additional
calculations for for ‘surfactants only’ showed increased sensitivity and decreased
specificity in comparison to the analysis for the entire data set. As the protocol uses a
short incubation period, one would expect that the predictive capacity for surfactants
would be greater than for the larger set of chemicals which would have also included
chemicals that required a longer exposure period to induce an effect. As there weremore surfactants tested in comparison to the number of alcohols, there was greater
evidence for the predictive capacity of the protocol and PM for surfactants rather than for‘alcohols only.’
The concordance of predicted and actual EU classifications was used to determine thepredictive capacity of INVITTOX Protocol No. 71 for solids. Of the sixty test chemicals,
20 were tested in a solid form. Thiorea was tested as a solid and in vivo ocular irritation
data for this chemical were not reported as the test animals died (Balls et al., 1995).
Analyses for the effect of solids on the predictive capacity of the protocol were carriedout using EU Risk Phrase classifications. The study criteria were not for in vivo-basedclassifications to be assigned to the following chemicals; chlorhexidine, fomesafen, Laspartic acid, maneb, quiniacrine, sodium oxalate, potassium cyanate. Of the remaining
12 solid test materials the only chemical classified as a Not Classified,tetraaminopyrimidine sulphate had its potential irritancy predicted to be either a Not
Classified/R36 irritant. Only three R36 chemicals were tested; 4-carboxybenzaldehyde
was predicted to be either a Not Classified/R36 irritant by all four test laboratories,
ammonium nitrate was assigned the correct classification by one laboratory but underpredicted by the remaining three laboratories and dibenzyl phosphate could not beclassified by one lab, was classified as either a Not Classified/R36 irritant and as a R41
by another laboratory. There were eight R41 test chemicals of which one had thecorrect irritancy classification assigned by all four test laboratories. The following
chemicals had their correct irritancy classification assigned by one or more test
laboratories; benzoyl-L-tartaric acid (one laboratory), imidazole (one laboratory),
promethazine HCl (three laboratories), sodium perborate (one laboratory). The
remaining test chemicals were under-predicted to be either Not Classified,
Not
Classified/R36, R36 or a classification could not be assigned due to the low greater-thanvalue. Overall, the data indicated that INVITTOX Protocol No. 71 and the PM had agreater predictive capacity for the Not Classified and R36 chemicals rather than the R41
chemicals tested as solids. This finding is in accordance with the predictive capacity ofthe protocol and PM calculated for chemicals that were tested as liquids. Subsequently
202

Fluorescein Leakage Assay Background Review Document

the data did not support the hypothesis that solid test chemicals adversely affected thepredictive capacity of INVITTOX Protocol No. 71 and the respective PM. Due to therelatively few solid test chemicals for which definitive predictive classifications based on
INVITTOX Protocol No. 71 data and in vivo-based classifications were available, it isacknowledged that findings reported here can only be considered potentially indicativeof the effect of solid test chemicals on the predictive capacity of this protocol andrespective PM.
The concordance of predicted and actual EU classifications was used to determine thepredictive capacity of the FL assay for coloured chemicals. There were nine materials
tested as part of the EC/HO study that were known to be coloured. As there were too
few test chemicals that had definitive classifications, observations are made forchemicals that had mixed classifications, i.e. Not Classified/R36. Two of thesechemicals (quiniacrine, maneb) did not have EU classifications as the study criteria were
not met.
The two Not Classified chemicals (tetraaminopyrimidine sulphate,tricholoroacetic acid 3%) were classified correctly or as Not Classified/R36 chemicals by
the various laboratories. There were no R36 coloured chemicals according to the in vivo
data. Of the five R41 chemicals, all had their irritancy under-predicted by all four
laboratories with the following exceptions; imidazole (one laboratory) and promethazine
HCl (three laboratories). There were too few coloured chemicals tested to definitively
determine the predictive capacity of the assay for coloured chemicals although thesefindings did seem to indicate that the predictive capacity of INVITTOX Protocol No. 71
was not good for coloured chemicals. Many chemicals that were not coloured also had
their irritancy over-predicted or under-predicted. In general, the results indicated thatthe ability of the protocol to test and predict the irritancy potential of coloured materials
needs to be investigated further.
Overall, the EC/HO study data indicated that the predictive capacity of INVITTOX
Protocol No. 71 varied according to the type of test material, i.e. the predictive capacity
was better for surfactants rather than alcohols. For all classification systems, theprotocol had a greater predictive capacity for the non-irritants rather than the irritants foranalyses of the entire data set and ‘alcohols only’ and the contrarty for ‘surfactants only’.
Irritant materials that are not detected are of greater concern than non-irritant materials
that are misclassified as irritants. Further work is needed to increase both specificity
and sensitivity of the protocol and/or PM. In addition, formulations also need to betested with this protocol as it is known that different PMs or modifications to the testing
protocol, from those used for chemicals, can sometimes be required when predicting theirritancy of formulations.
From the data collected for this section, both INVITTOX Protocol No. 71 and No. 120
produced results that spanned five orders of magnitude. An advantage of such a large
range of values is that wide ranges can be used to distinguish different classes of
irritation. The PM devised for INVITTOX Protocol No. 71 for this BRD used relatively
wide ranges of values to distinguish three levels of irritancy. In comparison, the PM for
INVITTOX Protocol No. 120 used smaller ranges of values to distinguish threeclassifications of irritancy; this entails that INVITTOX Protocol No. 120 reproducibility
needs to be high.
INVITTOX Protocol No. 120 and the PM were only used to test surfactants andsurfactant-based formulations that were soluble in HBSS. Therefore, only the predictive
capacity of this protocol for this limited but relevant chemical class could be determined.
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Fluorescein Leakage Assay Background Review Document

There was a higher incidence of chemicals that were misclassified in comparison to thenumber of misclassified formulations. This finding indicated that the predictive capacityof INVITTOX Protocol No. 71 for formulations could also differ to the predictive capacity
calculated in this BRD for chemicals only. The predictive capacity of INVITTOX Protocol
No. 120 for surfactants and surfactant-based formulations was greater than that for
INVITTOX Protocol No. 71 for ‘all chemicals’ and ‘surfactants only’ when theconcordance values for the EU, GHS and EPA classification systems were compared foranalyses of ‘non-irritant versus the rest.’ In the absence of ‘severe irritants versus therest’ analyses for INVITTOX Protocol No. 71 for surfactants only, it was considered
useful to compare the available results, i.e. severe irritants versus the rest for INVITTOX
Protocol No. 71 for all chemicals and INVITTOX Protocol No. 120 for surfactants only for
the GHS and EPA classification systems. Comparisons of the GHS and EPA results for‘severe irritants versus the rest’ analyses showed that the concordance values werevery similar but higher for INVITTOX Protocol No. 120 rather than INVITTOX Protocol
No. 71 for the GHS classifications and lower for the EPA classifications. Sensitivity was
100% for INVITTOX Protocol No. 120 and only approximately 45% for INVITTOX
Protocol No. 71 for the GHS and EPA classification systems. Overall, these findings
suggested that the predictive capacity of INVITTOX Protocol No. 120 was greater than
INVITTOX Protocol No. 71 for surfactants. There are significant differences between
the protocols including the test materials exposure duration and the time-point FL ismeasured. Zanvit et al., (1999) stated that the COLIPA FL assay protocol (INVITTOX
Protocol No. 120) is purposely performed four hours after the chemical exposure as theresults for surfactants are more predictive of in vivo effects at this time-point rather than
immediately following the exposure.
Further testing is required to determine thepredictive capacity of INVITTOX Protocol No. 120 for other types of materials.
It is to be noted that the findings reported above were based on different data sets,
which varied in the amount and quality of in vitro and in vivo data. For example, there
were approximately 100% more data analysed for INVITTOX Protocol No. 71 incomparison to INVITTOX Protocol No. 120. Both chemicals and formulations were
included in the data set for INVITTOX Protocol No. 120 whereas only chemicals were
tested using INVITTOX Protocol No. 71. Additionally, the PMs used with the various
protocols were developed differently. The PM established for INVITTOX Protocol No.
71 was devised based on the EC/HO FL study data and the EU classifications provided
by ECVAM, therefore one would expect the protocol and PM to have a good predictive
capacity as the PM was challenged by the same data set used to define its thresholds.
In comparison, a PM was devised for INVITTOX Protocol No. 120 before the testing of
the COLIPA test materials. The PM was based on historical data supplied by Company# 4 for surfactants and surfactant-based formulations. This PM was adapted by theauthors of this BRD so that the threshold values correlated to the three principal classesfeatured in the EU, GHS and EPA classification systems; the PM could not distinguishCategory III and Category IV EPA classified test materials. The predictive capacity of
INVITTOX Protocol No. 120 and the PM was good as it was used to measure the effectsof the same class of materials it was devised to test.
Taking into account thesedifferent factors, makes it difficult to judge which protocol and PM were challenged to agreater extent by the various data sets.
In comparison to the data analysed for INVITTOX Protocol No. 71 and INVITTOX
Protocol No. 120, the quality and amount of data was less for INVITTOX Protocol No.
82. The in vivo based EU classifications used in the publication concerning this protocol
were devised by considering historical in vivo data from a number of different sources.
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These data were not available in a form compatible with the ECVAM template v6.
Therefore the EU classifications featured in the publication (Clothier et al., 1994) wereused to determine the predictive capacity of this protocol and PM for this classification
system. There were no analyses for the predictive capacity of the assay for the GHS
and EPA classification systems. Twenty-one chemicals were tested at a concentration
previously noted as being able to distinguish irritants and non-irritants. The authors
stated that consideration of 72h recovery data enabled some R36 irritants to be
distinguished from R41 irritants. Although the amount of recovery or deterioration at the
72h time-point was not stated in the publication of Clothier et al., (1994), a 30%
difference in 72h values respective to the initial FL assay values was used by theauthors of this BRD to indicate a substantial difference for analyses for this BRD. Using
this criterion, the concordance value was the highest recorded for any data set analysedin this BRD for analyses of ‘non-irritants versus the rest.’ Sensitivity was not as high as
recorded for INVITTOX Protocol No. 120 but specificity was higher than that recorded
for any other data set for analyses of ‘non-irritants versus the rest.’ Further work would
be required to determine if the predictive capacity was as good for a larger set of test
materials that also included formulations. As the data indicated that the recovery data
increased the predictive capacity of this assay, it would be interesting to investigate if
recovery data could increase the predictive capacity of other FL assay protocols.
A preliminary analysis of the effect of recovery data on the predictive capacity of
INVITTOX Protocol No. 71 was carried out. Based on the results of INVITTOX Protocol
No. 71 tested in the EC/HO study, a PM was proposed in the publication of Balls et al.,(1995) which distinguishes R36 and R41 irritants on the basis of recovery or
deterioration 72h following the initial exposure. There was no data set which contained
recovery FL assay data and accompanying in vivo data for ocular irritation
classifications. Subsequently, in vitro data from the publications of Shaw et al., (1991)
and Ward et al., (1997a) were matched to in vivo data taken from a number of different
sources. Due to a lack of chemical information the suitability of the paired in vitro and invivo data was unknown. Therefore, the findings of this analysis can only be interpreted
as indicators of the protocol’s and PMs predictive capacity. A wide range of chemicalclasses were tested although there were only a maximum of 20 data points for the EUclassification system and fewer for the GHS and EPA classification systems (results forsome chemicals were reported by both Shaw et al., (1991) and Ward et al., (1997a)).
INVITTOX Protocol No. 71 results which led to mixed classifications (i.e. R36/R41) were
not included in the data analyses. The concordance values ranged from 72.7 to 84.6%
for the different classification systems for analyses of ‘non-irritants versus the rest.’ For
analyses of ‘non-irritants versus the rest’ the concordance rates for all threeclassification systems were substantially better for the recovery data set in comparison
to INVITTOX Protocol No. 71 without recovery data, although the recovery data set was
smaller. To definitively determine the predictive capacity of this protocol and PM, bothchemicals and formulations need to be tested for which there are existing high quality invivo data.
Overall, there were certain types of data (i.e., chemical classes, chemicals, formulations)
missing for all protocols which prevented the predictive capacity of each protocol frombeing fully determined. There was greater evidence for the predictive capacity of
INVITTOX Protocol No. 71 as it had been used in more laboratories, to test a greaternumber of test materials belonging to a wider range of different chemical classes. In
comparison, INVITTOX Protocol No. 120 had only been used to test surfactants andsurfactant-based formulations known to be soluble in HBSS in two laboratories. The
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predictive capacity for surfactants and surfactant-based formulations tested with
INVITTOX Protocol No. 120 was greater than INVITTOX Protocol No. 71. The
predictive capacity of INVITTOX Protocol No. 71 for formulations requires investigation.
Modifications to the PM could potentially increase the predictive capacity of INVITTOX
Protocol No. 71 for the chemicals. Insufficient high quality in vitro and in vivo data has
been obtained for INVITTOX Protocol No. 82 to be able to determine its predictive
capacity. INVITTOX Protocol No. 86 was analysed in section 6.1.4.
b. Possible rationale for differences observed
Protocol differences
There are some fundamental protocol differences which would have impacted on thepredictive capacity calculated for the various FL assay protocols. In comparison to
INVITTOX Protocol No. 71, INVITTOX Protocol No. 120 has a different MDCK cellstrain, a longer chemical exposure, and the amount of FL is measured 4h after thechemical exposure. The cell strain used for INVITTOX Protocol No. 120 forms a tighter
barrier than the cell strain used in INVITTOX Protocol No. 71, therefore a longer
chemical exposure period is required for INVITTOX Protocol No. 120 for damage to themonolayer to be measured. It was possible that the 4h FL measurements used by
INVITTOX Protocol No. 120 could have increased the predictive capacity of this protocol
relative to INVITTOX Protocol No. 71. In addition the different PMs used in conjunction
with the various protocols will have affected the predictive capacity. It should be notedthat the PM for INVITTOX Protocol No. 120 was devised and used to test surfactants
and surfactant-based formulations only.
Quality of in vivo data
The quality of the in vitro and in vivo data sets differed per protocol. The EC/HO study
and the COLIPA study tested the same chemicals and formulations in the in vitro test
and in the in vivo tests. In comparison, the analysis of INVITTOX Protocol No. 82 wascarried out using EU classifications based on historical in vivo data from a number ofdifferent sources. It is assumed that those involved in analysing this protocol knew thesuitability of the paired in vivo based classifications with the in vitro data although this is
not documented in the publication of Clothier et al., (1994). For the analysis of
INVITTOX Protocol No. 71 with recovery data, the in vitro data were paired with in vivo
classifications by the authors of this BRD and the suitability of such pairings were
unknown as information regarding the CAS number and purity were not available for thein vitro data.
For complete data sets, INVITTOX Protocol No. 71 and INVITTOX Protocol No. 120
differed as to which classification systems the protocols and PMs had the highest andlowest concordance rates. For all three classification systems, INVITTOX Protocol No.
71 and the respective PM had a higher predictive capacity for the non-irritants and mild
irritants whilst INVITTOX Protocol No. 120 and the PM had a greater predictive capacity
for the mild and severe irritants.

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Number of test materials
For all analyses, the mean in vitro results/classifications produced by each laboratory
were analysed. INVITTOX Protocol No. 71 was evaluated by testing 59 chemicals infour laboratories although only the definitive classifications were considered in theanalyses. INVITTOX Protocol No. 120 was evaluated by testing 33 chemicals in twolaboratories (not all the same chemicals were tested in each laboratory). For the otherprotocols tested; there were fewer in vitro data available, the in vitro data were produced
in a single laboratory and the in vivo data were of a lower quality. INVITTOX Protocol
No. 120 had a higher predictive capacity than INVITTOX Protocol No. 71 which could
have been due to the fewer test materials. The amount of data often entails that the
protocol has been more extensively tested by a greater range of different types ofmaterials. However, the data set for INVITTOX Protocol No. 120 was purposely limited
to only surfactants and surfactant-based formulations, therefore in this case the numberof test materials was not related to the number of different types of materials tested.
Types of test materials
With the exception of INVITTOX Protocol No. 120, a wide range of chemicals was tested
using the different protocols analysed in this section. INVITTOX Protocol No. 120 wasused to test surfactants and surfactant-based formulations only. Of all the data sets,
INVITTOX Protocol No. 71 was used to test the widest range of different chemicals,
although no formulations were tested.
Potency range of test materials
The GHS in vivo based classifications of the data sets for INVITTOX Protocol No. 71(EC/HO study) and INVITTOX Protocol No. 120 were compared for potency ranges as
there were a greater number of GHS classifications in comparison to the number of EU
and EPA classifications. For INVITTOX Protocol No. 120 there were a similar number of
No Category and Category 1 test materials and fewer Category 2 irritants. For theclassifications used in the analyses for INVITTOX Protocol No. 71, there were similar
numbers of Category 1 irritants and No Category chemicals and more Category 2. Thisdata does not represent the proportions of the different classifications for the entire data
sets (not analysed due to mixed classifications). The number of No Category andCategory 1 chemicals was similar for each protocol and INVITTOX Protocol No. 71 hada higher proportion of Category 2 chemicals in comparison to INVITTOX Protocol No.
120. Category 2 (mild irritants) are known to be difficult to detect using both in vitro andin vivo methods and consequently one could expect the predictive capacity calculated
for INVITTOX Protocol No. 71 would be lower than that calculated for INVITTOX
Protocol No. 120 which was assessed using a data set containing relatively fewer
Category 2 chemicals.
The data sets for INVITTOX Protocol No. 82 and INVITTOX Protocol No. 71 withrecovery data, consisted of significantly fewer data in comparison to those for INVITTOX
Protocol No. 71 and INVITTOX Protocol No. 120.
The ranges of potency varied
between these smaller data sets. Although one could expect these different potency
ranges to affect the predictive capacity calculated for these protocols, differences in theamount and the quality of the in vivo data were hypothesised to have affected thepredictive capacity to a greater extent.

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6.3.1. Attempt to combine the data using weight-of-evidence approaches
A weight-of-evidence analysis was predominately carried-out for the FL assay
INVITTOX Protocols which had the greatest amounts of in vitro and in vivo data
available, to enable the predictive capacity to be determined. The weight-of-evidence
analysis applied was devised by the authors of this BRD and acknowledged to be apreliminary study.
INVITTOX Protocol No. 86
INVITTOX Protocol No. 86 was the only protocol that had raw in vitro and in vivo data
available. In a weight-of-evidence approach, the results from this analysis would receive
greater weighting due to the quality of the data, i.e. the same highly relevant
formulations were tested in both the in vitro and in vivo tests and compositions were
known. However, the data set was only comprised of the surfactant-based formulations
tested in the CTFA study Phase III (Gettings et al., 1996). Data were available for 23
out of 25 formulations that were tested in a single laboratory. This was considered asmall data set and would receive little weighting based on this criterion. As no
chemicals were tested, the predictive capacity for chemicals could not be ascertained.
Therefore, the limited range of test materials adds little weight in evidence of the various
applicability domains of this protocol.
The PM for INVITTOX Protocol No. 86 was only capable of distinguishing irritants from
non-irritants. In addition, there was also a range of TEP assay values that could not be
used to classify formulations as either irritants or non-irritants. For the formulations, thepredictive capacity for the EU, GHS and EPA classifications differed but all showed agreater predictive capacity for the irritants rather than the non-irritants in all threeclassification systems. These findings contrasted to those reported in the publication of
Gettings et al., (1996) where the assay was compared to FHSA classifications. A posthoc threshold value enabled 14/18 of the FHSA irritants and all of the non-irritants to becorrectly identified. Although the in vivo classification systems have different criteria forlabeling materials as irritants or non-irritants, as these two findings are contradictory, theliterature does not give any further weighting to the results calculated for this BRD.
Preliminary analyses of the effects of certain physical properties on the predictive
capacity of INVITTOX Protocol No. 86 were inconclusive regarding the affect of coloured
materials, and indicated that viscosity did not adversely affect the protocol’s predictive
capacity.
In comparison to the analyses for other INVITTOX Protocols which allowed threeirritancy classifications to be predicted, there was no evidence of the protocol’s andPM’s ability to distinguish mild and severe ocular irritants. Comparisons of the predictive
capacity of INVITTOX Protocol No. 86 with other INVITTOX protocols analysed in thissection showed that the concordance values for INVITTOX Protocol No. 86 for ‘nonirritants versus the rest’ was comparable to the respective values for INVITTOX Protocol
No. 71 for ‘all chemicals’ and ‘surfactants only’ and lower than the respective values for
INVITTOX Protocol No. 120. As INVITTOX Protocol No. 86 was only analysed for its
ability to distinguish irritants from non-irritants and comparisons of predictive capacity
were made with protocols that determined different levels of irritancy one would haveexpected the predictive capacity of INVITTOX Protocol No. 86 to be higher. Due to thedifferent sizes and quality of the data sets analysed for the various protocols, there wasrelatively little weight to support the findings for INVITTOX Protocol No. 86.
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INVITTOX Protocol No. 71
The largest data set available for analyses was for INVITTOX Protocol No. 71 which
was comprised of chemicals with a wide range of mechanistic actions and potencies.
Subsequently, in comparison to the smaller data sets available for the other protocols
greater weighting was given to the results from these analyses. INVITTOX Protocol No.
71 data were available for 59 chemicals tested in four different laboratories. This data
set did not contain any values for formulations so only the predictive capacity forchemicals could be determined. Only raw in vivo data were available for this study.
Mean INVITTOX Protocol No. 71 data for each chemical tested in each laboratory werecompared with EU, GHS and EPA classifications. For all three classification systems
there was a greater predictive capacity for the irritants rather than the non-irritants.
There was inconclusive evidence to determine if solid materials reduced the predictive
capacity of the protocol. The viscosity of the test materials was unknown and therefore
the effect of viscosity on the predictive capacity of INVITTOX Protocol No. 71 could not
be ascertained. Based on a limited amount of data for the very few coloured materials
tested, the findings did seem to indicate that the predictive capacity of INVITTOX
Protocol No. 71 for coloured chemicals was reduced. However, there were a large
number of chemicals that were not coloured that also had their irritancy over- or underpredicted. There were too few coloured chemicals tested to give weighting to thisanalysis although the results indicated that the ability of the protocol to test and predict
the irritancy potential of materials with these physicochemical properties needs to beinvestigated further. As there were more solid chemicals tested in relation to thenumber of coloured test chemicals, there was greater weight-of-evidence for the effectof solid materials on the predictive capacity of the protocol.
INVITTOX Protocol No. 71 with recovery data
Based on the results from the EC/HO study for INVITTOX Protocol No. 71, Balls et al.,(1995) proposed a PM that utilised recovery data to distinguish mild and severe irritants.
The PM took into account the recovery or deterioration of effects 72h following thechemical exposure. Recovery data from two publications were utilised to generatepredicted EU, GHS and EPA classifications. There were no in vivo data generated bythese studies so in vivo classifications were taken from other studies. As the paired invitro and in vivo classifications were based on chemical name and concentration only,
the results from analyses of these data receive little weighting due to the quality of the‘paired’ data. Additionally, little weighting was given to the results due to the size of thedata set and the absence of data for formulations. For the EU, GHS and EPA
classification systems, the protocol and PM had a good predictive capacity for thesevere irritants (R41, Category 1 and Category I) rather than the non-irritants and there
were too few mild irritants in the data set to comment on the predictive capacity formaterials with this level of irritancy.
INVITTOX Protocol No. 120
INVITTOX Protocol No. 120 data were generated by testing COLIPA surfactants andsurfactant-based formulations known to be soluble in HBSS. A modified version of the
PM featured in the COLIPA study was used to generate predicted EU, GHS and EPA
classifications based on INVITTOX Protocol No. 120 data. Raw in vivo data were used
to generate the EU, GHS and EPA classifications. The quality of the data set wascomparable to that used to analyse INVITTOX Protocol No. 71 but there were less thanhalf the data available for the analysis of INVITTOX Protocol No. 120. The test
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materials for INVITTOX Protocol No. 120 were limited to surfactants only, but bothchemicals and formulations were tested. Overall, this data set was considered to haveless weighting than the result for INVITTOX Protocol No. 71, but greater weighting than
the results for the other INVITTOX Protocols analysed for this BRD.
The predictive capacity of INVITTOX Protocol No. 120 for the EU, GHS and EPA
classifications did not vary greatly. For all three classification systems, INVITTOX
Protocol No. 120 had a better predictive capacity for surfactants in comparison to
INVITTOX Protocol No. 71 for ‘surfactants only’ for analyses of ‘non-irritants versus therest.’ Both protocols had a higher predictive capacity for the mild and severe irritants
rather than the non-irritants as determined by analyses of ‘non-irritants versus the rest’
although there were fewer R36 irritants in the INVITTOX Protocol No. 120 data set incomparison to the data set for INVITTOX Protocol No. 71. The data set for INVITTOX
Protocol No. 120 was; larger than INVITTOX Protocol No. 71 for ‘surfactants only’,
generated by two laboratories and comprised of data for both chemicals andformulations. The INVITTOX Protocol No. 71 data for ‘surfactants only’ was generated
by four laboratories and consisted of fewer test surfactants which were limited tochemicals. Therefore, INVITTOX Protocol No. 120 had greater weighting in support ofits better predictive capacity for surfactants than INVITTOX Protocol No. 71. The
greater predictive capacity of INVITTOX Protocol No. 120 for surfactants was supportedby the publication of Zanvit et al., (1999). Results for the nine surfactants common to
the COLIPA and the EC/HO study were compared to in vivo MMAS data by Pearson’s
correlation and Spearman’s rank coefficients. INVITTOX Protocol No. 120 (COLIPA)
was found to show a greater correlation to the in vivo data in comparison to INVITTOX
Protocol No. 71 (EC/HO study protocol) (Zanvit et al., 1999).
The results from the literature where surfactants or surfactant-based formulations were
tested (Zanvit et al., (1999), Cottin and Zanvit (1997), (Southee, 1998)), ascertained thepredictive capacity of INVITTOX Protocol No. 120 by comparing the in vitro data with
Draize MAS, MMAS scores or classifications. The results from these analyses
supported the findings for the concordance analyses performed here; that the assay had
a greater tendency to over-predict rather than under-predict the irritancy of surfactants.
These findings from multiple studies support and give extra weighting to the findings
regarding the predictive capacity of the assay for EU, GHS and EPA classifications.
There was insufficient information about the physical state of the test materials todetermine if the predictive capacity of INVITTOX Protocol No. 120 was affected bymaterials that were coloured, solid, etc.
INVITTOX Protocol No. 82.
Based on concordance values for analyses of ‘non-irritants versus the rest,’ thepredictive capacity of INVITTOX Protocol No. 82 was greater than for the other
INVITTOX Protocols analysed in this section. EU classifications were reported in theliterature and used for the concordance analyses featured in this BRD. Although the 21
test chemicals covered a range of different chemical classes, little weighting can begiven to the results due to; the relatively few test chemicals, the absence of data forformulations, and the quality of the historical in vivo data.
Jones et al., (2001) used a protocol similar to INVITTOX Protocol No 82 to test hair careformulations. The protocol had a tendency to over-predict rather than under-predict theirritancy potential of the test shampoos and conditioners. Little weighting can be given
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to this finding as; there was no information regarding the formulations compositions, thedata set was small, and the in vivo data used was not transparent. In view of thecontradictory findings for these two studies featuring INVITTOX Protocol No 82, overall
there was little evidence in support of the predictive capacity of this protocol.
In the following sections, additional factors were investigated in further detail todetermine how the different data sets potentially affected the predictive capacity
calculated for each protocol.
Potency of test materials
For each FL assay protocol assessed, the potency ranges of the test materials weresimilar, as the aim of most studies was to establish the predictive capacity of the FL
assay protocols for test materials with mild or moderate ocular irritation potentials.
Quantitative scores rather than qualitative classifications were used to compare thepotency ranges. Greater weighting could be given to the findings for INVITTOX Protocol
No. 71 that featured in the EC/HO study as the test materials covered the entire range of
MMAS values equally. In comparison, the COLIPA study tested materials that covered
the entire range of MMAS values although the majority had MMAS values below 50.
The range of MAS values for INVITTOX Protocol No. 86 data was also limited to asimilar range (e.g. 10-43 MAS). The Fixed Dose FL assay (INVITTOX protocol No. 82)
was only evaluated in a single study where the majority of test chemicals were classified
as non-irritants or as R41 irritants (Clothier et al., 1994). Jones et al., (2001) used asimilar protocol but used an in-house qualitative classification scheme for classifying theformulations. Subsequently there was little weight in support of this protocol as there
were very few materials with mild irritancy that the FL assay was designed to measure.
Mechanisms
The in vitro data collected for this BRD were predominately produced by testingchemicals that exert toxicity via membrane lysis, and to a lesser extent coagulation;some materials had both mechanisms of action. Saponification was a mechanism of
toxicity for only a few test chemicals. There was no indication that any of the test
materials specifically affected tight junctions. There are chemicals that are known toaffect tight junction formation, i.e. Calphostin C, and this has been tested with humancorneal cells and MDCK cells but preliminary experiments showed no direct effects on
fluorescein leakage (Clothier and Limb, personal communication). If a test material wascapable of damaging the tight junctions, this would tend to occur before membrane
damage. FL assay results alone cannot distinguish damage to the tight junctions fromdamage to the cell membranes. Therefore, it would seem that appropriate materials, i.e.
those that cause membrane damage, have been tested in the FL assay in order todetermine its predictive capacity. If a cell viability assay was also used to test thematerials, the difference between specific tight junction damage and cell membrane
damage could be potentially ascertained (Clothier and Samson, 1997)
To conclude, there was insufficient data to equally analyse the four INVITTOX Protocols.
The proposed PM for INVITTOX Protocol No. 71 performed relatively well for predicting
EU, GHS and EPA classifications for ocular irritation. The predictive capacity for theseclassification systems agreed with the findings reported in the literature where theperformance was measured against other types of in vivo data. This indicated that the
PM defined specifically for this BRD and used for these analyses was appropriate.
Overall, there was greater evidence for the predictive capacity of INVITTOX Protocol
No. 71 in comparison to the other INVITTOX Protocols analysed in this BRD. The PM
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applied to INVITTOX Protocol No. 120 data for the BRD analyses was a modified
version of the one featured in the COLIPA study. The predictive capacity of INVITTOX
Protocol No. 120 for the EU, GHS and EPA classification systems was not dissimilar tothat reported in the literature where the predictive capacity was measured against othertypes of in vivo data. The predictive capacity of INVITTOX Protocol No. 120 was better
than INVITTOX Protocol No. 71 when comparing data for surfactants and surfactantbased formulations. Although it can be stated that INVITTOX Protocol No. 71 waschallenged by materials with a wider range of mechanisms, both protocols have beenequally challenged by the materials they were designed to test, i.e. materials withmembrane lysis as the mode of action. At the low concentrations often employed forformulations, these types of materials can cause disruption to the tight junctions;
damage caused by mild irritants can be repaired within hours. Only INVITTOX Protocol
No. 120 tested both chemicals and formulations and the protocol and PM appeared tobe appropriate for both surfactants and surfactant-based formulations. INVITTOX
Protocol No. 86 had high quality in vitro and in vivo data but the test materials werelimited to surfactant-based formulations only. Comparisons with the results for the other
INVITTOX Protocols showed that the concordance of INVITTOX Protocol No. 86
predicted classifications with the in vivo classifications was comparable to the otherprotocols for different data sets.

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6.3.2. Modifications to the assay if designed today
The FAL investigated using a transfected human corneal cell line for the FL assay.
Japanese human corneal epithelial cells transfected with the simian virus 40 (J-HCET)
were used. Human corneal cell cultures were hypothesised to predict potential humanin vivo ocular irritation more accurately than cell types originating from different tissue
types and species, i.e. MDCK cells. A human corneal cell line is currently used atGillette Medical Evaluation Laboratories (USA) for the FL assay although few clones ofthis cell line remain. Subsequently, investigations were conducted to determine theability of the J-HCET cell line to grow on Nunc 0.45µm polycarbone inserts and to formtight junctions that cause an impermeable monolayer to sodium-fluorescein dye. In
comparison to the cell model used at Gillette Medical Evaluation Laboratories, anadvantage of the J-HCET cell line is that it can be cultured for up to 400 passages(Araki-Sasaki et al., 1995) and cells up to 100 passages have been genetically typed by
the FAL (Wilkinson, 2006). In comparison, the Gillette Medical Evaluation Laboratories
model only expresses properties similar to human corneal cells for up to approximately
20 passages. The J-HCET cell line can be cultured in serum-free medium when
supplemented with plant-derived products. Although both cell lines are capable ofgrowing in stratified layers, which model the in vivo cornea rather than a monolayer, the
J-HCET cells do not form a impermeable layer suitable for the FL assay. Therefore, the
J-HCET cell line was used as a monolayer for the FL assay; as is used with the MDCK
cells. Further research is required to find a human corneal cell line capable of forming
stratified layers which models the in vivo situation more closely as stocks of the Gillette
Medical Evaluation Laboratories clone which does form stratified layers, are being
depleted. Work would be required to interpret the effect of stratified layers on FL assay
results as 3D skin models using keratinocytes grown in multiple layers have revealed
that cells grown in stratified layers can affect FL even in the absence of tight junctions(personal communication, R Clothier). In addition, multiple layers can cause problems
with the resazurin assay which has been found to be a valuable addition for interpreting
FL assay results. The resazurin assay has difficultly to penetrate all of the layers andcan be difficult to remove which causes inaccurate results. Further work is necessary
to evaluate a 3D corneal model with the FL assay. Preliminary experiments have shownthat if the calcium concentration is high enough the tight junctions are correctly formed.
Annex IVa

Information available on the chemicals tested to assess FL assay
predictivity of in vivo eye irritation

Annex IVb

Information available on the products/formulations tested toassess FL assay predictivity of in vivo eye irritation

Annex Vai

CTFA Phase III data set containing predicted and actual EU,
GHS and EPA classifications for each formulation tested using the
TEP assay/ INVITTOX Protocol No. 86. Raw in vitro and in vivo
data were available

Annex Vaii

Example of a data set containing predicted and actual EU,
GHS and EPA classifications for each test material. Data sets containedsummarised in vitro data. -Entire in vitro and in vivo
Data sets provided on CD

Annex Vb

In vivo reference data as reported in the literature for each substance213

Fluorescein Leakage Assay Background Review Document

tested using the FL assay –Example given but all data provided on CD
Annex Vc

Example of the in vivo reference data as submitted by Company # 3 for 17
formulations -Entire in vitro and in vivo data sets provided on CD

Annex VI

Table presenting the studies where FL assay results were used toassess the assay’s predictive capacity for in vivo ocular irritation

Annex VII

Raw in vivo data as entered into ECVAM v6 template -provided on CD

Annex A

CTFA Study Phase III formulation compositions (from draft HET-CAM
BRD: Appendix C2 (ICCVAM/NICEATM, 2004)

Annex B

COLIPA Study test chemicals and formulations compositions (fromCOLIPA)

Annex C

Formulations compositions from Company # 3

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7. Applicability Domain
The applicability domain of the FL assay and the various protocols was investigated.
The FL assay was developed specifically to model loss of trans-epithelial impermeabilityof the conjunctiva and cornea (INVITTOX Protocol No. 71), which occurs duringchemical-induced ocular irritation. The FL assay models ocular irritation caused bydamage to the inter-cellular junctions and the cell membranes of the corneal andconjunctival epithelia. The FL assay is useful for testing mild to moderate irritantmaterials within a defined range where it can measure mechanistic damage to theadhesion molecules. Various FL assay protocols have featured in studies that
evaluated their ability to model and predict in vivo ocular irritation. In order to test thepredictive capacity of the FL assay protocols for in vivo ocular irritation, a range of
relevant chemicals and formulations have been tested. These were predominately
cosmetic ingredients and formulations that are the materials that are most likely to enter
into the human eye as a result of frequent use.
The applicability domain(s) for the various FL assay protocols have been determined byevaluating the predictive capacity of various FL assay protocols for EU, GHS and EPA
classifications based on in vivo data. The PMs were devised according to descriptionsin Section 6 of this BRD. The predictive capacities of all the protocols discussed above
did not vary greatly according to whether they were compared to EU, GHS or EPA
classifications.
The amount, type, and quality of in vivo data used to determine the applicability domainof the different protocols varied for each protocol. For the larger data sets analysed, thepredictive capacities of the protocols and PMs for the EU, GHS and EPA classification
systems did not vary greatly. For the smaller data sets, a single classification affected toa greater extent the overall predictive capacity of the protocols for the three classification
systems. The type and quality of the data for each protocol are discussed below. The
applicability domains for the various protocols were then discussed in the final
paragraphs of this section.
INVITTOX Protocol No. 71
The EC/HO study evaluated the predictive capacity of INVITTOX Protocol No. 71 by
testing a wide range of pure chemicals (Balls et al., 1995). The conclusions formed for
INVITTOX Protocol No. 71 were based on the results for which definitive classifications
were predicted (i.e., greater-than results which prevented definitive classifications frombeing assigned were not analysed). The results indicated that the predictive capacity of
the assay varied for different types of chemicals and that the assay was more suited to
predicting the effects of surfactants rather than alcohols. The predictive capacity ofsurfactants was similar to when data for all the test chemicals were analysed. No further
analyses to determine the predictive capacity for different chemical classes were
performed, as there were too few chemicals representative of each chemical class. The
protocol and/or PM required further work to increase the overall predictive capacity, andfor the non-irritants in particular, for the EU, GHS and EPA classification systems.
INVITTOX Protocol No. 71 with recovery data
Using a limited data set, the use of recovery data generated by INVITTOX Protocol No.
71 to predict potential ocular irritancy was investigated. This data set was of the lowest
quality analysed due to the unknown suitability of the paired in vitro and in vivo data forthe test chemicals. Despite this concern, the concordance rates of predicted and actual215

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EU, GHS and EPA classifications were higher than those for INVITTOX Protocol No. 71
without recovery data, but this was hypothesised to be due to the relatively smaller data
set rather than a greater predictive capacity of the PM.
INVITTOX Protocol No. 82
The quality of INVITTOX Protocol No. 82 (Fixed Dose FL assay) data was low incomparison to the data sets used to assess the applicability domain of the other
INVITTOX protocols. The modified PM was used to assess the predictive capacity ofthis assay for the test chemicals featured in the Clothier et al., (1994) publication. The
concordance value was the highest one recorded for any data set analysed in this BRD.
Sensitivity was not as high as recorded for INVITTOX Protocol No. 120 but specificity
was higher than that recorded for any other data set. This result was good in
consideration of the nature of the in vivo data and that a number of different types ofchemicals had been tested. Further work would be required to determine if thepredictive capacity was as high for a larger set of test materials that also included
formulations.
INVITTOX Protocol No. 86
INVITTOX Protocol No. 86 (TEP assay) was tested in the CTFA study Phase III and thiswas the only data set available to assess the predictive capacity of this protocol. As thetest materials were all surfactant-based formulations, the applicability domain of thisprotocol can only be ascertained for these types of materials. Based on relatively few
data, the predictive capacity of INVITTOX Protocol No. 86 displayed a greater predictive
capacity for the irritants rather than the non-irritants.
INVITTOX Protocol No. 120
EU, GHS and EPA classifications based on raw in vivo data from the COLIPA study
were matched against mean INVITTOX Protocol No. 120 values to allow the predictive
capacity and applicability domain of this protocol to be ascertained. Surfactants andsurfactant-based formulations were tested with this protocol; the applicability domain of
this protocol for only these types of test materials could be ascertained. INVITTOX
Protocol No. 120 had a better predictive capacity than INVITTOX Protocol No. 71 and
INVITTOX Protocol No. 86 for surfactants and surfactant-based formulations. In
comparison to INVITTOX Protocol No. 71, INVITTOX Protocol No. 120 has a longer
chemical exposure and FL is measured 4h after the exposure rather than immediately.
INVITTOX Protocol No. 86 has the same exposure duration as INVITTOX Protocol No.
120 but FL is measured immediately following the exposure and the EC50 (%) isrecorded The longer exposure duration is used for INVITTOX Protocol No. 120 as thecell strain and inserts used cause a more impermeable monolayer that requires a longer
chemical exposure for effects to be measured. Zanvit et al., (1999) stated that FL ismeasured 4h following the chemical exposure to enable the effects of cationicsurfactants to be measured correctly. It is stated that cationic surfactants cause thecells to stick together which affects the rate of FL measured immediately following theexposure; the 4h results were reported to more accurately reflect the irritancy of thesetest materials. This finding was supported by the greater predictive capacity of
INVITTOX Protocol No. 120 in comparison to INVITTOX Protocol No. 86.
Potency of test materials
The potency of the materials used to assess the predictive capacity of the various FL
assay protocols would have affected the predictive capacities calculated. The GHS
classifications for INVITTOX Protocol No. 71 and INVITTOX Protocol No. 120 data sets216

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were compared as there were slightly more GHS classifications in comparison to thenumber of EU and EPA classifications. For both INVITTOX Protocol No. 71 and
INVITTOX Protocol No. 120 there were fewer Category 2 irritants in comparison to thenumber of No Category and Category 1 test materials; there were a similar number of
No Category and Category 1 test materials. Therefore, both protocols were equallychallenged by data sets with similar ranges of potency.
The data sets for INVITTOX Protocol No. 82 and INVITTOX Protocol No. 71 withrecovery data, consisted of significantly fewer data in comparison to those for INVITTOX
Protocol No. 71 and INVITTOX Protocol No. 120. The proportion of materials with mild
irritancy according to the in vivo data was lower for these smaller data sets incomparison to the data sets for INVITTOX Protocol No. 71 and INVITTOX Protocol No.
120. Although one could expect the different potency ranges to affect the predictive
capacity calculated for these protocols, differences in the quality of the in vivo data weremore likely to have affected the predictive capacity to a greater extent.
Physical state of test materials
Various properties of test materials that were known to affect the ability of the assay toaccurately measure induced toxicity were investigated to determine how they affected
the predictive capacities calculated for the different FL assay protocols. Data for
INVITTOX Protocol No. 71 were predominately used as this data set contained thehighest number of chemicals for which detailed chemical information could be attained.
There was insufficient information regarding the pH of the test materials to determine its
effect on the predictive capacity of the FL assay.
-Solid materials
Data evaluations were performed to determine if the predictive capacity of INVITTOX
Protocol No. 71 was affected by testing solid chemicals. Solids can be difficult to test as
the concentration in contact with the cells cannot be assumed to be equal to that placedon the cell monolayer, nor uniformly distributed. Also, solids cannot be easily removed
from the cell surface following the short exposure period (Balls et al., 1995). The
concordance of predicted and actual EU classifications did not support the hypothesisthat the predictive capacity of the protocol was adversely affected by solid test
chemicals, however it was acknowledged that the test set of chemicals contained
relatively few solids.
-Viscous materials
Viscous materials can be difficult to remove from the monolayer following the short
exposure period. Due to the short exposure period, mildly irritating materials often need
to be tested neat in order to produce a response which can be measured. Therefore theproblems associated with viscous materials cannot be reduced by dilution. The effect ofviscosity on the predictive capacity of INVITTOX Protocol No. 71 could not be
ascertained as the viscosity of the test materials was unknown. CTFA study Phase III
data did not indicate that viscosity affected the predictive capacity of INVITTOX Protocol
No. 86. In comparison to INVITTOX Protocol No. 71, this protocol has a longer
chemical exposure and 5x more washing steps following the removal of the test
material. One could hypothesise that the predictive capacity of INVITTOX Protocol No.
71 for viscous materials could be lower than that of INVITTOX Protocol No. 86 due tothe shorter exposure duration which would be more greatly affected by any difficulties inremoving the test chemical. This would impact on protocol reproducibility, andpotentially predictive capacity.
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Fluorescein Leakage Assay Background Review Document-Coloured materials
There were nine EC/HO study test chemicals that were known to have colour. This wasconsidered to be too few to determine the predictive capacity of INVITTOX Protocol No.
71 for chemicals with colour, although observations of the results did seem to indicatethat the predictive capacity of the protocol was not good for coloured chemicals.
The results for INVITTOX Protocol No. 86 which tested 23 formulations, of which 19
were coloured, did not indicate that the predictive capacity of the protocol was affectedby materials with colour. As the problems associated with measuring the effects ofcoloured materials would be similar for all protocols, based on this result no FL assay
protocols should have their predictive capacities adversely affected by coloured
materials if the test materials were fully removed following the exposure period.
However, in comparison to other INVITTOX Protocols, INVITTOX Protocol No. 86 hasmore washing steps following the removal of the test material which could reduce theimpact of coloured materials on the predictive capacity of this protocol in comparison tothat for INVITTOX Protocol No. 71. Further work is required to determine the effects ofthe physical states of test materials on the predictive capacity of the various FL assay
protocols.
-Solubility of test materials
The FL assay generally had a good predictive capacity for materials that are watersoluble and/or the toxic effect is not affected by dilution. It is important that the basic
toxic mechanism is not affected by dilution as the formation of micelles by surfactants
can unpredictably alter cellular responses over a concentration range that impacts on
the predictive ability of in vitro assays.
Test materials that are not soluble in HBSS or distilled water can only be reproducibly
tested in the FL assay if they form a stable suspension or emulsion. Emulsions andsuspensions will not be as homogeneous as a solubilised material and it is not easy toestablish that an emulsion or suspension is uniform or stable. However, the shortchemical exposure duration provides less time for the emulsion or suspension todegrade. Materials that are insoluble or do not form stable aqueous emulsions can besolubilised or suspended in mineral oil to reduce evaporation and ensure that the test
concentrations remain in contact with the cells for the specified exposure period. The
PM featured in INVITTOX Protocol No. 120 was only applied to data generated by
testing surfactants and surfactant-based formulations that were soluble in HBSS.
Although the effect of solid and viscous materials were not definitively determined for
INVITTOX Protocol No. 71 it is possible that the predictive capacity of INVITTOX
Protocol No. 71 for EU, GHS and EPA classifications could have been affected byunknown solubilities of the chemicals or the use of solvents other than HBSS. The
EC/HO study published Pearson’s correlation coefficients and Spearman’s rankcorrelation coefficients for in vitro data compared with in vivo data for; the entire dataset, the chemicals soluble in water, chemicals insoluble in water, surfactants, solids,
liquids and solutions (Balls et al., 1995). Due to extensive between-laboratory variation
for the four participating laboratories, it was not possible to observe any definitive effectsof these factors on the predictive capacity calculated, e.g., in comparison to thepredictive capacities for the entire data set, the predictive capacity for only thosechemicals that were soluble in water was better for one laboratory, only very slightly
better for two laboratories and worse for another.

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Fluorescein Leakage Assay Background Review Document-Test materials binding to the insert membrane
Certain test materials can bind to the insert membrane, thus making their removal very
difficult. Chemical binding to the insert membrane, is more common for cationicsurfactants, such as benzalkonium chloride, which are attracted to the positively
charged membrane (Balls et al., 1995). Negatively charged cell surface proteins canalso attract positively charged surfactants. Chemical binding to the insert membrane orcell surfaces, increases the chemical exposure duration but can also physically block thepassage of sodium-fluorescein dye through the insert. Also, test materials with highmolecular weights that are not fully removed, can physically block the passage of thesodium-fluorescein dye through the insert, which could cause chemical effects to beunder-estimated. Ward et al., (1997a) found that the different properties of the insert
membranes affected the interactions with various test chemicals.
To counter the effects of coloured, viscous and solid materials and those that bind to themonolayer or insert membrane, on the predictive capacity of the FL assay, sufficientwashing steps are needed to ensure the full removal of the test materials at the end of
the exposure period. However, many washing steps increases the likelihood of damage
caused to the monolayer and/or insert membrane, thus producing erroneous results.
INVITTOX Protocols No. 71, 82 and 86 have one or two washing steps after the removalof the test materials whereas INVITTOX Protocol No. 86 has ten washing steps.
Types of test materials
The majority of data used to assess the predictive capacities of the various FL
INVITTOX Protocols were generated by testing surfactants and surfactant-based
formulations. Most cosmetic formulations and some ingredients are surfactant-based
and surfactants respectively. Therefore, the various protocols have been evaluated by
testing relevant materials, i.e. those that are humans are likely to be routinely exposed
to, and which are potentially able to enter the eye and cause ocular irritation. Cosmetic
formulations or ingredients at the concentrations intended for use tend to range from
non-irritants to moderate irritants that cause membrane damage as their primarymechanism of irritancy/toxicity. The following chemical classes were proposed by the
ECVAM Eye Irritation Meeting (2005) as having membrane lysis as their primarymechanism of eye irritation; surfactants, organic solvents, ketones, alcohols, volatileliquids, ethers, polyethers, esters, aromatics, amines. At lower concentrations thanthose causing membrane damage, these types of chemicals could also cause damage
to the inter-cellular junctions. Therefore, the FL assay has the potential to measure thetoxicity exerted by a range of materials, and to predict their in vivo ocular irritation
effects. A number of chemicals belonging to these various chemical classes were
tested using INVITTOX Protocol No. 71 as part of the EC/HO study, but there were too
few in each class to determine the predictive capacity of this protocol for each chemicalclass. Further testing could determine the predictive capacity of the protocols for thesedifferent chemical classes. The choice of test chemicals representing these various
chemical classes requires careful consideration in order to allow the effects of otherphysical properties such as viscosity and colour to be distinguished.
Conclusions
There was insufficient data to equally analyse the four INVITTOX Protocols. The largest
high quality data sets (i.e. raw in vivo data used to calculate EU, GHS and EPA
classifications) were available for INVITTOX Protocol No. 71, INVITTOX Protocol No.
120, and to a lesser extent INVITTOX Protocol No. 86. With the exception of INVITTOX
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Protocol No. 71, all other FL INVITTOX protocols showed a greater predictive capacity
for the irritants rather than the non-irritants. None of the protocols showed a significantly
greater predictive capacity for any particular classification system. As the difference inpredictive capacities for the various classification systems was slight, the protocols
differed as to which classification system they had a slightly greater predictive capacityfor. The predictive capacity of INVITTOX Protocol No. 86 for surfactant-based
formulations was higher than that calculated for INVITTOX Protocol No. 71 for‘surfactants only.’ INVITTOX Protocol No. 120 had the greatest predictive capacity forsurfactants and surfactant-based formulations in comparison to INVITTOX Protocol No.
86 and INVITTOX Protocol No. 71 for ‘surfactants only.’ The greater predictive capacity
was hypothesised to be due to the measurement of FL 4h after, rather than immediately
after, the chemical exposure. INVITTOX Protocol No. 71 had a greater predictive
capacity for the surfactants rather than the alcohols tested.
Preliminary analyses for the effect of coloured test materials on the predictive capacityof the protocols indicated that INVITTOX Protocol No. 86 may be less adversely affected
than INVITTOX Protocol No. 71. This was hypothesised to be due to the greaternumber of washing steps following removal of the test material in INVITTOX Protocol
No. 86, in comparison to INVITTOX Protocol No. 71. Increased washing steps in other
INVITTOX Protocols could potentially enhance the removal of other types of test
materials and increase the reproducibility and robustness of the protocols.
As a result of the findings regarding the predictive capacities of the INVITTOX Protocols
analysed for this BRD, two recommendations can be made according to the intended
use of the FL assay. Firstly, if the FL assay is to be used to test surfactants andsurfactant-based formulations only, it is recommended that INVITTOX Protocol No. 120
is investigated further. If the FL assay is to be used to test a wider range of materials
from many different chemical classes, further testing could be carried-out using
INVITTOX Protocol No. 71 to increase the existing data set for a wide range ofchemicals. As INVITTOX Protocol No. 120 measures FL 4h following the chemical
exposure to increase the predictive capacity of the assay for cationic surfactants, further
work could investigate the effect of FL measured at this time-point on the predictive
capacity of INVITTOX Protocol No. 71. It would also be interesting to discover if
modifications to the PM for INVITTOX Protocol No. 71 would increase the predictive
capacity of this protocol. For any protocol examined in the future, further work is needed
to determine the effects of physical properties of the test chemicals such as colour andviscosity on the predictive capacity.
The principal advantage of the FL assay is that it can measure recovery and delayed
effects from the initial acute test material exposure for up to 96h on the same set ofcells. Recovery from effects is an important element in the EU, GHS and EPA
classification systems for ocular irritation. Use of the FL assay to measure recovery anddelayed effects is only stated as part of INVITTOX Protocol No. 82 although other FL
assay INVITTOX Protocols have also been used to measure recovery. However, there
were no large data sets with high quality in vitro recovery data and corresponding in vivo
data available for analyses for this BRD. Inclusion of recovery data for the classificationof some types of chemicals could potentially increase the predictive capacity of the FL
assay protocols. The time-point of recovery to be considered for the classification of test
chemicals could vary according to chemical class (Cottin and Zanvit, 1997). The
authors of this BRD suggest that further work could investigate the use of recovery datafor increasing the predictive capacity of either INVITTOX Protocol No. 71 or INVITTOX
220

Fluorescein Leakage Assay Background Review Document

Protocol No. 120 which already have existing large data sets for immediate chemicalinduced effects.

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8. Supporting materials
8.1. Relevant publications, other scientific reports and review (in chronologicalorder).
Refer to separate volume222

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8.2. Relevant unpublished data223

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