皮膚感作性試験代替法 rLLNA

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

1

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

7

ICCVAM Test Method Evaluation Report
The Reduced Murine Local Lymph Node Assay: An Alternative Test Method
Using Fewer Animals to Assess the Allergic Contact Dermatitis Potential ofChemicals and Products----------------------------------------------------------------------------------------------------- 15

皮膚感作性試験代替法 Reduced Local Lymph Node Assay (rLLNA)の評価会議報告書

JaCVAM 評価会議

平成 24 年（2012 年）7 月 4 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

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皮膚感作性試験代替法 Reduced Local Lymph Node Assay (rLLNA)について、第三者評価委員会
からの報告を受け１）、以下の 9 項目について審議した。本 2～8 項目は OECD ガイダンス文書 No．34に示された検討項目である２）。なお、本動物実験代替法の利用にあたっては、適用範囲を十分に配慮した上で使用されるべきである。
＜審議内容＞
１．検討対象の試験法は、日本のどの法規制やガイドラインに関係しているか。
Reduced local lymph node assay (rLLNA) は、化学物質の皮膚感作性を評価する試験法である。
rLLNAの原理、試験手順及び判定基準は、既にOECDテストガイドラインに収載されているLocal
lymph node assay (LLNA) と同一で、一用量のみで試験する点だけが異なる。
rLLNAは、医薬品、医療機器、医薬部外品又は化粧品の原料、その他の物質又は製品に関する法
規制又はガイドラインが求めている皮膚感作性に関係する。
２．検討対象の試験法とその妥当性を示すデータは、透明で独立な評価を受けているか。
ICCVAM は、第三者評価委員会を組織し、11 施設で実施された LLNA の試験成績を基に、rLLNAの試験法及び妥当性を評価した。その組織及び評価結果は ICCVAM のホームページで公表されている。
また、我が国の rLLNA 第三者評価委員会は、上記 ICCVAM の結論を評価し、その結果を評価会
議に提出した。この結果も、評価会議での審議が終了後公開される予定である。
よって、rLLNA は透明で独立な評価を受けていると判断する。
３．当該試験法で得られるデータは、対象毒性を十分に評価あるいは予測できるものであるか。デー
タは、当該試験法と従来の試験法の、代替法としての繋がりを示しているか。あるいは（同時に）そのデータは、当該試験法と、対象としているあるいはモデルとしている動物種についての影響との繋がりを示しているか。
皮膚感作性物質は、適用部位の所属リンパ節を活性化する。従来のLLNAはこの原理に基づき、マウスに種々の濃度の被験物質を適用し、所属リンパ節細胞の増殖反応を[3H-methyl] thymidineの取り込みを指標として求め、皮膚感作性の有無を評価する。rLLNAも同様の原理に基づく試験法である。
LLNAでは適切な用量範囲で試験した場合、反応強度との間に用量相関性があるとされている。当
該試験法で得られるデータは、最も反応が強く表れると想定される一用量での作用をとらえるものであるが、適切な用量が設定されれば、LLNAと同様に、ヒトに対する被験物質の皮膚感作性を十分に評価あるいは予測できるものである。
４．当該試験法は、ハザードあるいはリスク、あるいはその両方を評価するのに有用であるか。
rLLNAは、被験物質の皮膚感作性の有無のみを評価するものであり、適用用量と皮膚感作性の程度との関係を調べるものでは無い。
５．当該試験法とその妥当性を示すデータは、その試験法で安全性を保証しようとする、行政上のプログラムあるいは関係官庁が対象としている化学物質や製品を、十分広く対象としたものとなっ3

ているか。当該試験法が適用できる条件及び適用できない条件が明確であるか。
rLLNA試験法とその妥当性を示すデータは、様々な用途で使用されている被験物質465物質について行われた11施設でのLLNAの試験成績を元にICCVAMが作成したものであり、その整合性を我が国のrLLNA第三者評価委員会で評価したものである。これらのデータによりrLLNAの皮膚感作性試
験としての妥当性が示されている。よって、上記製品又は原料を幅広くカバーするデータが提供されていると見なせる。
当該試験法においては、過度の局所刺激性や明白な全身毒性を示さない最高用量で試験するが、LLNAと比較して1.9%（6/315）の偽陰性がみられている。その原因としては、設定された最高用量での反応性が低下したことが考えられる。
偽陰性の疑いがある場合は被験物質の毒性や物性等の情報を集め、また用量反応情報が必要な場合
はLLNAを実施すべきである。
６．当該試験法は、プロトコルの微細な変更に対して十分頑健で、適切な訓練経験を持つ担当者と適切な設備のある施設において、技術習得が容易なものであるか。
LLNA法と原理的に同じであることから、rLLNAの精度、施設内及び施設間再現性及び頑健性は
LLNAと同等と考えられるが、一用量のみを用いることから、その用量の選定や被験物質の溶解性等により評価がばらつく可能性がある。よって本試験法は、ICCVAMが推奨する最新の試験計画書に準拠する必要がある。
適切な訓練経験を持つ担当者と適切な設備のある施設において、上記の留意点以外においては、
rLLNAはLLNAと比べて技術的に困難になるところはなく、技術習得は容易である。
７．当該試験法は、時間的経費的に有用性があり、行政上で用いられやすいものであるか。
rLLNAは、LLNAと同じ試験手順をとることから、LLNAに対して時間的な優位性は小さいが、使用動物数を最大40%まで削減でき、経費的には優位である。
化学物質の皮膚感作性の有無を区別するために、まずrLLNAを実施し、偽陰性の可能性がある場
合は被験物質の毒性や物性等の情報を集め、用量反応情報を得るためのLLNAの実施を考慮すべきである。
陽性反応が得られた場合において、行政上、その用量依存性が求められたときは追加試験が必要となる。
８．当該試験法は、従来の試験法と比べて、科学的・倫理的・経済的に、新しい試験法あるいは改訂
試験法であることが正当化されているか。
rLLNAは、被験物質を一用量のみで試験することにより従来のLLNAに比べて使用動物数を削減でき、かつ同等の精度を有する改訂試験法であることが、ICCVAM及び我が国のrLLNA第三者評価委員会で評価されている。
９. 安全性評価のための行政的資料として、 受け入れ可能な試験法であるか。
rLLNAは、医薬品、医療機器、医薬部外品又は化粧品の原料、並びにその他の物質又は製品の皮
膚感作性の有無を評価するために有用である。
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rLLNAでは用量反応情報は得られないため、リスクアセスメントを行うための評価用資料として利用するには不十分であるが、rLLNAで陰性と判定された物質は、他の科学的情報から皮膚感作性が疑われる可能性がある場合を除き、これ以上の皮膚感作性に関する試験を求めないとすることも可能である。
以上の審議の結果、JaCVAM 評価会議は、皮膚感作性試験代替法 rLLNA について以下のように結論した。
本試験法は、従来の LLNA を一用量で行い使用動物数を削減する改訂試験法であり，LLNA と同等の検出感度を有する。
本試験法は、最新の ICCVAM が推奨する試験計画書に準拠し、偽陰性（最高用量での反応低下等）の可能性を理解して適切に試験を実施し、結果を評価するとき、行政上のプログラムあるいは関係官
庁が広く対象としている化学物質や製品の皮膚感作性の有無を科学的に評価できる。
参考文献
１．皮膚感作性試験代替法 Reduced Local Lymph Node Assay (rLLNA)の第三者評価報告書
２．OECD (2005) OECD Series on testing and assessment Number 34, Guidance document on thevalidation and international acceptance of new or updated test methods for hazard
assessment, ENJ/JM/MONO(2005) 14
３．Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM),
National Toxicology Program (NTP), et al. Background Review Document:“in vitro
Cytotoxicity Test Methods for Estimating Acute Oral systemic Toxicity”, NIH Publication
No: 07-4518

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Reduced Local Lymph Node Assay (rLLNA、単群局所リンパ節試験＊)の第三者評価報告書（＊ 試験法の邦文名は、提案名称）

平成 23 年 11 月 19 日

皮膚感作性試験代替法評価委員会

委員長

金澤由基子（医薬品機構）委員

牧栄二（元・安評センター）1

皮膚感作性試験法としては、皮膚外用剤として用いる医薬品ならびに化粧品原料を含む
化学物質等の皮膚での接触感作性のリスクを動物で予測する試験法としてモルモットを用いる Maximization Test（GPMT）法があり、その試験成績は皮膚外用剤の臨床での皮膚感
作性と良い相関性を有することが示されている。また、GPMT の代替試験法として、マウスを用いる Local Lymph Node Assay (LLNA，局所リンパ節試験)があり、その予測率は、
GPMT に劣らないとされ、国際的に認知されている。両者の違いは、GPMT が感作誘発期の皮膚反応を肉眼的に判定するのに対して、LLNA は感作に基づく耳介リンパ節の細胞増殖反応を[3H-methyl]-thymidine（3H-TdR）の取り込み量を測定することで定量的かつ客観的に判定するところにある。また、2010 年に改定された OECD テストガイドライン（TG）
429 には、LLNA と Reduced Local Lymph Node Assay (rLLNA)が記載されている。
rLLNA は、その手法において元の LLNA の試験法を踏襲するものであり、異なる点は、
LLNA が 3 用量の被験物質で検討されるのに対し、rLLNA は 1 用量の被験物質で皮膚感作性を検討するところにある。また、rLLNA は、動物福祉の 3R の原則（動物実験代替法の活用（replacement）、使用動物数の削減（reduction）、苦痛の軽減（refinement））に沿ったものであり、使用動物数においては、元の LLNA と比較して、最大（注解 40％まで動物数が削減される（注解：陽性対照群が設定しない場合）。
本報告では、本邦において未だ検証されていない rLLNA の試験法について、その有用性と限界に関する ICCVAM の勧告（2008）を基に編纂を行う。
１． 試験法
1-1

原理

rLLNA の試験法の原理は、LLNA の試験法の原理と同じである。即ち、皮膚感作性を有する低分子化合物が経皮投与されると、皮膚組織中のタンパク質と結合し、感作抗原として皮膚の樹状細胞に認識される。その後、樹状細胞は活性化しながら皮膚から所属リンパ節へ遊走し、抗原提示を行い抗原特異的な T リンパ球細胞の増殖を誘導する。この一連の生体応答が感作誘導期である。LLNA および rLLNA では、感作誘導期のリンパ節における抗原特異的な T リンパ球細胞の増殖を、3H-TdR の核酸成分への取り込みを指標として検出する。LLNA と rLLNA の試験法における唯一の違いは、被験物質の試験に用いる用量数にある。LLNA においては、最大溶解度で過度の局所刺激性を示さず、更には、全身毒性を示さない濃度を最高用量とし、各被験物質に対して少なくとも 3 用量が試験に使用されるが、rLLNA においては、LLNA で示す最高用量の 1 用量のみが試験に使用される。
1-2

試験手順および判定

1-2-1

試験手順

8-12 週齢の CBA/J あるいは CBA/Ca 系の雌マウスを用い、マウスの両耳に被験物質を各々25μL、3 日間塗布し、その 3 日後に 3H-TdR を尾静脈投与する。その 5 時間後に耳介
2

リンパ節を摘出し、その耳介リンパ節細胞中に取り込まれた 3H-TdR 量を測定する。
1-2-2

判定

溶媒対照群に対する被験物質投与群の 3H-TdR 取り込み量の比（Stimulation index：SI）
が 3 倍を越えた際に、陽性と判定する。判定においては、陰性対照群および陽性対照群の反応も考慮する。LLNA においては、結果が明確でない場合、用量相関性の強さ、統計学的有意差も考慮されるが、rLLNA では実施できない。
２．rLLNA の精度
潜在的に皮膚感作性を有する物質を確認するための rLLNA の精度は、LLNA で実施された 11 施設の試験成績を基に、retrospective に評価された。
LLNA では 471 被験物質のうち、318 被験物質が陽性であり、153 被験物質が陰性であった。或る被験物質が同じ溶媒で 1 回以上試験されている場合を 1 試験として数えると、465 被験物質が評価の対象となり、その中の 315 被験物質が感作性として分類され、150
被験物質が非感作性として分類された。6 被験物質は、LLNA において最高用量以外の用量の SI 値が 3 以上を示したため陽性と評価された。換言すれば、rLLNA においては偽陰性となる。rLLNA は、最高用量でのみ試験され、評価されるため、これら 6 被験物質
（2-Methyl-2H-isothiazole-3-one、C19-azlactone、Azithromycin、Camphorquinone、
Nickel sulfate および Non-ionic surfactant 2）は、LLNA と比較すると、非感作性物質として位置づけられた。
３． rLLNA の施設間再現性
rLLNA の施設間再現性は、2 或いは 3 施設において、次の 5 被験物質について同じ溶媒を用いて個々に試験され、LLNA と比較して評価された。
・Dinitrochlorobenzene (DNCB)・Hexyl cinnamic aldehyde (HCA)・Linalool alcohol
・Methyl salicylate
・Potassium dichromate
各施設における全ての試験は、DNCB と potassium dichromate を感作性物質として、
methyl salicylate を非感作性物質として分類した（一致率 100％）。
HCA と linalool alcohol は、2 施設で試験され、LLNA 試験では感作性物質として分類され、rLLNA では非感作性物質として分類された。これら 2 試験法における一致しない結果を検証してみると、試験された最高用量での両試験における結果の違いによるものであった。しかしながら、rLLNA と LLNA は同一の試験計画書を使用しており、両者の精度を評価するために使用した一連のデータは類似しているため、rLLNA の施設内および施設間信
頼性は、LLNA に類似するものと考えられる。
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４． 試験法の有用性と限界
rLLNA の科学的な価値は、十分に評価されており、最新の ICCVAM が推奨した LLNA
試験計画書（ICCVAM 2008a）に準拠して行われたとき、rLLNA の成績は、用量-反応情報を必要としない場合には、皮膚感作性物質と非感作性物質を区別するに十分であると、
ICCVAM は結論している。
・ LLNA と比較すると、rLLNA は、各々の試験に対して最大 40％まで動物数を減らすことができる。
・ 化学物質や製品のアレルギー性接触皮膚炎 (ACD) の潜在性を調べるために LLNA を行う前に、先ず rLLNA を実施することを ICCVAM は勧めている。
－ rLLNA において陽性と評価された物質は、感作性物質として分類できる。
－ 用量-反応情報が必要な場合、用量段階を持つ LLNA において試験されなければならない。
－ ACD を誘発すると考えられる物質については、rLLNA よりむしろ LLNA を最初から実施して評価すべきである。
・ LLNA と比較して rLLNA に 1.9％ (6/318)の偽陰性の結果を示す可能性がある。
－ この情報は rLLNA の結果を評価するとき考慮されるべきであり、陰性の結果は常に補足情報（例えば、高用量での反応低下の可能性、類似物質による試験結果、タンパク結合活性、分子量、その他の試験データ）を統合して評価することを促すべきである。
－ もし偽陰性の結果が示唆されるならば、LLNA 或いは他の公認の皮膚感作性試験法による確認試験が考慮されるべきである。
５． 試験計画書
rLLNA は、ICCVAM が推奨する最新の LLNA 試験計画書に従って実施されることを
ICCVAM は勧めている。その重要な点は、以下の通りである。
・ 使用される最高濃度は、過度の局所刺激性や明白な全身毒性を示さない、最大溶解濃度であること。
・ 動物の成績は、個々に収集されること。
－ これは、偽陰性や偽陽性の結果を引き起こす外れ値の識別や棄却に備えるためである。
－ 動物の成績を個々に収集することは（合算して得た成績と比較して）、被験物質の反応が溶媒対照群の反応と有意に異なっているか否かを決めるための統計解析に備えるためである。
・ 投与群には最低 4 匹の動物が使用されること。
－ OECD TG 429 の LLNA では、投与群には少なくとも 4 匹の動物が必要であるとし4ている。
－ 統計解析によれば、投与群の動物数が 4 匹でも、統計解析の結果に有意な影響はな
いようである。
・ 陽性対照物質は、試験毎に使用されること。
－ これは、この試験の実施手順や試験系の全てが、陽性反応を生ずるために適切に反応しているかを明らかにする。
６． 今後の検討事項
ICCVAM は、潜在的な皮膚感作性を確認するための rLLNA を更に特徴付け、その有用性と応用性を高めるために下記の追加の検討を推奨している。
・ 今回の評価において、LLNA と比較し、rLLNA に偽陰性の結果を生じさせた６被験物
質の異常な用量反応の原因を明らかにするために努力すること。
－ この情報は、LLNA と比較して rLLNA の精度を高める方法を見つけるために役立つ。
－ 市販後調査や職業上の暴露に関する情報も含め、LLNA において異常な用量反応を示すこれらの物質やその他の物質のモルモットやヒトにおける成績も一緒に含めて考えるよう努めること。
・ LLNA および rLLNA の試験で、動物の成績は個々に収集すること。
－ 個別に集めれば外れ値があった場合、棄却することができる。
・ 試験法の精度を低下させずに投与群当たりの動物数を減らすため、動物からの成績は個々に集められ、解析されるべきである。
－ ICCVAM が推奨する最新の LLNA 試験計画書には、そのような判断のために必要な統計解析手法が組み込まれている。
－ また、最新の試験計画書には、一緒に設定する陽性対照群の動物数を、その施設の陽性対照の背景データを評価することにより、減らすことができるか決めるためのガイダンスも盛り込まれている。
７． rLLNA の試験計画書
Day 1 – 3：投与毎に 4 匹のマウスの各々の両耳の背面に適当な溶媒を用いた被験物質（LLNA で試験される最高用量）或いは対照物質の 25 μL
を塗布する。
↓
Day 4 – 5：無処置↓
Day 6：各々の動物の尾静脈に 20 μCi 3H-TdR 若しくは 2 μCi
125I-iododeoxyuridine と 10-5 M fluorodeoxyuridine を投与する。

5↓5 時間後：耳介リンパ節を個別に摘出し、押しつぶして単離細胞懸濁液を調製する。
↓
単離細胞懸濁液をリン酸緩衝生理食塩水で 2 回洗浄し、4℃で 18 時間、
5% trichloroacetic acid で DNA を沈殿させる。
↓
Trichloroacetic acid にペレットを再懸濁し、3H 測定にはシンチレーショ
ン溶液を加え、125I 測定には再懸濁したペレットをガンマー線測定チュー
ブに加え、放射能測定に備える。
↓
放射能測定：対照群と処置群の平均 dpm（毎分崩壊数）を測定する。
取り込み量（Stimulation index: SI）は、次の式より算出；
処置群の平均 dpm／対照群の平均 dpm
SI > 3：被験物質を感作性物質として分類
SI < 3：被験物質を非感作性物質として分類
８． まとめ・ rLLNA は、最新の ICCVAM が推奨する LLNA 試験計画書に準拠して行われるとき、皮膚感作性物質と非感作性物質を十分に区別することができると、ICCVAM は結論している。
・ 化学物質や製品の ACD の潜在性を調べるために、LLNA を行う前に rLLNA を実施す
ることを ICCVAM は勧めている。
・ LLNA と比較して、rLLNA では使用動物数を各試験当たり最大 40％まで削減できる。
・ rLLNA には 1.9％ (6/318)の偽陰性の結果を示す可能性がある。もし偽陰性の結果が示
唆されるならば、LLNA 或いは他の公認の皮膚感作性試験法による確認試験を考慮すべきである。
・ 用量-反応情報が必要な場合は、用量段階を持つ LLNA において試験されなければならない。
９． rLLNA 編纂委員会としての結論
・ rLLNA は、化学物質の皮膚感作性を評価することが可能な試験方法である。

6

・ rLLNA の結果が２ < SI < ３の場合、その判定に当たっては、被験物質に関する情報（例えば、最高用量での反応低下の可能性、タンパク結合性、類似化合物における皮膚感作性など）に基づいて慎重に評価する必要がある。
・ rLLNA の結果が SI > ３であれば、これ以上の皮膚感作性試験は不要である。
・ LLNA や GPMT により非感作性物質と評価された原薬（化学物質）で製造された製品の評価には、rLLNA のみの実施でよい。
・ rLLNA の邦文名として「単群局所リンパ節試験」を提案する。
１０． 文献ICCVAM 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing
the Allergic Contact Dermatitis Potential of Chemical/Compounds. NIH
Publication No. 99-4494. Research Triangle Park, NC: National Institute ofEnvironmental Health Sciences.
Available: http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
ICCVAM 2008a. Recommended Performance Standards. Murine Local Lymph Node
Assay. NIH Publication No. 07-4518. Research Triangle Park. NC: National
Institute of Environmental Health Sciences. Available: http://iccvam.niehs.nih.gov/
ICCVAM 2008b. The Reduced Murine Local Lymph Node Assay: An Alternative
Test Method Using Fewer Animals to Assess the Allergic Contact Dermatitis
Potential of Chemicals and Products. Research Triangle Park. NC: National
Institute of Environmental Health Sciences. Available: http://iccvam.niehs.nih.gov/
Kimber I, Deaman RJ, Betts CJ, Gerberick GF, Ryan CA, Kem PS, et al. 2006. The
local lymph node assay and skin sensitization: a cut-down screen to reduce animal
requirements? Contact Dermatitis. 54: 181-185
OECD 2002: Test guideline 429. Skin Sensitization: Local Lymph Node Assay,
adopted April 24, 2002. In: OECD Guidelines for Testing of Chemicals. Paris:
OECD. Available:
http://lysander.sourceoecd.org/vl=1706714/cl=11/nw=1/rpsv/cw/vhosts/oecdjournals/
1607310x/v1n4/contp1-1.htm
OECD 2010: Test guideline 429. Skin Sensitization: Local Lymph Node Assay,
adopted July 22, 2010. In: OECD Guidelines for Testing of Chemicals. Available:
7http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/OECD/OECD-TG429-2010.pdf
Wind M, Matheson J, Jacobs A, Tice R, Stokes W: ICCVAM Test Method
Recommendations for the Reduced LLNA (rLLNA): An Alternative Test Method
Using Fewer Animals to Assess the Allergic Contact Dermatitis Potential ofChemicals and Products. Available:
http://ICCVAM.niehs.nih.gov/meetings/SOT09/posters/Wind.pdf

8

ICCVAM TEST METHOD EVALUATION REPORTThe Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and Products

NIH Publication Number 09-6439

ICCVAM TEST METHOD EVALUATION REPORTThe Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and Products

U.S. Department of Health and Human Services

NIH Publication Number 09-6439

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

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

About the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM)andThe National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM)
In 1997, the National Institute of Environmental Health Sciences (NIEHS), one of the National
Institutes of Health, established ICCVAM to:¾¾

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

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

x
x
x
x
x

x
x
x
x

Consumer Product Safety Commission
Department of Agriculture
Department of Defense
Department of Energy
Department of Health and Human Services- Centers for Disease Control and Prevention

Agency for Toxic Substances and Disease Registry
National Institute of Occupational Safety and Health- Food and Drug Administration- National Institutes of Health
Office of the Director
National Cancer Institute
National Institute of Environmental Health Sciences
National Library of Medicine
Department of the Interior
Department of Labor- Occupational Safety and Health Administration
Department of Transportation
Environmental Protection Agency

The NICEATM-ICCVAM graphic symbolizes the important role of new andalternative toxicological methods in protecting and advancing the health of people,
animals, and our environment.

ICCVAM Test Method Evaluation Report
The Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and ProductsInteragency Coordinating Committee on the
Validation of Alternative Methods

National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods

National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

2009
NIH Publication No. 09-6439

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

ICCVAM Test Method Evaluation Report

This document is available electronically at
http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-LD/TMER.htm
When referencing this document, please cite as follows:
Interagency Coordinating Committee on the Validation of Alternative Methods. 2009.
ICCVAM Test Method Evaluation Report. The Reduced Murine Local Lymph Node Assay: An
Alternative Test Method Using Fewer Animals to Assess the Allergic Contact Dermatitis
Potential of Chemicals and Products. NIH Publication Number 09-6439. Research Triangle
Park, NC: National Institute of Environmental Health Sciences.

ICCVAM Test Method Evaluation Report

Table of Contents
List of Tables ......................................................................................................................iv
List of Abbreviations and Acronyms..................................................................................v
Interagency Coordinating Committee on the Validation of Alternative Methods:
Agency Representatives ....................................................................................................vii
Acknowledgements.............................................................................................................ix
Preface ............................................................................................................................... xv
Executive Summary .........................................................................................................xix
1.0

Introduction .............................................................................................................1

2.0

ICCVAM Recommendations for the rLLNA Test Method ...................................3

3.0

2.1

ICCVAM Recommendations: Test Method Usefulness and Limitations.........3

2.2

ICCVAM Recommendations: Test Method Protocol ......................................4

2.3

ICCVAM Recommendations: Future Studies .................................................6

2.4

ICCVAM Recommendations: Performance Standards....................................7

Validation Status of the rLLNA Test Method ........................................................9
3.1

Test Method Description ................................................................................93.1.1 General Test Method Procedures ........................................................93.1.2 Similarities and Differences between the Protocols
for the Traditional LLNA and the rLLNA...........................................9

4.0

3.2

Validation Database ..................................................................................... 10

3.3

Reference Test Method Data ........................................................................ 11

3.4

Test Method Accuracy ................................................................................. 12

3.5

Test Method Reliability................................................................................ 13

3.6

Animal Welfare Considerations: Reduction, Refinement, andReplacement ................................................................................................ 14

ICCVAM Consideration of Public and SACATM Comments ............................ 15
4.1

Public Comments in Response to 72 FR 27815 (May 17, 2007) ................... 15

4.2

Public Comments in Response to 72 FR 52130 (September 12, 2007).......... 16

4.3

Public Comments in Response to 73 FR 1360 (January 8, 2008) .................. 17

4.4

Public Comments in Response to 73 FR 29136 (May 20, 2008) ................... 18

4.5

Public and SACATM Comments: SACATM Meeting on June 18-19,
2008............................................................................................................. 18i

ICCVAM Test Method Evaluation Report

5.0

References.............................................................................................................. 19

Appendix A ICCVAM Evaluation of the Reduced Local Lymph Node Assay
Method – Timeline ................................................................................... A-1
Appendix B ICCVAM-Recommended Protocol (Updated 2008): The Murine
Local Lymph Node Assay: A Test Method for Assessing the Allergic
Contact Dermatitis Potential of Chemicals and Products...................... B-1
Appendix C Evaluating the Impact of Reducing the Sample Size from Five toFour Animals per Group on the Performance of the Ratio Rule of
SI >3 in LLNA Testing............................................................................. C-1
Appendix D Final Background Review Document: Reduced Murine Local Lymph
Node Assay ............................................................................................... D-1
Appendix E ESAC Statement on the Validity of the rLLNA ..................................... E-1
Appendix F Independent Scientific Peer Review Panel Assessment .......................... F-1
F1

Peer Review Panel Report: Validation Status of New Versions andApplications of the Murine Local Lymph Node Assay: A Test Method forAssessing the Allergic Contact Dermatitis Potential of Chemicals andProducts......................................................................................................F-3

F2

Summary Minutes of Independent Scientific Peer Review Panel Meetingon March 4-6, 2008.................................................................................F-161

Appendix G Federal Register Notices and Public Comments......................................G-1
G1
Federal Register Notices ............................................................................ G-3
G1-1 72 FR 27815 (May 17, 2007): The Murine Local Lymph Node
Assay: Request for Comments, Nominations of Scientific Experts, andSubmission of Data....................................................................................G-5
G1-2 72 FR 52130 (September 12, 2007): Draft PerformanceStandards for the Murine Local Lymph Node Assay: Request forComments..................................................................................................G-9
G1-3 73 FR 1360 (January 8, 2008): Announcement of an Independent
Scientific Peer Review Panel Meeting on the Murine Local Lymph Node
Assay; Availability of Draft Background Review Documents; Request
for Comments ..........................................................................................G-11
G1-4 73 FR 29136 (May 20, 2008): Peer Review Panel Report on the
Validation Status of New Versions and Applications of the Murine
Local Lymph Node Assay (LLNA): A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemicals and Products:
Notice of Availability and Request for Public Comments.........................G-15
G2

Public Comments Received in Response to Federal Register Notices....... G-17

G3

SACATM Comments: SACATM Meeting on June 18-19, 2008 .............. G-51
ii

ICCVAM Test Method Evaluation Report

Appendix H Relevant Skin Sensitization Regulations and Testing Guidelines ..........H-1
H1 Relevant Skin Sensitization Test Regulations............................................................H-3
H2

OECD Guideline 429: Skin Sensitisation – Local Lymph Node Assay......................H-5

H3

OECD Guideline 406: Skin Sensitisation................................................................H-15

H4

EPA Health Effects Test Guidelines OPPTS 870.2600: Skin Sensitization..............H-27

iii

ICCVAM Test Method Evaluation Report

List of Tables
Table 1

Performance of the rLLNA in Predicting Skin Sensitizers Compared to
the Traditional LLNA ................................................................................ xxii

Table 3-1

Chemical Classes Represented in the Current Traditional LLNA Database .. 11

Table 3-2

Evaluation of the Performance of the rLLNA in Predicting Skin
Sensitizers Compared to the Traditional LLNA............................................ 13

Table 3-3

Interlaboratory Reproducibility of Skin Sensitization Outcome forthe rLLNA ................................................................................................... 14iv

ICCVAM Test Method Evaluation Report

List of Abbreviations and Acronyms
ACD
AOO
BAuA
BRD
CPSC
DMSO
DNCB
dpm
EC
EC3
ECPA
ECVAM
EFfCI
EPA
EPR
ESAC
FR
GP
GPMT
3
H
HCA
ICCVAM
ILS
125
IU
IWG
JaCVAM
LLNA
LNC
NC
NICEATM
OECD
PBS
rLLNA

Allergic contact dermatitis
Acetone: olive oil (4:1 by volume)
Federal Institute for Occupational Safety and Health (Germany)
Background review document
U.S. Consumer Product Safety Commission
Dimethyl sulfoxide
Dinitrochlorobenzene
Disintegrations per minuteEmulsion concentrate
Estimated concentration needed to produce a stimulation index of 3
European Crop Protection Association
European Centre for the Validation of Alternative Methods
European Federation for Cosmetic Ingredients
U.S. Environmental Protection Agency
Epoxy resin
ECVAM Scientific Advisory Committee
Federal Register
Guinea pig
Guinea Pig Maximization Test
Tritiated
Hexyl cinnamic aldehyde
Interagency Coordinating Committee on the Validation of Alternative
Methods
Integrated Laboratory Systems
125
I-iododeoxyuridine
Immunotoxicity Working Group
Japanese Center for the Validation of Alternative Methods
Murine local lymph node assay
Lymph node cells
Not calculated
National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods
Organisation for Economic Co-operation and Development
Phosphate-buffered saline
Reduced murine local lymph node assayv

ICCVAM Test Method Evaluation Report

SACATM
SC
SD
SI
TCA
TG
U.K.
U.S.
U.S.C.

Scientific Advisory Committee on Alternative Toxicological Methods
Suspension concentrateStandard deviation
Stimulation index
Trichloroacetic acid
Test Guideline
United Kingdom
United States
United States Codevi

ICCVAM Test Method Evaluation Report

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

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

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

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

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

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

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

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

National Institute for Occupational Safety and
Health• Paul Nicolaysen, V.M.D.
◊ K. Murali Rao, M.D., Ph.D.
National Institutes of Health• Margaret D. Snyder, Ph.D.
National Library of Medicine• Pertti (Bert) Hakkinen, Ph.D.
◊ Jeanne Goshorn, M.S.

• Principal agency representative◊ Alternate principal agency representative

Occupational Safety and Health Administration• Surender Ahir, Ph.D.
vii

ICCVAM Test Method Evaluation Report[This Page Intentionally Left Blank]viii

ICCVAM Test Method Evaluation Report

Acknowledgements
The following individuals are acknowledged for their contributions to the evaluation of the
reduced LLNA.
Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) Immunotoxicity Working Group (IWG)
U.S. Consumer Product Safety
Commission
Joanna Matheson, Ph.D. (IWG Co-Chair)
Marilyn Wind, Ph.D.

National Institute of Environmental
Health Sciences
Dori Germolec, Ph.D.
William S. Stokes, D.V.M., D.A.C.L.A.M.
Raymond R. Tice, Ph.D.

U.S. Environmental Protection Agency
Office of Pesticide Programs
Masih Hashim, D.V.M., Ph.D.
Marianne Lewis
Deborah McCall
Timothy McMahon, Ph.D.
Amy Rispin, Ph.D.
Office of Prevention, Pesticides, and Toxic
Substances
Ronald Ward, Ph.D.
Office of Research and Development
Marsha Ward, Ph.D.
Office of Science Coordination and Policy
Karen Hamernik, Ph.D.

National Institute for Occupational
Safety and Health
B. Jean Meade, D.V.M., Ph.D.
European Centre for the Validation of
Alternative Methods – Liaison
Silvia Casati, Ph.D.
Japanese Center for the Validation of
Alternative Methods – Liaison
Hajime Kojima, Ph.D.

U.S. Food and Drug Administration
Center for Devices and Radiological HealthDaniel Lyle, Ph.D.
Vasant G. Malshet, Ph.D., D.A.B.T.
Jeffrey Toy, Ph.D.
Center for Drug Evaluation and ResearchPaul Brown, Ph.D.
Abigail Jacobs, Ph.D. (IWG Co-Chair)
Jiaqin Yao, Ph.D.
Center for Veterinary Medicine
Ruth Barratt, Ph.D., D.V.M.

ix

ICCVAM Test Method Evaluation Report

Murine Local Lymph Node Assay
Independent Scientific Peer Review Panel(March 4–6, 2008)
Michael Luster, Ph.D. (Panel Chair)
Senior Consultant to the National Institutefor Occupational Safety and Health
Health Effects Laboratory
Morgantown, WV

Dagmar Jírová, M.D., Ph.D.
Toxicologist, Research Manager
Head of Reference Center for Cosmetics andReference Laboratory for Experimental
Immunotoxicology
National Institute of Public HealthPrague, Czech Republic

Nathalie Alépée, Ph.D.
Associate Research Fellow
Pfizer PDRD
MCT Laboratory
Amboise, FranceDavid Lovell, Ph.D.
Reader in Medical Statistics
Postgraduate Medical School
University of Surrey
Guildford, Surrey, U.K.

Anne Marie Api, Ph.D.
Vice President, Human Health Sciences
Research Institute for Fragrance Materials
Woodcliff Lake, NJ

Howard Maibach, M.D.
Professor, Department of Dermatology
University of California – San Francisco
San Francisco, CA

Nancy Flournoy, M.S., Ph.D.
Professor and Chair
Department of Mathematics and Statistics
University of Missouri – Columbia
Columbia, MO

James McDougal, Ph.D.
Professor and Director of Toxicology Research
Department of Pharmacology and
Toxicology
Boonshoft School of Medicine
Wright State University
Dayton, OH

Thomas Gebel, Ph.D.
Regulatory Toxicologist
Federal Institute for Occupational Safety &HealthDortmund, Germany

Michael Olson, Ph.D.
Director of Occupational Toxicology
Corporate Environment, Health and Safety
GlaxoSmithKline
Research Triangle Park, NC

Sidney Green, Ph.D.*
Professor of Pharmacology
College of Medicine
Howard University
Washington, DC

Raymond Pieters, Ph.D.
Associate Professor
Immunotoxicology Group Leader
Institute for Risk Assessment Sciences
Utrecht University
Utrecht, The Netherlands

Kim Headrick, B.Admin., B.Sc.
International Harmonization and Senior
Policy Advisor
Policy and Programme Service Office
Health Canada
Ottawa, Ontario, Canada

Jean Regal, Ph.D.
Professor, Department of Pharmacology
University of Minnesota Medical School
Duluth, MNx

ICCVAM Test Method Evaluation Report

Jonathan Richmond, MB ChB, FRCSEd*
Head, Animal (Scientific) Procedures
DivisionHome OfficeLondon, U.K.

Michael Woolhiser, Ph.D.
Technical Leader – Immunotoxicology
Senior Toxicology Specialist
Dow Chemical
Midland, MI

Peter Theran, V.M.D.
Consultant
Massachusetts Society for the Prevention of
Cruelty to Animals
Novato, CA

Takahiko Yoshida, M.D., Ph.D.
Professor, Department of Health Science
Asahikawa Medical College
Hokkaido, Japan

Stephen Ullrich, Ph.D.
Professor of Immunology
Graduate School of Biomedical Sciences
University of Texas
M.D. Anderson Cancer Center – Houston
Houston, TX* Drs. Green and Richmond were unable to attend the public meeting on March 4-6, 2008. However, they wereinvolved in the peer review of the background review documents and concur with the conclusions andrecommendations included in the Independent Scientific Peer Review Panel Report - Validation Status of New
Versions and Applications of the Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic
Contact Dermatitis Potential of Chemicals and Products.

xi

ICCVAM Test Method Evaluation Report

National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
William Stokes, D.V.M., D.A.C.L.A.M.
Director; Project Officer
Raymond Tice, Ph.D.
Deputy Director
Deborah McCarley
Special Assistant; Assistant Project Officer
NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)David Allen, Ph.D.
Eleni Salicru, Ph.D.
Thomas Burns, M.S.
Catherine Sprankle
Linda Litchfield
Judy Strickland, Ph.D., D.A.B.T.
Gregory Moyer, M.B.A.
Linda Wilson
Michael Paris
Statistical Consultant for ILS, Inc.
Joseph Haseman, Ph.D.

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ICCVAM Test Method Evaluation Report

Other Acknowledgements
ICCVAM and NICEATM gratefully acknowledge the following individuals and institutions that
submitted data to NICEATM for the evaluation of the reduced LLNA.
David Basketter, Ph.D.
Unilever Safety and Environmental
Assurance Centre
Sharnbrook, U.K.

Ian Kimber, Ph.D.
Syngenta Central Toxicology Laboratory
Macclesfield, U.K.
Michael J. Olson, Ph.D.
GlaxoSmithKline
Research Triangle Park, NC

Phil Botham, Ph.D.
European Crop Protection Association
Brussels, Belgium

K. Skirda
TNO Quality of Life
Delft, Netherlands

Eric Debruyne, Ph.D.
Bayer CropScience SA, Sophia Antipolis
Cedex, France
G. Frank Gerberick, Ph.D.
Procter and Gamble Company
Cincinnati, OH

Peter Ungeheuer, Ph.D.
European Federation for Cosmetic
Ingredients
Frankfurt, Germany

Dori Germolec, Ph.D.
National Toxicology Program
Research Triangle Park, NC

Hans Werner Vohr, Ph.D.
Bayer HealthCare
Wuppertal-Elberfeld, Germanyxiii

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ICCVAM Test Method Evaluation Report

Preface
Allergic contact dermatitis (ACD) is an adverse health effect that frequently develops inworkers and consumers exposed to skin-sensitizing chemicals and products. ACD results inlost workdays1 and can significantly diminish quality of life (Hutchings et al. 2001; Skoet et al.
2003). To minimize the occurrence of ACD, regulatory authorities require testing to identify
substances that may cause it. Sensitizing substances must be labeled with a description of thepotential hazard and the precautions necessary to avoid development of ACD.
Skin sensitization testing has typically required the use of guinea pigs (Buehler 1965;
Magnusson and Kligman 1970). However, in 1998, the Interagency Coordinating Committee
on the Validation of Alternative Methods (ICCVAM) evaluated an alternative known as themurine (mouse) local lymph node assay (traditional LLNA2). ICCVAM concluded that thetraditional LLNA provided several advantages over the commonly accepted guinea pig test
methods, including elimination of potential pain and distress, use of fewer animals, less timeto perform, and availability of dose-response information. ICCVAM recommended the LLNA
as an alternative test method for assessing the skin sensitization potential of most types ofsubstances. United States and international regulatory agencies subsequently accepted the
traditional LLNA as a valid alternative test method for ACD testing.
In 2007, the U.S. Consumer Product Safety Commission requested that ICCVAM evaluate
several modifications of the traditional LLNA, 3 including the “reduced LLNA” (rLLNA), also
referred to as the “cut-down” or “limit dose” LLNA. ICCVAM assigned this activity a highpriority after considering comments from the public and ICCVAM’s Scientific Advisory
Committee on Alternative Toxicological Methods (SACATM). As part of their ongoingcollaboration with ICCVAM, scientists from the European Centre for Validation of Alternative
Methods and the Japanese Center for Validation of Alternative Methods served as liaisons to
the ICCVAM Immunotoxicity Working Group (IWG). A detailed timeline of the rLLNA test
method evaluation is included with this report.
This Test Method Evaluation Report provides ICCVAM’s recommendations regarding theusefulness and limitations of the rLLNA for assessing the ACD potential of substances. Whendeemed appropriate for use, the rLLNA can reduce by 40% the number of animals used foreach test compared to the traditional LLNA. The report also provides the updated ICCVAMrecommended LLNA test method protocol, which addresses the rLLNA procedure. The
database of substances used to validate the rLLNA is discussed and summarized.
ICCVAM carefully compiled and assessed all available data and arranged an independentscientific peer review. ICCVAM and the IWG solicited and considered public comments andstakeholder involvement throughout the rLLNA evaluation process. The National Toxicology
Program Interagency Center for the Evaluation of Alternative Methods (NICEATM),ICCVAM, and the IWG began the process by preparing a draft background review document(BRD) describing the validation status of the rLLNA test method, including its reliability and1

http://www.bls.gov/IIF
The “traditional LLNA” refers to the validated ICCVAM-recommended LLNA test method protocol, whichmeasures lymphocyte proliferation based on incorporation of tritiated thymidine into the cells of the draining
auricular lymph nodes.
3
Available at http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
2xv

ICCVAM Test Method Evaluation Reportaccuracy for the substances evaluated, and draft test method recommendations for usefulness
and limitations. ICCVAM released these documents to the public for comment on January 8,
2008, at which time ICCVAM also announced a meeting of the international independentscientific peer review panel (Panel) (Federal Register 73 FR 13604).
The Panel met in public session on March 4–6, 2008, to review the ICCVAM draft BRD forcompleteness and accuracy. The Panel then evaluated (1) the extent to which the draft BRD
addressed established validation and acceptance criteria and (2) the extent to which the BRD
supported ICCVAM’s draft test method recommendations. Before concluding theirdeliberations, the Panel considered written comments and comments made at the meeting by
public stakeholders. The final Panel report was made available to the public for comment on
May 20, 2008.5
ICCVAM provided SACATM with the draft BRD and draft Test Method Evaluation Report,
the Panel report, and all public comments for discussion at their meeting on June 18-19, 2008,
where public stakeholders were given another opportunity to comment.
After SACATM’s meeting, ICCVAM considered the SACATM comments, the Panel report,
and all public comments before finalizing the ICCVAM Test Method Evaluation Report andBackground Review Document, which is provided as an appendix to this report. The
consolidated document will be provided to U.S. Federal regulatory agencies for consideration
and be made available to the public. The ICCVAM Authorization Act requires that Federal
agencies respond to ICCVAM within 180 days after receiving the ICCVAM test method
recommendations. Agency responses will be posted on the NICEATM–ICCVAM website6 asthey become available.
We gratefully acknowledge the many individuals who contributed to the preparation, review,and revision of this report. We especially recognize the Panel members for their thoughtful
evaluations and generous contributions of time and effort. Special thanks are extended to
Dr. Michael Luster for serving as the Panel Chair and to Dr. Michael Woolhiser, Dr. Michael
Olson, and Ms. Kim Headrick for their service as Evaluation Group Chairs. We thank the IWG
for assuring a meaningful and comprehensive review. We especially thank Dr. Joanna
Matheson (Consumer Product Safety Commission) and Dr. Abigail Jacobs (U.S. Food andDrug Administration Center for Drug Evaluation and Research) for serving as Co-Chairs of the
IWG. Integrated Laboratory Systems, Inc., the NICEATM support contractor, provided
excellent scientific and operational support, for which we thank Dr. David Allen, Mr. Thomas
Burns, Ms. Linda Litchfield, Mr. Michael Paris, Dr. Eleni Salicru, Ms. Catherine Sprankle, Dr.
Judy Strickland, and Ms. Linda Wilson; and Dr. Joseph Haseman, ILS consulting statistician,for statistical support. We also acknowledge Dr. Raymond Tice, Deputy Director of
NICEATM, for his efforts. Finally, we thank Dr. Silvia Casati and Dr. Hajime Kojima, theIWG liaisons from the European Centre for the Validation of Alternative Methods and theJapanese Center for the Validation of Alternative Methods, respectively, for their participationand contributions.

4

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_25553.pdf
Announced in 73 FR 29136 (http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E8-11195.pdf);
available at http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf
6
http://iccvam.niehs.nih.gov/methods/immunotox/rLLNA.htm
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ICCVAM Test Method Evaluation Report

This comprehensive ICCVAM evaluation of the rLLNA should facilitate regulatory agency
decisions on the acceptability of the method. Following regulatory acceptance, use of themethod by industry can be expected to significantly reduce the number of animals required for
ACD testing while continuing to support the protection of human health.
William S. Stokes, D.V.M., D.A.C.L.A.M
Rear Admiral/Assistant Surgeon General, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM
Marilyn Wind, Ph.D.
Deputy Associate Executive Director
Directorate for Health Sciences
U.S. Consumer Product Safety Commission
Chair, ICCVAM

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ICCVAM Test Method Evaluation Report

Executive Summary
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
evaluated the validation status of the reduced murine local lymph node sssay (rLLNA), a test
method for assessing the potential of substances to cause allergic contact dermatitis (ACD).
ACD is an allergic skin reaction characterized by redness, swelling, and itching that can result
from contact with a sensitizing chemical or product. This Test Method Evaluation Reportprovides ICCVAM’s recommendations regarding the usefulness and limitations of the rLLNA
as an alternative to the traditional murine local lymph node assay (LLNA). When deemed
appropriate for use, the rLLNA can reduce by 40% the number of animals used for each testcompared to the traditional LLNA. This report also includes the updated ICCVAMrecommended LLNA test method protocol, the final rLLNA background review document(BRD), and recommendations for future studies and performance standards.
The National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM), ICCVAM, and the ICCVAM Immunotoxicity Working
Group prepared a draft BRD and draft test method recommendations, which were provided to
an international independent scientific peer review panel (Panel) and the public for comment.
The BRD evaluated data from 471 traditional LLNA studies, including the 211 substances fromthe 1998 ICCVAM evaluation of the traditional LLNA (ICCVAM 1999), and 246 from thepeer-reviewed literature and submissions to NICEATM in response to a May 17, 2007, Federal
Register request for comments (72 FR 278157). A detailed timeline of the rLLNA test method
evaluation is included with this report.
The Panel met in public session on March 4–6, 2008, to discuss their peer review of the
ICCVAM draft BRD and to provide conclusions and recommendations on the current
validation status of the rLLNA test method. The Panel also reviewed how well the information
contained in the draft BRD supported ICCVAM’s draft test method recommendations. In
finalizing this Test Method Evaluation Report and the BRD, which is included as an appendix,
ICCVAM considered the conclusions and recommendations of the Panel and comments fromICCVAM’s Scientific Advisory Committee on Alternative Toxicological Methods and the
public.
ICCVAM Recommendations: Test Method Usefulness and Limitations
ICCVAM concludes that the scientific validity of the rLLNA has been adequately evaluatedand that the performance of the rLLNA, when conducted in accordance with the updated
ICCVAM-recommended LLNA protocol, is sufficient to distinguish between skin sensitizers
and non-sensitizers in cases that do not require dose-response information. ICCVAM also
concludes that, compared to the traditional LLNA, the rLLNA will reduce animal use by 40%
for each test. Accordingly, ICCVAM recommends that the rLLNA test method should be usedroutinely to determine the ACD potential of chemicals and products before conducting the
traditional LLNA. Negative substances can be classified as non-sensitizers, and positive
substances can be classified as sensitizers.
In cases that require dose-response information, positive substances must be tested in the
traditional multiple-dose LLNA. Therefore, if dose-response information is required for a
7

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
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ICCVAM Test Method Evaluation Report

substance that, after consideration of all available information, is also suspected of having thepotential to produce ACD, it should be evaluated initially using the traditional LLNA.
There is a small possibility of a false negative result (1.9% [6/318]) in the rLLNA compared tothe traditional LLNA. This information should be considered when evaluating results from therLLNA, and negative results should always prompt a weight-of-evidence evaluation of
supplemental information (e.g., possibility of downturn in response at the high dose, test resultswith similar substances, peptide-binding activity, molecular weight, other testing data). If false
negative results are suggested, confirmatory testing in the traditional LLNA or another acceptedskin sensitization test method should be considered.
ICCVAM Recommendations: Test Method Protocol
The updated LLNA test method protocol recommended by ICCVAM is included as anappendix to this report. In the traditional LLNA, at least three dose levels of each test substanceare evaluated. The rLLNA evaluates only the highest dose of the test substance along with theconcurrent vehicle- and positive-control groups. ICCVAM recommends testing only the highest
concentration, defined as the maximum soluble concentration that does not induce excessive
local irritation and/or overt systemic toxicity.
ICCVAM recommends that individual animal data should be collected in order to permit
identification and exclusion of outlier values that could cause false negative or false positive
results. Collection of individual animal data (versus pooled) also allows for statistical analysis
to determine whether the test-substance response is significantly different from that of thevehicle control.
The ICCVAM-recommended LLNA test method protocol has been revised to require aminimum of four animals per dose group. Data analysis indicated that reducing dose groups
from five animals to four is unlikely to significantly affect the results of an LLNA study.
Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 429
for the LLNA currently requires at least five animals per dose group if individual animal data
are collected but only four animals in each dose group if lymph nodes from all animals in thegroup are pooled into one sample for data collection (OECD 2002). To determine if theserequirements could be harmonized without diminishing accuracy, NICEATM evaluated datafrom 83 LLNA studies (275 dose groups) from six different laboratories. This revision isimportant because many national regulations and policies require that the minimum number ofanimals be used for studies. Therefore, once TG 429 is updated with the revision, thecollection of individual animal data will be consistent with this requirement.
ICCVAM also recommends including a positive-control substance with each test to ensure thatall protocol procedures are conducted properly and all aspects of the test system work properly
such that they can produce a positive response. However, similar to OECD TG 429, the updated
ICCVAM-recommended LLNA test method protocol states that laboratories that conduct the
LLNA at least once per month and that have a history of and a documented proficiency for
obtaining consistent results with positive controls may consider testing positive control
substances at intervals of no more than six months.

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ICCVAM Test Method Evaluation Report

ICCVAM Recommendations: Future Studies
ICCVAM recommends additional studies to further characterize and potentially improve theusefulness and applicability of the rLLNA for identifying potential skin sensitizers.
•

Additional efforts should be made to understand the basis for abnormal dose
responses for six substances in this evaluation that would have resulted in false
negative results using the rLLNA compared to the traditional LLNA. This
information should help identify ways to improve the accuracy of the rLLNA
compared to the traditional LLNA. Efforts should also be made to identify data fromguinea pigs and humans for substances that exhibit abnormal dose responses in the
traditional LLNA. Information from post-marketing surveillance and/or
occupational exposures should be collected and assessed.

•

All future traditional LLNA and rLLNA studies should collect individual animal
data. This will allow detection of outliers and avoidance of false negative results thatcan occur from pooling data that include one or more abnormally low values.
Existing LLNA studies using data pooled from all animals in a dose group, such as
four of the six false negative rLLNA results in this evaluation, should be evaluatedfurther with data obtained from individual animals within each dose group to
determine if pooling of data may have led to false negative outcomes.

•

Data from individual animals should be collected and analyzed to identify
opportunities to use fewer animals per dose group without compromising test
method accuracy. The updated ICCVAM-recommended LLNA test method protocol
incorporates statistical procedures necessary for such determinations. This includes
evaluating the laboratory’s historical positive-control database to determine if thenumber of animals in the concurrent positive-control group can be reduced.

ICCVAM Recommendations: Performance Standards
The ICCVAM-recommended test method performance standards for the traditional LLNA8 may
be used to evaluate the performance of modified test methods, including the rLLNA, that arefunctionally and mechanistically similar to the traditional LLNA. Modified protocols for the
rLLNA that adhere to the traditional LLNA performance standards would be considered
acceptable for hazard identification purposes.
Validation Status of the rLLNA Test Method
ICCVAM (1999) compared the accuracy and reliability of traditional LLNA results to resultsfrom guinea pig tests (EPA 2003) and results obtained from the human maximization test andsensitizing substances included in human patch test allergen panels. ICCVAM concluded that
the LLNA was a valid alternative to currently accepted guinea pig test methods for most testing
situations and that the LLNA reduces the number of animals required for testing while also
refining the procedure by eliminating animal pain and distress. The LLNA was subsequently
accepted by U.S. regulatory agencies as an alternative to the guinea pig tests (e.g., Guinea Pig
Maximization Test and Buehler Test) for assessing the potential of substances to cause ACD.

8

Available at http://iccvam.niehs.nih.gov/methods/immunotox/PerfStds/llna-ps.htm
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ICCVAM Test Method Evaluation Report

The only difference between the test method protocols for the traditional LLNA and the rLLNA
is the number of dose levels tested for a test substance. In the traditional LLNA, at least threedose levels are tested for each substance, with the highest dose based on maximum solubility
and the avoidance of excessive local irritation and/or systemic toxicity. In contrast, only thehighest dose of a substance is tested in the rLLNA (Kimber et al. 2006). Because the criteria forchoosing the highest dose in the traditional LLNA and in the rLLNA are the same, themaximum dose level tested in the traditional LLNA and that tested in the rLLNA should be thesame. Thus, the accuracy and reliability of the rLLNA test method should be similar for thesame substances tested in the traditional LLNA, although the accuracy was slightly different
based on available data described below.
Accuracy and Reliability of the rLLNA
The accuracy of the rLLNA for identifying potential skin sensitizers was compared to that of
the traditional LLNA. In the 471 traditional LLNA studies, 318 results were positive and
153 were negative. When studies in which substances were tested more than once in the samevehicle were combined to yield an overall skin sensitization classification, 465 studies withunique combinations of substances and vehicles were evaluated, with 315 classified assensitizers and 150 classified as non-sensitizers.
As shown in Table 1, compared to the traditional LLNA, the rLLNA has an accuracy of 98.7%(465/471), a sensitivity of 98.1% (312/318), a specificity of 100% (153/153), a false positive
rate of 0% (0/153), and a false negative rate of 1.9% (6/318). When only unique combinationsof substances and vehicles are considered, the rLLNA has an accuracy of 98.7% (459/465), asensitivity of 98.1% (309/315), a specificity of 100% (150/150), a false positive rate of 0%(0/150), and a false negative rate of 1.9% (6/315).
Table 1

Performance of the rLLNA in Predicting Skin Sensitizers Compared to the
Traditional LLNA
False
Positive

False
Negative100%(42/42)0% (0/42)

1.8%(3/169)98.1%(312/318)100%(153/153)

0% (0/153)

1.9%(6/318)98.1%(309/315)100%(150/150)

0% (0/150)

1.9%(6/315)

Data

N

Accuracy

Sensitivity

Specificity

Kimber et al. (2006)

211

98.6%
(208/211)98.2%(166/169)

rLLNA

471

98.7%
(465/471)

rLLNA(substances repeated in
the same vehicle
considered together)

465

98.7%
(459/465)

Abbreviation: N = number of tests
Accuracy = the percentage of correct outcomes (positive and negative) of a test method
Sensitivity = the percentage of all positive substances that are classified as positive
Specificity = the percentage of all negative substances that are classified as negative
False positive rate = the percentage of all negative substances that are falsely identified as positive
False negative rate = the percentage of all positive substances that are falsely identified as negative

Interlaboratory reproducibility of the rLLNA was assessed with traditional LLNA data forfive substances tested independently in the same vehicle at two or three laboratories:
dinitrochlorobenzene (DNCB), hexyl cinnamic aldehyde (HCA), linalool alcohol, methylxxii

ICCVAM Test Method Evaluation Reportsalicylate, and potassium dichromate. All studies classified DNCB, methyl salicylate, andpotassium dichromate as sensitizers or non-sensitizers (i.e., 100% concordance). HCA andlinalool alcohol, which were tested independently in two laboratories, were classified assensitizers by one traditional LLNA study and as non-sensitizers by the other study. Review ofthese two studies indicates that the discordant results were due to differences in the highest
dose levels tested. However, because the rLLNA and traditional LLNA use identical protocols
and the data sets used to evaluate their accuracy are similar, the intra- and interlaboratory
reliability of the rLLNA is deemed to be similar to that of the traditional LLNA.

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1.0

Introduction

The murine local lymph node assay (traditional LLNA9) is an alternative skin sensitization test
method that requires fewer animals and less time than currently accepted guinea pig tests (e.g.,
the Guinea Pig Maximization Test and the Buehler Test). It can also eliminate animal pain anddistress. The LLNA measures cell proliferation in the draining auricular lymph nodes of themouse by analyzing incorporation of a radioactive marker into newly synthesized DNA. The
LLNA was the first alternative test method evaluated and recommended by the U.S.
Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM).
International regulatory authorities have now recognized the traditional LLNA as an
acceptable alternative to guinea pig tests for most testing situations.
The reduced murine local lymph node assay (rLLNA), also referred to as the “cut-down” or“limit dose” LLNA, was one of several modified versions of the LLNA nominated by the U.S.
Consumer Product Safety Commission (CPSC) for evaluation by ICCVAM and the National
Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM).10 (The term “reduced LLNA” has been adopted in this document to beconsistent with the terminology used for this test method in Europe.) The proposed rLLNA
could reduce the number of animals used for skin sensitization testing by 40% for each testcompared to the traditional LLNA.
The ICCVAM Authorization Act of 2000 (Public Law 106-545, 42 United States Code 285l-3)
charged ICCVAM with coordinating the technical evaluations of new, revised, and alternative
test methods with regulatory applicability. After considering comments from the public and
ICCVAM’s advisory committee, the Scientific Advisory Committee on Alternative
Toxicological Methods (SACATM), ICCVAM members unanimously agreed that the rLLNA
should have a high priority for evaluation. A detailed timeline of the rLLNA test method
evaluation is provided in Appendix A. The updated ICCVAM-recommended LLNA test
method protocol, accompanying statistical evaluation, and final rLLNA background review
document (BRD) are provided in Appendices B, C, and D, respectively.
The ICCVAM Immunotoxicity Working Group (IWG) was formed to work with NICEATM in
evaluating the test methods. Dr. Silvia Casati was the European Centre for the Validation of
Alternative Methods (ECVAM) liaison, and Dr. Hajime Kojima was the Japanese Center for the
Validation of Alternative Methods (JaCVAM) liaison to the IWG.
To facilitate peer review of the validation status of the rLLNA, the IWG and NICEATM, whichadministers ICCVAM and provides scientific support for ICCVAM activities, prepared acomprehensive BRD that provided information and data from validation studies and scientific
literature. A May 17, 2007, Federal Register (FR) notice (72 FR 2781511) requested data andinformation on these test methods and nominations of individuals to serve on an international
independent scientific peer review panel (Panel). The request was also disseminated via the
ICCVAM electronic mailing list and through direct requests to over 100 stakeholders. Eight
9

The “traditional LLNA” refers to the validated ICCVAM-recommended LLNA protocol (ICCVAM 1999;
Dean et al. 2001), which measures lymphocyte proliferation based on incorporation of tritiated thymidine into
the cells of the draining auricular lymph nodes.
10
Available at http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
11
Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
1

ICCVAM Test Method Evaluation Reportindividuals submitted data and three individuals or organizations nominated members to the
Panel.
ICCVAM examined data from 471 traditional LLNA studies (318 sensitizers and 153 nonsensitizers) representing 457 unique substances. ICCVAM built on a recent assessment of thisprocedure by the ECVAM Scientific Advisory Committee (ESAC; ESAC 2007), which useddata from 211 traditional LLNA studies (211 unique substances) (Kimber et al. 2006). In an
April 2007 statement, ESAC concluded “that the peer reviewed and published information is ofa quality and nature to support the use of the rLLNA within tiered-testing strategies to reliably
distinguish between chemicals that are skin sensitisers and non-sensitisers…” (Appendix E)
On January 8, 2008, ICCVAM announced the availability of the ICCVAM draft BRD and apublic Panel meeting to review the validation status of the rLLNA (and other modifications to
the traditional LLNA) (73 FR 136012). The ICCVAM draft BRD and draft test method
recommendations were posted on the NICEATM–ICCVAM website. 13 All of the information
provided to the Panel and all public comments received prior to the Panel meeting were madeavailable on the NICEATM–ICCVAM website.
The Panel met in public session on March 4–6, 2008, to review the rLLNA’s validation status
and the completeness and accuracy of the ICCVAM draft BRD. The Panel evaluated (1) the
extent to which the draft BRD addressed established validation and acceptance criteria and(2) the extent to which the BRD supported ICCVAM’s draft proposed test method uses,
recommended protocols, draft test method performance standards, and proposed future studies.
Interested stakeholders from the public were provided opportunities to comment at the Panelmeeting. The Panel considered these comments as well as those submitted prior to the meeting
before concluding their deliberations. On May 20, 2008, ICCVAM posted a report of the
Panel’s recommendations14 (see Appendix F) on the NICEATM–ICCVAM website for public
review and comment (announced in 73 FR 2913615).
ICCVAM provided SACATM with the draft BRD and draft test method recommendations, thePanel report, and all public comments for discussion at their meeting on June 18–19, 2008,
where public stakeholders were given another opportunity to comment.
ICCVAM and the IWG considered the SACATM comments, the Panel report, and all publiccomments when finalizing the test method recommendations provided in this report. As
required by the ICCVAM Authorization Act, ICCVAM will make this Test Method Evaluation
Report and the accompanying final BRD available to the public and to U.S. Federal agencies
for consideration. Federal agencies must respond to ICCVAM within 180 days after receiving
ICCVAM test method recommendations. Agency responses will be made available to the public
on the NICEATM–ICCVAM website as they are received.

12

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_25553.pdf
http://iccvam.niehs.nih.gov/methods/immunotox/llna-panelDocs.htm
14
Available at http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf
15
Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E8-11195.pdf
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2.0

ICCVAM Recommendations for the rLLNA Test Method

ICCVAM evaluated the validation status of the rLLNA test method as a reduction alternative tothe traditional LLNA. The rLLNA should be used for the hazard identification of skinsensitizing substances if dose-response information is not needed (e.g., for a compound
presumed to be a strong sensitizer), provided there is adherence to all other LLNA protocol
specifications as described in the updated ICCVAM-recommended LLNA test method protocol(available in Appendix B and at the NICEATM–ICCVAM website16). To further reduce animaluse, the rLLNA should be used routinely as an initial test to determine allergic contact
dermatitis (ACD) potential of chemicals and products before conducting the traditional LLNA.
Negative substances can be classified as non-sensitizers, and positive substances can beclassified as sensitizers.
Where dose-response information is required (e.g., for a compound presumed to be a weak orborderline sensitizer), positive substances must be tested in the traditional multidose LLNA.
Accordingly, those substances for which dose-response information will be required and thatare also suspected of having allergic contact dermatitis potential following consideration of allavailable information should be initially evaluated using the traditional LLNA.
2.1

ICCVAM Recommendations: Test Method Usefulness and Limitations

NICEATM and ICCVAM conducted a retrospective evaluation of rLLNA data to determine thetest method’s ability to distinguish between skin sensitizers and non-sensitizers. The
performance assessment for the 465 unique substance and vehicle combinations evaluated in
the study is provided in Section 3.0. Based on a review of the available data and comparison
with the traditional LLNA, the scientific validity of the rLLNA has been adequately evaluated.
ICCVAM concluded that, when conducted in accordance with the updated ICCVAMrecommended LLNA test method protocol specifications included in Appendix B, therLLNA’s performance is sufficient to distinguish between skin sensitizers and non-sensitizers
when dose-response information is not required. This recommendation is based on its
performance compared to that of the traditional LLNA. ICCVAM also concludes that use of therLLNA can reduce by 40% the number of animals used for each test.
There is a small possibility of a false negative result (1.9% [6/318]) when compared to the
traditional LLNA. This information should be considered when evaluating results from therLLNA, and negative results should always be subjected to a weight-of-evidence evaluation of
supplemental information (e.g., possibility of downturn in response at the high dose, test resultswith similar substances, peptide-binding activity, molecular weight, other testing data). If false
negative results are suggested, confirmatory testing in the traditional LLNA or another acceptedskin sensitization test method should be considered.
All of the testing limitations that apply to the traditional LLNA apply to the rLLNA also. For
example, the rLLNA may not be suitable for use with certain types of test substances, such as
nickel salts, mixtures, high-molecular weight compounds that cannot penetrate the stratum
corneum, strong dermal irritants, or chemicals whose pharmacodynamic activity is to releasedermal cytokines that cause local lymph node proliferation (e.g., certain pharmaceuticals such16

http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAprotocol2008.pdf
3

ICCVAM Test Method Evaluation Report

as imiquimod [Gaspari 2007]). Additionally, the rLLNA may not be suitable for test substancesthat do not adhere for an acceptable period of time when applied to the dorsum of the ear.
Independent Peer Review Panel Conclusions and Recommendations
The Panel agreed that the available data support ICCVAM’s draft recommendation that therLLNA should be routinely recommended for hazard identification when dose-response
information is not required. The Panel also agreed that to further reduce animal use the rLLNA
should be routinely recommended as the initial test to identify sensitizers even if dose-response
information is required, because negative results would not require additional testing. This isapplicable in the occupational and public health setting in which obtaining hazard information
is of critical importance. Subsequent traditional LLNA testing of substances that were positivein the rLLNA will provide dose-response information to assure detection of hazardous
substances and allow potency estimates. The benefits of screening out the negatives, which do
not require dose-response information, are clear; however, the animal welfare gains will dependon the proportion of test substances in any class that turn out to be non-sensitizers. The possible
consequences of delays from another round of testing of those materials identified as sensitizers
should also be considered.
The Panel agreed that the draft test method recommendations adequately addressed the low
false negative rate by giving cautionary and weight-of-evidence consideration to the negative
substances (and any possible false positive results). Furthermore, the Panel concluded thatinterspecies differences between the animal model and humans would probably make the false
negative rate unimportant.
2.2

ICCVAM Recommendations: Test Method Protocol

ICCVAM recommends basing the protocol for rLLNA testing on the updated ICCVAMrecommended LLNA protocol, which addresses the rLLNA procedure (Appendix B). The only
difference between the traditional LLNA and the rLLNA test methods is that the middle- andlow-dose groups are omitted in the rLLNA. On the basis of Panel comments, ICCVAM updated
the traditional LLNA test method protocol to provide guidance on identifying the appropriate
maximum dose for testing. In the rLLNA, in addition to the concurrent vehicle and positivecontrol groups, each test substance is tested at only one dose level (the high dose), whereas in
the traditional LLNA each test substance is tested at a minimum of three dose levels. The test
substance concentration should be the highest soluble concentration that does not induce overt
systemic toxicity and/or excessive local irritation. Any other approach, such as one based on apre-established threshold dose level, is inappropriate. For example, Kimber et al. (2006)
proposed a 10% threshold concentration at which all negative results would be consideredvalid. However, 51 (16% [51/315]) of the test substances evaluated were non-sensitizers atconcentrations of at least 10%17 but were sensitizers at higher concentrations.
In the traditional LLNA test method protocol, a stimulation index (SI) is calculated as the ratioof the mean incorporation of 3H-thymidine or 125I-iododeoxyuridine by the auricular lymph
nodes of the treated animals and that of the vehicle control animals. In the rLLNA, as in the
traditional LLNA, the threshold for classifying a substance as a skin sensitizer is an SI ≥ 3.
17

An initial dose was tested at 10% or greater and resulted in a stimulation index (SI) < 3, while a subsequent
higher dose resulted in an SI ≥ 3.
4

ICCVAM Test Method Evaluation Report

In the updated LLNA test method protocol (Appendix B), ICCVAM recommends collecting
individual animal data in order to allow identification and exclusion of outlier values that could
result in false negative or false positive results. This is especially important to help avoid false
negative results for weaker sensitizers (i.e., substances that induce an SI just above 3). The U.S.
Environmental Protection Agency (EPA) Health Effects Test Guideline 870.2600 (EPA 2003)
also requires the collection of individual animal data for the assessment of interanimal
variability and a statistical comparison of test- and control-group measurements. While theOrganisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 429(OECD 2002) allows for both the collection of individual animal measurements and the poolingof the lymph nodes for each treatment group, the latter eliminates any measure of interanimal
variability and/or identification of outlier values, as well as statistical identification of apositive/negative response.
OECD TG 429 requires that each dose group consist of at least four animals if pooled animal
data are collected and a minimum of five animals if individual animal data are collected (OECD2002). To determine if the required number of animals for individual animal data collection
could be the same as the required number for pooled data without diminishing accuracy,
NICEATM evaluated data from 83 LLNA studies (275 dose groups) from six different
laboratories (Appendix C). This is important because most animal-use regulations require thatthe minimum number of animals be used in studies, which currently results in many countries
collecting only pooled data because doing so requires fewer animals. This evaluation indicatedthat a reduction in the sample size from five to four animals per group is unlikely to have asignificant impact on the results of an LLNA study; therefore, the ICCVAM-recommended
LLNA test method protocol (Appendix B) was revised to require a minimum of four animals
per dose group.
The updated ICCVAM-recommended LLNA test method protocol (Appendix B) also
recommends that each test include a concurrent positive-control substance. Use of a positivecontrol substance can ensure that all protocol procedures are conducted properly and that all
aspects of the test system work properly such that they produce a positive response. However,similar to OECD TG 429 (OECD 2002), the updated ICCVAM-recommended test method
protocol states that testing of the positive-control substance at intervals of no more than six
months may be considered in laboratories that conduct the LLNA at least once per month andthat have a history and a documented proficiency for obtaining consistent results with positive
controls.
Users should be aware that the decision to include a positive control only periodically instead ofconcurrently could affect the adequacy and acceptability of negative study results generated
without a concurrent positive control. For example, if a false negative result is obtained in the
periodic positive-control test, all negative test-substance results obtained since the last
acceptable periodic positive-control test and the unacceptable periodic positive-control test
could be questioned. In order to demonstrate that the prior negative test-substance results are
acceptable, a laboratory could be expected to repeat all negative tests, which would require
additional expense and increased animal use.
Independent Peer Review Panel Conclusions and Recommendations
The Panel agreed with ICCVAM’s draft test method recommendations and recommended
adherence to the ICCVAM-recommended LLNA protocol (with modifications omitting the5

ICCVAM Test Method Evaluation Report

middle- and low-dose groups) for future rLLNA testing. The Panel also advised collecting
individual animal data for future studies because it would allow an estimate of interanimal
variability and conducting a statistical analysis to determine if the test substance is significantly
different from the control substance.
The Panel agreed that the current recommendation to select a maximum applied dose for the
rLLNA based on the absence of overt systemic toxicity and/or excessive local irritation isappropriate. The Panel also agreed that the data did not support establishment of a uniform
concentration threshold for the maximum concentration to be tested. Thus, it seemed justifiable
that preliminary experimentation (as would be typically performed during a dose range-finding
study) should be conducted for vehicle selection, test substance solubility, and stability in thevehicle.
2.3

ICCVAM Recommendations: Future Studies

ICCVAM recommends additional studies to further characterize and potentially improve theusefulness and applicability of the rLLNA for identifying potential skin sensitizers. For
instance, to improve the predictive performance of the rLLNA compared to the traditional
LLNA, ICCVAM recommends investigating the basis for abnormal dose responses for six
substances that would have resulted in false negative results using the rLLNA rather than the
traditional LLNA. This information should help identify ways to improve the accuracy of therLLNA compared to the traditional LLNA.
Efforts should also be made to identify data from guinea pigs and humans for substances likethese that exhibit abnormal dose responses in the traditional LLNA. Information from postmarketing and/or occupational exposures should be collected and assessed.
ICCVAM recommends that all future LLNA studies should collect and analyze individual
animal data. This will allow detection of outliers and avoidance of false negative results thatcan occur from pooling data that include one or more abnormally low values. Existing LLNA
studies using data pooled from all animals in a dose group, such as four of the six false negativerLLNA results in this evaluation, should be evaluated further with data obtained fromindividual animals within each dose group to determine if data pooling may have led to false
negative outcomes.
ICCVAM also recommends that users identify opportunities to use fewer animals per dosegroup without compromising test method accuracy. Thus, laboratories conducting the LLNA
should collect and analyze data from individual animals. The updated ICCVAM-recommended
LLNA test method protocol includes statistical procedures necessary for such determinations(Appendix B). This includes evaluating the laboratory’s historical positive-control database todetermine if the number of animals in the concurrent positive-control group can be reduced.
Independent Peer Review Panel Conclusions and Recommendations
The Panel indicated that, though limited in scope, the available data supported ICCVAM’s draft
test method recommendations for additional studies. The Panel agreed that attempts should bemade to investigate if maximum solubility was achieved (e.g., use of chemical-specific methods
to document solubility). For hazard assessment, it was troublesome that there were so many
vehicle choices, because the vehicle could have a significant effect on whether (and how much)a test substance penetrated the skin barrier. Observed vehicle effects may relate to dermal
6

ICCVAM Test Method Evaluation Reportpenetration as well as to immunomodulation. The Panel considered it desirable to follow thehierarchy of vehicles recommended in the ICCVAM-recommended LLNA protocol. The Panelsuggested that it might be informative to test both known mild and severe sensitizers
concurrently in all recommended vehicles to evaluate whether a specific vehicle choice(s)might influence the results.
2.4

ICCVAM Recommendations: Performance Standards

ICCVAM developed performance standards for the traditional LLNA, which may in turn beapplied to the rLLNA.18 These test method performance standards are proposed to evaluate
modified LLNA test methods that are functionally and mechanistically similar to the traditional
LLNA. Thus, modified rLLNA test method protocols that adhere to the LLNA performancestandards would be considered acceptable for hazard identification purposes.

18

Available at http://iccvam.niehs.nih.gov/methods/immunotox/PerfStds/llna-ps.htm
7

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8

ICCVAM Test Method Evaluation Report

3.0

Validation Status of the rLLNA Test Method

The following is a synopsis of the information in the final ICCVAM BRD (Appendix D),
which reviews the available data and information for the rLLNA test method. The ICCVAM
BRD describes the current validation status of the rLLNA test method, including what is known
about its reliability and accuracy, the scope of the substances tested, and standardized protocols
used for the validation study.
3.1

Test Method Description

The purpose of the rLLNA test method is to identify potential skin sensitizers by quantifying
lymphocyte proliferation. The mechanistic basis is identical to that of the traditional LLNA,
which measures the magnitude of lymphocyte proliferation, which in turn correlates with theextent to which sensitization develops after a topical induction exposure to a skin-sensitizing
substance.
With one exception, the technical aspects of the rLLNA are identical to those of the traditional
LLNA (ICCVAM 1999). The traditional LLNA tests three dose levels of each test substance forskin-sensitizing activity. In the rLLNA, only one dose of the test substance is tested: theconcentration that provides maximum solubility without causing overt systemic toxicity and/or
excessive skin irritation (Kimber et al. 2006). Guidance for evaluating local irritation andsystemic toxicity in the LLNA is provided in the updated ICCVAM-recommended LLNA
protocol (Appendix B).
3.1.1 General Test Method Procedures
The rLLNA measures lymphocyte proliferation after topical exposure to a potential skinsensitizing substance. The test substance is administered topically on three consecutive days tothe ears of mice at a concentration that provides maximum solubility of the test substancewithout systemic toxicity and/or excessive local irritation. Two days after the final applicationof the test substance, 3H-thymidine or 125I-iododeoxyuridine (in phosphate-buffered saline; 250µL/mouse) is administered via the tail vein. Five hours later the draining auricular lymph nodes
are excised, and a single-cell suspension from the lymph nodes of each animal is prepared forquantifying the incorporation of radioactivity, which correlates with lymph node cellproliferation.
The incorporation of radioactive 3H-thymidine or 125I-iododeoxyuridine for each mouse isexpressed in disintegrations per minute (dpm). The SI is calculated as the ratio of the mean
dpm/mouse for each treatment group against the mean dpm/mouse for the vehicle control
group. The threshold for a positive response is an SI ≥ 3.
3.1.2 Similarities and Differences between the Protocols for the Traditional LLNA and therLLNA
As mentioned above, the only difference between the traditional LLNA (ICCVAM 1999) andthe rLLNA is that only one test substance dose is included in rLLNA, while three doses aretested in the traditional LLNA. All other procedures are identical.

9

ICCVAM Test Method Evaluation Report

3.2

Validation Database

Data were obtained from 11 different sources, including published reports and unpublished datasubmitted to NICEATM in response to a May 17, 2007, FR notice (72 FR 2781519). The
rLLNA database consisted of the results for the highest doses tested in these studies.
The resulting database consisted of 457 unique substances tested in a total of 471 traditional
LLNA studies (Table 3-1), 211 of which were included in the original ICCVAM evaluation of
the traditional LLNA (ICCVAM 1999). Fourteen of the 457 unique substances20 were repeatedfrom two to five times in different LLNA studies. Specifically, nine of the 14 substances were
evaluated two to five times in different vehicles, and five of the 14 substances were evaluated
two to five times in the same vehicle. Two of the five substances evaluated in the same vehicle(hexyl cinnamic aldehyde [HCA] and potassium dichromate) were also tested using different
vehicles (one study for HCA and two studies for potassium dichromate). Due to the small
number of repeated studies (5% of total studies), all studies were treated independently for the
purpose of this accuracy evaluation. When the studies for the substances repeated in the samevehicle were considered together to yield an overall skin sensitization classification, there were
465 studies with unique substance and vehicle combinations.
Table 3-1 provides the chemical class information for these test substances. The table
distinguishes the chemical classifications of the 211 substances included in the original
evaluation of the rLLNA (Kimber et al. 2006; ESAC 2007) and the chemical classifications of
the additional substances received in response to the FR notice. Of the 211 substances initiallyevaluated by Kimber et al. (2006), the chemical classes with the greatest number of substanceswere carboxylic acids (29) and halogenated hydrocarbons (27). Of the additional
246 substances included in this evaluation, the chemical classes with the greatest number ofsubstances tested were pharmaceutical chemicals (125), carboxylic acids (15), and lipids (14).
Of the substances included in this evaluation, 10 were formulations. Seventy substances could
not be assigned to a specific chemical class due to incomplete available information (e.g., thelack of a Chemical Abstracts Service Registry Number or structure).

19
20

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
Some substances were tested in more than one vehicle. In such instances, each substance–vehicle
combination was considered separately, thus a total of 465 unique substance–vehicle combinations were usedin the performance evaluation.
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ICCVAM Test Method Evaluation Report

Table 3-1 Chemical Classes1 Represented in the Current Traditional LLNA Database
Chemical Class
Alcohols

Number of
Substances Original2
9

Number of
Substances Additional2
4

Number of
Substances Original0

Number of
Substances Additional
2

Aldehydes

21

4

Amides40Isocyanates

10Ketones

50

Amidines10Lactones2

2

Amines14

7

Lipids7

14

Anhydrides

10

Macromolecular
substances305

Carbohydrates

3

2

Nitriles1

1

Carboxylic acids

29

15

Nitro compounds

20Esters30Nitrosocompounds

30

EthersChemical Class
Inorganic
Chemicals

142Onium compounds

1

0

3

0

10

Pharmaceuticalchemicals4

0125Heterocyclic
compounds

18

4

Phenols18

2

Hydrocarbons,
Acyclic2

1

Polycyclic
compounds

5

3

Hydrocarbons,
Cyclic14

7

Quinones1

1

Hydrocarbons,
halogenated27

1

Sulfur compounds

20

2

Hydrocarbons,
other

7

8

Urea

3

0

Imines0

1

Unknown

28

42

Formulations

1

Total number of substances assigned to chemical classes does not equal the total number of substances evaluated becausesome substances were assigned to more than one class and some substances were not assigned to a specific chemicalclass.
2
Number of substances - Original represents the substances evaluated in Kimber et al. (2006).
Number of substances - Additional represents the substances received in response to the released Federal Register notice(72 FR 27815, May 17, 2007) available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf.
3
No chemical class could be assigned. The terms “formulation” and “macromolecular substances” were used to classify thesesubstances.
4
The chemical classification of “pharmaceutical chemicals” for the GlaxoSmithKline (GSK) substances was suggested by Dr.
Michael Olson of GSK to capture three types of pharmaceutical substances (actives, intermediates, and starting materials).

3.3

Reference Test Method Data

The traditional LLNA data used for evaluation of the rLLNA include the results for all tested
doses of each substance. In addition to calculated SI values for each of the tested doses, thevehicles tested and EC3 values (estimated concentration needed to produce an SI value of 3) for
substances classified as sensitizers were provided in Gerberick et al. (2005). The data received
in response to the May 2007 FR notice included calculated SI values for the vehicle and each of
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ICCVAM Test Method Evaluation Report

the tested doses. If EC3 values were not included in the data source, they were calculated,
where possible, using either interpolation or extrapolation (Dearman et al. 2007). This
information and the complete database (by each source) are provided in Annex III of the BRD(Appendix D).
3.4

Test Method Accuracy

The ability of the rLLNA to correctly identify potential skin sensitizers was compared to that of
the traditional LLNA. Of the 471 studies, 318 detected skin sensitizers, and 153 detected nonsensitizers. When studies of the substances tested more than once in the same vehicle wereconsidered together to yield an overall skin sensitization classification, 465 unique substance–
vehicle combinations resulted. Of these, 315 were identified as sensitizers and 150 as nonsensitizers.
Based on the available study data, the rLLNA has an accuracy of 98.7% (465/471), a sensitivityof 98.1% (312/318), a specificity of 100% (153/153), a false positive rate of 0% (0/153), and afalse negative rate of 1.9% (6/318) when compared to the traditional LLNA (Table 3-2). Whensubstances tested more than once in the same vehicle were considered together, the resulting
465 studies had an accuracy of 98.7% (459/465), a sensitivity of 98.1% (309/315), a specificityof 100% (150/150), a false positive rate of 0% (0/150), and a false negative rate of 1.9%
(6/315).
This analysis of the rLLNA yielded six false negative results. A review of the data for these six
substances indicates that the traditional LLNA classification of the substances as skinsensitizers was based not on the highest tested dose but on a low- or mid-dose level that
produced an SI ≥ 3, while the highest dose tested produced an SI < 3. Because the rLLNA tests
substances at only the highest dose level, all six substances would be incorrectly identified as
non-sensitizers (i.e., false negatives). Four of the six substances that resulted in false negatives
using the rLLNA compared to the traditional LLNA came from LLNA studies that used pooleddata. There were no patterns of consistency for these substances with regard to physicochemical
properties.

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ICCVAM Test Method Evaluation Report

Table 3-2 Evaluation of the Performance of the rLLNA in Predicting Skin Sensitizers
Compared to the Traditional LLNA
Data

False
Positive

False
Negative100%(42/42)0% (0/42)

1.8%(3/169)98.1%(312/318)100%(153/153)

0% (0/153)

1.9%(6/318)98.1%(309/315)100%(150/150)

0% (0/150)

1.9%(6/315)N

Accuracy

Sensitivity

Specificity

Kimber et al. (2006)

211

98.6%
(208/211)98.2%(166/169)

rLLNA

471

98.7%
(465/471)

rLLNA approach(substances repeated in
the same vehicle
considered together)

465

98.7%
(459/465)

Abbreviation: N = number of tests
Accuracy = the percentage of correct outcomes (positive and negative) of a test method
Sensitivity = the percentage of all positive substances that are classified as positive
Specificity = the percentage of all negative substances that are classified as negative
False positive rate = the percentage of all negative substances that are falsely identified as positive
False negative rate = the percentage of all positive substances that are falsely identified as negative

3.5

Test Method Reliability

The BRD assessed interlaboratory reproducibility of the rLLNA with traditional LLNA data forfive substances that had been tested independently in the same vehicle at multiple laboratories.
These five substances were dinitrochlorobenzene (DNCB), HCA, linalool alcohol, methyl
salicylate, and potassium dichromate. Table 3-3 summarizes the responses obtained by therLLNA. All studies classified DNCB, methyl salicylate, and potassium dichromate (3/5 = 60%)
as sensitizers or non-sensitizers (i.e., 100% concordance). HCA and linalool alcohol, whichwere tested independently in two laboratories, were each classifed as a sensitizer by one
traditional LLNA study and as a non-sensitizer by the other traditional LLNA study. Review ofthe studies indicates that the discordant results were due to differences in the highest dose levels
tested. However, because the rLLNA and traditional LLNA use identical protocols and the data
sets used to evaluate their accuracy are similar, the intra- and interlaboratory reliability of therLLNA is deemed to be similar to that of the traditional LLNA (see ICCVAM 1999 for thesestatistics).

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ICCVAM Test Method Evaluation Report

Table 3-3 Interlaboratory Reproducibility of Skin sensitization Outcome for the rLLNA
Substance
1-Chloro-2dinitrobenzene
Hexyl cinnamic
aldehyderLLNA Dose(%)/SIrLLNAClassification1

0.25/38.00+

0.25/7.10+

50/17.00+

10/2.84–

100/8.30+

30/1.30–

20/0.90–

Data submitted by D. Germolec

20/1.72–

Gerberick et al. (2005)

0.5/16.10+

0.25/3.39+

0.5/10.10+

Data Source

VehicleGerberick et al. (2005)

AOO

Data submitted by D. Germolec
Gerberick et al. (2005)

AOO

Data submitted by H.W. Vohr
Gerberick et al. (2005)

Linalool alcohol

Methyl salicylate

Potassium dichromate

Data submitted by D. Basketter,
I. Kimber, and F. Gerberick
Gerberick et al. (2005)

Data submitted by D. Germolec
Ryan et al. (2002)

AOO

AOO

DMSO

Abbreviations: AOO = Acetone: olive oil; DMSO = Dimethyl sulfoxide; rLLNA = Reduced murine local lymph node assay;
SI = stimulation index
1– = non-sensitizer, + = sensitizer

3.6

Animal Welfare Considerations: Reduction, Refinement, and Replacement

Compared to the traditional LLNA, the rLLNA will reduce the number of animals used toassess skin sensitization. Becuse the rLLNA tests only the highest dose level of the test
substance in addition to the concurrent control groups, the number of animals tested would bedecreased by at least 40% for each test. Ryan et al. (2008) described the impact of reducing thenumber of animals per group from five to two on the performance of the rLLNA and concluded
that such a small number of animals per group was inadequate for hazard identification of skinsensitizers.

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ICCVAM Test Method Evaluation Report

4.0

ICCVAM Consideration of Public and SACATM Comments

ICCVAM received 27 public comments in response to four FR notices released between
May 2007 and May 2008 (see Appendix G). Comments received in response to or related to
the FR notices are also available on the NICEATM–ICCVAM website.21 The following
sections, delineated by FR notice, briefly discuss the public comments received.
4.1

Public Comments in Response to 72 FR 27815 (May 17, 2007): The Murine
Local Lymph Node Assay: Request for Comments, Nominations of ScientificExperts, and Submission of Data

NICEATM requested the following:
1. Public comments on the appropriateness and relative priority of evaluation of thevalidation status ofa. The LLNA as a stand-alone assay for determining potency (including
severity) for the purpose of hazard classificationb. The rLLNA approachc. Non-radioactive LLNA methods
d. The use of the LLNA for testing mixtures, aqueous solutions, and metals
e. The current applicability domain
2. Nominations of expert scientists to consider as members of a possible peer review
panel
3. Submission of data for the LLNA and/or modified versions of the LLNA
In response to this FR notice, NICEATM received 17 comments. Six comments included
additional data and information, while two others offered data and information upon request.
Three nominated four potential panelists for consideration. Three commenters suggested
reference publications for consideration during the Panel evaluation. NICEATM provided thedata and suggested references to the Panel for evaluation.
Three comments remarked specifically on the rLLNA.
One commenter suggested rearranging the priority sequence of test method evaluation frommost to least pressing: a, e, d, b, and c (see list above). ICCVAM did not establish a relative
priority for these activities because they were all considered to be high-priority activities.
Accordingly, all LLNA-related activities described above were discussed at the March 2008
Panel meeting.
Another commenter noted that ESAC issued a statement supporting the use of the rLLNA“within tiered-testing strategies to reliably distinguish between chemicals that are skinsensitisers and non-sensitisers” (Appendix E), thereby reducing animal use by as much as 50%.
The ESAC statement also notes the following limitations: “the test results provided by therLLNA do not allow the determination of the potency of a sensitising chemical” and “negative
21

Available at http://ntp-apps.niehs.nih.gov/iccvampb/searchPubCom.cfm
15

ICCVAM Test Method Evaluation Reporttest results associated with testing using concentrations of less than 10% should undergo further
evaluation.” The commenter states that ICCVAM should (1) expeditiously review and endorse
the ESAC peer review and circulate harmonized testing recommendations regarding this assay
to U.S. agencies before the end of the year, and (2) NICEATM should collaborate with
ECVAM to address the question of concentration threshold.
As indicated in Section 1.0, ICCVAM and NICEATM collaborated with liaisons from ECVAM
and JaCVAM to update with 260 additional LLNA studies the Kimber et al. (2006) evaluation
upon which the ESAC statement was based. This comprehensive evaluation was expedited forinclusion in the publicly transparent ICCVAM peer review process, which included the March
2008 Panel meeting.
A third commenter stated that ESAC considered the rLLNA to be scientifically validated but
only when used as a screening test to distinguish between sensitizers and non-sensitizers andwith due regard to the conditions set forth in the official ESAC statement of April 27, 2007.
This statement was based on the outcome of a review of LLNA data for 211 chemicals (Kimber
et al. 2006). The review of existing and newly provided LLNA data proposed by NICEATM–ICCVAM therefore presents an ideal opportunity to assess further the validity of the rLLNA for
screening purposes. The ICCVAM test method recommendations detailed in Section 2.0
describe the usefulness and limitations of the rLLNA based on the comprehensive ICCVAM
evaluation of an expanded database of 471 LLNA studies.
4.2

Public Comments in Response to 72 FR 52130 (September 12, 2007): Draft
Performance Standards for the Murine Local Lymph Node Assay: Request forComments

NICEATM requested public comments on the initial ICCVAM-recommended draft LLNA
performance standards developed to facilitate evaluation of modified LLNA protocols withregard to the traditional LLNA. In response to this FR notice, NICEATM received fourcomments, two of which suggested clarifications to the text. Another recommended that test
substances chosen for testing in the various LLNA methods should be pure, with conclusive
structures, and should not be mixtures.
The ICCVAM review of the rLLNA, in which only the highest dose is used to assign apositive/negative result for a test substance, was a retrospective evaluation of available LLNA
studies with which to compare the outcome of the traditional protocol (in which all doses are
considered and any positive result, regardless of concentration, can be used to establish asensitizing substance). Therefore, although the validation status of the LLNA for testing
mixtures is still under review, ICCVAM and NICEATM considered it appropriate to include allavailable data in the evaluation of the rLLNA.
The fourth commenter addressed the rLLNA in general. The commenter supported thedevelopment of performance standards that expedite the validation of new protocols similar topreviously validated methods but was disappointed that NICEATM–ICCVAM has chosen todevelop performance standards for such a narrow scope of applicability (i.e., modifications of
the standard LLNA that involve incorporation of non-radioactive methods of detecting
lymphocyte proliferation). The commenter suggested that limited resources available to
NICEATM and ICCVAM would be better spent on activities that would have greater impact on
16

ICCVAM Test Method Evaluation Report

the reduction, refinement, or replacement of animal use, such as evaluating the use of humancell lines or one of the available in vitro skin models as a replacement for the LLNA.
ICCVAM considered the comment and concluded that the proposed modifications to the LLNA
protocol and expanded applications have significant potential to further reduce and refine
animal use. ICCVAM is also interested in in vitro models and non-animal approaches forassessing allergic contact dermatitis; however, no in vitro replacements for the LLNA have yet
been nominated or submitted to ICCVAM for evaluation.
4.3

Public Comments in Response to 73 FR 1360 (January 8, 2008): Announcementof an Independent Scientific Peer Review Panel Meeting on the Murine Local
Lymph Node Assay; Availability of Draft Background Review Documents;
Request for Comments

NICEATM requested public comments on the draft BRDs, draft ICCVAM test
recommendations, draft test method protocols, and revised draft LLNA performance standards
for an international independent scientific peer review panel meeting to evaluate modifications
and new applications for the LLNA. NICEATM received six comments in response to this FR
notice. Four commenters focused on the traditional LLNA and two commenters provided
comments specific to the rLLNA.
One commenter agreed with ICCVAM’s recommendation of the rLLNA for hazard
identification purposes, noting that Kimber et al. (2006) did not propose a 10% concentrationthreshold as the absolute cutoff for defining non-sensitizing chemicals. Gerberick et al. (2005)
showed that for some compounds tested the highest concentration was at least 20% and did not
induce a positive response at any concentration tested; these compounds were categorized as
non-sensitizing. Cockshott et al. (2006) reported that a negative result obtained with the highest
concentration tested at 10% would be considered a valid result if the positive control, a mild tomoderate sensitizer, gave a positive response (i.e., a chemical that is negative at a top
concentration of 10% does not represent a significant human sensitization hazard). This issimilar to the definition of a non-sensitizing chemical in the Guinea Pig Maximization Test
(GPMT) or Buehler Test as one that induces responses lower than 30% or 15%, respectively.
Therefore, if a chemical elicits positive responses in 20% or 25% of the test animals in a
GPMT, it would be considered a non-sensitizer from a regulatory perspective.
ICCVAM and the Panel agreed that the maximum applied dose for the rLLNA should be basedon the absence of overt systemic toxicity and/or excessive local irritation. The available data did
not support establishment of a uniform concentration threshold for the maximum concentration
to be tested.
Another commenter’s response referred first to the April 2007 ESAC statement:“…supporting the use of the rLLNA ‘within tiered-testing strategies to reliably
distinguish between chemicals that are skin sensitisers and non-sensitisers,’ thereby
reducing animal use by as much as 50%. In spite of the ESAC recommendation,
ICCVAM conducted its own data call-in and data review. The reviewed database iscomprehensive and contains a broad cross-section of the chemical universe. The
performance characteristics were all above 95% (false negative and positive rates arevery low or zero). Even though this additional review was largely unnecessary, [the17

ICCVAM Test Method Evaluation Reportcommenter was] pleased that ICCVAM’s draft recommendations concluded
favorably for the rLLNA procedure…”
The commenter urged the Panel to concur. As reflected in the Independent Scientific Review
Panel Assessment (Appendix F), the Panel generally agreed with ICCVAM’s test method
recommendations for the rLLNA, which have been updated to reflect comments from the Panel,
SACATM, and the public.
4.4

Public Comments in Response to 73 FR 29136 (May 20, 2008): Peer Review
Panel Report on the Validation Status of New Versions and Applications of the
Murine Local Lymph Node Assay (LLNA): A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemicals and Products: Notice of
Availability and Request for Public Comments

NICEATM requested submission of written public comments on the Independent ScientificPeer Review Panel Assessment. No comments were received in response to this FR notice.
4.5

Public and SACATM Comments: SACATM Meeting on June 18-19, 2008

The June 18-19, 2008, SACATM meeting included a discussion of the ICCVAM review of therLLNA test method.
There were no public comments specific to the rLLNA.
One SACATM member concurred with the recommendation that the rLLNA protocol should
discuss how to determine the maximum dose if only a single dose is to be used in a screening
process. An investigator must be able to define excessive irritation; otherwise, the testing may
produce a bell-shaped response curve. In response to this comment and the Panel’s
recommendation, ICCVAM added to the updated LLNA test method protocol specific guidanceon how to determine the maximum concentration to be tested so as to avoid overt systemic
toxicity and/or excessive local irritation (Appendix B, Annex III).
Another SACATM member suggested that the rLLNA appeared favorable because 100%
(153/153) of the non-sensitizing agents and 98.1% (312/318) of the sensitizing agents werecorrectly predicted. ICCVAM agrees that this high level of agreement between the traditional
LLNA and the rLLNA supports routine use of the rLLNA as recommended by ICCVAM.

18

ICCVAM Test Method Evaluation Report

5.0

References

Buehler EV. 1965. Delayed contact hypersensitivity in the guinea pig. Arch Dermatol 91:171177.
Cockshott A, Evans P, Ryan CA, Gerberick GF, Betts CJ, Dearman RJ, et al. 2006. The local
lymph node assay in practice: A current regulatory perspective. Hum Exp Toxicol 25:387–394.
Dean J, Twerdok L, Tice R, Sailstad D, Hattan D, Stokes WS. 2001. ICCVAM evaluation of
the murine local lymph node assay. Conclusions and recommendations of an independentscientific peer review panel. Regul Toxicol Pharmacol 34:258–273.
Dearman RJ, Betts CJ, Farr C, McLaughlin J, Berdasco N, Wiench K, et al. 2007. Comparative
analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl
acrylate, and ethylhexyl acrylate) using the local lymph node assay. Contact Dermatitis 57:242–
247.
EPA. 2003. Health Effects Test Guideline, OPPTS 870.2600. Skin Sensitization EPA 712–C–
03–197. Washington, DC: U.S. Environmental Protection Agency. Available:
http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/870-2600.pdf.
ESAC. 2007. Statement on the Reduced Local Lymph Node Assay (rLLNA). European
Commission Directorate General, Joint Research Centre, Institute for Health and Consumer
Protection, European Centre for the Validation of Alternative Methods. April 2007. Available:
http://ecvam.jrc.it/ft_doc/ESAC26_statement_rLLNA_20070525-1.pdf.
Gaspari AA. 2007. Mechanism of action and other potential roles of an immune response
modifier. Cutis 79:36–45.
Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, et al. 2005. Compilationof historical local lymph node data for evaluation of skin sensitization alternative methods.
Dermatitis 16:157–202.
Hutchings CV, Shum KW, Gawkrodger DJ. 2001. Occupational contact dermatitis has anappreciable impact on quality of life. Contact Dermatitis 45:17–20.
ICCVAM 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemical/Compounds. NIH Publication No. 99-4494.
Research Triangle Park, NC: National Toxicology Program. Available:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf.
ICCVAM Authorization Act. 2000. Public Law 106-545, 42 U.S.C. 285l-3.
Kimber I, Dearman RJ, Betts CJ, Gerberick GF, Ryan CA, Kern PS, et al. 2006. The local
lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?
Contact Dermatitis 54:181–185.
Magnusson B, Kligman AM. 1970. Allergic Contact Dermatitis in the Guinea Pig.
Springfield, IL: Charles C. Thomas.
OECD. 2002. Test Guideline 429. Skin Sensitisation: Local Lymph Node Assay, adopted April
24, 2002. In: OECD Guidelines for Testing of Chemicals. Paris: OECD. Available:
19

ICCVAM Test Method Evaluation Report

http://titania.sourceoecd.org/vl=7033968/cl=16/nw=1/rpsv/cw/vhosts/oecdjournals/1607310x/v1
n4/contp1-1.htm
Ryan CA, Chaney JG, Kern PS, Patlewicz GY, Basketter DA, Betts CJ, et al. 2008. The
reduced local lymph node assay: the impact of group size. J Appl Toxicol 28:518–523.
Ryan CA, Cruse LW, Skinner RA, Dearman RJ, Kimber I, Gerberick GF. 2002. Examination of
a vehicle for use with water soluble materials in the murine local lymph node assay. Food Chem
Toxicol 40:1719–1725.
Skoet R, Zachariae R, Agner T. 2003. Contact dermatitis and quality of life: a structured reviewof the literature. Br J Dermatol 149:452–456.

20

ICCVAM Test Method Evaluation Report: Appendix A

Appendix A
ICCVAM Evaluation of the Reduced Local Lymph Node Assay
Test Method – Timeline

A-1

ICCVAM Test Method Evaluation Report: Appendix A[This Page Intentionally Left Blank]

A-2

ICCVAM Test Method Evaluation Report: Appendix A

January 10, 2007

January 2007

ICCVAM receives a letter from the CPSC nominating six LLNA
review activities for evaluation,22 including the reduced murine
local lymph node assay (rLLNA).
The ICCVAM IWG is re-established to work with NICEATM tocarry out LLNA evaluations.

January 24, 2007

ICCVAM endorses the six CPSC-nominated LLNA review
activities, including evaluation of the rLLNA.

May 17, 2007

Federal Register notice (72 FR 27815) – The Murine Local
Lymph Node Assay: Request for Comments, Nominations of
Scientific Experts, and Submission of Data

June 12, 2007

SACATM endorses with high priority the six CPSC-nominated
LLNA review activities, including evaluation of the rLLNA.

November 12–13, 2007

ECVAM Workshop on Alternative Methods (Reduction,
Refinement, Replacement)

January 8, 2008

Federal Register notice (73 FR 1360) – Announcement of an
Independent Scientific Peer Review Panel Meeting on the Murine
Local Lymph Node Assay; Availability of Draft Background
Review Documents; Request for Comments

March 4–6, 2008

Independent Scientific Peer Review Panel holds a public meeting,with opportunity for oral public comments, at CPSC Headquarters
in Bethesda, MD, to discuss LLNA review activities, including
the rLLNA. The Panel was charged with reviewing the current
validation status of the rLLNA and commenting on the extent towhich the information in the draft BRD supported the draft
ICCVAM test method recommendations.

May 20, 2008

Federal Register notice (73 FR 29136) – Announcement of thePeer Review Panel Report on the Validation Status of New
Versions and Applications of the Murine Local Lymph Node
Assay (LLNA): A Test Method for Assessing the Allergic
Contact Dermatitis Potential of Chemicals and Products: Noticeof Availability and Request for Public Comments23

June 18–19, 2008

SACATM public meeting for comments on the Panel report
ICCVAM endorses the TMER for the rLLNA test method, which
includes the final rLLNA BRD.

October 29, 2008

Abbreviations: BRD = Background review document; CPSC = U.S. Consumer Product Safety Commission;
ECVAM = European Centre for the Validation of Alternative Methods; ICCVAM = Interagency Coordinating
Committee on the Validation of Alternative Methods; IWG = ICCVAM Immunotoxicity Working Group;
LLNA = murine local lymph node assay; NICEATM = National Toxicology Program Interagency Center for
the Evaluation of Alternative Toxicological Methods; rLLNA = Reduced murine local lymph node assay;
22
23

http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf
A-3

ICCVAM Test Method Evaluation Report: Appendix A

SACATM = Scientific Advisory Committee on Alternative Toxicological Methods; TMER = Test method
evaluation report

A-4

ICCVAM Test Method Evaluation Report: Appendix B

Appendix B
ICCVAM-Recommended Protocol (Updated 2008)
The Murine Local Lymph Node Assay:24 A Test Method for Assessing the Allergic
Contact Dermatitis Potential of Chemicals and Products
Annex IAn Approach to Dissection and Identification of the Draining(“Auricular”) Lymph Nodes..................................................................... B-15

Annex II

An Example of How to Reduce the Number of Animals in theConcurrent Positive Control Group of the Local Lymph Node Assay ... B-19

Annex III

Evaluating Local Irritation and Systemic Toxicity in the Local
Lymph Node Assay ................................................................................... B-21

24

Based on ICCVAM (1999) and Dean et al. (2001)
B-1

ICCVAM Test Method Evaluation Report: Appendix B[This Page Intentionally Left Blank]

B-2

ICCVAM Test Method Evaluation Report: Appendix B

Preface
The murine local lymph node assay (LLNA) is a test method developed to assess whether achemical has the potential to induce allergic contact dermatitis (ACD) in humans. In 1998,
the LLNA was submitted to the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) for evaluation as an alternative (i.e., stand-alone) test
method to the guinea pig (GP) sensitization tests accepted by U.S. regulatory agencies. In
1999, based on a comprehensive evaluation of the LLNA by an independent scientific peer
review panel (Panel),25 ICCVAM concluded that the LLNA is an acceptable alternative to the
GP test methods to assess the ACD hazard potential of most substances (Dean et al. 2001).
The Panel also concluded that the LLNA offers animal welfare advantages compared to useof the traditional GP methods, in that it provides for animal use refinement (i.e., eliminationof distress and pain) and reduces the total number of animals required. An ICCVAM
Immunotoxicity Working Group (IWG) reviewed the 1999 Panel report and developed
recommendations applicable to the regulatory use of the LLNA. The IWG then worked with
the National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) to produce a recommended test method protocol(ICCVAM 2001)26 that would accurately reflect the ICCVAM and Panel recommendations(ICCVAM 1999).
In March 2008, ICCVAM and NICEATM convened an independent scientific peer review
panel (Panel) to evaluate new versions and applications of the LLNA. The Panel provided
conclusions and recommendations in their report, many of which were applicable to the
traditional LLNA test method protocol.27 ICCVAM subsequently considered the Panel’s
conclusions and recommendations, as well as comments from the Scientific Advisory
Committee on Alternative Toxicological Methods (SACATM) and public, and updated the2001 ICCVAM-recommended LLNA test method protocol. The updated ICCVAMrecommended LLNA test method protocol will be forwarded with the Panel’s report toagencies for their consideration.
The updated ICCVAM-recommended test method protocol for the LLNA is based on
evaluation of previous experience and scientific data. It is provided to Federal agencies fortheir consideration as a standardized test method protocol recommended for generation ofdata for regulatory purposes. Prior to conducting a LLNA test to meet a regulatory
requirement, it is recommended that the appropriate regulatory agency be contacted for theircurrent guidance on the conduct and interpretation of this assay. Additional information on
the ICCVAM LLNA review process and deliberations of the Panel can be found at the
ICCVAM website (http://iccvam.niehs.nih.gov) or in the Panel report (ICCVAM 2008a).
We want to express our sincere appreciation to the ICCVAM IWG for their careful
deliberations and efforts in updating the LLNA test method protocol, and especiallyappreciate the efforts of the Working Group Co-Chairs, Abigail Jacobs, Ph.D., from the U.S.
Food and Drug Administration and Joanna Matheson, Ph.D., from the U.S. Consumer
Products Safety Commission. We also want to acknowledge the outstanding support25

http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/LLNAProt.pdf
27
http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf
26

B-3

ICCVAM Test Method Evaluation Report: Appendix B

provided by NICEATM and the Integrated Laboratory Systems, Inc., support staff. Lastly,
we appreciate the efforts of the Panel members for their diligent review, and the comments
provided by SACATM and numerous stakeholders, including the public.
William S. Stokes, D.V.M., D.A.C.L.A.M.
Rear Admiral/Assistant Surgeon General, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM
Marilyn Wind, Ph.D.
Deputy Associate Executive Director
Directorate for Health Sciences
U.S. Consumer Product Safety Commission
Chair, ICCVAM

B-4

ICCVAM Test Method Evaluation Report: Appendix B

1.0

General Principle of Detection of Skin Sensitization Using the Local
Lymph Node Assay

The basic principle underlying the murine local lymph node assay (LLNA) is that sensitizers
induce proliferation of lymphocytes in the lymph node draining the site of substanceapplication. Under appropriate test conditions, this proliferation is proportional to the dose
applied, and provides a means of obtaining an objective, quantitative measurement ofsensitization. The test measures cellular proliferation as a function of in vivo radioisotope
incorporation into the DNA of dividing lymphocytes. The LLNA assesses this proliferation in
the draining lymph nodes proximal to the application site (see Annex I). This effect occurs as adose response in which the proliferation in test groups is compared to that in the concurrent
vehicle-treated control group. A concurrent positive control is added to each assay to provide anindication of appropriate assay performance.

2.0

Description of the Local Lymph Node Assay

2.1

Sex and strain of animals

Young adult female mice (nulliparous and non-pregnant) of the CBA/Ca or CBA/J strain arerecommended.28 Females are used because most data in the existing database were generatedusing mice of this gender. At the start of the study, mice should be age 8–12 weeks. All miceshould be age matched (preferably within a one-week time frame). Weight variations between
the mice should not exceed 20% of the mean weight.
2.2

Preparation of animals

The temperature of the experimental animal room should be 21°C (±3°C) and the relative
humidity 30%–70%. When artificial lighting is used, the light cycle should be 12 hours light:
12 hours dark. For feeding, an unlimited supply of standard laboratory mouse diets and drinking
water should be used. The mice should be acclimatized for at least five days prior to the start of
the test (ILAR 1996). Mice should be housed in small groups unless adequate scientificrationale for housing mice individually is provided (ILAR 1996). Healthy mice are randomly
assigned to the control and treatment groups. The mice are uniquely identified prior to being
placed in the study. The method used to mark the mice should not involve identification via theear (e.g., marking, clipping, or punching of the ear). All mice should be examined prior to theinitiation of the test to ensure that there are no skin lesions present.
2.3

Preparation of doses

Solid test substances should be dissolved in appropriate solvents or vehicles and diluted, if
appropriate, prior to dosing of the mice. Liquid test substances may be dosed directly (i.e.,
applied neat) or diluted prior to dosing. Fresh preparations of the test substance should be
prepared daily unless stability data demonstrate the acceptability of storage.

28

Male mice or other strains of mice may be used if it is sufficiently demonstrated that these animals perform as
well as female CBA mice in the LLNA.
B-5

ICCVAM Test Method Evaluation Report: Appendix B

2.4

Test Conditions

2.4.1 Solvent/vehicle
The selected solvent/vehicle must not interfere with or bias the test result and should be selectedon the basis of maximizing the test concentrations while producing a solution/suspension
suitable for application of the test substance. In order of preference, recommended
solvents/vehicles are acetone: olive oil (4:1 v/v), N,N-dimethylformamide, methyl ethyl ketone,
propylene glycol, and dimethyl sulfoxide, but others may be used (Kimber and Basketter 1992).
Particular care should be taken to ensure that hydrophilic materials are incorporated into avehicle system that wets the skin and does not immediately run off. Thus, wholly aqueous
vehicles may need to be avoided. It may be necessary for regulatory purposes to test thesubstance in the clinically relevant solvent or product formulation.
2.4.2 Controls
Concurrent negative (solvent/vehicle) controls should be included in each test to ensure that thetest system is functioning properly and that the specific test is valid. In some circumstances(e.g., when using a solvent/vehicle not recommended in Section 2.4.1), it may be useful to
include a naïve control. Except for treatment with the test substance, the mice in the negative
control groups should be handled in an identical manner to the mice of the treatment groups.
Concurrent positive controls are used to ensure the appropriate performance of the assay bydemonstrating that the test method is responding with adequate and reproducible sensitivity to asensitizing substance for which the magnitude of the response is well characterized. Inclusionof a concurrent positive control is also important since it can confirm technical competence inperforming the test and can demonstrate intra- and interlaboratory reproducibility andcomparability. The positive control should produce a positive LLNA response (i.e., astimulation index [SI] ≥ 3 over the negative control group). In particular, for negative LLNA
studies, the concurrent positive control must induce a SI ≥ 3 relative to its vehicle-treatedcontrol. The positive control dose should be chosen such that the induction is reproducible but
not excessive (i.e., SI > 20). Preferred positive control substances are hexyl cinnamic aldehydeor mercaptobenzothiazole. There may be circumstances where, given adequate justification,other positive control substances may be used.
Although the positive control substance should be tested in the same vehicle as the test
substance, there may be certain regulatory situations where it is necessary to test the positive
control substance in both a standard and a non-standard vehicle (e.g., a clinically/chemically
relevant formulation) to test for possible interactions.
Inclusion of a positive control with each test is recommended to ensure that all test method
protocol procedures are being conducted properly and that all aspects of the test system areworking properly such that they are capable of producing a positive response. However,periodic testing (i.e., at intervals ≤6 months) of the positive control substance may beconsidered in laboratories that conduct the LLNA regularly (i.e., conduct the LLNA at afrequency of no less than once per month) and that have a history and a documented proficiency
for obtaining consistent results with positive controls. Adequate proficiency with the LLNA canbe successfully demonstrated by generating consistent results with the positive control in atleast 10 independent tests conducted within a reasonable period of time (i.e., less than one
B-6

ICCVAM Test Method Evaluation Report: Appendix Byear). A positive control group should always be included when there is a procedural change tothe LLNA (i.e., change in trained personnel, change in test method materials and/or reagents,change in test method equipment, change in source of test animals, etc.), and such changes
should be documented in laboratory reports. Consideration should be given to the impact ofthese changes on the adequacy of the previously established historical database in determining
the necessity for establishing a new historical database to document consistency in the positive
control results. Users should be aware that the decision to only include a positive control on aperiodic basis instead of concurrently will have ramifications on the adequacy and acceptabilityof negative study results generated without a concurrent positive control during the interval
between each periodic positive control study. For example, if a false negative result is obtainedin the periodic positive control study, all negative test substance results obtained in the interval
between the last acceptable periodic positive control study and the unacceptable periodic
positive control study will be questioned. In order to demonstrate that the prior negative test
substance study results are acceptable, a laboratory would be expected to repeat all negative
studies, which would require additional expense and increased animal use. These implications
should be carefully considered when determining whether to include concurrent positive
controls or to only conduct periodic positive controls. Consideration should also be given tousing fewer animals in the concurrent positive control group when this is scientifically justified,
as discussed below and in Annex II.
Benchmark controls may be useful to demonstrate that the test method is functioning properly
for detecting the skin sensitization potential of substances of a specific chemical class or a
specific range of responses, or for evaluating the relative skin sensitization potential of a test
substance. Appropriate benchmark controls should have the following properties:

2.5•

Structural and functional similarity to the class of the substance being tested•

Known physical/chemical characteristics•

Supporting data on known effects in animal models•

Known potency for sensitization response

Methodology

A minimum of four animals per dose group is recommended. The collection of lymph nodes
from individual mice is necessary in order to identify if any of the individual animal responses
are outliers (e.g., in accordance with statistical tests such as Dixon’s test). This will aid in
avoiding false negative results for weaker sensitizers (i.e., substances that normally would
induce an SI just above 3 might be incorrectly classified as negative due to a low outlier value,
because the resulting mean SI may be less than 3 if an outlier is not identified and excluded).
Individual animal measurements allow for the assessment of interanimal variability, a statisticalcomparison of the difference between test substance and vehicle control group measurements,
and the evaluation of statistical power for different group sizes. Finally, evaluating thepossibility of reducing the number of mice in the positive control group is only feasible when
individual animal data are collected.
As noted above, concurrent negative and positive control groups should be included, unless alaboratory can demonstrate adequate proficiency that would support the use of a periodic
positive control study. The number of mice in the concurrent positive control group might be
B-7

ICCVAM Test Method Evaluation Report: Appendix B

reduced compared to the vehicle and test substance groups, if the laboratory demonstrates,
based on laboratory-specific historical data,29 that fewer mice can be used without substantially
increasing the frequency with which studies will need to be repeated. An example of how to
reduce the number of mice in the concurrent positive control group is provided in Annex II.
Test substance treatment dose levels should be based on the recommendations given in Kimber
and Basketter (1992) and in the ICCVAM Panel Report (ICCVAM 1999). Dose levels are
selected from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc. The
maximum concentration tested should be the highest achievable level while avoiding excessive
local irritation and overt systemic toxicity (Annex III). Efforts should be made to identify
existing information that may aid in selecting the appropriate maximum test substance doselevel. In the absence of such information, an initial prescreen test, conducted under identicalexperimental conditions except for not conducting an assessment of lymph node proliferative
activity, may be necessary. In order to have adequate information on which to select amaximum dose level to use in the definitive test and to identify a dose-response relationship,data should be collected on at least three test substance dose levels with two mice per dosegroup, in addition to the concurrent solvent/vehicle control group.
The LLNA experimental procedure is performed as follows:
Day 1. Identify and record the weight of each mouse before applying the test
substance. Apply 25 µL/ear of the appropriate dilution of the test substance, or the
positive control, or the solvent/vehicle only, to the dorsum of both ears of eachmouse.
Days 2 and 3. Repeat the application procedure as carried out on Day 1.
Days 4 and 5. No treatment.
Day 6. Record the weight of each mouse. Inject 250 µL of sterile phosphatebuffered saline (PBS) containing 20 µCi of tritiated (3H)-methyl thymidine or
250 µL PBS containing 2 µCi of 125I-iododeoxyuridine (125IU) and 10-5 M
fluorodeoxyuridine into each mouse via the tail vein (Kimber et al. 1995;
Loveless et al. 1996). Five hours later, each mouse is euthanized and the draining(“auricular”) lymph nodes of both ears are collected and placed in PBS (onecontainer per mouse). Both bilateral draining lymph nodes must be collected (seediagram and description of dissection in Annex I). A single-cell suspension oflymph node cells (LNC) is prepared for each individual mouse. The single-cellsuspension is prepared in PBS by either gentle mechanical separation through200-mesh stainless steel gauze or another acceptable technique for generating asingle-cell suspension. LNC are washed twice with an excess of PBS and the
DNA precipitated with 5% trichloroacetic acid (TCA) at 4°C for approximately
18 hours.
For the 3H-methyl thymidine method, pellets are resuspended in 1 mL TCA andtransferred to 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine
29

A robust historical dataset should include at least 10 independent tests, conducted within a reasonable periodof time (i.e., less than one year), with a minimum of four mice per negative and positive control groups.
B-8

ICCVAM Test Method Evaluation Report: Appendix B

is measured by β-scintillation counting as disintegrations per minute (dpm) for
each mouse and expressed as dpm/mouse. For the 125IU method, the 1 mL TCA
pellet is transferred directly into gamma-counting tubes. Incorporation of 125IU isdetermined by gamma counting and also expressed as dpm/mouse.
2.6

Observations

Mice should be carefully observed for any clinical signs, either of local irritation at theapplication site or of systemic toxicity (Annex III). Weighing mice prior to treatment and at thetime of necropsy will aid in assessing systemic toxicity. All observations are systematicallyrecorded and records maintained for each individual mouse. Animal monitoring plans must
include criteria to promptly identify mice exhibiting systemic toxicity or excessive irritation or
corrosion of skin for euthanasia.

3.0

Calculation of Results

Results for each treatment group are expressed as the mean SI. Each SI is the ratio of the mean
dpm/mouse within each test-substance treatment group or the positive control treated groupagainst the mean dpm/mouse for the solvent/vehicle treated control group. However, theinvestigator should be alert to possible outlier responses for individual mice within a group that
may necessitate analysis both with and without the outlier.
In addition to a formal assessment of the magnitude of the SI, a statistical analysis for presence
and degree of dose response may be conducted, which is possible only with the use ofindividual animals. Any statistical assessment should include an assessment of the doseresponse relationship as well as suitably adjusted comparisons of test groups (e.g., pair-wise
dosed group versus concurrent solvent/vehicle control comparisons). Analyses may include, forinstance, linear regression, William’s test to assess dose-response trends, or Dunnett’s test for
pairwise comparisons. In choosing an appropriate method of statistical analysis, the investigatorshould be aware of possible inequality of variances and other related problems that may
necessitate a data transformation or a non-parametric statistical analysis.

4.0

Evaluation and Interpretation of Results

In general, when the SI for any single treatment dose group is ≥ 3, the test substance is regarded
as a skin sensitizer (Kimber et al. 1994; Basketter et al. 1996; ICCVAM 1999) and a test
substance not meeting this criterion is considered a non-sensitizer in this test. However, themagnitude of the observed SI should not be the sole factor used in determining the biologicalsignificance of a skin sensitization response. Additional factors that could be considered include
the outcomes of statistical analyses, the strength of the dose-response relationship, chemical
toxicity, and solubility. For instance, a quantitative assessment may be performed by statisticalanalysis of individual mouse data and may provide a more complete evaluation of the test
substance’s ability to act as a sensitizer (see Section 3.0). Equivocal results (e.g., the SI does
not reach 3, but it is near 3 and there is a positive dose-response relationship) should beclarified by performing statistical analysis, and by considering structural relationships, available
toxicity information, and dose selection.

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ICCVAM Test Method Evaluation Report: Appendix B

5.0

Data and Reporting

5.1

Data

Individual animal dpm data should be presented in tabular form, along with the group mean
dpm/mouse, its associated error term, and the mean SI (and associated error term) for each dosegroup compared against the concurrent solvent/vehicle control group.
5.2

Test Report

The test report should contain the following information:
Test Substances and Control Substances• Identification data and Chemical Abstracts Service Registry Number, if known• Physical nature and purity• Physiochemical properties relevant to the conduct of the study• Stability of the test substance, if known• Lot number of the test substance
Solvent/Vehicle:• Justification for choice of solvent/vehicle• Solubility and stability of the test substance in the solvent/vehicle
Test Animals:• Strain of mice used
• Number, age, and sex of mice
• Source, housing conditions, diet, etc.
• Individual weight of the mice at the start and end of the test, including bodyweight range, as well as mean and associated error term for each group• Microbiological status of the mice
Test Conditions:• Concurrent and historical positive and negative (solvent/vehicle) control data
• Data from range-finding study, if conducted• Rationale for dose-level selection• Details of test substance preparation• Details of the administration of the test substance• Details of food and water quality• Detailed description of treatment and sampling schedules• Methods for measurement of toxicity• Criteria for considering studies as positive, negative, or equivocal
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ICCVAM Test Method Evaluation Report: Appendix B

Results:• Signs of systemic toxicity and/or local irritation• Values for dpm/mouse for each mouse within each treatment group• Mean and associated error term for dpm/mouse for each treatment group andthe results of outliner analysis for each dose group should be provided• Calculated SI and an appropriate measure of variability that takes into accountthe interanimal variability in both the test substance dosed and control groups• Dose-response relationship• Statistical analyses and method applied• Concurrent and historical positive and negative (solvent/vehicle) control data as
established in the test laboratory• Concurrent positive control data or, if not done, the date and laboratory reportfor the most recent periodic positive control and a report detailing the historical
positive control data for the laboratory justifying the basis for not conducting aconcurrent positive control.
Discussion of the Results
Conclusion
A Quality Assurance Statement for GLP-compliant Studies• This statement should indicate all inspections made during the study and thedates any results were reported to the Study Director. This statement should
also confirm that the final report reflects the raw data.

6.0

References

Basketter DA, Gerberick GF, Kimber I, Loveless SE. 1996. The local lymph node assay – a
viable alternative to currently accepted skin sensitisation tests. Food Chem Toxicol 34:985-997.
Dean JH, Twerdok LE, Tice RR, Sailstad DM, Hattan DG, Stokes WS. 2001. ICCVAM
evaluation of the murine local lymph node assay. Conclusions and recommendations of an
independent scientific peer review panel. Regul Toxicol Pharmacol 34:258-273.
Ehling G, Hecht M, Heusener A, Huesler J, Gamer AO, van Loveren H, et al. 2005. A
European inter-laboratory validation of alternative endpoints of the murine local lymph node
assay: first round. Toxicology 212:60-68.
EPA. 1998. Health Effects Test Guidelines: OPPTS 870.1200 - Acute Dermal Toxicity. EPA
712–C–98–192. Washington, DC: U.S. Environmental Protection Agency. Available:
http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Series/870-1200.pdf.
Hayes BB, Gerber PC, Griffey SS, Meade BJ. 1998. Contact hypersensitivity todicyclohexylcarbodiimide and diisopropylcarbodiimide in female B6C3F1 mice. Drug Chem
Toxicol 21:195-206.
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ICCVAM Test Method Evaluation Report: Appendix B

Hayes BB, Meade BJ. 1999. Contact sensitivity to selected acrylate compounds in B6C3F1
mice: relative potency, cross reactivity, and comparison of test methods. Drug Chem Toxicol
22:491-506.
Homey B, von Schilling C, Blumel J, Schuppe H-C, Ruzicka T, Jürgen AH, et al. 1998. An
integrated model for the differentiation of chemical-induced allergic and irritant skin reactions.
Toxicol Appl Pharmacol 153:83-94.
ICCVAM. 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemicals/Compounds. The Results of an IndependentPeer Review Evaluation Coordinated by the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and the National Toxicology Program
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). NIH
Publication No. 99-4494. Research Triangle Park, NC: National Institute of Environmental
Health Sciences. Available: http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf.
ICCVAM. 2001. Protocol: Murine Local Lymph Node Assay (LLNA). Purpose: Assessment ofAllergic Contact Dermatitis Potential. Recommended by: ICCVAM Immunotoxicity Working
Group – based on an Independent Expert Peer Review Panel Evaluation of the LLNA. Research
Triangle Park, NC: National Institute of Environmental Health Sciences. Available:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/LLNAProt.pdf.
ICCVAM. 2008a. Independent Scientific Peer Review Panel Report. Validation Status of New
Versions and Applications of the Murine Local Lymph Node Assay: A Test Method forAssessing the Allergic Contact Dermatitis Potential of Chemicals and Products. Research
Triangle Park, NC: National Institute of Environmental Health Sciences. Available:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf.
ICCVAM. 2008b. Non-radioactive Murine Local Lymph Node Assay: Flow Cytometry Test
Method Protocol (LLNA: BrdU-FC) Draft Background Review Document. Research Triangle
Park, NC: National Institute of Environmental Health Sciences. Available:
http://iccvam.niehs.nih.gov/methods/immunotox/llna-BRD.htm.
ICCVAM. In press. Report on the ICCVAM-NICEATM/ECVAM/JaCVAM Scientific
Workshop on Acute Chemical Safety Testing: Advancing In Vitro Approaches and Humane
Endpoints for Systemic Toxicity Evaluations. Research Triangle Park, NC: National Institute ofEnvironmental Health Sciences. Available:
http://iccvam.niehs.nih.gov/methods/acutetox/Tox_workshop.htm.
Institute of Laboratory Animal Research (ILAR). 1996. Guide for the Care and Use ofLaboratory Animals. 7th ed. Washington, DC: National Academies Press.
Kimber I, Basketter DA. 1992. The murine local lymph node assay; collaborative studies andnew directions: a commentary. Food Chem Toxicol 30:165-169.
Kimber I, Dearman RJ, Scholes EW, Basketter DA. 1994. The local lymph node assay:
developments and applications. Toxicology 93:13-31.
Kimber I, Hilton J, Dearman RJ, Gerberick GF, Ryan CA, Basketter DA, et al. 1995. Aninternational evaluation of the murine local lymph node assay and comparison of modified
procedures. Toxicology 103:63-73.
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ICCVAM Test Method Evaluation Report: Appendix B

Loveless SE, Ladics GS, Gerberick GF, Ryan CA, Basketter DA, Scholes EW, et al. 1996.
Further evaluation of the local lymph node assay in the final phase of an international
collaborative trial. Toxicology 108:141-152.
OECD. 1987. Guideline For Testing of Chemicals – Test Guideline 402: Acute Dermal
Toxicity. Paris: Organisation for Economic Co-operation and Development. Available:
http://www.oecd.org/document/40/0,2340,en_2649_34377_37051368_1_1_1_1,00.html.
Patterson RM, Noga E, Germolec D. 2007. Lack of evidence for contact sensitization by
Pfiesteria extract. Environ Health Perspect 115:1023-1028.
Reeder MK, Broomhead YL, DiDonato L, DeGeorge GL. 2007. Use of an enhanced local
lymph node assay to correctly classify irritants and false positive substances. Toxicologist 96(S-1):235.
Tilney NL. 1971. Patterns of lymphatic drainage in the adult laboratory rat. J Anat 109:369383.
Vohr, HW, Jürgen AH. 2005. The local lymph node assay being too sensitive? Arch Toxicol
79:721-728.
Woolhiser MR, Hayes BB, Meade BJ. 1998. A combined murine local lymph node andirritancy assay to predict sensitization and irritancy potential of chemicals. Toxicol Meth 8:
245-256.

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ICCVAM Test Method Evaluation Report: Appendix B, Annex I

Annex I:An Approach to Dissection and Identification of the Draining(“Auricular”) Lymph Nodes
1.0

Background

Although minimal technical training of the murine local lymph node assay (LLNA) is required,extreme care must be taken to ensure appropriate and consistent dissection of the lymph nodes.
It is recommended that technical proficiency in the dissection and identification of the lymph
nodes draining the ear be achieved by practice on mice that have been (a) injected with acolored agent (dye) and/or (b) sensitized with a strong positive sensitizer. Brief descriptions ofthese practice dissections are provided below. Recognizing that nodes from vehicle-treated andnaïve mice are smaller, laboratories performing the LLNA must also gain proficiency in thedissection of these nodes. It may be helpful for laboratories inexperienced in this procedure to
request guidance from laboratories that have successfully performed the LLNA.

2.0

Training and Preparation for Node Identification

2.1

Identification of the Draining Node – Dye Treatment

There are several methods that can be used to provide color identification of the draining nodes.
These techniques may be helpful for initial identification and should be performed to ensure
proper isolation of the appropriate node. Examples of such treatments are listed below. It should
be noted that other such protocols might be used effectively.
Evan’s Blue Dye treatment:
Inject approximately 0.1 mL of 2% Evan’s Blue Dye (prepared in sterile saline)
intradermally into the pinnae of an ear. Euthanize the mouse after several minutes
and continue with the dissection as noted below.
Colloidal carbon and other dye treatments:
Colloidal carbon and India ink are examples of other dye treatments that may be
used (Tilney 1971).
2.2

Identification of the Draining Node – Application of Strong Sensitizers

For the purpose of node identification and training, a strong sensitizer is recommended. This
agent should be applied in the standard acetone: olive oil vehicle (4:1). Suggested sensitizers forthis training exercise include 0.1% oxazolone, 0.1% (w/v) 2,4-dinitrochlorobenzene, and 0.1%(v/v) dinitrofluorobenzene. After treating the ear with a strong sensitizer, the draining node will
dramatically increase in size, thus aiding in identification and location of the node.
Using a procedure similar to that described in the test method protocol, apply the agent to thedorsum of both ears (25 µL/ear) for 3 consecutive days. On the fourth day, euthanize themouse. Identification and dissection (listed below) of the node should be performed in theseanimals prior to practice in non-sensitized or vehicle-treated mice, where the node issignificantly smaller.
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ICCVAM Test Method Evaluation Report: Appendix B, Annex I

Please note: Due to the exacerbated response, the suggested sensitizers are not recommended ascontrols for assay performance. They should only be used for training and node identification
purposes.

3.0

Dissection Approach

3.1

Lateral Dissection (Figure B-1)

Although lateral dissection is not the conventional approach used to obtain the nodes draining
the ear, it may be helpful as a training procedure when used in combination with the ventral
dissection. Perform this approach bilaterally (on both sides of the mouse). After euthanizing themouse, place it in a lateral position. Wet the face and neck with 70% ethanol. Use scissors andforceps to make an initial cut from the neck area slightly below the ear. Carefully extend theincision toward the mouth and nose. Angle the tip of the scissors slightly upward during thisprocedure to prevent the damage of deeper tissue. Gently retract the glandular tissue in the area
using the forceps. Using the masseter muscle, facial nerves, blood vessels, and the bifurcationof the jugular vein as landmarks, isolate and remove the draining node (Figure B-1). The
draining node (“auricular”) will be positioned adjacent to the masseter muscle and proximal toand slightly above the jugular bifurcation.
3.2

Ventral Dissection (Figure B-2)

The most commonly used dissection approach is from the ventral surface of the mouse. Thisapproach allows both right and left draining nodes to be obtained without repositioning themouse. With the mouse ventrally exposed, wet the neck and abdomen with 70% ethanol. Usescissors and forceps to carefully make the first incision across the chest and between the arms.
Make a second incision up the midline perpendicular to the initial cut, and then cut up to thechin area. Reflect the skin to expose the external jugular veins in the neck area. Take care to
avoid salivary tissue at the midline and nodes associated with this tissue. The nodes draining theear (“auricular”) are located distal to the masseter muscle, away from the midline, and near thebifurcation of the jugular veins.

4.0

Accuracy in Identification

The nodes can be distinguished from glandular and connective tissue in the area by the
uniformity of the nodal surface and a shiny translucent appearance. Application of sensitizing
agents (especially the strong sensitizers used in training) will cause enlargement of the nodesize. If a dye is injected for training purposes, the node will take on the tint of the dye.

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ICCVAM Test Method Evaluation Report: Appendix B, Annex I

Figure B-1

Lateral Dissection

Figure B-2

Ventral Dissection

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ICCVAM Test Method Evaluation Report: Appendix B, Annex II

Annex II:
An Example of How to Reduce the Number of Animals in the Concurrent
Positive Control Group of the Local Lymph Node Assay
As stated in the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) Murine Local Lymph Node Assay (LLNA) test method protocol (Section 2.4.2 ofAppendix B), a concurrent positive control is recommended to ensure the appropriate
performance of the assay. Appropriate performance is demonstrated when the test method
responds with adequate and reproducible sensitivity to a sensitizing substance for which themagnitude of the response is well characterized. The number of mice in the concurrent positive
control group may possibly be reduced if the laboratory demonstrates, based on laboratoryspecific historical data, that fewer mice can be used without compromising the integrity of thestudy (i.e., positive control results should be always be positive compared to the vehicle control
results). As illustrated in the example and accompanying explanation below, reducing thenumber of animals in the positive control group is only feasible when individual animal data arecollected.
The stimulation index (SI) results for each positive control test can be used to generate mean SI
values for every possible combination of SI values for as few as two animals. The mean SI
values for every combination of numbers for each group size can then be used to calculate thefailure rate of the positive control for each group size (i.e., the percentage of the combinations
for which the mean SI < 3). Table B-1 provides an example of positive control results fromfour tests in one laboratory of 30% hexyl cinnamic aldehyde (HCA) using six CBA/J mice pergroup. In these tests, with six animals, HCA produced “borderline” positive results (i.e., themean SI values were marginally greater than 3). To determine whether the number of animals
can be reduced, sample size reductions (i.e., N = 5, 4, 3, or 2) can be evaluated by taking all
possible samples from the six values for each test given in Table B-1, which can occur in thefollowing ways: N = 2 (15 samples), N = 3 (20 samples), N = 4 (15 samples), and N = 5 (sixsamples).
Table B-1 Example of SI Results from Four Local Lymph Node Assay Positive Control
Studies with 30% HCA
Test

1

2

3

4

Animal 1

2.13

3.56

4.68

0.78

Animal 2

4.55

1.54

4.44

9.16

Animal 3

3.64

3.00

5.41

6.66

Animal 4

1.98

3.87

3.32

3.02

Animal 5

3.09

3.79

2.89

2.32

Animal 6

3.77

3.96

1.81

2.91

Mean SI

3.19

3.29

3.76

4.14

Abbreviations: HCA = hexyl cinnamic aldehyde; SI = stimulation index

The failure rate of the positive control was then calculated using the SI results for each group of
two, three, four, or five values to determine the likelihood of obtaining a mean SI < 3. The
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ICCVAM Test Method Evaluation Report: Appendix B, Annex II

results for these four “borderline” HCA tests were then added to the results from an additional
12 robust positive control tests included in this laboratory’s historical database to determine theoverall likelihood of obtaining a mean SI < 3 for the positive control substance (Table B-2).
The failure rate reflects the frequency with which a positive control test will fail, which would
result in retesting the positive control and any concurrent test substances. Each laboratory is
encouraged to determine the lowest number of animals to use in the positive control groupbased on the highest failure rate considered acceptable by the laboratory.
Table B-2 Example of Positive Control Failure Rate for 30% HCA Based on Data
Collected in Single Laboratory
Number of
Animals

HCA
Test 1
17%

HCA
Test 20%

HCA
Test 30%

HCA
Test 4
0%

Results from
Other Tests10%Overall Likelihoodof a Mean SI < 3
1%

(1/6)

(0/6)

(0/6)

(0/6)

(0/72)(1/96)

427%(4/15)

13%(2/15)

0%

7%

0%

3%

(0/15)(1/15)(0/180)(7/240)340%(8/20)

30%(6/20)

5%

20%

0%

6%

(1/20)(4/20)(0/240)(19/320)2

47%(7/15)

33%(5/15)

13%

40%

1%

9%

(2/15)(6/15)(1/180)(21/240)5Abbreviations: HCA = hexyl cinnamic aldehyde; SI = stimulation index
1
These represent 12 positive control studies in the same laboratory where all mice in the positive control
groups treated with 30% HCA produced an SI ≥ 3.

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ICCVAM Test Method Evaluation Report: Appendix B, Annex III

Annex III:
Evaluating Local Irritation and Systemic Toxicity in the Local
Lymph Node Assay
As noted in the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) Murine Local Lymph Node Assay (LLNA) protocol, at least three dose levels of atest substance should be evaluated. The highest dose level tested should be a concentration of
100% (i.e., neat substance for liquid substances) or the maximum soluble concentration (forsolids), unless available information suggests that this concentration induces systemic toxicity
or excessive local irritation after topical application.
In the absence of such information, a prescreen test should be performed using three dose levelsof the test substance, in order to define the appropriate dose level to test in the LLNA. Six mice
(two per concentration) are used, and the prescreen is conducted under identical conditions as
the main LLNA study, except there is no assessment of lymph node proliferation. All mice will
be observed daily for any clinical signs of systemic toxicity or local irritation at the applicationsite. For example, observations might occur before and after treatment on Days 1, 2, and 3.
Body weights are recorded pre-test and prior to termination (Day 6). Both ears of each mouse
are observed for erythema (and scored using Table B-3). Ear thickness measurements are takenusing a thickness gauge (e.g., digital micrometer or Peacock Dial thickness gauge) on Day 1(pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6.
Excessive local irritation is indicated by an erythema score ≥3 and/or ear swelling of ≥25%.
Table B-3 Erythema Scores
Observation

Value

No visual effect0

Slight erythema (barely perceptible)

1

Well-defined erythema

2

Moderate to severe erythema (beet redness)3

Eschar (i.e., piece of dead tissue that is cast off
from the surface of the skin)4

A 25% increase in ear swelling has been used as an initial step to identify substances that causea skin reaction due to an irritant response rather than sensitization (Reeder et al. 2007;
ICCVAM 2008b). A statistically significant difference from control animals has also been used
to delineate irritants from non-irritants in the LLNA (Hayes et al. 1998; Homey et al. 1998;
Woolhiser et al. 1998; Hayes and Meade 1999; Ehling et al. 2005; Vohr and Jürgen 2005;
Patterson et al. 2007). While these statistical differences often occur when ear swelling is lessthan 25%, they have not been associated specifically with excessive irritation (Woolhiser et al.
1998; Ehling et al. 2005; Vohr and Jürgen 2005; Patterson et al. 2007). Additionally, an
adequately robust statistical comparison would require that a vehicle control group be included
and that more than two animals per group be tested. Both of these requirements would
substantially increase the number of animals used for this prescreen test. For this reason, athreshold increase in ear swelling above pre-dosing levels is recommended for this prescreen
test.
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ICCVAM Test Method Evaluation Report: Appendix B, Annex III

Test guidelines for assessing acute systemic toxicity recommend a number of clinicalobservations for assessing systemic toxicity (OECD 1987; EPA 1998). The following
observations, which are based on test guidelines and current practices (ICCVAM in press), may
indicate systemic toxicity when used as part of an integrated assessment and therefore may
indicate that the maximum dose recommended for the LLNA has been exceeded:•••

Clinical signs:–

Changes in nervous system function (e.g., piloerection, ataxia, tremors,and convulsions)–

Changes in behavior (e.g., aggressiveness, change in grooming activity,
marked change in activity level)–

Changes in respiratory patterns (i.e., changes in frequency and intensity ofbreathing such as dyspnea, gasping, and rales)–

Changes in food and water consumption–

Lethargy and/or unresponsiveness–Any clinical signs of more than slight or momentary pain and distress

Reduction in body weight >10% from Day 1 to Day 6
Mortality

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ICCVAM Test Method Evaluation Report: Appendix C

Appendix C
Evaluating the Impact of Reducing the Sample Size from Five to Four Animals perGroup on the Performance of the Ratio Rule of SI > 3 in LLNA Testing

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ICCVAM Test Method Evaluation Report: Appendix C

1.0

Introduction

Test Guideline 429 issued by the Organisation for Economic Co-operation and Development(OECD; OECD 2002) states that “A minimum of four animals is used per dose group, with aminimum of three concentrations of the test substance, plus a negative control group treated
only with the vehicle for the test substance, and a positive control, as appropriate. In thosecases in which individual animal data are to be collected, a minimum of five animals per dosegroup are used.” This analysis was undertaken to determine if the number of animals requiredfor individual animal data collection could be harmonized with that required for pooled data
without diminishing accuracy. This is important because most animal-use regulations requirethat the minimum number of animals be used in studies, which currently results in only pooleddata being collected in many countries because it currently requires fewer animals.
Therefore, the issue under investigation in the evaluation that follows is the impact ofmodifying the murine local lymph node assay (LLNA) test method protocol by reducing thenumber of individual animals per group from 5 to 4. More specifically, the evaluation considers
how often this reduction in animal usage would have an impact on the overall LLNA outcome
when the decision criterion used to determine a sensitizer from a non-sensitizer is a stimulationindex (SI) greater than or equal to 3 (i.e., the “Ratio Rule”). Since the “true” underlying
sensitizer status for individual substances is generally not known, this investigation will focus
on the degree of disagreement rather than on which observed outcome is the “correct” one. Thisevaluation focused primarily on the Ratio Rule, although the possible use of a formal statistical
test will also be considered.
The results of the following analyses indicate that a reduction in the sample size from 5 to 4
animals per group is unlikely to have any significant impact on the results of the LLNA testwhen using the Ratio Rule. If using statistics, the power for detecting LLNA effects will be
reduced slightly when using 4 animals per group relative to using 5 animals per group.
However, the practical impact of this power difference may be minimal, in that the power
difference appears to be small for detecting effects above the Ratio Rule cutoff point of SI = 3.
Importantly, this analysis also indicates that a statistical test based on 4 animals per group will
identify more sensitizers than using the Ratio Rule based on 5 animals per group.

2.0

Methods

The database evaluated includes three different strains of animals: CBA, BALB/c, and B6C3F1.
This report evaluates in detail only the CBA database; the data from the other two strains are
summarized (Section 4.0 and Table C-7) and may be evaluated more definitively in due
course. The CBA database consists of 83 individual studies, each with three or four dosed
groups and a control group. There are not 83 distinct substances, because some substances aretested in multiple studies. The number of individual animals per group in these studies ranged
from 2 to 9. There were a total of 277 dosed groups, two of which were excluded from theagreement-disagreement analysis since there were only 2 or 3 animals per group. Study results
were evaluated on a dose-by-dose basis as well as on a study-by-study basis, recognizing thatthe doses within a study used a common control group. Also, for certain labs, a common control
group was used for multiple substances.
For each study having 5 animals per group (i.e., N = 5), all possible random samples of size 4(responses measured as disintegrations per minute [dpm] of a radiolabeled tracer compound)
C-3

ICCVAM Test Method Evaluation Report: Appendix C

were taken from both the control and experimental groups (25 possible combinations), and theresults of the Ratio Rule were compared for each of the samples with that of the full data set of
5 animals. The level of agreement was then determined.
For those studies having more than 5 animals per group, a similar procedure was applied, but inthis case random samples were taken for both the N = 5 and N = 4 protocols, and there were far
more combinations of samples to deal with (8100 rather than 25). Once again, the level ofagreement between the N = 5 and N = 4 protocols were determined.

3.0

Results

Using the Ratio Rule criterion, the CBA mouse database consisted of a mix of sensitizers(49 studies) and non-sensitizers (33 studies), with one study (discussed in more detail below)
producing a borderline effect. Table C-1 shows the frequency of the various SI values in the
275 usable (for agreement-disagreement analysis) dosed groups, together with the average
agreement seen between samples of N = 5 and N = 4. As can be seen in the table, thedisagreement in study results is limited to SIs in the 2.1 to 4.7 range, with the disagreement
increasing as the SI approaches 3. The overall average agreement between N = 4 and N = 5
studies is quite good: 97.5%. Moreover, as discussed in more detail below, the disagreement inoutcome is due primarily to the inherent variability in the data (and the closeness of the SI to 3),
not to the reduction in sample size.
The individual study results for the CBA strain are summarized in Annex I.
Although the primary focus of this evaluation is on the Ratio Rule (i.e., SI > 3), it is possiblethat a formal statistical test may be used in addition to (or possibly even in place of) the Ratio
Rule. For this reason, a simple Student’s t test (based on the logged dpm data) was also used tocompare each dosed group with its concurrent control. The results of this analysis are
summarized in Table C-2. It is clear that using a formal statistical test will identify far more“positives” than the Ratio Rule, i.e., statistical significance (p < 0.05) was achieved for some
dosed groups producing an SI well below 3. This matter is discussed in more detail below.

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ICCVAM Test Method Evaluation Report: Appendix C

Table C-1 Breakdown of Individual Dosed Group SIs: CBA StrainSI<2.1
2.1 – 2.5
2.6
2.7
2.8
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
4.0 – 4.7
>4.7
Total

Agreement between N = 5 andN = 4 samples
100.00%90.10%
85.00%
73.30%
64.00%
56.00%
55.50%
73.50%
88.00%
68.00%
84.00%
90.00%
100.00%97.90%
100.00%97.50%

Frequency
154
16
2
3
2
1
2
4
1
1
1
1
1
16
70
275

Abbreviations: N = number of animals per dose group; SI = stimulation index

Table C-2 Distribution of Statistically Significant (p < 0.05) SIs: CBA StrainSI<1.7
1.7 – 1.9
2.0 – 2.5
2.6 – 3.0
> 3.0
Total

Percentage of statistically significant(p < 0.05) SIs
0.00%
52.20%
88.00%
85.70%
100.00%Frequency
131
23
17
7
1
277

Abbreviation: SI = stimulation index

4.0

Discussion

It was known in advance that the reduction in sample size from N = 5 to N = 4 would haveessentially no impact on study results for “strong sensitizers” and for “clear non-sensitizers,”
and this is confirmed in Table C-1. What was not known was (1) how frequently suchoutcomes are seen in practice; (2) the specific range of SI values in which some impact on study
outcome may be evident; (3) the magnitude of the impact for those studies having an SI close to
3; and (4) whether the disagreement in study outcome was due primarily to the reduction insample size or to the inherent variability in the data (and the closeness of the SI to 3). The
current investigation addresses all of these issues.
With regard to the first issue, for the CBA mouse database, only 34 of the 275 dosed groups(12%) had less than 100% agreement between N = 5 and N = 4 outcomes. Thus, for most dosed
groups, the reduced sample size will not even be an issue when using the Ratio Rule.
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ICCVAM Test Method Evaluation Report: Appendix C

Moreover, the reduced sample size becomes an issue only for a relatively narrow range of
SI values. The range of SI values in this database producing less than 100% agreement was 2.1
to 4.7, but this may be somewhat misleading in that many studies in this range produced 100%agreement (see Table C-1 and Annex I).
As the SI approaches 3, the disagreement between a sample of N = 5 and N = 4 increases
notably (Table C-1). However, and this may be the single most important “take home” messageof this entire analysis, the disagreement is far more a function of the animal-to-animal
variability than it is to the reduction in sample size. That is, a second sample of 5 animals would
show almost the same level of disagreement with the first sample of 5 animals, as would asample of 4 animals. Thus, the reduction in sample size is a relatively small contributor to thisdifference. This important concept is illustrated below with two examples from the CBA mouse
database, the first showing an SI of 2.8 (Table C-3), just below the Ratio Rule threshold of
SI = 3, the second showing an SI of 3.2 (Table C-4), just above the Ratio Rule threshold.
The first example is the high dose of the third hexyl cinnamic aldehyde study, which had an SIof 2.8 for N = 6 (Table C-3). This is the one study noted above with a borderline effect. Since
N = 6, this required selection of samples of size 5 from both the control and dosed groups, andsome of these samples did not give the same result as that seen for the full six animal sample.
The results are summarized below and compared with the N = 4 strategy.
Table C-3 Example Showing Effect of Sample Size on Agreement of Results for a Test
Substance with SI = 2.8

Agreement (SI > 3)
Agreement (SI < 3)
Disagreement (one SI > 3; one SI < 3)

Two N = 5 samples
7.7% (10/36) (10/36)
52.2% (26/36) (26/36)
40.1% (by subtraction)

One N = 5 sample andone N = 4 sample
10.5% (10/36) (85/225)
44.9% (26/36) (140/225)
44.6% (by subtraction)

Abbreviations: N = number of animals per dose group; SI = stimulation index

As can be seen from these calculations (see also Annex I), the agreement between N = 5 andN = 4 strategies is “only” 55%. However, the disagreement is not due primarily to a reduction
in sample size, since the agreement is very similar to that found for two N = 5 samples (60%).
In other words, only 4.5% of the observed 45% disagreement is due to the reduction in sample
size. The rest is due to the inherent variability among animals (and the closeness of the SI to 3)
that would be evident even if a second sample of size 5 were used.
The second example is the mid-dose of the dipropylene triamine study, which had an SI of 3.2
also for N = 6 (Table C-4). The results are summarized below and compared with the N = 4
strategy.

C-6

ICCVAM Test Method Evaluation Report: Appendix C

Table C-4 Example Showing Effect of Sample Size on Agreement of Results for a Test
Substance with SI = 3.2

Agreement (SI > 3)
Agreement (SI < 3)
Disagreement (one SI > 3; one SI < 3)

One N = 5 sample and
Two N = 5 samples
one N = 4 sample
56.25% (27/36) (27/36) 50.67% (27/36) (152/225)
6.25% (9/36) (9/36)
8.11% (9/36) (73/225)
37.50% (by subtraction) 41.22% (by subtraction)

Abbreviations: N = number of animals per dose group; SI = stimulation index

The results are very similar to those of the first example, in that most of the 41% disagreement
between the N = 4 sample and the N = 5 sample is due to the inherent variability of the data andthe closeness of the SI to 3, not to the reduction in sample size.
Another point that should be noted: in the instances in which there is disagreement, the N = 4
strategy may actually have a higher likelihood of producing an SI > 3 result than using a sampleof size 5. This occurs when the underlying SI is close to but below 3. For instance, consider the
first example given above in which the observed SI = 2.8. A sample of size 4 would have a 38%
chance (85/225) of producing an SI > 3 compared with only 28% (10/36) when using N = 5. Inthat sense, N = 4 could be regarded as having greater “power” than N = 5 for these data.
However, use of the Ratio Rule implicitly assumes that an SI less than 3 is biologicallyunimportant and thus should not be detected. Thus, the increased likelihood of exceeding the
Ratio Rule criterion using N = 4 in the example above could be regarded as an increase in thefalse positive rate, rather than an increase in power. Importantly, as N increases, the likelihoodof detecting SI = 2.8 by the Ratio Rule approaches zero, with maximum “power” occurring for
N = 1.
However, some investigators may regard an SI of 2.8 as biologically important, especially ifseen at the top dose, as was the case in this study. Consequently, these investigators might
actually prefer the performance of N = 4 rather than N = 5 in this example. Of course, if SI < 3
responses are considered important, it would make far more sense to carry out a formal
statistical test to detect them rather than using the Ratio Rule, which will likely not detect them.
Although not detected by the Ratio Rule, the SI = 2.8 effect noted above in the high dose hexyl
cinnamic aldehyde study is highly significant (p < 0.01) by Student’s t test.
Moreover, it is likely that this particular SI = 2.8 is a “real” effect, not only because it is highly
significant statistically, but also because in four other studies with this compound, the SIs
produced for this dose were 2.2, 4.1, 4.2, and 6.6, with higher doses producing even greater
effects (see Annex I). Without these additional studies, it is possible that this effect would be“missed” since SI = 2.8 does not satisfy the Ratio Rule criterion of SI > 3, and without
individual animal data, it would not be possible to determine whether or not this effect was
statistically significant. This is another illustration of the value of individual animal data andalso the value of using a formal statistical test. It also shows that in some cases a sample of
N = 4 is actually more likely to produce the “correct” conclusion than N = 5 when using the
Ratio Rule.

C-7

ICCVAM Test Method Evaluation Report: Appendix C

As can be seen in Table C-2, a formal statistical test will identify as statistically significant (p <0.05) many responses that would not be detected by the Ratio Rule. In some cases, statisticalsignificance is achieved for SI values as low as 1.7 (see Annex I and Table C-2). Normally,
this “increased power” would be considered very desirable, but apparently it is possible thatcertain SIs in the 1.7 to 3.0 range, while truly different from controls, may be reflecting“irritation” rather than a true sensitizing effect, and thus may not be indicative of a meaningful
human risk. Discussion of this matter is beyond the scope of this investigation, but it is logicalto assume that since the Ratio Rule is widely used for LLNA data, while a formal statistical test
is not, there must be concern that a formal statistical test will produce too many “significant
effects” for SIs in the 2 to 3 range. That is, SIs below 3 may be statistically significant andreflect “real” dosed group effects, but responses in this range are considered biologicallyunimportant. As can be seen in Table C-2, most of the SIs in the 2 to 3 range are in fact
statistically significant. Use of the Ratio Rule also implicitly assumes that false positives are
more important than false negatives.
Any consideration of statistical power must take into account the variability in response among
animals. To illustrate this, consider the 17 CBA mouse studies carried out at BASF (seeTable C-11 in Annex I). The mean control dpm response across these 17 studies was 552.3.
The mean standard deviation (SD; based on the logged dpm responses) among the control
animals was 0.4077. Based on this information, we can carry out a power calculation, which issummarized in Table C-5.
To explain further: Power is primarily a function of (1) the magnitude of the difference between
the dosed and control groups, (2) the underlying variability among animals, and (3) the sample
size. In the table below, “difference” is the size (on a log scale) of the “fold increase” that is tobe detected. The SD is the assumed underlying standard deviation among animals (on a log
scale) as determined by the data from BASF (see Table C-11 in Annex I). This SD is assumed
to be the same in the dosed and control groups, an assumption consistent with the data frommultiple labs obtained to date. Delta is the standardized (by SD) difference to be detected and is
the key input variable into the power calculation program. The power calculations given below
are based on a two-sided Student’s t test, and assume an underlying normal distribution for thelogged data. The specific power calculations were taken fromhttp://www.danielsoper.com/statcalc/calc49.aspx. In this program “Cohen’s d” is just thestandardized difference, Delta. This is a very simple program to use, and alternative power
calculations can easily be made.

C-8

ICCVAM Test Method Evaluation Report: Appendix C

Table C-5 Post-hoc Power Calculations Based on the BASF Control Data

Assumed control response
Log (Control response)
Dosed group response
Log (Dosed group response)
Difference (log scale)
Assumed SD (log scale)
Delta = Difference/SD
Power for N = 5
Power for N = 4

Dosed Group Increase Relative to Controls
3.5-fold
3-fold
2.5-fold
2-fold
552.3
552.3
552.3
552.3
6.314
6.314
6.314
6.314
1933.05
1656.901380.75
1104.60
7.567
7.413
7.230
7.007
1.253
1.099
0.916
0.693
0.40770.40770.40770.4077
3.07
2.70
2.25
1.70
99.0%
96.4%
87.9%
65.8%
95.7%
89.8%
76.8%
53.0%

Abbreviations: N = number of animals per dose group; SD = standard deviation

From these calculations, the conclusion is that if the underlying variability among control
animals is similar to that seen in an average BASF study, then there is an excellent chance thatan underlying SI of 2.5 will be detected as statistically significant (p < 0.05), although thislikelihood is higher for N = 5 (87.9%) than for N = 4 (76.8%). This power calculation is also
consistent with the empirical results summarized in Table C-2. An underlying SI of 2.5 would
almost certainly not be detected by the Ratio Rule, nor would one want it to be detected, sinceuse of the Ratio Rule implicitly assumes that such an effect is of no consequence, as noted
earlier.
From the website given above, a general power curve can be constructed for N = 5 and N = 4
by specifying different values of Delta, which could reflect different “-fold increases (i.e., SI
values),” different underlying variabilities, or a combination of these two factors. Such powercomparisons are summarized below in Table C-6 and Figure C-1 and include the four from
Table C-5.

C-9

ICCVAM Test Method Evaluation Report: Appendix C

Table C-6 Selected Power Comparisons for N = 5 and N = 4 Samples Based on BASF
Control Data
SI
4.34
4.25
4.00
3.75
3.69
3.50
3.25
3.13
3.00
2.75
2.66
2.50
2.26
2.25
2.00
1.92
1.75
1.63
1.50
1.25
1.00

Delta
3.60
3.55
3.40
3.24
3.20
3.07
2.89
2.80
2.70
2.48
2.40
2.25
2.00
1.99
1.70
1.60
1.37
1.20
0.99
0.55
0.00

N=5
99.9%
99.9%
99.7%
99.5%
99.4%
99.0%
98.0%
97.4%
96.4%
93.2%
91.6%
87.9%
79.5%
79.1%
65.8%
60.5%
47.9%
38.6%
28.0%
11.6%
2.5%

N=4
99.1%
98.9%
98.3%
97.2%
96.9%
95.7%
93.3%
91.8%
89.8%
84.3%
81.9%
76.8%
66.8%
66.3%
53.0%
48.2%
37.4%
30.0%
21.9%
9.7%
2.5%

Abbreviations: N = number of animals per dose group; SI = stimulation index

Figure C-1 Power Curve for N = 5 and N = 4 Samples Based on BASF Control Data
110
100
90
80
70
60
50
40
30
20
10
0

N=5
N=4

0

1

2

3

4

5

SI
Abbreviations: N = number of animals per dose group; SI = stimulation index
C-10

ICCVAM Test Method Evaluation Report: Appendix C

Although these particular “Deltas” could result from different combinations of –fold increases
and assumed variability, the power calculations for the BASF data indicate that the mostnotable differences in power between N = 5 and N = 4 occur for SIs below 3, a range for which
detection of an effect is apparently viewed as a “false positive” as discussed earlier. That is, theRatio Rule implicitly assumes that SIs less than 3 should not be detected, so the fact thatsamples of N = 5 are more likely than samples with N = 4 to detect significant effects for SIs
below 3 could be viewed as a disadvantage rather than an advantage of a larger sample size. For
SI = 3.5 (at least for the BASF data), the power is high and similar for N = 5 and N = 4 (99.0%
vs. 95.7%).
Note also from Table C-6 that there will be far more sensitizers identified by a statistical test
based on 4 animals per group than would be identified by the Ratio Rule using 5 animals pergroup. For example, a formal statistical test with N = 4 would have approximately 90% powerfor detecting an SI = 3, compared with only 50% power by using the Ratio Rule (regardless ofN).
Although this report focuses on the large CBA mouse database, there are two smaller LLNA
databases involving BALB/c and B6C3F1 mice. Although these other databases were notevaluated in detail, the pattern of LLNA response seen in these two strains was very similar tothat seen in the CBA database. This comparison is summarized in Table C-7 below. In thistable, the percentage of positive studies is the percentage of studies having SI > 3 in at least one
dosed group. As can be seen in Table C-7, there is little evidence of a strain difference in thepattern of LLNA response, and thus there is very little likelihood that a detailed evaluation ofthese other two strains would change the conclusions of this report.
Table C-7 Comparison of CBA, BALB/c, and B6C3F1 Databases
StrainCBABALB/cB6C3F1

No. ofStudies
83
41
10

No. of % Positive
Doses
Studies277
59 (49/83)
133
63 (26/41)
28
70 (7/10)

Distribution of SIs<1.7
1.7 – 1.9 2.0 – 2.5 2.6 – 3.0
131 (47%) 23 (8%) 17 (6%)
7 (3%)
67 (50%) 12 (9%)
8 (6%)
6 (5%)
15 (54%)1 (4%)
1 (4%)
2 (7%)> 3.0
99 (36%)40 (30%)9 (32%)

Abbreviation: No. = number; SI = stimulation index

There is one B6C3F1 mouse study that deserves special mention: the National Toxicology
Program 2,4,5-trichlorophenoxyacetic acid study, which used a sample size of 6 animals pergroup. The top dose in this study produced a mean SI response of 3.03, which is the weakest“Ratio Rule positive” of any study in the three databases (control dpm responses were 63-6975-90-119-133 compared with 213-229-244-249-325-405 in the top dosed group). The impactof reducing the sample size from 6 to 5 or 4 animals per group is summarized below.

C-11

ICCVAM Test Method Evaluation Report: Appendix C

Table C-8 Example Showing Effect of Sample Size on Agreement of Results for a Test
Substance with SI = 3.03

Agreement (SI > 3)
Agreement (SI < 3)
Disagreement (one SI > 3; one SI < 3)

Two N = 5 samples
25.0% (18/36) (18/36)
25.0% (18/36) (18/36)
50.0% (by subtraction)

One N = 5 sample andone N = 4 sample
26.4% (18/36) (119/225)
23.6% (18/36) (106/225)
50.0% (by subtraction)

Abbreviations: N = number of animals per dose group; SI = stimulation index

For these data, there is 50% disagreement between samples of size 4 and samples of size 5, butthere is also 50% disagreement between two samples of size 5. This is a somewhat extreme
example of the point made earlier, namely that most of the disagreement in Ratio Rule results
observed between samples of size 5 and samples of size 4 shown in Table C-1 is not due to the
reduction in sample size, but rather due to the variability in response among animals and thecloseness of the SI to the cutoff point of 3.
Finally, it is important to understand that Table C-1 is not measuring accuracy; it is measuringagreement. That is, Table C-1 assesses the reliability of N = 5 and N = 4 samples to produce
the same classification outcome using the Ratio Rule; it does not assess the ability of N = 5 andN = 4 samples to produce the correct sensitizer classification (which for most substances is notknown in any case). As illustrated in this report, as SI approaches 3, different samples may
produce different classifications using the Ratio Rule, regardless of sample size, because ofnaturally occurring variability among animals. Importantly, most of the discordance between
N = 5 and N = 4 samples shown in Table C-1 is not due to the reduction in sample size.
With regard to accuracy of classification using the Ratio Rule, for 90% (75/83) of the CBA
studies, there is no difference in accuracy using N = 5 and N = 4, based on the top dose group
SI response. For eight studies, each with a top dose SI close to 3, there are slight differences inagreement, as shown in Table C-9.

C-12

ICCVAM Test Method Evaluation Report: Appendix C

Table C-9 Likelihood of SI > 3 for All CBA Studies Showing Less than Complete
Agreement for the Top Dose Response Using N = 5 and N = 4 Samples
Substance
Formulation 54
Hexyl cinnamic aldehyde
Formulation 39
Bakelite EPR 161
Formulation 55
Potassium dichromate
Formulation 51
1,6-(Bis(2-3-epoxypropoxy)hexane

Top Dose SI
2.3
2.8
3.3
3.5
3.7
4.1
4.51
4.7

Likelihood of SI > 3 (%)
N=5
N=40 (0/36)
7 (16/225)
28 (10/36)
38 (85/225)
92 (33/36)
78 (175/225)
83 (30/36)
77 (174/225)
100 (36/36)
90 (202/225)
100 (1/1)
92 (23/25)
100 (36/36)
96 (215/225)
100 (36/36)
94 (211/225)

Abbreviations: N = number of animals per dose group; SI = stimulation index
1
Maximum response seen at mid-dose rather than top dose.

It is not known with certainty whether or not these eight substances are truly sensitizers. The
one exception may be hexyl cinnamic aldehyde, which was confirmed in four other studies tobe positive, with three showing SI > 4 at this dose. Thus, for this one compound the N = 4
sample may actually be more likely to be “accurate” than the N = 5 sample using the Ratio
Rule.
If we assume that the Ratio Rule classifies all other substances correctly, and thus all six
substances in Table C-9 with SI > 3 are sensitizers, then there is a small loss in power byreducing the sample size per group from 5 to 4. However, this difference in power is small, andfor all six substances, the likelihood is still quite high (77% - 96%) that the substance will beidentified as a sensitizer using a sample of size 4. Recall also that these are “worst cases” andthat for 90% of the CBA studies there is no difference in power at all between samples of N = 5
and N = 4. Thus, not only does the reduction in sample size from N = 5 to N = 4 have little
impact on reliability using the Ratio Rule, it also appears to have little impact on the accuracyof classification.

5.0

Conclusion

For strong sensitizers and for obvious non-sensitizers, the reduction in sample size from 5 to 4
will have essentially no impact on the observed study outcome using the Ratio Rule. For those
substances having an SI between (approximately) 2 and 4, the outcomes may be different,
especially as SI approaches 3, but any such differences reflect primarily the inherent variability
among animals and the closeness of the SI to 3 rather than the impact of reducing the sample
size. Empirical examination of data from 83 CBA LLNA studies confirms that it is very
unlikely that a reduction in sample size from 5 to 4 animals per group would have any impact
on the overall interpretation of study results using the Ratio Rule.
Although the BALB/c and B6C3F1 databases were not evaluated in detail, the pattern of LLNA
response seen in these strains is very similar to that seen in the larger CBA database, so a moredefinitive analysis of these other two strains would almost certainly not change the conclusionsof this report. We conclude that a reduction in the sample size from 5 to 4 animals per group isunlikely to significantly impact the results of the LLNA test when using the Ratio Rule.
C-13

ICCVAM Test Method Evaluation Report: Appendix C

If a formal statistical test is used rather than (or in addition to) the Ratio Rule, the effect of
reducing the sample size from N = 5 to N = 4 is to decrease the power slightly. However, for
SI > 3, the power differences between samples of N = 5 and N = 4 are minimal. Moreover, astatistical test based on 4 animals per group will identify more sensitizers than using the Ratio
Rule based on 5 animals per group. Thus, even if a formal statistical test is used rather than (orin addition to) the Ratio Rule, the practical impact of reducing the sample size from 5 to 4
animals per group on the interpretation of experimental results appears to be minimal.

C-14

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Annex I:
Summary of Study Results – CBA Mouse Database
Table C-10

Experiments Conducted at ECPA Laboratories

Study1
Dincocap EC 0.8

ControlN
5

Control
Mean
175

ControlSD
50

Experimental
N
5

Experimental
Mean
471

Experimental
SD
198

SI
2.73

Agreement(%)2
88 (22/25)

Dincocap EC 4.0

5

175

50

5

4007

1578

22.93

100

1863

40.5

3

1004

Dincocap EC 10.0

5

175

50

4

7088

Formaldehyde-1 1.0

5

163

59

5

125

12

0.8

100

Formaldehyde-1 5.0

5

163

59

5

208

147

1.3

100

3

100

Formaldehyde-1 20.0

5

163

59

5

781

439

4.8

Formaldehyde-2 1.0

5

844

513

5

838

737

1.0

100

Formaldehyde-2 5.0

5

844

513

5

1824

1341

2.2

92 (23/25)

Formaldehyde-2 20.0

5

844

513

5

5188

2845

6.13

100

HCA-1 3.0

5

430

154

5

571

153

1.3

100

3

100

HCA-1 10.0

5

430

154

5

955

368

2.2

HCA-1 30.0

5

430

154

5

1870

376

4.33

100

649

1.9

3

100

4.2

3

100

3

100

HCA-2 3.0
HCA-2 10.0

5
5

708
708

172
172

5
5

1353
2981

1422

HCA-2 30.0

5

708

172

5

6525

4014

9.2

Oxyfluorfen EC 1

5

192

117

5

238

67

1.2

100

Oxyfluorfen EC 7

5

192

117

5

234

162

1.2

100

3

100

Oxyfluorfen EC 33

5

192

117

5

1043

311

5.4

Potassium dichromate 0.02

5

153

84

5

260

139

1.7

100

Potassium dichromate 0.10

5

153

84

5

234

135

1.5

100

3

Potassium dichromate 0.50

5

153

84

5

626

390

4.1

Quinoxyfen/
cyproconazole 7

5

226

86

5

283

102

1.3

100

Quinoxyfen/
cyproconazole 33

5

226

86

5

1470

276

6.53

100

Quinoxyfen/
cyproconazole 100

5

226

86

5

3075

621

13.63

100

Trifluralin EC 7

5

194

46

5

357

163

1.83

100

349

3

100

3

100

Trifluralin EC 33
Trifluralin EC 100

5
5

194
194

46
46

5
5

1585
3965

1456

8.2

20.5

92 (23/25)

Abbreviations: EC = emulsion concentrate; ECPA = European Crop Protection Association; HCA = hexyl cinnamic aldehyde; N = numberof animals per dose group; SD = standard deviation; SI = stimulation index
1
Test substance and dose tested (%)
2
Agreement (%) between N = 5 and N = 4 for the Ratio Rule. When agreement is less than 100%, numbers in parentheses indicate theproportion of the total number of N = 4 and N = 5 dose group combinations that agree with respect to whether SI < 3 or SI > 3. This iscalculated by multiplying the proportion of N = 5 dose groups yielding SI > 3 with the proportion of N = 4 dose groups yielding SI > 3 andthen adding the product of the proportion of N = 5 dose groups yielding SI < 3 with the proportion of N = 4 dose groups yielding SI < 3.
3
These SIs are significantly different (p < 0.05) from 1 based on a Student’s t test applied to the logged disintegrations per minute data.
4
Although N = 4 for the experimental group, the responses in this particular group clearly would have shown 100% concordance between
the outcomes for N = 5 and N = 4.

C-15

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Table C-11

Experiments Conducted at BASF Laboratories

Study1
SC-1 3ControlN
6

Control
Mean
626

Control
SD
216

ExperimentalN
6SC-1 10

6

626

216

SC-1 30

6

626

216

HCA-3 2.5

6

1322

HCA-3 5

6

1322

HCA-3 10
HCA-4 3
HCA-4 10

6
6
6

1322
703
703

Experimental
Mean
511

Experimental
SD
124

6

789

6

1168

465

6

465

6

465
197
197

6
5
6

SI
0.8

Agreement
(%)2
100

245

1.3

100

414

1.93

100

1479

161

1.1

100

1571

921

1.2

100

1791

2.8

3

554

4.6

3

100

6.6

3

100

3

100

3749
3209
4659

1479
1409

HCA-4 30

6

703

197

6

6929

1187

9.9

HCA-5 10

5

176

26

5

711

240

4.13

100

611

7.8

3

100

3

100

HCA-5 30

5

176

26

5

1362

HCA-5 50

5

176

26

5

849

422

4.8

1,6-Bis(2,3-epoxypropoxy)hexane
0.3

6

967

454

6

913

81

0.9

100

1,6-Bis(2,3-epoxypropoxy)hexane
1.0

6

967

454

6

1611

584

1.7

100

1,6-Bis(2,3-epoxypropoxy)hexane
3.0

6

967

454

6

4500

3061

4.73

94
(211/225)m-Phenylenebis (methylamine)
0.3

6

468

154

6

900

440

1.93

100

m-Phenylenebis (methylamine)
1.0

6

468

154

6

4256

1298

9.13

100

m-Phenylenebis (methylamine)
3.0

6

468

154

6

20691

6436

44.23

100

Oxirane, mono((C12-14-alkyloxy)
methyl) derivs 0.3

6

218

96

6

512

218

2.33

92
(208/225)

Oxirane, mono((C12-14-alkyloxy)
methyl) derivs 1.0

6

218

96

6

908

598

4.23

92
(206/225)

Oxirane, mono((C12-14-alkyloxy)
methyl) derivs 3.0

6

218

96

6

4963

1861

22.73

100

1,2-Diaminocyclohexane 0.1

5

446

327

6

528

114

1.2

100

1,2-Diaminocyclohexane 0.3

5

446

327

6

810

290

1.8

100

3

100

1,2-Diaminocyclohexane 1.0

5

446

327

6

3736

1982

8.4

Trimethylhexamine diamine 1.0

6

742

448

6

1599

400

2.23

885

Trimethylhexamine diamine 3.0

6

742

448

6

2972

1191

4.03

93
(209/225)

Trimethylhexamine diamine 10.0

6

742

448

6

6581

1250

8.93

100

1-(2,3-epoxypropoxy)-2,2bis[(2,3-epoxypropoxy)
methylbutane 1.0

6

388

310

6

797

392

2.13

816

1-(2,3-epoxypropoxy)-2,2bis[(2,3-epoxypropoxy)
methylbutane 3.0

6

388

310

6

2531

1812

6.53

100

C-16

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

ControlN

Control
Mean

ControlSD

Experimental
N

Experimental
Mean

Experimental
SD

SI

Agreement
(%)2

6

388

310

6

4644

2150

12.03

100

3-Aminomethyl-3,5,5trimethylcyclohexylamine 0.3

6

309

85

6

384

134

1.2

100

3-Aminomethyl-3,5,5trimethylcyclohexylamine 1.0

6

309

85

6

806

248

2.63

867

3-Aminomethyl-3,5,5trimethylcyclohexylamine 3.0

6

309

85

6

6597

1867

21.43

100

Dipropylene triamine 0.3

6

349

101

6

753

228

2.23

100

254

3

598

3

100

Study1
1-(2,3-epoxypropoxy)-2,2bis[(2,3-epoxypropoxy)
methylbutane 10.0

Dipropylene triamine 1.0

6

349

101

6

1106

3.2

Dipropylene triamine 3.0

6

349

101

6

4344

1350

12.4

N-(2-Hydroxyethyl)ethylendiamine 3.0

6

445

179

6

891

277

2.03

100

N-(2-Hydroxyethyl)ethylendiamine 10.0

6

445

179

6

766

230

1.73

100

N-(2-Hydroxyethyl)ethylendiamine 30.0

6

445

179

6

2937

626

6.63

100

p-tert-Butylphenyl 1-(2,3epoxy)propyl ether 0.1

6

406

83

6

553

148

1.4

100

p-tert-Butylphenyl 1-(2,3epoxy)propyl ether 0.3

6

406

83

6

681

230

1.73

100

p-tert-Butylphenyl 1-(2,3epoxy)propyl ether 1.0

6

406

83

6

5780

3279

14.23

100

Bakelite EPR 161

6

770

189

6

789

108

1

100

0.1

3

Bakelite EPR 161

0.3

6

770

189

6

1825

733

2.4

Bakelite EPR 161

1.0

6

770

189

6

2694

1652

3.53

689

Bakelite EPR 162

0.3

6

591

251

6

6225

3285

10.53

100

Bakelite EPR 162

1.0

6

591

251

6

11790

4292

19.93

100

3469

39.9

3

100

3

100

Bakelite EPR 162

3.0

6

591

251

6

23583

99
(222/225)

Bakelite EPR 164

0.3

6

463

208

6

2920

1049

6.3

Bakelite EPR 164

1.0

6

463

208

6

8427

1833

18.23

100

7000

3

100

Bakelite EPR 164

3.0

6

463

208

6

10387

22.4

Abbreviations: EPR = epoxy resin; N = number of animals per dose group; SC = suspension concentrate; SD = standard deviation; SI =stimulation index
1
Test substance and dose tested (%)
2
Agreement (%) between N = 5 and N = 4 for the Ratio Rule. When agreement is less than 100%, numbers in parentheses or footnoted
indicate the proportion of the total number of N = 4 and N = 5 dose group combinations that agree with respect to whether SI < 3 or
SI > 3. This is calculated by multiplying the proportion of N = 5 dose groups yielding SI > 3 with the proportion of N = 4 dose groups
yielding SI > 3 and then adding the product of the proportion of N = 5 dose groups yielding SI < 3 with the proportion of N = 4 dosegroups yielding SI < 3.
3
These SIs are significantly (p < 0.05) different from 1 based on a Student’s t test applied to the logged disintegrations per minute data.
4
55% = (26/36 x 140/225) + (10/36 x 85/225)
588% = (35/36 x 204/225) + (1/36 x 21/225)
681% = (33/36 x 195/225) + (3/36 x 30/225)
786% = (35/36 x 198/225) + (1/36 x 27/225)
859% = (27/36 x 152/225) + (9/36 x 73/225)
968% = (30/36 x 174/225) + (6/36 x 51/225)

C-17

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Table C-12

Experiments Conducted at DuPont Laboratories

Study1DU-1A 5ControlN
5

Control
Mean
506

ControlSD
185

Experimental
N
5

Experimental
Mean
284

Experimental
SD
122

SI
0.6

Agreement(%)2
100

DU-1A 25

5

506

185

5

596

166

1.2

100

DU-1A 50

5

506

185

5

354

198

0.7

100

DU-1A 100

5

506

185

5

526

313

1.0

100

DU-1B 1

5

1067

301

5

635

202

0.6

100

DU-1B 5

5

1067

301

5

1165

386

1.1

100

DU-1B 10

5

1067

301

5

1413

1145

1.3

100

DU-1B 25

5

1067

301

5

1144

388

1.1

100

DU-1C 5

5

617

265

5

419

156

0.7

100

DU-1C 25

5

617

265

4

883

517

1.4

1003

DU-1C 50

5

617

265

5

1075

432

1.7

100

DU-1C 100

5

617

265

4

779

262

1.3

1003

DU-1D 5

5

1067

301

5

755

196

0.7

100

DU-1D 10

5

1067

301

5

1019

266

1.0

100

DU-1D 25

5

1067

301

5

1337

493

1.3

100

DU-1D 50

5

1067

301

4

1086

281

1.0

1003

DU-2A 5

5

992

446

5

4132

815

4.24

100

4

100

DU-2A 25

5

992

446

5

5422

939

5.5

DU-2A 50

5

992

446

5

6604

1282

6.74

100

4

100

DU-2A 100

5

992

446

5

6482

724

6.5

DU-2E 5

5

452

219

5

433

169

1.0

100

DU-2E 25

5

452

219

5

370

142

0.8

100

DU-2E 50

5

452

219

5

509

285

1.1

100

DU-2E 100

5

452

219

5

623

200

1.4

100

DU-3 5

5

917

533

5

531

231

0.6

100

DU-3 10

5

917

533

5

720

306

0.8

100

DU-3 25

5

917

533

5

699

174

0.8

100

DU-3 50

5

917

533

5

538

179

0.6

100

DU-4 5

5

516

114

5

439

203

0.9

100

DU-4 25

5

516

114

5

505

257

1.0

100

DU-4 50

5

516

114

5

500

200

1.0

100

DU-4 100

5

516

114

5

538

65

0.9

100

DU-5A 5

5

589

317

5

1576

504

2.74

76 (19/25)

DU-5A 25

5

589

317

5

903

534

1.5

100

DU-5A 50

5

589

317

5

915

223

1.6

100

DU-5A 100

5

589

317

5

499

230

0.8

100

DU-5B 5

5

1057

256

5

835

406

0.8

100

DU-5B 25

5

1057

256

5

1168

352

1.1

100

DU-5B 50

5

1057

256

5

1087

200

1.0

100

DU-5B 100

5

1057

256

5

1200

394

1.1

100

C-18

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Study1
DU-5C 1

ControlN
5

Control
Mean
354

ControlSD
140

Experimental
N
5

Experimental
Mean
491

Experimental
SD
136

SI
1.4

Agreement(%)2100

DU-5C 5

5

354

140

5

692

313

2.04

100

DU-5C 25

5

354

140

5

429

195

1.2

100

DU-5C 100

5

354

140

5

312

124

0.9

100

DU-6 5

4

468

290

5

503

300

1.1

1003

DU-6 25

4

468

290

5

381

106

0.8

1003

DU-6 50

4

468

290

5

400

176

0.9

1003

DU-6 80

4

468

290

5

440

211

0.9

1003

DU-7 5

5

721

191

5

1394

1154

1.9

100

DU-7 25

5

721

191

5

846

331

1.2

100

DU-7 50

5

721

191

5

817

286

1.1

100

DU-7 80

5

721

191

5

915

249

1.3

100

DU-8A 1

9

486

186

4

680

178

1.4

1003

DU-8A 10

9

486

186

5

658

261

1.4

100

DU-8A 50

9

486

186

4

391

184

0.8

1003

DU-8A 100

9

486

186

5

473

263

1.0

100

DU-8B 5

5

786

312

5

916

460

1.2

100

DU-8B 25

5

786

312

5

1515

621

1.9

100

DU-8B 50

5

786

312

5

1121

764

1.4

100

DU-8B 100

5

786

312

5

1422

921

1.8

100

4

DU-9A 5

5

677

307

5

2405

1569

3.6

DU-9A 25

5

677

307

5

3354

1463

5.04

100

773

4

100

4

100

DU-9A 50

5

677

307

5

5975

8.8

84 (21/25)

DU-9A 100

5

677

307

5

9118

3211

13.5

DU-9B 5

5

1049

285

5

809

362

0.8

100

DU-9B 25

5

1049

285

5

822

195

0.8

100

DU-9B 50

5

1049

285

5

622

242

0.6

100

DU-9B 100

5

1049

285

5

493

88

0.5

100

DU-10 0.5

5

177

67

5

174

25

1.0

100

DU-10 1.0

5

177

67

5

230

73

1.3

100

DU-10 2.5

5

177

67

5

265

55

1.5

100

DU-10 5.0

5

177

67

3

289

122

1.6

NC5

DU-11B 5

5

984

210

5

1362

561

1.4

100

DU-11B 25

5

984

210

5

639

449

0.6

100

DU-11B 50

5

984

210

5

651

531

0.7

100

DU-11B 100

5

984

210

5

1016

1032

1.0

100

DU-11C 5

5

769

310

5

1168

472

1.5

100

DU-11C 25

5

769

310

5

871

217

1.1

100

DU-11C 50

5

769

310

5

719

133

0.9

100

DU-11C 100

5

769

310

5

1113

300

1.4

100

DU-12 1

5

617

265

5

479

132

0.8

100

C-19

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Study1
DU-12 5ControlN
5

Control
Mean
617

ControlSD
265

Experimental
N
5

Experimental
Mean
749

Experimental
SD
378

SI
1.2

Agreement(%)2100

DU-12 25

5

617

265

5

477

253

0.8

100

DU-12 50

5

617

265

5

872

497

1.4

100

DU-13A 5

5

621

455

5

284

67

0.5

100

DU-13A 25

5

621

455

5

276

93

0.4

100

DU-13A 50

5

621

455

5

322

167

0.5

100

DU-13A 100

5

621

455

5

370

56

0.6

100

DU-13B 1

5

578

161

5

703

450

1.2

100

DU-13B 10

5

578

161

5

551

179

1.0

100

DU-13B 50

5

578

161

5

413

117

0.7

100

DU-13B 100

5

578

161

5

376

201

0.7

100

Abbreviations: DU = DuPont; N = number of animals per dose group; NC = not calculated; SD = standard deviation; SI = stimulation index
1
Test substance and dose tested (%)
2
Agreement (%) between N = 5 and N = 4 for the Ratio Rule. When agreement is less than 100%, numbers in parentheses indicate theproportion of the total number of N = 4 and N = 5 dose group combinations that agree with respect to whether SI < 3 or SI > 3. This iscalculated by multiplying the proportion of N = 5 dose groups yielding SI > 3 with the proportion of N = 4 dose groups yielding SI > 3and then adding the product of the proportion of N = 5 dose groups yielding SI < 3 with the proportion of N = 4 dose groups yielding
SI < 3.
3
Although N = 4 for the experimental group, the responses in this particular group clearly would have shown 100% concordance between
the outcomes for N = 5 and N = 4.
4These SIs are significantly (p < 0.05) different from 1 based on a Student’s t test applied to the logged disintegrations per minute data.
5
Agreement could not be assessed, since N < 4.

C-20

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Table C-13

Experiments Conducted at EFfCI Laboratories

Study1

ControlN

Control
Mean

5

327

Fumaric Acid 5Control Experimental Experimental Experimental
SD
N
Mean
SD

85

5

419

Agreement
(%)2

SI

126

1.3

100

3

100

Fumaric Acid 10

5

327

85

5

742

284

2.3

Fumaric Acid 25

5

327

85

5

479

201

1.5

100

Linoleic Acid 10

5

223

133

5

326

176

1.5

100

303

7.0

3

100

9.1

3

100

3.1

3

56 (14/25)

9.3

3

100

1174

10.3

3

100

934

3

100

16.1

3

100

2304

16.0

3

100

1446

5.6

3

100

8.8

3

100

3

1004

Linoleic Acid 25
Linoleic Acid 50
Linoleic Acid 10
Linoleic Acid 25
Linoleic Acid 50
Maleic Acid 10
Maleic Acid 25
Maleic Acid 50
Octinol 10
Octinol 25

5
5
5
5
5
5
5
5
5
5

223
223
223
223
223
327
327
327
1120
1120

133
133
133
133
133
85
85
85
512
512

5
5
5
5
5
5
5
5
5
5

1567
2025
699
2075
2290
2186
5262
5244
6327
9833

601
301
344

686

2523

6.7

Octinol 50

5

1120

512

4

12594

1250

11.2

Oleic Acid 10

5

223

133

5

581

408

2.63

1688

14.9

3

100

897

6.9

3

100

3.8

3

100

6.9

3

100

3

100

Oleic Acid 25
Oleic Acid 50
Squalene 10
Squalene 25

5
5
5
5

223
223
223
223

133
133
133
133

5
5
5
5

3336
1550
839
1536

245
209

84 (21/25)

Squalene 50

5

223

133

5

1821

327

8.2

Succinic Acid 5

5

327

85

5

376

146

1.1

100

Succinic Acid 10

5

327

85

5

407

113

1.2

100

Succinic Acid 25

5

327

85

5

420

243

1.3

100

3

80 (20/25)

Undecylenic
Acid 10

5

223

133

5

556

140

2.5

Undecylenic
Acid 25

5

223

133

5

736

250

3.33

84 (21/25)

Undecylenic
Acid 50

5

223

133

5

991

149

4.43

100

Abbreviations: EFfCI = European Federation for Cosmetics Ingredients; N = number of animals per dose group; SD = standard deviation;
SI = stimulation index
1
Test substance and dose tested (%)
2
Agreement (%) between N = 5 and N = 4 for the Ratio Rule. When agreement is less than 100%, numbers in parentheses indicate theproportion of the total number of N = 4 and N = 5 dose group combinations that agree with respect to whether SI < 3 or SI > 3. This iscalculated by multiplying the proportion of N = 5 dose groups yielding SI > 3 with the proportion of N = 4 dose groups yielding SI > 3 andthen adding the product of the proportion of N = 5 dose groups yielding SI < 3 with the proportion of N = 4 dose groups yielding SI < 3.
3
These SIs are significantly (p < 0.05) different from 1 based on a Student’s t test applied to the logged disintegrations per minute data.
4
Although N = 4 for the experimental group, the responses in this particular group clearly would have shown 100% concordance between
the outcomes for N = 5 and N = 4.

C-21

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Table C-14

Experiments Conducted at BAuA Laboratories
ControlN

Control
Mean

ControlSD

Experimental
N

Experimental
Mean

Experimental
SD

SI

Agreement(%)2

Yellow E-JD 3442 1

5

70

21

5

70

19

1.0

100

Yellow E-JD 3442 3

5

70

21

5

52

9

0.8

100

Yellow E-JD 3442 9

5

70

21

5

60

32

0.9

100

Yellow E-JD 3442 15

5

70

21

5

61

16

0.9

100

147

4.8

3

100

3.4

3

88 (22/25)

4.4

3

100

4.6

3

100

2.4

3

100

2.5

3

96 (24/25)

1.9

3

100

2.5

3

100

3

100

Study1

CI Reactive Red 231 1
CI Reactive Red 231 3
CI Reactive Red 231 9
CI Reactive Red 231 15
P-46 1
P-46 3P-46 9P-46 15

5
5
5
5
5
5
5
5

70
70
70
70
70
70
70
70

21

5

21

5

21

5

21

5

21

5

21

5

21

5

21

5

334
234
305
317
167
175
135
175

78
121
105
86
73
39
45

CI Reactive Yellow
174 1

5

70

21

5

288

62

4.1

CI Reactive Yellow
174 3

5

70

21

5

231

70

3.33

80 (20/25)

CI Reactive Yellow
174 9

5

70

21

5

385

242

5.53

100

CI Reactive Yellow
174 15

5

70

21

5

539

114

7.83

100

Navy 14 08 723 1

5

70

21

5

353

54

5.13

100

116

4.8

3

100

5.7

3

100

3

100

Navy 14 08 723 3
Navy 14 08 723 9

5
5

70
70

21

5

21

5

335
398

102

Navy 14 08 723 15

5

70

21

5

361

90

5.2

Dispersionsrot 2754 1

5

70

21

5

68

27

1.0

100

Dispersionsrot 2754 3

5

70

21

5

65

19

0.9

100

Dispersionsrot 2754 9

5

70

21

5

67

40

1.0

100

Abbreviations: BAuA = Federal Institute for Occupational Safety and Health (Germany); N = number of animals per dose group; SD =standard deviation; SI = stimulation index
1
Test substance and dose tested (%)
2
Agreement (%) between N = 5 and N = 4 for the Ratio Rule. When agreement is less than 100%, numbers in parentheses indicate theproportion of the total number of N = 4 and N = 5 dose group combinations that agree with respect to whether SI < 3 or SI > 3. This iscalculated by multiplying the proportion of N = 5 dose groups yielding SI > 3 with the proportion of N = 4 dose groups yielding SI > 3 andthen adding the product of the proportion of N = 5 dose groups yielding SI < 3 with the proportion of N = 4 dose groups yielding SI < 3.
3
These SIs are significantly (p < 0.05) different from 1 based on a Student’s t test applied to the logged disintegrations per minute data.

C-22

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Table C-15

Experiments Conducted at Dow AgroSciences Laboratories

Study1

Control Control Control Experimental Experimental Experimental
N
Mean
SD
N
Mean
SD

SI

Agreement(%)2

Formulation 29 5

6

567

305

6

1036

663

1.8

100

Formulation 29 25

6

567

305

6

913

200

1.6

100

Formulation 29 100

6

567

305

6

823

373

1.5

100

253

1.8

3

100

7.2

3

100

3

100

Formulation 30 5
Formulation 30 25

6
6

536
536

258
258

6
6

947
3839

736

Formulation 30 100

6

536

258

6

7269

1014

13.6

Formulation 31 5

6

385

121

5

393

223

1.0

100

215

1.9

3

100

1.8

3

100

6.5

3

100

44.7

3

100

3

100

Formulation 31 25
Formulation 31 100
Formulation 32 5
Formulation 32 25

6
6
6
6

385
385
332
332

121
121
346
346

5
6
6
6

724
696

262

2136
14833

737
6139

Formulation 32 100

6

332

346

6

22965

5480

69.3

Formulation 33 5

6

672

249

6

479

194

0.7

100

Formulation 33 25

6

672

249

6

913

496

1.4

100

Formulation 33 100

6

672

249

6

843

303

1.3

100

Formulation 34 5

6

385

121

6

713

331

1.9

100

Formulation 34 25

6

385

121

6

528

227

1.4

100

Formulation 34 100

6

385

121

6

581

216

1.5

100

Formulation 35 5

6

332

346

6

360

294

1.1

100

Formulation 35 25

6

332

346

6

383

158

1.2

100

Formulation 35 100

6

332

346

6

412

317

1.3

100

Formulation 37 1

6

744

359

6

1008

525

1.4

100

Formulation 37 5

6

744

359

6

1999

1687

2.7

564

Formulation 37 15

6

744

359

6

5586

4162

7.53

100

Formulation 38 5

6

889

520

6

960

515

1.1

100

1541

3

100

2102

12.7

3

100

268

1.7

3

100

3

845

Formulation 38 25
Formulation 38 100
Formulation 39 1

6
6
6

889
889
627

520
520
256

6
6
6

4098
11232
1076

4.6

Formulation 39 5

6

627

256

6

1551

650

2.5

Formulation 39 25

6

627

256

6

2083

259

3.33

736

Formulation 40 1

5

8217

263

6

1481

621

1.8

100

5

821

7

401

2.8

3

73 (55/75)

821

7

5.7

3

100

3

100

Formulation 40 5
Formulation 40 25

5

263
263

6
6

2316
4646

1833

Formulation 41 5

6

1017

325

6

1936

1024

1.9

Formulation 41 25

6

1017

325

6

1891

1133

1.9

100

7

2750

5.6

3

100

250

0.7

100

Formulation 41 100
Formulation 49 5

6

1017

325

5

5653

5

7

298

6

442

626

C-23

ICCVAM Test Method Evaluation Report: Appendix C, Annex I

Study1

Control Control Control Experimental Experimental Experimental
N
Mean
SD
N
Mean
SD

5

6267

Formulation 49 100

5

626

7

298

5

Formulation 50 5

6

1208

882

Formulation 50 25

6

1208

882

Formulation 49 25

298

6

880

Agreement
(%)2

SI

444

1.4

100

2958

489

4.7

3

100

6

796

183

0.7

100

6

786

436

0.7

100

3

100
100

Formulation 50 100

6

1208

882

6

9439

4239

7.8

Formulation 51 5

6

863

526

6

1346

537

1.6
3

Formulation 51 25

6

863

526

6

3893

2120

4.5

96
(215/225)

Formulation 51 100

6

863

526

6

2084

1725

2.4

668

Formulation 53 2.5

5

3927

159

6

596

317

1.5

100

Formulation 53 7.5

5

392

7

159

6

1240

987

3.2

3

529

Formulation 53 15

5

3927

159

4

2609

1494

6.73

10010

Formulation 54 5

6

438

143

6

551

357

1.3

100

Formulation 54 25

6

438

143

6

502

262

1.2

100

Formulation 54 100

6

438

143

6

1016

583

2.3

93
(209/225)

Formulation 55 5

6

529

238

6

781

602

1.5

100

3

6811

Formulation 55 25

6

529

238

6

1348

947

2.5

Formulation 55 100

6

529

238

6

1972

758

3.73

90
(202/225)

Formulation 56 5

6

529

238

6

1726

831

3.33

5712

Formulation 56 25

6

529

238

6

3217

1996

6.13

100

3

NC13

Formulation 56 100

6

529

238

2

2064

21

3.9

Abbreviations: N = number of animals per dose group; NC = not calculated; SD = standard deviation; SI = stimulation index
1
Test substance and dose tested (%)
2
Agreement (%) between N = 5 and N = 4 for the Ratio Rule. When agreement is less than 100%, numbers in parentheses or footnoted
indicate the proportion of the total number of N = 4 and N = 5 dose group combinations that agree with respect to whether SI < 3 or
SI > 3. This is calculated by multiplying the proportion of N = 5 dose groups yielding SI > 3 with the proportion of N = 4 dose groups
yielding SI > 3 and then adding the product of the proportion of N = 5 dose groups yielding SI < 3 with the proportion of N = 4 dosegroups yielding SI < 3.
3
These SIs are significantly (p < 0.05) different from 1 based on a Student’s t test applied to the logged disintegrations per minute data.
4
56% = (26/36 x 142/225) + (10/36 x 83/225)
584% = (35/36 x 194/225) + (1/36 x 31/225)
673% = (33/36 x 175/225) + (3/36 x 50/225)
7
Data reflects elimination of one control outlier (4258) in Formulation 40, one dosed group outlier (428) in Formulation 41, one control
outlier (3) and one dosed group outlier (6273) in Formulation 49, and one control outlier (3172) in Formulation 53.
866% = (29/36 x 172/225) + (7/36 x 53/225)
952% = (4/6 x 42/75) + (2/6 x 33/75)
10Although N = 4 for the experimental group, the responses in this particular group clearly would have shown 100% concordance between
the outcomes for N = 5 and N = 4.
1168% = (31/36 x 168/225) + (5/36 x 57/225)
1257% = (26/36 x 150/225) + (10/36 x 75/225)
13
Agreement could not be assessed, since N < 4.

C-24

ICCVAM Test Method Evaluation Report: Appendix D

Appendix D
Final Background Review Document:
Reduced Murine Local Lymph Node Assay

D-1

ICCVAM Test Method Evaluation Report: Appendix D[This Page Intentionally Left Blank]

D-2

ICCVAM Test Method Evaluation Report: Appendix D

Background Review Document
Reduced Murine Local Lymph Node Assay

Interagency Coordinating Committee on the
Validation of Alternative Methods

National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods

National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

2009

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

D-3

ICCVAM Test Method Evaluation Report: Appendix D[This Page Intentionally Left Blank]

D-4

ICCVAM Test Method Evaluation Report: Appendix D

Table of Contents
List of Tables ...................................................................................................................D-8
List of Figures..................................................................................................................D-9
List of Abbreviations and Acronyms............................................................................D-10
Interagency Coordinating Committee on the Validation Of Alternative Methods:
Agency Representatives ................................................................................................D-13
Acknowledgements........................................................................................................D-15
Preface ...........................................................................................................................D-21
Executive Summary ......................................................................................................D-23
1.0

Introduction and Rationale for the Proposed Use of the Reduced Murine
Local Lymph Node Assay (rLLNA) to Identify Skin Sensitizers.....................D-27
1.1

2.0

Introduction ................................................................................................D-27
1.1.1

Historical Background ...............................................................D-27

1.1.2

Allergic Contact Dermatitis .......................................................D-27

1.1.3

U.S. Consumer Product Safety Commission (CPSC)
Nomination................................................................................D-28

1.1.4

Description of the rLLNA .........................................................D-28

1.1.5

Results of an ECVAM Peer Review of the rLLNA ...................D-29

1.2

Regulatory Rationale and Applicability of the rLLNA ................................D-29

1.3

Scientific Basis for the rLLNA....................................................................D-29
1.3.1

Purpose and Mechanistic Basis .................................................D-29

1.3.2

Applicability Domain ................................................................D-30

1.4

Test Method Validation...............................................................................D-30

1.5

Selection of Citations for the rLLNA BRD..................................................D-30

rLLNA Protocol Components.............................................................................D-31
2.1

Overview ....................................................................................................D-31

2.2

Basis for Test Method Selection..................................................................D-31

2.3

Proprietary Test Method Components .........................................................D-31

2.4

Basis for the Number of Mice per Dose Group............................................D-31

2.5

Study Acceptance Criteria...........................................................................D-31

2.6

Basis for Selection of the Test Substance Dose ...........................................D-31

D-5

ICCVAM Test Method Evaluation Report: Appendix D

3.0

4.0

5.0

6.0

Substances Used for Validation of the rLLNA ................................................. D-33
3.1

Rationale for the Substances or Products Included in the Evaluation ........... D-33

3.2

Rationale for the Number of Substances Included in the Evaluation............ D-33

3.3

Detailed Description of Substances Included in the Evaluation ................... D-33

3.4

Coding Procedures...................................................................................... D-35

Comparative In Vivo Reference Data – the Traditional LLNA ........................ D-37
4.1

The Traditional LLNA Protocol Used to Generate Comparative In Vivo
Reference Data ........................................................................................ D-37

4.2

Comparative Traditional LLNA Reference Data Used ................................ D-37

4.3

Availability of Original Records for Comparative Traditional LLNA
Reference Data ........................................................................................... D-37

4.4

Quality of Comparative Traditional LLNA Reference Data......................... D-37

4.5

Accuracy and Reliability of the Traditional LLNA...................................... D-38
4.5.1

Accuracy ................................................................................... D-38

4.5.2

Reliability.................................................................................. D-38

rLLNA Test Method Data and Results .............................................................. D-39
5.1

Description of the rLLNA Test Method Protocol Used to Generate Data .... D-39

5.2

Availability of Original rLLNA Data Used to Evaluate Accuracy andReliability................................................................................................... D-39

5.3

Description of the Statistical Procedure Used to Evaluate rLLNA Data....... D-39

5.4

Summary of Results.................................................................................... D-39

5.5

Use of Coded Substances............................................................................ D-40

5.6

Lot-to-Lot Consistency of Test Substances ................................................. D-40

5.7

Availability of Original Data for External Audit ......................................... D-40

Accuracy of the rLLNA ...................................................................................... D-41
6.1

Performance Statistics ................................................................................ D-41

6.2

Discordant Results ...................................................................................... D-43

7.0

Reliability of the rLLNA..................................................................................... D-47

8.0

rLLNA Data Quality........................................................................................... D-49
8.1

Adherence to National and International GLP Guidelines ........................... D-49

8.2

Data Quality Audits .................................................................................... D-49

8.3

Impact of Deviations from GLP Guidelines ................................................ D-49

8.4

Availability of Laboratory Notebooks or Other Records ............................. D-49
D-6

ICCVAM Test Method Evaluation Report: Appendix D

9.0

Other rLLNA Scientific Reports and Reviews .................................................D-51
9.1

Reports in the Peer-Reviewed Literature .....................................................D-51
9.1.1

Ryan et al. (2008)...........................................................................D-51

10.0 Animal Welfare Considerations .........................................................................D-53
10.1 How the rLLNA will Refine, Reduce, or Replace Animal Use ....................D-53
10.2 Requirements for the Use of Animals..........................................................D-53
11.0 Practical Considerations.....................................................................................D-55
11.1 Transferability of the rLLNA ......................................................................D-55
11.2 rLLNA Training Considerations..................................................................D-55
11.3 Cost Considerations ....................................................................................D-55
11.4 Time Considerations ...................................................................................D-55
12.0 References............................................................................................................D-57
13.0 Glossary ...............................................................................................................D-61
Annex IECVAM Scientific Advisory Committee Statement on the Validityof the rLLNA..........................................................................................D-65

Annex II

Physicochemical Properties of Substances Evaluated in the rLLNA ..D-71

Annex III

Traditional LLNA Data Used for the Performance Analysis of therLLNA .................................................................................................. D-109

Annex IV

Substances in the NICEATM LLNA Database for which an Initial
Dose of 10% or Greater Elicited a Negative Result but a
Subsequent Higher Dose Elicited a Positive Response........................ D-145

D-7

ICCVAM Test Method Evaluation Report: Appendix D

List of Tables
Table D-1
Table D-2
Table D-3
Table D-4
Table D-5
Table D-6

Summary of Traditional LLNA Data Sources and Rationale for
Substance Selection ................................................................................. D-34
Chemical Classes Represented in the Current Traditional LLNA
Database .................................................................................................. D-35
Performance of the rLLNA in Predicting Skin Sensitizers Compared to
the Traditional LLNA .............................................................................. D-42
Traditional LLNA Data for Substances Identified as False Negatives
by the rLLNA .......................................................................................... D-43
Summary of Available Physicochemical Properties for False
Negatives, as Identified by the rLLNA..................................................... D-45
rLLNA Responses for Repeated Studies .................................................. D-48

D-8

ICCVAM Test Method Evaluation Report: Appendix D

List of Figures
Figure D-1

Dose-Response Curves for Substances Identified as Sensitizers by the
Traditional LLNA but as Non-Sensitizers by the rLLNA..........................D-44

D-9

ICCVAM Test Method Evaluation Report: Appendix D

List of Abbreviations and Acronyms
ACD
ACE
AOO
BGIA
BRD
CASRN
CESIO

Conc.
CPSC
DBP
DEP
DMF
DMSO
DNCB
EC3
ECPA
ECVAM
EFfCI
EPA
ESAC
FDA
FR
GLP
GPMT
HCA
ICCVAM
ILS
ISO
IWG
Kow
LLNA

Allergic contact dermatitis
AcetoneAcetone: olive oil (4:1 by volume)
Berufsgenossenschaftliches Institut fur Arbeitsschutz (German Institutefor Occupational Safety and Health)
Background review document
Chemical Abstracts Service Registry Number
Comite Europeen des Agents de Surface et de Leurs Intermediaires
Organiques (European Committee of Surfactants and Their Organic
Intermediates)
Concentration tested
U.S. Consumer Product Safety Commission
Dibutyl phosphate
Diethyl phthalate
Dimethyl formamide
Dimethyl sulfoxide
Dinitrochlorobenzene
Estimated concentration needed to produce a stimulation index of 3
European Crop Protection Association
European Centre for the Validation of Alternative Methods
European Federation for Cosmetic Ingredients
U.S. Environmental Protection Agency
European Centre for the Validation of Alternative Methods Scientific
Advisory Committee
U.S. Food and Drug Administration
Federal Register
Good Laboratory PracticeGuinea Pig Maximization Test
Hexyl cinnamic aldehyde
Interagency Coordinating Committee on the Validation of Alternative
Methods
Integrated Laboratory Systems
International Organization for Standardization
Immunotoxicity Working Group
Octanol-water partition coefficient
Murine local lymph node assay
D-10

ICCVAM Test Method Evaluation Report: Appendix D

MEK
NA
NC
ND
NICEATM
NIEHS
NTP
OECD
OPPTS
PA
PG
RIFM
rLLNA
SACATM
SI
SOT
TG
TNO
U.K.
U.N.
U.S.
w/v

Methyl ethyl ketone
Not applicableNot calculated
No data
National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods
National Institute of Environmental Health Sciences
National Toxicology Program
Organisation for Economic Co-operation and Development
Office of Prevention, Pesticides and Toxic Substances
Pluronic acid
Propylene glycol
Research Institute for Fragrance Materials
Reduced murine local lymph node assay
Scientific Advisory Committee on Alternative Toxicological Methods
Stimulation index
Society of Toxicology
Test guideline
TNO Nutrition and Food Research Institute (Netherlands)
United Kingdom
United Nations
United States
Weight-to-volume ratio

D-11

ICCVAM Test Method Evaluation Report: Appendix D[This Page Intentionally Left Blank]

D-12

ICCVAM Test Method Evaluation Report: Appendix DInteragency Coordinating Committee on the Validation of
Alternative Methods: Agency Representatives
Agency for Toxic Substances and Disease
Registry• Moiz Mumtaz, Ph.D.

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

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

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

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

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

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

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

National Institute for Occupational Safety and
Health• Paul Nicolaysen, V.M.D.
◊ K. Murali Rao, M.D., Ph.D.
National Institutes of Health• Margaret D. Snyder, Ph.D.
National Library of Medicine• Pertti (Bert) Hakkinen, Ph.D.
◊ Jeanne Goshorn, M.S.

• Principal agency representative◊ Alternate principal agency representative

Occupational Safety and Health Administration• Surender Ahir, Ph.D.
D-13

ICCVAM Test Method Evaluation Report: Appendix D[This Page Intentionally Left Blank]

D-14

ICCVAM Test Method Evaluation Report: Appendix D

Acknowledgements
The following individuals are acknowledged for their contributions to the evaluation of the
reduced LLNA.
Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) Immunotoxicity Working Group (IWG)

U.S. Consumer Product Safety
Commission
Joanna Matheson, Ph.D. (IWG Co-Chair)
Marilyn Wind, Ph.D.

National Institute of Environmental
Health Sciences
Dori Germolec, Ph.D.
William S. Stokes, D.V.M., D.A.C.L.A.M.
Raymond R. Tice, Ph.D.

U.S. Environmental Protection Agency
Office of Pesticide Programs
Masih Hashim, D.V.M., Ph.D.
Marianne Lewis
Deborah McCall
Timothy McMahon, Ph.D.
Amy Rispin, Ph.D.
Office of Prevention, Pesticides, and Toxic
Substances
Ronald Ward, Ph.D.
Office of Research and Development
Marsha Ward, Ph.D.
Office of Science Coordination and Policy
Karen Hamernik, Ph.D.

National Institute for Occupational
Safety and Health
B. Jean Meade, D.V.M., Ph.D.
European Centre for the Validation of
Alternative Methods — Liaison
Silvia Casati, Ph.D.
Japanese Center for the Validation of
Alternative Methods — Liaison
Hajime Kojima, Ph.D.

U.S. Food and Drug Administration
Center for Devices and Radiological HealthDaniel Lyle, Ph.D.
Vasant G. Malshet, Ph.D., D.A.B.T.
Jeffrey Toy, Ph.D.
Center for Drug Evaluation and ResearchPaul C. Brown, Ph.D.
Abigail Jacobs, Ph.D. (IWG Co-Chair)
Jiaqin Yao, Ph.D.
Center for Veterinary Medicine
Ruth Barratt, Ph.D., D.V.M.

D-15

ICCVAM Test Method Evaluation Report: Appendix D

Reduced Murine Local Lymph Node Assay
Independent Scientific Peer Review Panel(March 4–6, 2008)
Michael Luster, Ph.D. (Panel Chair)
Senior Consultant to the National Institutefor Occupational Safety and Health
Health Effects Laboratory
Morgantown, WV

Dagmar Jírová, M.D., Ph.D.
Toxicologist, Research Manager
Head of Reference Center for Cosmetics andReference Laboratory for Experimental
Immunotoxicology
National Institute of Public HealthPrague, Czech Republic

Nathalie Alépée, Ph.D.
Associate Research Fellow
Pfizer PDRD
MCT Laboratory
Amboise, FranceDavid Lovell, Ph.D.
Reader in Medical Statistics
Postgraduate Medical School
University of Surrey
Guildford, Surrey, U.K.

Anne Marie Api, Ph.D.
Vice President, Human Health Sciences
Research Institute for Fragrance Materials
Woodcliff Lake, NJ

Howard Maibach, M.D.
Professor, Department of Dermatology
University of California – San Francisco
San Francisco, CA

Nancy Flournoy, M.S., Ph.D.
Professor and Chair
Department of Mathematics and Statistics
University of Missouri – Columbia
Columbia, MO

James McDougal, Ph.D.
Professor and Director of Toxicology Research
Department of Pharmacology and
Toxicology
Boonshoft School of Medicine
Wright State University
Dayton, OH

Thomas Gebel, Ph.D.
Regulatory Toxicologist
Federal Institute for Occupational Safety &HealthDortmund, Germany

Michael Olson, Ph.D.
Director of Occupational Toxicology
Corporate Environment, Health and Safety
GlaxoSmithKline
Research Triangle Park, NC

Sidney Green, Ph.D.*
Professor of Pharmacology
College of Medicine
Howard University
Washington, DC

Raymond Pieters, Ph.D.
Associate Professor
Immunotoxicology Group Leader
Institute for Risk Assessment Sciences
Utrecht University
Utrecht, The Netherlands

Kim Headrick, B.Admin., B.Sc.
International Harmonization and Senior
Policy Advisor
Policy and Programme Service Office
Health Canada
Ottawa, Ontario, Canada

Jean Regal, Ph.D.
Professor, Department of Pharmacology
University of Minnesota Medical School
Duluth, MN

D-16

ICCVAM Test Method Evaluation Report: Appendix D

Jonathan Richmond, MB ChB, FRCSEd*
Head, Animal (Scientific) Procedures
DivisionHome OfficeLondon, U.K.

Michael Woolhiser, Ph.D.
Technical Leader – Immunotoxicology
Senior Toxicology Specialist
Dow Chemical
Midland, MI

Peter Theran, V.M.D.
Consultant
Massachusetts Society for the Prevention of
Cruelty to Animals
Novato, CA

Takahiko Yoshida, M.D., Ph.D.
Professor, Department of Health Science
Asahikawa Medical College
Hokkaido, Japan

Stephen Ullrich, Ph.D.
Professor of Immunology
Graduate School of Biomedical Sciences
University of Texas
M.D. Anderson Cancer Center – Houston
Houston, TX* Drs. Green and Richmond were unable to attend the public meeting on March 4–6, 2008; however, they took
part in the scientific peer review and concur with the conclusions and recommendations included in the
Independent Scientific Peer Review Panel Report - Validation Status of New Versions and Applications of the
Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential ofChemicals and Products.

D-17

ICCVAM Test Method Evaluation Report: Appendix D

National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
William Stokes, D.V.M., D.A.C.L.A.M.
Director; Project Officer
Raymond Tice, Ph.D.
Deputy Director
Deborah McCarley
Special Assistant; Assistant Project Officer
NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)David Allen, Ph.D.
Eleni Salicru, Ph.D.
Thomas Burns, M.S.
Catherine Sprankle
Linda Litchfield
Judy Strickland, Ph.D., D.A.B.T.
Michael Paris

D-18

ICCVAM Test Method Evaluation Report: Appendix D

Other Acknowledgements
ICCVAM and NICEATM gratefully acknowledge the following individuals and institutions that
submitted data to NICEATM used for the evaluation of the rLLNA.
David Basketter, Ph.D.
Unilever Safety and Environmental
Assurance Centre
Sharnbrook, U.K.

Ian Kimber, Ph.D.
Syngenta Central Toxicology Laboratory
Macclesfield, U.K.
Michael J. Olson, Ph.D.
GlaxoSmithKline
Research Triangle Park, NC, U.S.

Phil Botham, Ph.D.
European Crop Protection Association
Brussels, Belgium

K. Skirda
TNO Quality of Life
Delft, Netherlands

Eric Debruyne, Ph.D.
Bayer CropScience SA, Sophia Antipolis
Cedex, France
G. Frank Gerberick, Ph.D.
Procter and Gamble Company
Cincinnati, OH, U.S.

Peter Ungeheuer, Ph.D.
European Federation for Cosmetic
Ingredients
Frankfurt, Germany

Dori Germolec, Ph.D.
National Toxicology Program
Research Triangle Park, NC, U.S.

Hans Werner Vohr, Ph.D.
Bayer HealthCare
Wuppertal-Elberfeld, Germany

D-19

ICCVAM Test Method Evaluation Report: Appendix D[This Page Intentionally Left Blank]

D-20

ICCVAM Test Method Evaluation Report: Appendix D

Preface
In 1998, the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with the National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) evaluated the validation statusof the murine local lymph node assay (traditional LLNA) as an alternative to guinea pig test
methods (e.g., the Guinea Pig Maximization Test and the Buehler Test) for assessing theallergic contact dermatitis (ACD) potential of substances. ICCVAM subsequently
recommended that the LLNA could be used as a valid substitute for the accepted guinea pig test
methods in most ACD testing situations (ICCVAM 1999).
Based on the ICCVAM recommendations, the ICCVAM member agencies that require
regulatory submission of ACD data accepted the LLNA, with identified limitations, as analternative to guinea pig tests for assessing the potential of substances to cause ACD. In 2002,
the LLNA was adopted as Test Guideline 429 by the 30 member countries of the Organisation
for Economic Co-operation and Development (OECD; OECD 2002).
The reduced murine local lymph node assay (rLLNA), also referred to as the “cut-down” or“limit dose” LLNA, was one of several modified versions of the LLNA nominated by the U.S.
Consumer Product Safety Commission (CPSC) for evaluation by ICCVAM.30 (The term
“reduced LLNA” has been adopted in this document to be consistent with the terminology used
for this test method in Europe.) The proposed rLLNA could reduce the number of animals forskin sensitization testing by 40% for each test compared with the traditional LLNA. ICCVAM
assigned this activity a high priority following consideration of comments from the public and
ICCVAM’s advisory committee, the Scientific Advisory Committee on Alternative
Toxicological Methods (SACATM).
The ICCVAM Immunotoxicity Working Group (IWG) and NICEATM (1) prepared a draft
background review document (BRD) that described the validation status of the rLLNA test
method, including its reliability and accuracy, the substances evaluated, and the availability of astandardized protocol and (2) developed draft test method recommendations based on thisevaluation. An international independent scientific peer review panel (Panel) met on March 4–
6, 2008, to assess the current validation status of the rLLNA. The Panel also reviewed thecompleteness and accuracy of the draft ICCVAM BRD and the extent to which the information
therein supported the ICCVAM draft test method recommendations for proposed test method
uses, recommended protocol, test method performance standards, and future studies.
ICCVAM considered the conclusions and recommendations of the Panel, as well as comments
received from the public and SACATM, when finalizing ICCVAM’s BRD and test method
recommendations on the usefulness and limitations of the rLLNA.
We gratefully acknowledge the organizations and scientists who provided data and information
for this BRD. We would also like to recognize the efforts of the individuals who contributed toits preparation, review, and revision. We especially recognize the Panel members for theirthoughtful evaluations and generous contributions of time and effort. Special thanks areextended to Dr. Michael Luster for serving as the Panel Chair and to Dr. Michael Woolhiser,
Dr. Michael Olson, and Ms. Kim Headrick for their service as Evaluation Group Chairs. We
30

Available at http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
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ICCVAM Test Method Evaluation Report: Appendix D

thank the IWG for assuring a meaningful and comprehensive review. We especially thank Dr.
Joanna Matheson (CPSC) and Dr. Abigail Jacobs (U.S. Food and Drug Administration Center
for Drug Evaluation and Research) for serving as Co-Chairs of the IWG, as well as the IWG
members and ICCVAM representatives who subsequently reviewed the BRD and provided
comments.
Integrated Laboratory Systems, Inc., the NICEATM support contractor, provided excellent
scientific and operational support for which we thank Dr. David Allen, Mr. Thomas Burns, Ms.
Linda Litchfield, Mr. Michael Paris, Dr. Eleni Salicru, Ms. Catherine Sprankle, and Dr. Judy
Strickland. We also acknowledge Dr. Raymond Tice, Deputy Director of NICEATM, for his
contributions to this project. Finally, we want to thank Dr. Silvia Casati and Dr. Hajime
Kojima, the IWG liaisons from the European Centre for the Validation of Alternative Methods
and the Japanese Center for the Validation of Alternative Methods, respectively for their
participation.
William S. Stokes, D.V.M., D.A.C.L.A.M.
Rear Admiral/Assistant Surgeon General, U.S. Public Heath Service
Director, NICEATM
Executive Director, ICCVAM
Marilyn Wind, Ph.D.
Deputy Associate Executive Director
Directorate for Health Sciences
U.S. Consumer Product Safety Commission
Chair, ICCVAM

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ICCVAM Test Method Evaluation Report: Appendix D

Executive Summary
In 1999, the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) recommended the murine local lymph node assay (traditional LLNA) as a valid
substitute for currently accepted guinea pig test methods to assess allergic contact dermatitis(ACD) potential of substances in most ACD testing situations. The recommendation was basedon a comprehensive evaluation that included an independent scientific peer review panel(Panel) assessment of the validation status of the LLNA. The Panel report and the ICCVAM
recommendations (ICCVAM 1999) are available at the NICEATM–ICCVAM website.31
ICCVAM forwarded to U.S. Federal agencies its recommendation that the traditional LLNA
should be considered for regulatory acceptance or other non-regulatory applications forassessing the ACD potential of substances, while recognizing that some testing situations would
still require the use of traditional guinea pig test methods (ICCVAM 1999). The LLNA wassubsequently incorporated into national and international test guidelines for the assessment ofskin sensitization (International Organization for Standardization [ISO] 10993-10: Tests for
Irritation and Sensitization [ISO 2002]; Organisation for Economic Co-operation andDevelopment Test Guideline [TG] 429 [OECD 2002]; U.S. Environmental Protection Agency
Health Effects Test Guideline OPPTS 870.2600: Skin Sensitization [EPA 2003]).
In 2007, the U.S. Consumer Product Safety Commission (CPSC) nominated the rLLNA (alsoreferred to as the “cut-down” or “limit dose” LLNA) as one of several modified versions of the
LLNA for evaluation by ICCVAM. The proposed rLLNA could reduce the number of animals
for skin sensitization testing by 40% per test compared with the traditional LLNA. The term
“reduced LLNA” has been adopted in this document to be consistent with the terminology used
for this test method in Europe.
ICCVAM assigned this activity a high priority; and the National Toxicology Program
Interagency Committee on the Evaluation of Alternative Methods (NICEATM), along with the
ICCVAM Immunotoxicity Working Group (IWG), collaborated closely with liaisons from theEuropean Centre for the Validation of Alternative Methods and the Japanese Center for the
Validation of Alternative Methods to facilitate the evaluations requested by the CPSC.
NICEATM and the ICCVAM IWG prepared this background review document (BRD), whichsummarizes the current validation status of the rLLNA for assessing the skin sensitization
potential of substances. It includes detailed information about the reliability and relevance ofthe rLLNA, and the scope of the substances that were evaluated. It provides a comprehensive
review of available data and information on the use of the rLLNA for hazard classification.
This information summarized in this BRD is from a retrospective review of traditional LLNA
data. The database considered was obtained from 12 different sources and included 457 uniquesubstances32 tested in a total of 471 traditional LLNA studies. ICCVAM had considered 211 of
the substances during its 1998 evaluation of the traditional LLNA (ICCVAM 1999). An
additional 246 substances were obtained from the peer-reviewed literature published after that
evaluation and from data submitted to NICEATM in response to a 2007 Federal Register (FR)31
32

Available at http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
Some substances were tested in more than one vehicle. In such instances, each substance-vehicle combination
was considered separately, and thus there were a total of 465 unique substance-vehicle combinations thatwere used in the performance evaluation.
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ICCVAM Test Method Evaluation Report: Appendix D

notice (72 FR 27815, May 17, 200733). Specifically, three sources were published journal
articles and eight were responses to the May 2007 FR notice. Due to the small number of
repeated studies (5% of total studies), all studies were treated independently for the purpose ofthis accuracy evaluation.
The 1999 ICCVAM-recommended LLNA protocol accepted by U.S. regulatory agencies is
consistent with procedures described in OECD TG 429 and was used as the basis fordevelopment of the OECD test guideline. Still, TG 429 allows for more procedural variation
than the 1999 ICCVAM-recommended protocol (ICCVAM 1999). The protocol for the rLLNA
is identical to that for the traditional LLNA (ICCVAM 1999), except that the traditional LLNA
tests a substance at three dose levels, with the highest dose level being that which does not
induce systemic toxicity and/or excessive skin irritation. In the rLLNA, a substance is tested atonly a single dose level, which is the highest dose level that would have been tested in the
traditional LLNA. As in the traditional LLNA, the threshold for classifying a substance as askin sensitizer in the rLLNA is a stimulation index (SI) ≥ 3.
Information on chemical classes for each substance was retrieved from the National Library ofMedicine’s ChemIDplus® database or assigned for each test substance using a standard
classification scheme based on the National Library of Medicine Medical Subject Headings
classification system.34 Chemical class information is included to indicate the variety ofstructural elements in the evaluated substances. One hundred and twenty-five complex
substances were identified simply as pharmaceuticals. Ten substances were formulations.
Seventy substances could not be assigned to a specific chemical class due to incomplete
information (e.g., no Chemical Abstracts Service Registry Number or structure provided).
The ability of the rLLNA to correctly identify potential skin sensitizers was compared to that of
the traditional LLNA. In the 471 studies, 318 detected skin sensitizers, and 153 detected nonsensitizers. When studies for substances tested more than once in the same vehicle (i.e., 465
unique substance and vehicle combinations) were considered together to yield an overall skinsensitization classification, 315 were classified as sensitizers, and 150 were classified as nonsensitizers.
Based on the data available from the 471 studies, the rLLNA has an accuracy of 98.7%(465/471), a sensitivity of 98.1% (312/318), a specificity of 100% (153/153), a false positive
rate of 0% (0/153), and a false negative rate of 1.9% (6/318) when compared to the traditional
LLNA. Based on the 465 unique substance and vehicle combinations, the rLLNA has an
accuracy of 98.7% (459/465), a sensitivity of 98.1% (309/315), a specificity of 100%
(150/150), a false positive rate of 0% (0/150), and a false negative rate of 1.9% (6/315).
Six substances yielded false negative results in the rLLNA (i.e., the substances were classified
as sensitizers in the traditional LLNA but as non-sensitizers in the rLLNA). A review of thedata for these six substances indicates that the traditional LLNA classification of the substances
as skin sensitizers was based not on the highest dose level tested, which induced an SI < 3 but
on a low- or mid-dose level that produced an SI ≥ 3. Because the rLLNA only tests substancesat the highest dose level, all six substances would be incorrectly identified as non-sensitizers(i.e., false negatives). Four of the six substances that resulted in false negatives using the33
34

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
Available at http://www.nlm.nih.gov/mesh/meshhome.html
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ICCVAM Test Method Evaluation Report: Appendix D

rLLNA compared to the traditional LLNA came from LLNA studies that used pooled data.
There were no patterns of consistency for these substances with regard to physicochemical
properties.
Interlaboratory reproducibility of the rLLNA was assessed with data for five substances tested
independently in the same vehicle at multiple laboratories. Among these five substances, three(60%) were classified as sensitizers or non-sensitizers in all studies (i.e., 100% concordance).
Each of the other two substances, tested independently in two laboratories, was classified as asensitizer by one traditional LLNA study and as a non-sensitizer by the other traditional LLNA
study. Review of the studies indicates that the discordant results were due to differences in thehighest dose levels tested. However, because the traditional LLNA and the rLLNA use identical
protocols and the data sets used to evaluate their accuracy are similar, the reliability of the two
methods would be expected to be similar. That is, the intra- and interlaboratory reliability of therLLNA would be expected to be the same as that of the traditional LLNA (see ICCVAM 1999
for these statistics).
A review of published literature on the rLLNA revealed only one published report in addition tothat of Kimber et al. (2006). Ryan et al. (2008) described the impact of reducing the number ofanimals per group from five to two on the performance of the rLLNA and concluded that thesensitivity is inadequate for hazard identification of skin sensitizers.
Compared to the traditional LLNA, the rLLNA will reduce the number of animals used toassess skin sensitization. Because the rLLNA tests only the highest dose level of the test
substance in addition to the concurrent control groups, the number of animals tested would
decrease by at least 40% for each test.
The database included in this BRD will be updated as additional information becomes available
during future use of the traditional LLNA and the rLLNA.

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ICCVAM Test Method Evaluation Report: Appendix D

1.0

Introduction and Rationale for the Proposed Use of the Reduced
Murine Local Lymph Node Assay (rLLNA) to Identify Skin
Sensitizers

1.1

Introduction

1.1.1 Historical BackgroundIn 1999, the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) recommended the murine local lymph node assay (traditional LLNA35) as a valid
substitute for currently accepted guinea pig test methods to assess allergic contact dermatitis(ACD) potential of most types of substances. ICCVAM based its recommendation on acomprehensive evaluation that included an independent scientific peer review panel (Panel)assessment of the validation status of the LLNA. The Panel report and the ICCVAM
recommendations (ICCVAM 1999) are available at the NICEATM–ICCVAM website.36
ICCVAM forwarded to U.S. Federal agencies its recommendation that the traditional LLNA
should be considered for regulatory acceptance or other non-regulatory applications forassessing the ACD potential of substances, while recognizing that some testing situations would
still require the use of traditional guinea pig test methods (ICCVAM 1999). The LLNA wassubsequently incorporated into national and international test guidelines for the assessment ofskin sensitization (International Organization for Standardization [ISO] 10993-10: Tests for
Irritation and Sensitization [ISO 2002]; Organisation for Economic Co-operation andDevelopment Test Guideline [TG] 429 [OECD 2002]; U.S. Environmental Protection Agency[EPA] Health Effects Test Guideline OPPTS 870.2600: Skin Sensitization [EPA 2003]).
1.1.2 Allergic Contact Dermatitis
ACD is a frequent occupational health problem. According to the U.S. Department of Labor
Bureau of Labor Statistics, in 2005, 980 cases of ACD involved days away from work.37
ACD develops in two phases, induction and elicitation. The induction phase occurs when asusceptible individual is exposed topically to a skin-sensitizing substance. Induction depends
on the substance passing through the epidermis, where it forms a hapten complex with dermal
proteins. Langerhans cells, the resident antigen-presenting cells in the skin, process the hapten
complex. The processed hapten complex then migrates to the draining lymph nodes. Antigen
presentation to T-lymphocytes follows, which leads to the clonal expansion of these cells. At
this point, the individual is sensitized to the substance (Basketter et al. 2003; Jowsey et al.
2006). Studies have shown that the magnitude of lymphocyte proliferation correlates with theextent to which sensitization develops (Kimber and Dearman 1991, 1996).
During the elicitation phase, the individual is again topically exposed to the substance. As in the
induction phase, the substance penetrates the epidermis, is processed by the Langerhans cells,
and is presented to circulating T-lymphocytes. The T-lymphocytes are then activated, which
35

The “traditional LLNA” refers to the validated ICCVAM-recommended LLNA (ICCVAM 1999), whichmeasures lymphocyte proliferation based on incorporation of tritiated thymidine into the cells of the draining
auricular lymph nodes.
36
Available at http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
37
Available at http://www.bls.gov/IIF
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ICCVAM Test Method Evaluation Report: Appendix D

causes release of cytokines and other inflammatory mediators. This release produces a rapid
dermal immune response that can lead to ACD (ICCVAM 1999; Basketter et al. 2003; Jowsey
et al. 2006).
1.1.3 U.S. Consumer Product Safety Commission (CPSC) Nomination
On January 10, 2007, the CPSC formally requested that ICCVAM and the National Toxicology
Program Interagency Center for the Evaluation of Alternative Toxicological Methods(NICEATM) evaluate several activities related to the LLNA.38 The nominated activities
included the following:•

The LLNA as a stand-alone assay for potency determination (including severity) for
classification purposes•

Non-radioactive LLNA protocols•

The reduced LLNA (rLLNA) (also known as the ‘‘cut-down’’ or ‘‘limit dose’’
LLNA procedure)•

The use of the LLNA to test mixtures, aqueous solutions, and metals

ICCVAM unanimously agreed that the nominated activities should have a high priority forevaluation. ICCVAM’s advisory committee, the Scientific Advisory Committee on Alternative
Toxicological Methods (SACATM), also recommended that the nominated activities be
undertaken with a high priority.
As ICCVAM and NICEATM collaborate closely with the European Centre for the Validationof Alternative Methods (ECVAM) and the Japanese Center for the Validation of Alternative
Methods, both organizations identified liaisons to the ICCVAM Immunotoxicity Working
Group to facilitate the evaluations requested by the CPSC.
1.1.4 Description of the Reduced Murine Local Lymph Node Assay
Kimber and colleagues initially discussed the rLLNA in a 2006 publication (Kimber et al.
2006). The rLLNA was also discussed in two posters (Basketter et al. 2007; Chaney et al. 2007,
subsequently published as Ryan et al. 2008) and one platform presentation (Basketter 2007) atthe Society of Toxicology (SOT) Annual Meeting in Charlotte, NC, on March 25–29, 2007.
The protocol for the rLLNA is identical to that of the traditional LLNA (as described in the1999 ICCVAM-recommended protocol) with one exception. In the traditional LLNA, threedose levels of each test substance are tested, while in the rLLNA only the highest dose levelthat does not induce systemic toxicity and/or excessive skin irritation is tested for skinsensitizing activity (Kimber et al. 2006).
The term “limit dose,” sometimes used to refer to the rLLNA, accurately depicts a modified
LLNA that tests only the highest dose level that does not induce local irritation and/or systemic
toxicity. The terms “cut-down” and “reduced” LLNA also accurately describe the reduction in
the number of doses tested and emphasize the reduction in the number of animals used toperform the test. For consistency with the terminology presented in the publications that firstdescribed this version of the LLNA, the term “reduced LLNA” (rLLNA) will be used.
38

Available at http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
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ICCVAM Test Method Evaluation Report: Appendix D

1.1.5 Results of an ECVAM Peer Review of the rLLNA
The ECVAM Scientific Advisory Committee (ESAC) established a review panel toretrospectively analyze the published LLNA data to determine if limiting the number of test
substance dose levels to only the highest dose level could successfully reduce the number ofanimals used per test. The review was based on the evaluation published by Kimber et al.
(2006). At its semi-annual meeting on April 26–27, 2007, ESAC reviewed the rLLNA.
The ESAC statement on the rLLNA, dated April 27, 2007 (Annex I), states that:“… the peer reviewed and published information is of a quality and nature to support
the use of the rLLNA within tiered-testing strategies to reliably distinguish between
chemicals that are skin sensitisers and non-sensitisers, and that animal use can be
minimised providing:• The concentration used to evaluate sensitisation potential is the maximum
consistent with solubility and the need to avoid local and other systemic adverse
effects, and that this principle rather than strict adherence to the specificrecommended absolute concentrations as in OECD TG 429 should be used.
• Negative test results associated with testing using concentrations of less than 10%
should undergo further evaluation.
• Positive and negative (vehicle) control groups are used, as appropriate, per OECD
TG 429.
• The full LLNA should be performed when it is known that an assessment ofsensitisation potency is required.”
The ESAC statement also recommends “that further work should be undertaken to determine if
the 10% concentration threshold referenced above is optimal.”
1.2

Regulatory Rationale and Applicability of the rLLNA

Current regulatory testing requires assessment of the potential skin sensitization hazard ofregulated substances/products. The rLLNA is being considered for use in identifying skinsensitizers in a weight-of-evidence strategy such as that proposed in the United Nations
Globally Harmonised System of Classification and Labelling of Chemicals (U.N. 2005). Unlike
the traditional LLNA, the rLLNA evaluates the ability of a substance to be a sensitizer based on
testing a single, highest-testable dose level; therefore, dose-response information is not
generated. Thus, the rLLNA is being proposed for “yes/no” identification of sensitization
hazards.
1.3

Scientific Basis for the rLLNA

1.3.1 Purpose and Mechanistic Basis
The purpose of the rLLNA is to identify potential skin sensitizers by quantifying lymphocyte
proliferation in the draining auricular lymph nodes after application of a test substance to theears of a mouse. The mechanistic basis is identical to that of the traditional LLNA (see Section
1.1.2).

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ICCVAM Test Method Evaluation Report: Appendix D

1.3.2 Applicability Domain
The applicability domain of the rLLNA should be identical to that of the traditional LLNA. The
traditional LLNA was not recommended for the testing of metals, mixtures/extracts,
pharmaceuticals, or strong dermal irritants (ICCVAM 1999).
1.4

Test Method Validation

The ICCVAM Authorization Act of 2000 (Sec. 4(c)) mandates that “[e]ach Federal Agency …
shall ensure that any new or revised … test method … is determined to be valid for its proposed
use prior to requiring, recommending, or encouraging [its use]” (Public Law 106-545, 42
United States Code 285l-3).
Validation is the process by which the reliability and relevance of an assay for a specificpurpose are established (ICCVAM 1997). Relevance is the extent to which an assay willcorrectly predict or measure the biological effect of interest (ICCVAM 1997). For the rLLNA,
relevance is determined by how well the assay identifies (1) substances capable of producing
skin sensitization in humans and (2) substances that should be assessed using a diverse set of
substances that represent both of the types of chemical and product classes to be tested and therange of responses to be identified.
Reliability is the reproducibility of a test method within and among laboratories. The validation
process provides data and information that allow U.S. Federal agencies to develop guidance on
the use of test methods in evaluating the skin sensitization potential of substances.
The first stage in this evaluation is the preparation of a draft background review document(BRD) that comprehensively reviews the relevant data and information about a test method,
including its mechanistic basis, proposed uses, reliability, and performance characteristics(ICCVAM 1997). The draft BRD is made available to the public and an independent scientificpeer review panel (Panel) for review and comment. ICCVAM considers these comments andthose of SACATM as they finalize the BRD. ICCVAM provides the final BRD to regulatory
agencies for consideration as part of the ICCVAM Test Method Evaluation Report.
1.5

Selection of Citations for the rLLNA BRD

The test method data summarized in this BRD were obtained from the original LLNA
evaluation (ICCVAM 1999), peer-reviewed scientific literature, the 2007 SOT Annual Meeting,and responses to a Federal Register (FR) notice requesting such data (72 FR 27815, May 17,200739). The terms “reduced LLNA,” “cut-down LLNA,” “limit dose LLNA,” and “limit test
LLNA” were used to search MEDLINE®, TOXLINE®, and Web of Science® for publications
relevant to the rLLNA test method. A review of these databases through December 2007
revealed two published reports (Kimber et al. 2006; Ryan et al. 2008 [published online ahead ofprint as Ryan et al. 2007]). The rLLNA was also represented at the 2007 SOT Annual Meetingin two posters (Basketter et al. 2007; Chaney et al. 2007, subsequently published as Ryan et al.
2008) and one platform presentation (Basketter 2007).

39

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ICCVAM Test Method Evaluation Report: Appendix D

2.0

rLLNA Protocol Components

2.1

Overview

The technical aspects of the rLLNA are identical to those of the traditional LLNA; the two
methods differ only in the number of test substance dose levels tested (Kimber et al. 2006). In
the traditional LLNA, each test substance is tested at a minimum of three dose levels. The
highest dose level is the maximum soluble concentration that does not cause systemic toxicity
and/or excessive local irritation (ICCVAM 1999). In the rLLNA, in addition to the concurrent
vehicle-control group, each test substance is tested at only the highest testable dose level(Kimber et al. 2006).
A Stimulation Index (SI) is calculated as the ratio of radioactivity incorporated into the cells ofdraining auricular lymph nodes of the treated animals to that of the vehicle-control animals. In
both the traditional LLNA and the rLLNA, the threshold for classifying a substance as a skinsensitizer is an SI ≥ 3.
2.2

Basis for Test Method Selection

The rLLNA was proposed by Kimber et al. (2006) in an effort to reduce the number of animals
used for skin sensitization testing and as a means of streamlining the LLNA for testing that will
be required under the Registration, Evaluation and Authorisation of Chemicals regulations(Kimber et al. 2006).
2.3

Proprietary Test Method Components

The rLLNA does not employ any proprietary components.
2.4

Basis for the Number of Mice per Dose GroupThe basis for the number of mice per dose group in the rLLNA is the same as that for the
traditional LLNA (ICCVAM 1999).
2.5

Study Acceptance Criteria

Similar to the traditional LLNA, in order for an rLLNA study to be considered acceptable, thepositive control must yield an SI ≥ 3 (ICCVAM 1999).
2.6

Basis for Selection of the Test Substance DoseAs noted in Section 2.1, the rLLNA tests each substance at only the highest testable dose level,in addition to the concurrent vehicle control. Consistent with the criteria for selecting thehighest dose level in the traditional LLNA (ICCVAM 1999), the dose level used to evaluatesensitization potential in the rLLNA should be the maximum soluble concentration that does
not cause systemic toxicity and/or excessive local irritation (ICCVAM 1999).

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ICCVAM Test Method Evaluation Report: Appendix D

3.0

Substances Used for Validation of the rLLNA

3.1

Rationale for the Substances or Products Included in the Evaluation

Data from 471 LLNA studies were obtained from 12 sources (Table D-1), including publishedreports and unpublished data submitted to NICEATM in response to 72 FR 27815.40
3.2

Rationale for the Number of Substances Included in the Evaluation

The database from the 471 traditional LLNA studies included 457 unique substances,41 211 ofwhich were included in the original ICCVAM evaluation of the traditional LLNA (ICCVAM
1999). Fourteen of the 457 unique substances were tested two to five times each in different
LLNA studies. Specifically, nine of the 14 substances were evaluated two to five times indifferent vehicles, and five of the 14 substances were evaluated two to five times in the samevehicle. Two of the five substances evaluated in the same vehicle (hexyl cinnamic aldehyde[HCA] and potassium dichromate) were also tested using different vehicles (one study for HCA
and two studies for potassium dichromate). Due to the small number of repeated studies (5% of
total studies), all were treated independently for accuracy evaluation. When the studies for thesubstances repeated in the same vehicle were considered together to yield an overall skinsensitization classification, there were 465 studies with unique substance–vehicle combinations.
3.3

Detailed Description of Substances Included in the Evaluation

Annex II provides information on the physicochemical properties (e.g., physical form tested),
Chemical Abstracts Service Registry Number (CASRN), and chemical class for each substancetested. This information was obtained from the published reports, submitted data, or literaturesearches.
When available, chemical classes for each substance were retrieved from the National Libraryof Medicine’s ChemIDplus® database. If chemical class information was not located, chemicalclasses were assigned for each test substance using a standard classification scheme based on
the National Library of Medicine Medical Subject Headings.42 A substance could be assigned
to more than one chemical class; however, no substance was assigned to more than threeclasses. Certain complex pharmaceuticals and pharmaceutical intermediates were simply
identified as pharmaceutical substances. Chemical class information is presented only toindicate the variety of structural elements present in the substances evaluated in this analysis;
it is not intended to evaluate the impact of structure on skin sensitization activity or potency.

40

May 17, 2007, available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
Some substances were tested in more than one vehicle. In such instances, each substance–vehicle combination
was considered separately, thus a total of 465 unique substance–vehicle combinations were evaluated.
42
Available at http://www.nlm.nih.gov/mesh/meshhome.html
41

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ICCVAM Test Method Evaluation Report: Appendix D

Table D-1 Summary of Traditional LLNA Data Sources and Rationale for SubstanceSelection
Number ofStudiesPrimary Data Source and Substance Selection Rationale

Data Source
Gerberick et al. (2005)1

210

Compiled from previously conducted studies (published literatureand unpublished sources) on substances with varying skinsensitization potential

124

Pharmaceuticals, pharmaceutical intermediates

31

Compiled from previously conducted studies (published literatureand unpublished sources) on substances with varying skinsensitization potential

K. Skirda/CESIO (TNO Report
V7217)

18

Data were provided by CESIO member companies for use in apaper titled “Limitations of the Local Lymph Node Assay (LLNA)
as preferred test for skin sensitisation: concerns about false positiveand false negative test results” (TNO report V7217)

Lalko and Api (2006)17

Original research conducted on essential oils, which wererepresentative of the oils commonly used in perfumery. Eachcontains significant amounts of one or more known skinsensitizers.

H.W. Vohr/BGIA

16

Original research with epoxy resin components as part of avalidation effort for non-radioactive versions of the local lymph
node assay

Ryan et al. (2002)

15

Original research with known water-soluble haptens and known
skin sensitizers to assess the usefulness of a novel vehicle

D. Germolec/NIEHS

15

Substances evaluated by the National Toxicology Program forskin sensitization potential

E. Debruyne/Bayer CropScienceSA

10

Original research on different pesticide types and formulations

P. Ungeheur/EFfCI

9

Data for selected unsaturated chemicals were provided in thereport entitled “Comparative Experimental Study on the Skin
Sensitising Potential of Selected Unsaturated Chemicals as
Assessed by the Murine Local Lymph Node Assay (LLNA) and
the Guinea Pig Maximisation Test (GPMT)”

P. Botham/ECPA

6

Plant protection products (i.e., pesticides) were evaluated in thelocal lymph node assay with a novel vehicle to assess its
usefulness

Basketter et al., 2007

1

Original research that re-evaluated resorcinol in the local lymph
node assay, which identified resorcinol as a sensitizer.

Total

4713

M.J. Olson/GlaxoSmithKline
Basketter, Gerberick, and Kimber

2

Abbreviations: BGIA = Berufsgenossenschaftliches Institut fur Arbeitsschutz; CESIO = Comite Europeen des Agents deSurface et de Leurs Intermediaires Organiques; ECPA = European Crop Protection Association; EFfCI = European Federation
for Cosmetic Ingredients; NIEHS = National Institute for Environmental Health Sciences: TNO = TNO Nutrition and Food
Research
1
These data were submitted to ICCVAM in 1998 for the original evaluation of the validation status of the LLNA (ICCVAM
1999) and were evaluated by the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory
Committee in its evaluation of the rLLNA (Gerberick et al. 2005).
2
Data were included in a submission to ECVAM for the validation of the traditional LLNA as a stand-alone assay for potency
determination.
3
The total number of studies does not take into account the fact that some substances were tested more than once (seeSection 3.2)

D-34

ICCVAM Test Method Evaluation Report: Appendix D

Table D-2 provides chemical class information for the test substances in this rLLNA
evaluation. The table distinguishes the chemical classifications of the 211 substances in theoriginal evaluation of the rLLNA (Kimber et al. 2006; ESAC 2007) and the chemicalclassifications of the additional substances received in response to 72 FR 27815.43 Of the 211
substances initially evaluated by Kimber et al. (2006), the known chemical classes with thegreatest number of substances were carboxylic acids (29) and halogenated hydrocarbons (27).
Of the additional 246 substances in this evaluation, the known chemical classes with thegreatest number of substances tested were pharmaceutical chemicals (125), carboxylic acids(15), and lipids (14). Ten of the substances included in this evaluation were formulations.
Seventy substances could not be assigned to a specific chemical class due to incomplete
information (e.g., the lack of a CASRN or structure).
3.4

Coding Procedures

Neither the previous evaluation of these 211 substances (ICCVAM 1999) nor any additional
studies used in this evaluation describe coding of substances to avoid potential scoring bias.
Table D-2 Chemical Classes1 Represented in the Current Traditional LLNA Database
Number of
Substances Original2
9

Number of
Substances Additional2
4

Number of
Substances Original0

Number of
Substances Additional
2

Aldehydes

21

4

Isocyanates

1

0

Amides4

Amidines

10Ketones500Lactones2

2

AminesAnhydrides

14

7

Lipids

7

14

1

0

Macromolecular
Substances305

Carbohydrates

3

2

Nitriles1

1

Carboxylic Acids

29

15

Nitro Compounds

20Esters30Nitroso Compounds

30

Ethers14

2

Onium Compounds

10Formulations30

10Pharmaceuticalchemicals4

0125Heterocyclic Compounds

18

4

Phenols18

2

Hydrocarbons, Acyclic

2

1

Polycyclic Compounds

5

3

Hydrocarbons, Cyclic

14

7

Quinones1

1

Hydrocarbons, Halogenated27

1

Sulfur Compounds

20

2

Hydrocarbons, Other

7

8

Urea

3

0

Imines0

1

Unknown

28

42

Chemical Class
Alcohols

Chemical Class
Inorganic Chemicals

1

Total number of substances assigned to chemical classes does not equal the total number of substances evaluated because some substanceswere assigned to more than one class and some substances were not assigned to a specific chemical class.
2
Number of substances - original represents the substances evaluated in Kimber et al. (2006).
Number of substances - additional represents the substances received in response to 72 FR 27815 (May 17, 2007) (see below)
3
No chemical class could be assigned. The terms “formulation” or “macromolecular substance” was used to identify these substances.
4
The chemical classification of "pharmaceutical chemicals" for the GlaxoSmithKline (GSK) substances was suggested by Dr. Michael Olsonof GSK to capture three types of pharmaceutical substances (actives, intermediates, and starting materials).

43

May 17, 2007, available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
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D-36

ICCVAM Test Method Evaluation Report: Appendix D

4.0

Comparative In Vivo Reference Data – the Traditional LLNA

4.1

The Traditional LLNA Protocol Used to Generate Comparative In Vivo
Reference Data

As described in Section 2.1, the traditional LLNA protocol was consistent with the original
ICCVAM-recommended protocol (ICCVAM 1999). That original LLNA test method protocol
was accepted by U.S. regulatory agencies (e.g., 2003 EPA Health Effects Test Guidelines) andis itself consistent with procedures described in OECD TG 429, having served as the basis fordevelopment of the test guideline. Still, TG 429 allows for more procedural variation than the
ICCVAM-recommended protocol (ICCVAM 1999).
4.2

Comparative Traditional LLNA Reference Data Used

The traditional LLNA data used to evaluate the rLLNA were obtained from 12 sources(Table D-1). In addition to calculated SI values for each of the tested dose levels, the vehicle
tested and values for the estimated concentration needed to produce an SI of 3 (EC3) for
substances classified as sensitizers were provided in Gerberick et al. (2005). The data received
in response to 72 FR 27815 (May 17, 200744) included calculated SI values for each of thedose levels tested and the vehicle used. If EC3 values were not included in the data source,
they were calculated, where possible, using either interpolation or extrapolation (Dearman etal. 2007). This information and the database (by each source) follow in Annex III.
4.3

Availability of Original Records for Comparative Traditional LLNA Reference
Data

An attempt was made to obtain the original records for the traditional LLNA data through the
FR notice (72 FR 27815, May 17, 200744) and requests to specific stakeholders. Although theoriginal study records were not obtained for any of the studies, compiled in vivo reports and/ortranscribed results were obtained and/or are available for all studies included in this evaluation.
4.4

Quality of Comparative Traditional LLNA Reference Data

Good Laboratory Practice (GLP) guidelines are internationally recognized rules designed to
produce high-quality laboratory records (OECD 1998; EPA 2006a, 2006b; U.S. Food and Drug
Administration [FDA] 2007a). They provide an internationally standardized procedure for the
conduct of studies, reporting requirements, archiving of study data and records, and information
about the test protocol to ensure the integrity, reliability, and accountability of a study.
Ideally, all data supporting the validity of a test method should be obtained from studies
reported and conducted in accordance with GLP guidelines. The extent to which the traditional
LLNA studies complied with GLP guidelines is based on the information provided in publishedand submitted reports. Based on the available information, the following papers and datasubmissions were identified as originating from studies that followed GLP guidelines or useddata obtained according to GLP guidelines:•
44

H.W. Vohr/Berufsgenossenschaftliches Institut fur Arbeitsschutz (BGIA)

Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_9544.pdf
D-37

ICCVAM Test Method Evaluation Report: Appendix D•

P. Ungeheuer/European Federation for Cosmetic Ingredients (EFfCI)•

E. Debruyne/Bayer CropScience SA•

P. Botham/European Crop Protection Association (ECPA)•

M.J. Olson/GlaxoSmithKline (GSK)•

D. Germolec/National Institute for Environmental Health Sciences (NIEHS)

The publication by Gerberick et al. (2005) does not address the GLP compliance of any of thestudies discussed. Several of the substances listed in Gerberick et al. (2005) were included in
the original LLNA submission to ICCVAM (ICCVAM 1999). According to the submission,“Much of the data used here to support this submission and much of the data contained within
the publications cited in this document have been derived from audited Good Laboratory
Practice (GLP) compliant studies. Where this is not the case all investigations have been
conducted to the spirit of GLP or Good Research Practice in GLP compliant facilities”(reproduced in ICCVAM 1999). Furthermore, in response to requests from ICCVAM, records
were provided indicating compliance with GLP guidelines for some of the studies.
4.5

Accuracy and Reliability of the Traditional LLNA

4.5.1 Accuracy
ICCVAM (1999) reviewed the performance of the traditional LLNA with comparisons to(1) the Guinea Pig Maximization Test and the Buehler Test (EPA 2003) and (2) human results
obtained from the human maximization test45 and human patch test allergen46 panels. The
evaluation concluded that the LLNA demonstrated adequate accuracy (ICCVAM 1999).
4.5.2 Reliability
ICCVAM (1999) also reviewed the reliability of the traditional LLNA as assessed by intra- andinterlaboratory reproducibility. The evaluation concluded that the LLNA demonstrated
adequate intra- and interlaboratory repeatability and reproducibility (ICCVAM 1999).

45

The human maximization test involves application of occluded patches on the same skin site with a rest
period between each reapplication. Two weeks after the last induction patch, sensitization is evaluated using a
48-hour occluded patch test. The site is scored 24 and 48 hours after patch removal.
46
Allergen patch tests are diagnostic tests applied to the surface of the skin to identify the cause of contact
dermatitis. Chemicals and substances included in these tests (e.g., nickel, rubber, and fragrance mixes) are
known to cause contact dermatitis (i.e., skin sensitization) (http://www.fda.gov/cber/allergenics.htm).
D-38

ICCVAM Test Method Evaluation Report: Appendix D

5.0

rLLNA Test Method Data and Results

5.1

Description of the rLLNA Test Method Protocol Used to Generate Data

No specific rLLNA studies were conducted for this evaluation; rather, data from traditional
LLNA studies were evaluated retrospectively. The only difference in the test method protocols
between the traditional LLNA and the rLLNA is the number of dose levels tested. In the
traditional LLNA, at least three test-substance dose levels are tested, with the highest dose levelbased on maximum solubility and the avoidance of systemic toxicity and/or excessive local
irritation. In contrast, only the highest dose level of a substance is tested in the rLLNA (Kimber
et al. 2006). This retrospective evaluation assumes that the top dose level tested in the
traditional LLNA studies was in fact the maximum soluble concentration that did not cause
overt systemic toxicity and/or excessive local irritation. Because the criteria for choosing the
top dose in the traditional LLNA and in the rLLNA are the same, the maximum dose level
tested should be the same for both. However, it is important to consider that the highest possible
dose level selected in a prospective validation study may differ between the two versions of the
LLNA. Thus, the accuracy analysis of these same substances in a prospective rLLNA study
may differ from the accuracy analysis obtained in this retrospective rLLNA analysis.
5.2

Availability of Original rLLNA Data Used to Evaluate Accuracy and Reliability

While original study records were not obtained for any of the previously conducted studies,
compiled in vivo reports and/or transcribed results were obtained and/or available for all studies
included in this evaluation.47
5.3

Description of the Statistical Procedure Used to Evaluate rLLNA Data

The performance analysis in this BRD focuses on the ability of the rLLNA to identify potential
skin sensitizers as determined by the calculated SI for each test substance (see Section 2.1).
5.4

Summary of Results

The data evaluated here were obtained from 12 sources (Table D-1). Where available, thespecific information extracted for each substance includes its name, CASRN, physicochemical
properties (e.g., form tested, Log Kow), and chemical class48 (Annex II). Dose levels tested,along with calculated SI and/or EC3 values, sensitizing hazard classification, and the data
source are provided in Annex III. If EC3 values were not included in the data source, they were
calculated, where possible, using either interpolation or extrapolation (Dearman et al. 2007).
Other than the information provided in the submitted data, no additional attempt was made to
identify the source or purity of the test substance.

47

The LLNA data for several of the substances evaluated for this report were included in the database that was
submitted to ICCVAM in 1998 for the initial evaluation of LLNA (ICCVAM 1999). Therefore, some of theoriginal data for these substances were available for review.
48
Chemical classes were assigned by NICEATM based on the classification of the National Library ofMedicine’s Medical Subject Heading (available at http://www.nlm.nih.gov/mesh/meshhome.html).
D-39

ICCVAM Test Method Evaluation Report: Appendix D

5.5

Use of Coded Substances

Neither the previous evaluation of these 211 substances (ICCVAM 1999) nor any additional
studies used in this evaluation describe coding of substances to avoid potential scoring bias.
5.6

Lot-to-Lot Consistency of Test Substances

Ideally, a single lot of each substance is used during the validation of a test method. In
situations where multiple lots of a chemical must be used, the lot-to-lot consistency of a test
substance must be evaluated to ensure the consistency of the substance evaluated over thecourse of the study. The procedures used to evaluate lot-to-lot consistency are described in the
published reports. No attempt was made to review original records to assess the procedures
used to evaluate different batches.
Data submitted by P. Botham/ECPA, P. Ungheuer/EFfCI, and D. Germolec/NIEHS included
the source and the batch number of each tested substance.
5.7

Availability of Original Data for External Audit

The LLNA data included in the ICCVAM (1999) database were reviewed during the original
evaluation. The original data for the other studies included in this evaluation were not available.

D-40

ICCVAM Test Method Evaluation Report: Appendix D

6.0

Accuracy of the rLLNA

6.1

Performance Statistics

A critical component of a formal evaluation of the validation status of a test method is anassessment of the accuracy of the proposed tested method when compared to the current
reference test method (ICCVAM 2003). This aspect of assay performance is typically evaluatedby calculating:•Accuracy (concordance): the proportion of correct outcomes (positive and negative) of atest method•

Sensitivity: the proportion of all positive substances that are classified as positive•

Specificity: the proportion of all negative substances that are classified as negative•

Positive predictivity: the proportion of correct positive responses among substances
testing positive•

Negative predictivity: the proportion of correct negative responses among substances
testing negative•

False positive rate: the proportion of all negative substances that are falsely identified as
positive•

False negative rate: the proportion of all positive substances that are falsely identified as
negative

The ability of the rLLNA to correctly identify potential skin sensitizers was compared to that of
the traditional LLNA for 471 studies.49 Of the 471 studies, 318 detected skin sensitizers and
153 detected non-sensitizers.50 Classification of substances and complete data for each
substance are located in Annex III. When studies for the substances tested more than once inthe same vehicle were considered together to yield an overall skin sensitization classification,
465 unique substance–vehicle combination studies resulted. Of these, 315 detected sensitizers
and 150 detected non-sensitizers.
Based on the available study data, the rLLNA has an accuracy of 98.7% (465/471), a sensitivityof 98.1% (312/318), a specificity of 100% (153/153), a false positive rate of 0% (0/153), and afalse negative rate of 1.9% (6/318) when compared to the traditional LLNA. When substancestested more than once in the same vehicle were considered together, the resulting 465 studies
give an accuracy of 98.7% (459/465), a sensitivity of 98.1% (309/315), a specificity of 100%
(150/150), a false positive rate of 0% (0/150), and a false negative rate of 1.9% (6/315). The
performance characteristics of the rLLNA as discussed in Kimber et al. (2006) are presented inTable D-3.

49

Due to the small number of repeated studies (5%), all studies were treated independently for this accuracy
evaluation. When the studies for the substances repeated in the same vehicle were considered together to yieldan overall skin sensitization classification, there were 465 studies with unique substance–vehicle combinations.
50
For two of the repeated studies (HCA and linalool alcohol), the LLNA obtained discordant results. In bothcases, one study classified the substance as a non-sensitizer and the other classified it as a sensitizer. Reviewof the studies indicates differences in the highest dose levels tested. For each of the studies, the traditional
LLNA and the rLLNA both classified the substance as a sensitizer or as a non-sensitizer.
D-41

ICCVAM Test Method Evaluation Report: Appendix D

Table D-3 Performance of the rLLNA in Predicting Skin Sensitizers Compared to the Traditional LLNA
Accuracy

Sensitivity

Specificity

Positive
Predictivity

Negative
Predictivity

False
Positive

False
Negative

Data

N

%

No.

%

No.

%

No.

%

No.

%

No.

%

No.

%

No.

Kimber et al. (2006)

211

98.6

208/21198.2

166/169

100

42/42

100

166/166

93.3

42/45

0

0/42

1.8

3/169

rLLNA

471

98.7

465/47198.1

312/318100

153/153100

312/31296.2

153/159

0

0/153

1.9

6/318

rLLNA - substances
repeated in the samevehicle wereconsidered together

465

98.7

459/46598.1

309/315100

150/150100

309/30996.2

150/156

0

0/150

1.9

6/315

Abbreviations: N = number of studies; No. = numbers used to calculate percentage

D-42

ICCVAM Test Method Evaluation Report: Appendix D

Kimber et al. (2006) proposed that a minimum testing concentration be considered for the
purpose of judging the appropriateness of a non-sensitizing classification for a test substance. In
their evaluation, Kimber et al. proposed testing a minimum concentration of 10% in a dose
solution (2006). However, lack of sensitizing potential at 10% does not necessarily indicate thata substance will not elicit skin sensitization when tested at a higher concentration. In fact, 51
substances (16% [51/315]) within the current database were non-sensitizers at concentrations of
10%51 but were sensitizers at higher concentrations (see Annex IV).
According to the 1999 ICCVAM-recommended LLNA protocol, the maximum concentration
tested should be “the highest achievable level while avoiding overt systemic toxicity and/or
excessive local irritation.” Similar text is included in OECD TG 429 (2002). Thus, setting aminimum testing concentration is not advised because the maximum soluble concentration thatavoids systemic toxicity and/or excessive local irritation may be less than 10% with a nonsensitizing result.
6.2

Discordant Results

In the current analysis, six substances yielded false negative results in the rLLNA. The
discordant substances were 2-methyl-2H-isothiazol-3-one, C19-azlactone, azithromycin,
camphorquinone, nickel sulfate, and a substance designated as non-ionic surfactant 2. A reviewof the data for the false negatives indicates that the traditional LLNA classification of thesubstances as skin sensitizers was based on a low- or mid-dose level that produced an SI ≥ 3,
while the highest dose level tested produced an SI < 3 (see Table D-4). Because the rLLNA
evaluates only the highest dose level tested, all six substances were identified as non-sensitizers(i.e., false negatives). Four of the six substances that resulted in false negatives using therLLNA compared to the traditional LLNA came from LLNA studies that used pooled data.
Graphs of the dose-response curves for these six substances are provided in Figure D-1.
Table D-4 Traditional LLNA Data for Substances Identified as False Negatives by therLLNA

Substance
2-methyl-2H-isothiazol-3-one

VehicleAOO

Traditional LLNA Data Traditional LLNA Data
(Low- or Mid-Dose Level) (Highest Dose Level)
EC3 Dose (%)
SI
Dose (%)
SI
1.9
2.5
3.8
5
2.5

C19-azlactone

AOO

26
1

29.33

3.1

58.67

2.5

Azithromycin

Acetone

NC

10

3.7

40

2.1

Camphorquinone

AOO

10

10

3.0

25

1.7

Nickel sulfate

Pluronic L92
(1%)

2.5

2.5

3.0

5

2.3

Non-ionic surfactant 2
AOO
47.1
50
3.2
100
2.9
Abbreviations: AOO = acetone: olive oil (4:1 by volume); EC3 = estimated concentration needed to produce a
stimulation index of 3; NC = not calculated; SI = stimulation index
1
Data was not calculated because extrapolation between points that bracket an SI of 3 could not be done.

51

An initial dose was tested at a concentration of 10% or greater and resulted in an SI < 3, while a subsequent
higher concentration resulted in an SI ≥ 3.
D-43

ICCVAM Test Method Evaluation Report: Appendix D

Figure D-1

Dose-Response Curves for Substances Identified as Sensitizers by the
Traditional LLNA but as Non-Sensitizers by the rLLNA

2-Methyl-2H-isothiazol-3-one

C19-azlactone

4

4

3

3

2

2

1

1

0
0

1

2

3

4

5

0

6

0

10

20

Concentration (%)

Azithromycin
4

3

3

2

2

1

1

0

10

20

30

40

50

60

70

25

30

0

50

0

5

10

Concentration (%)

15

20

Concentration (%)

Non-ionic surfactant 2

Nickel sulfate
4

4

3

3

2

2

1

1

0

40

Camphorquinone

4

0

30

Concentration (%)

0
0

1

2

3

4

5

0

6

25

50

75

100

Concentration (%)

Concentration (%)

Note: The horizontal line in each figure indicates a stimulation index of 3, which is the threshold for a positive
response in the LLNA. Points on or above this line would indicate a positive (sensitizer) response, while
points below this line would indicate a negative (non-sensitizer) response.

Table D-5 provides a summary of the available physicochemical properties of these substances
and the vehicle used.
D-44

ICCVAM Test Method Evaluation Report: Appendix D

Table D-5 Summary of Available Physicochemical Properties for False Negatives, as
Identified by the rLLNA
Substance

CASRN

VehicleMolecular Weight(g/mol)

KOW1

2-Methyl-2H-isothiazol-3-one

2682-20-4

AOO

115.15

0.682

C19-azlactone—

AOO

379.63

5.212

Azithromycin

83905-01-5

Acetone748.993.243

Camphorquinone

465-29-2

AOO

166.22

2.152

Nickel sulfate

7786-81-4

Pluronic L92 (1%)

154.76-0.173

Non-ionic surfactant 2
—
AOO
—
—
Abbreviations: AOO = acetone: olive oil (4:1 by volume); CASRN = Chemical Abstracts Service Registry
Number
1
KOW represents the octanol-water partition coefficient (expressed on log scale).
2
KOW calculated by the method of Moriguchi et al. (1994) and provided in Gerberick et al. (2005).
3
KOW calculated by the method of Meylan and Howard (1995) and obtained from the web site
http://www.srcinc.com/what-we-do/databaseforms.aspx?id=385

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D-46

ICCVAM Test Method Evaluation Report: Appendix D

7.0

Reliability of the rLLNA

An assessment of test method reliability (intralaboratory repeatability and intra- andinterlaboratory reproducibility) is essential to evaluate the performance of an alternative test
method (ICCVAM 2003). Repeatability refers to the closeness of agreement between test
results obtained within a single laboratory when the procedure is performed on the same
substance under identical conditions within a given time period (ICCVAM 1997, 2003).
Intralaboratory reproducibility refers to the determination of the extent to which qualified
personnel within the same laboratory can replicate results using a specific test protocol at
different times. Interlaboratory reproducibility refers to the determination of the extent towhich different laboratories can replicate results using the same protocol and test substances,and indicates the extent to which a test method can be transferred successfully amonglaboratories.
In the data review, interlaboratory reproducibility of the rLLNA could be assessed withtraditional LLNA data available for only five substances that had been tested in the samevehicle at multiple labs (Annex III). These are dinitrochlorobenzene (DNCB), HCA, linalool
alcohol, methyl salicylate, and potassium dichromate. Table D-6 provides a summary of the
responses obtained by the rLLNA. Among these five substances, tested independently in two to
three laboratories, DNCB, methyl salicylate, and potassium dichromate (3/5 = 60%) wereclassified as sensitizers or non-sensitizers in all studies (i.e., 100% concordance). For the othertwo substances, HCA and linalool alcohol, tested independently in two laboratories, one
traditional LLNA study indicated each substance as a sensitizer and the other traditional LLNA
study indicated each substance as a non-sensitizer.
Review of the studies indicates that the discordant results were due to differences in the highest
dose levels tested. However, because the rLLNA and traditional LLNA use identical protocols
and use similar data sets to evaluate the accuracy of the rLLNA and traditional LLNA, thereliability of the two methods would be expected to be similar. That is, the intra- andinterlaboratory reliability of the rLLNA would be expected to be similar to that of the
traditional LLNA (see ICCVAM 1999 for these statistics).

D-47

ICCVAM Test Method Evaluation Report: Appendix D

Table D-6 rLLNA Responses for Repeated Studies
Traditional LLNA Response in Multiple Studies
Substance

Data Source

Vehicle1-Chloro-2dinitrobenzene

Gerberick et al. (2005)

AOO

0.01/1.50

0.025/1.8
00.05/2.4
00.1/8.90

0.25/38.00NA+0.01/1.17

0.025/1.1
2

0.05/1.9
3

0.1/1.95

0.25/7.10NA+

2.5/1.30

5/1.1010/2.50

25/10.00

50/17.00

NA+

2.5/1.10

5/1.2010/2.84

NA

NA

NA

-

NA

NA

NA

25/2.50

50/4.80

100/8.30+

1/1.0010/1.30

30/1.30

NA

NA

NA

-

1/1.002.5/1.10

5/1.6010/1.40

20/0.90NA-

1/0.862.5/1.19

5/1.16

10/1.41

20/1.72NA-

0.025/1.6
00.05/1.40

0.1/3.80

0.25/5.30

0.5/16.10

NA+

Data submitted by D.
Germolec

0.025/1.2
10.05/1.84

0.1/2.22

0.25/3.39

NA

NA

+

Ryan et al. (2002)

0.025/1.4
00.05/2.50

0.1/9.50

0.25/25.9
00.5/10.10

NA+

Data submitted by D.
Germolec
Hexyl cinnamic
aldehyde
Linalool alcohol

Gerberick et al. (2005)

AOO

Data Submitted by H.W. Vohr
Gerberick et al. (2005)

AOO

Data Submitted by D.
Basketter, I. Kimber, and F.
GerberickMethyl salicylate

Gerberick et al. (2005)

AOO

Data submitted by D.
Germolec
Potassium
dichromate

Gerberick et al. (2005)

DMSO

Dose
(%)/SI

Dose(%)/SI

Dose(%)/SI

Dose(%)/SI

Dose(%)/SIrLLNAClassification

Dose(%)/SI1

Abbreviations: AOO = acetone: olive oil; DMSO = dimethyl sulfoxide; NA = not applicable because dose level was not tested; SI = stimulation index
1- = non-sensitizer, + = sensitizer

D-48

ICCVAM Test Method Evaluation Report: Appendix D

8.0

rLLNA Data Quality

8.1

Adherence to National and International GLP Guidelines

The extent to which the LLNA studies complied with GLP guidelines is based on theinformation provided in published and submitted reports. Based on the available information,
the following papers and data submissions were identified as originating from studies that
followed GLP guidelines or used data obtained according to GLP guidelines: H.W. Vohr/BGIA,
P. Ungeheuer/EFfCI, E. Debruyne/Bayer CropScience SA, P. Botham/ECPA, M.J. Olson/GSK,
and D. Germolec/NIEHS.
8.2

Data Quality Audits

Formal assessments of data quality, such as quality assurance audits, generally involve asystematic and critical comparison of the data provided in a study report to the laboratory
records generated for a study.
Much of the data published by Gerberick et al. (2005) was conducted following GLP guidelines
or were conducted in GLP-compliant facilities. Therefore, it was previously inferred that data
audits were conducted on the data (ICCVAM 1999).
A formal assessment of the quality of the remainder of the LLNA data included in this BRD
was not feasible. The published data on the LLNA were limited to tested concentrations andcalculated SI and EC3 values. Auditing the reported values would require obtaining the originalindividual animal data for each LLNA experiment, which were not obtained. However, theconduct of many of the studies according to GLP guidelines implies that an independent quality
assurance audit was conducted.
8.3

Impact of Deviations from GLP Guidelines

The impact of deviations from GLP guidelines cannot be evaluated for the data reviewed in this
BRD, because no information on data quality audits was obtained.
8.4

Availability of Laboratory Notebooks or Other Records

The original records were not obtained for the studies included in this evaluation. Data wereavailable for several of the substances included in the ICCVAM 1999 evaluation, thus some of
the raw data for these substances were available for review.

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9.0

Other rLLNA Scientific Reports and Reviews

9.1

Reports in the Peer-Reviewed Literature

A search of the terms “reduced LLNA,” “cut-down LLNA,” “limit dose LLNA,” and “limit test
LLNA” in the MEDLINE®, TOXLINE®, and Web of Science® search engines throughDecember 2007 produced one relevant published report in addition to that of Kimber et al.
(2006). Three related presentations (two posters and one platform) were included in the 2007
SOT Annual Meeting held in Charlotte, NC, from March 25-29. One of the posters (Basketter
et al. 2007) and the platform presentation (Basketter 2007) detailed the evaluation that resulted
in the Kimber et al. (2006) publication and are therefore not discussed below. The informationin the second poster, Chaney et al. (2007), described the impact of reducing the number ofanimals per dose group on the performance of the rLLNA and is summarized below from thesubsequent publication (Ryan et al. 2008; published online ahead of print as Ryan et al. 2007).
9.1.1 Ryan et al. (2008)
Ryan et al. (2008) evaluated the impact of reducing the number of mice (from five animals totwo) on the performance characteristics using the rLLNA. Nineteen sensitizing and five nonsensitizing sustances were evaluated with 33 sensitizer datasets and eight non-sensitizer data
sets.
SI values were determined for all possible two-animal combinations for the control- and highdose groups. With 10 possible data combinations per experimental group, there were
100 possible sets of four values (two control animals and two high-dose animals) for each data
set. The 100 possible SI values, each based on a unique set of four values, were plotted for eachdata set, and the percentage of combinations that resulted in an SI ≥ 3 was calculated. Of thesensitizers evaluated, at least 96% of the combinations yielded an SI ≥ 3 for 76% (25/33) of thedata sets. Thirteen or fewer percent (≤13%) of the possible combinations of non-sensitizers(excluding three data sets for sodium lauryl sulfate) had an SI ≥ 3. For the data sets withthreshold SI values (2–4.9), however, 90% or more of the combinations resulted in SI ≥ 3 for
only 20% (4/20) of the sensitizers. Thirteen of the 20 (65%) sensitizer data sets had less than
75% of the combinations producing SI ≥ 3. The authors concluded that the decreased sensitivity
produced by using two mice per group was inappropriate for using the rLLNA to identify skinsensitization hazard.

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10.0 Animal Welfare Considerations
10.1

How the rLLNA will Refine, Reduce, or Replace Animal Use

Compared to the traditional LLNA, the rLLNA will reduce the number of animals used toassess skin sensitization. In addition to a concurrent vehicle-control group and a positivecontrol group, the traditional LLNA requires testing four to five mice with each of at least three
test-substance dose levels (ICCVAM 1999). Because the rLLNA tests only the highest doselevel of the test substance being evaluated, in addition to the concurrent control groups, thenumber of animals tested would be decreased by at least 40% for each test.
10.2

Requirements for the Use of Animals

The rationale for the use of animals and the basis for determining the number of animals used in
the rLLNA are the same as those for the traditional LLNA (ICCVAM 1999).

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11.0 Practical Considerations
Several issues in addition to performance evaluations must be taken into account whenassessing the practicality of an alternative test method in comparison to the existing test
method:• Assessments of the laboratory equipment and supplies needed to conduct thealternative test method• Level of personnel training• Labor costs• Time required to complete the test method
The time, personnel cost, and effort required to conduct the proposed test method(s) must be
considered reasonable in comparison to those of the test method it is intended to replace.
11.1

Transferability of the rLLNA

Test method transferability addresses the ability of a method to be performed accurately andreliably by multiple laboratories (ICCVAM 2003), including those experienced in the particular
type of procedure as well as laboratories with less or no experience in the particular procedure.
The degree of transferability of a test method can be evaluated by its interlaboratory
reproducibility. Section 7.0 discusses the minimum variability expected. The transferability of
the rLLNA is equal to that of the traditional LLNA (ICCVAM 1999), which includes
considerations for the required facilities, major fixed equipment, and any other necessary
supplies.
11.2

rLLNA Training Considerations

The level of training and expertise needed to conduct the rLLNA, and the training requirements
needed to demonstrate proficiency, are identical to that for the traditional LLNA (ICCVAM
1999).
11.3

Cost Considerations

The rLLNA uses the same basic protocol as the traditional LLNA. However, because fewer
animals are tested, the related test costs (e.g., animal care, radioactivity, scintillation fluid, etc.)
would be expected to be proportionally lower than the traditional LLNA.
11.4

Time Considerations

Because at least 40% fewer animals are tested in the rLLNA than in the traditional LLNA, theoverall time required to conduct the method (e.g., dosing mice, removing the auricular lymph
nodes from the animals) would be expected to decrease proportionally.

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12.0 References
Basketter D, Patlewicz G, Gerberick F, Ryan C, Kern P, Betts C, et al. 2007. Identification ofskin sensitizing chemicals in a reduced LLNA. [Abstract]. Toxicologist 96:236.
Basketter DA. 2007. Integrated systems and a modified local lymph node assay [Abstract].
Toxicologist 96:124.
Basketter DA, Sanders D, Jowsey IR. 2007. The skin sensitization potential of resorcinol:
Experience with the local lymph node assay. Contact Dermatitis 56:196–200.
Basketter DA, Smith Pease CK, Patlewicz GY. 2003. Contact allergy: The local lymph node
assay for the prediction of hazard and risk. Clin Exp Dermatol 28:218–221.
Chaney J, Ryan C, Kern P, Patlewicz G, Basketter D, Betts C, et al. 2007. The impact of
reducing animal numbers in the local lymph node assay. [Abstract]. Toxicologist 96:236.
Dearman RJ, Betts CJ, Farr C, McLaughlin J, Berdasco N, Wiench K, et al. 2007. Comparative
analysis of skin sensitization potency of acrylates (methyl acrylate, ethyl acrylate, butyl
acrylate, and ethylhexyl acrylate) using the local lymph node assay. Contact Dermatitis 57:242–
247.
EPA. 2003. Health Effects Test Guideline, OPPTS 870.2600. Skin Sensitization EPA 712–C–
03–197. Washington, DC: U.S. Environmental Protection Agency. Available:
http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/Revised/870r-2600.pdf.
EPA. 2006a. Good Laboratory Practice Standards. Toxic Substances Control Act. 40 CFR 792.
Available: http://www.access.gpo.gov/nara/cfr/waisidx_06/40cfr792_06.html.
EPA. 2006b. Good Laboratory Practice Standards. Federal Insecticide, Fungicide, andRodenticide Act. 40 CFR 160.
http://www.access.gpo.gov/nara/cfr/waisidx_06/40cfr160_06.html.
ESAC. 2007. Statement on the Reduced Local Lymph Node Assay (rLLNA). European
Commission Directorate General, Joint Research Centre, Institute for Health and Consumer
Protection, European Centre for the Validation of Alternative Methods, April 2007. Available:
http://ecvam.jrc.it/ft_doc/ESAC26_statement_rLLNA_20070525-1.pdf
FDA. 2007a. Good laboratory practice for nonclinical laboratory studies. 21 CFR 58.
Gerberick GF, Ryan CA, Kern PS, Schlatter H, Dearman RJ, Kimber I, et al. 2005. Compilationof historical local lymph node data for evaluation of skin sensitization alternative methods.
Dermatitis 16:157–202.
ICCVAM. 1997. Validation and Regulatory Acceptance of Toxicological Test Methods: A
Report of the ad hoc Interagency Coordinating Committee on the Validation of Alternative
Methods. NIH Publication No.: 97-3981. Research Triangle Park, NC: National Institute ofEnvironmental Sciences. Available: http://iccvam.niehs.nih.gov/docs/about_docs/validate.pdf.

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ICCVAM Test Method Evaluation Report: Appendix D

ICCVAM 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemical/Compounds. NIH Publication No. 99-4494.
Research Triangle Park, NC: National Institute of Environmental Sciences. Available:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf.
ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised,
and Alternative Test Methods. NIH Publication No: 03-4508. Research Triangle Park, NC:
National Institute of Environmental Sciences. Available:
http://iccvam.niehs.nih.gov/SuppDocs/SubGuidelines/SD_subg034508.pdf.
ICCVAM Authorization Act. 2000. Public Law 106-545, 42 U.S.C. 285l-3. Available:
http://iccvam.niehs.nih.gov/docs/about_docs/PL106545.pdf.
ISO. 2002. Biological evaluation of medical devices -- Part 10: Tests for irritation and delayedtype hypersensitivity. Available for purchase at: http://www.iso.org/iso/home.htm.
Jowsey IR, Basketter DA, Westmoreland C, Kimber I. 2006. A future approach to measuring
relative skin sensitising potency: a proposal. J Appl Toxicol 26:341–350.
Kimber I, Dearman RJ. 1991. Investigation of lymph node cell proliferation as a possible
immunological correlate of contact sensitizing potential. Food Chem Toxicol 29:125–129.
Kimber I, Dearman RJ. 1996. Contact hypersensitivity: immunological mechanisms. In:
Toxicology of Contact Hypersensitivity (Kimber I, Maurer T, eds). London: Taylor andFrancis, 4–25.
Kimber I, Dearman RJ, Betts CJ, Gerberick GF, Ryan CA, Kern PS, et al. 2006. The local
lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?
Contact Dermatitis 54:181–185.
Lalko J, Api AM. 2006. Investigation of the dermal sensitization potential of various essential
oils in the local lymph node assay. Food Chem Toxicol 44:739–746.
Meylan WM, Howard PH. 1995. Atom/fragment contribution method for estimating octanolwater partition coefficients. J Pharm Sci 84:83–92.
Moriguchi I, Hirono S, Nakagome I, Hirano H. 1994. Comparison of reliability of log P values
for drugs calculated by several methods. Chem Pharm Bull 42:976–978.
OECD. 1998. OECD Series on Principles of Good Laboratory Practice and Compliance
Monitoring Number 1: OECD Principles on Good Laboratory Practice (as revised in 1997).
ENV/MC/CHEM (98)17. Paris: OECD.
OECD. 2002. Test No. 429. Skin Sensitisation: Local Lymph Node Assay. Adopted April 24,
2002. In: OECD Guidelines for Testing of Chemicals. Paris: OECD. Available:
http://oberon.sourceoecd.org/vl=2675708/cl=14/nw=1/rpsv/cgibin/fulltextew.pl?prpsv=/ij/oecdjournals/1607310x/v1n4/s30/p1.idx.
Ryan CA, Chaney JG, Kern PS, Patlewicz GY, Basketter DA, Betts CJ, et al. 2008. The
reduced local lymph node assay: the impact of group size. J Appl Toxicol 28:518–523.
Ryan CA, Cruse LW, Skinner RA, Dearman RJ, Kimber I, Gerberick GF. 2002. Examination of
a vehicle for use with water soluble materials in the murine local lymph node assay. Food Chem
Toxicol 40:1719–1725.
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ICCVAM Test Method Evaluation Report: Appendix D

U.N. 2005. Globally Harmonised System of Classification and Labelling of Chemicals (GHS).
Geneva: United Nations Publications.

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13.0 Glossary
Accuracy: (a) The closeness of agreement between a test method result and an acceptedreference value. (b) The proportion of correct outcomes of a test method. It is a measure of test
method performance and one aspect of relevance. The term is often used interchangeably withconcordance (see also two-by-two table). Accuracy is highly dependent on the prevalence of
positives in the population being examined.*Allergic contact dermatitis (ACD): A Type IV allergic reaction of the skin that results fromrepeated skin contact with a skin sensitizer. Clinical signs include the development of erythema(redness) and edema (swelling), blistering, and itching. Also referred to as skin sensitization.
Assay: The experimental system used. Often used interchangeably with test and test method.*
Coded substances: Substances labeled by code rather than name so that they can be tested andevaluated without knowledge of their identity or anticipation of test results. Coded substances
are used to avoid intentional or unintentional bias when evaluating laboratory or test method
performance.
Concordance: The proportion of all substances tested that is correctly classified as positive or
negative. It is a measure of test method performance and one aspect of relevance. The term isoften used interchangeably with accuracy (see also two-by-two table). Concordance is highly
dependent on the prevalence of positives in the population being examined.*
EC3: The estimated concentration needed to produce a stimulation index of 3, as compared tothe concurrent vehicle control.
Essential test method component: Structural, functional, and procedural elements of a test
method that are used to develop the test method protocol. These components include uniquecharacteristics of the test method, critical procedural details, and quality control measures.
Adherence to essential test method components is necessary when the acceptability of a
proposed test method is being evaluated based on performance standards derived frommechanistically and functionally similar validated test method. [Note: Previously referred to as
minimum procedural standards.]*
False negative: A substance incorrectly identified as negative by a test method.*
False negative rate: The proportion of all positive substances falsely identified by a test
method as negative (see two-by-two table). It is one indicator of test method accuracy.*
False positive: A substance incorrectly identified as positive by a test method.*
False positive rate: The proportion of all negative substances that are falsely identified by atest method as positive (see two-by-two table). It is one indicator of test method accuracy.*
Good Laboratory Practices (GLP): Regulations promulgated by the U.S. Food and Drug
Administration and the U.S. Environmental Protection Agency, and principles and procedures
adopted by the OECD and Japanese authorities, which describe record keeping and quality
assurance procedures for laboratory records that will be the basis for data submissions tonational regulatory agencies.*
The definitions in this glossary are restricted to their uses with respect to the rLLNA and the traditional LLNA.

* Definition used by ICCVAM (ICCVAM 2003).
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ICCVAM Test Method Evaluation Report: Appendix D

Hazard: The potential for an adverse health or ecological effect. Hazard potential results only if
an exposure occurs that leads to the possibility of an adverse effect being manifested.*
Interlaboratory reproducibility: A measure of whether different qualified laboratories using
the same protocol and test substances can produce qualitatively and quantitatively similar
results. Interlaboratory reproducibility is determined during the prevalidation and validation
processes and indicates the extent to which a test method can be transferred successfully amonglaboratories.*
Intralaboratory repeatability: The closeness of agreement between test results obtained
within a single laboratory when the procedure is performed on the same substance underidentical conditions within a given time period.*
Intralaboratory reproducibility: The first stage of validation; a determination of whether
qualified people within the same laboratory can successfully replicate results using a specifictest protocol at different times.*
Immunological: Relating to the immune system and immune responses.
In vivo: In the living organism. Refers to assays performed in multicellular organisms.
Local lymph node assay (LLNA): An in vivo test method used to assess the skin sensitization
potential of a substance by measuring the proliferation of lymphocytes in the lymph nodes
draining the ears (i.e., auricular lymph nodes) of mice, subsequent to topical exposure on theear to the substance. The traditional LLNA measures lymphocyte proliferation by quantifying
the amount of tritiated thymidine (3H) incorporated into the cells of the draining lymph nodes.
Lymphocyte: A white blood cell found in the blood, lymph, and lymphoid tissues, which
regulates and plays a role in acquired immunity.
Negative predictivity: The proportion of correct negative responses among substances testingnegative by a test method (see two-by-two table). It is one indicator of test method accuracy.
Negative predictivity is a function of the sensitivity of the test method and the prevalence ofnegatives among the substances tested.*Non-sensitizer: A substance that does not cause skin sensitization after repeated skin contact.
Performance: The accuracy and reliability characteristics of a test method (see accuracy,
reliability).*
Positive control: A substance known to induce a positive response used to demonstrate thesensitivity of the test method and to allow for an assessment of variability in the conduct of theassay over time. For most test methods, the positive-control substance is tested concurrently
with the test substance and the vehicle/solvent control. However, for some in vivo test methods,
periodic studies using a positive-control substance is considered adequate by the OECD.
Positive predictivity: The proportion of correct positive responses among substances testing
positive by a test method (see two-by-two table). It is one indicator of test method accuracy.
The definitions in this glossary are restricted to their use with respect to the rLLNA and the traditional LLNA.

* Definition used by the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM 2003).
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ICCVAM Test Method Evaluation Report: Appendix D

Positive predictivity is a function of the sensitivity of the test method and the prevalence of
positives among the substances tested.*
Prevalence: The proportion of positives in the population of substances tested (see two-by-twotable).*
Protocol: The precise, step-by-step description of a test, including the listing of all necessary
reagents, criteria, and procedures for the evaluation of the test data.*
Quality assurance: A management process by which adherence to laboratory testing
standards, requirements, and record keeping procedures is assessed independently by
individuals other than those performing the testing.*
Reduction alternative: A new or modified test method that reduces the number of animals required.*
Reference test method: The accepted in vivo test method used for regulatory purposes toevaluate the potential of a test substance to be hazardous to the species of interest.*
Refinement alternative: A new or modified test method that refines procedures to lessen or
eliminate pain or distress in animals or enhances animal well-being.*
Relevance: The extent to which a test method correctly predicts or measures the biologicaleffect of interest in humans or another species of interest. Relevance incorporates considerationof the accuracy or concordance of a test method.*
Reliability: A measure of the degree to which a test method can be performed reproducibly
within and among laboratories over time. It is assessed by calculating intra- and interlaboratory
reproducibility and intralaboratory repeatability.*
Replacement alternative: A new or modified test method that replaces animals with nonanimal systems or one animal species with a phylogenetically lower one (e.g., a mammal with aninvertebrate).*Reproducibility: The consistency of individual test results obtained in a single laboratory(intralaboratory reproducibility) or in different laboratories (interlaboratory reproducibility)
using the same protocol and test substances (see intra- and interlaboratory reproducibility).*rLLNA (reduced LLNA): Also called the cut-down LLNA, limit test LLNA, or LLNA limit
dose procedure. A variant of the traditional LLNA that employs a single high dose level of thetest substance rather than multiple dose levels to determine its skin sensitization potential.
Sensitivity: The proportion of all positive substances that are classified correctly as positive ina test method. It is a measure of test method accuracy (see two-by-two table).*Skin sensitizer: A substance that induces an allergic response following skin contact (U.N. 2005).
Specificity: The proportion of all negative substances that are classified correctly as negative ina test method. It is a measure of test method accuracy (see two-by-two table).*Stimulation index (SI): A value calculated for the local lymph node assay to assess the skinsensitization potential of a test substance. The value is calculated as the ratio of radioactivity
The definitions in this glossary are restricted to their uses with respect to the rLLNA and the traditional LLNA.

* Definition used by ICCVAM (ICCVAM 2003).
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ICCVAM Test Method Evaluation Report: Appendix D

incorporated into the auricular lymph nodes of a group of treated mice to the radioactivity
incorporated into the corresponding lymph nodes of a group of vehicle-control mice. For the
traditional LLNA and the rLLNA, an SI ≥ 3 classifies a substance as a skin sensitizer.
Test: The experimental system used; used interchangeably with test method and assay.*
Test method: A process or procedure used to obtain information on the characteristics of a
substance or agent. Toxicological test methods generate information regarding the ability of a
substance or agent to produce a specified biological effect under specified conditions. Used
interchangeably with test and assay. See also validated test method and reference test.*
Transferability: The ability of a test method or procedure to be accurately and reliably
performed in different, competent laboratories.*
Two-by-two table: The two-by-two table can be used for calculating accuracy (concordance)([c+d]/[a+b+c+d]), negative predictivity (d/[c+d]), positive predictivity (a/[a+b]), prevalence([a+c]/[a+b+c+d]), sensitivity (a/[a+c]), specificity (d/[b+d]), false positive rate (b/[b+d]), andfalse negative rate (c/[a+c]).*New Test Outcome

Reference Test
Outcome

Positive

Positivea

Negative
c

Total
a+c

Negative

b

d

b+d

Total

a+b

c+d

a+b+c+d

Validated test method: An accepted test method for which validation studies have been
completed to determine the relevance and reliability of this method for a specific proposed use.*
Validation: The process by which the reliability and relevance of a procedure are established
for a specific purpose.*
Vehicle control: An untreated sample containing all components of a test system, including thevehicle that is processed with the test substance-treated and other control samples to establish thebaseline response for the samples treated with the test substance dissolved in the same vehicle.
Weight-of-evidence (process): The strengths and weaknesses of a collection of information
are used as the basis for a conclusion that may not be evident from the individual data.

The definitions in this glossary are restricted to their use with respect to the rLLNA and the traditional LLNA.

* Definition used by the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM 2003).
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Annex IECVAM Scientific Advisory Committee Statement on the Validity of the rLLNA

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ICCVAM Test Method Evaluation Report: Appendix D, Annex I
EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Instltute tor Health and Consume r Protection
European Centre for the Validation of Attemative Methods (ECVAM)ESAC Statement on the Reduced LocaJ Lymph Node Assay (rLLNA)th

At its 26 Meeting, held on 26-27 April 2007 at the European Centre for the Validationof Alternative Methods (BCVAM), Ispra, Italy, the non-Commission members of the
BCVAM Scientific Advisory Committee (ESAC)1 unanimously endorsed the following
statement
Skin sensitisation is an important toxicological endpoint with respect to human safety.
Having reviewed the final report of the independent peer review evaluation co-ordinatedby ICCVAM and NlCEATM2 , the report by the EMEA3, the pre-report of the
sccNFP", and evidence made available since the original submissions to ICCVAM, in
March 2000 the 14th meeting ofESAC stated:"Following a review of the scientific report and pUblications on the local lymph
node assay (LLNA) it is concluded that the LLNA is a scientifically validated
test which can be used to assess the skin sensitisation potential of chemicals. The
LLNA should be the preferred method, as it uses fewer animals and causes lesspain and distress than the conventional guinea-pig methods. In some instancesand for scientific reasons, the conventional methods can be used."
Since its acceptance for regulatory purposes, the LLNA has proved suitable for the
purposes of satisfying a range of ED and other regulatory requirements s.
The developers of the LLNA have now undertaken a retrospective analysis of publisheddata obtained with the LLNA 6.
They conclude that within a tiered testing strategy in the context of REACH a "reduced"
version of the LLNA (rLLNA), using only a negative control group and the equivalentof the high-dose group from the full LLNA, can be used as a screening test to
distinguish between sensitisers and non-sensitisers.
ESAC established a peer review panel to evaluate if there was the potential to minimise
animal use by employing the rLLNA as a screening test as part of a tiered-testing
strategy for chemicals.
Mindful that with the rLLNA:•
When compared with the full LLNA the rLLNA cannot and will not result in
additional false positives.
•
When compared with the full LLNA the rLLNA may produce a few false
negati ves (3: 169 in the reference document, reducing to 2: 169 when negative
results obtained with concentrations of <10% are considered invalid)•
The test results provided by the rLLNA do not allow the determination of thepotency of a sensitising chemical.
ESAC states that the peer reviewed and pubJished information is of a quality and nature
to support the use of the rLLNA within tiered-testing strategies to reliably distinguishbetween chemicals that are skin sensitisers and non-sensitisers, and that animal use canbe minimised providing:

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ICCVAM Test Method Evaluation Report: Appendix D, Annex I
EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE

Instltuie lor Health and Consu mer Protection
European Centre for the Validation of Akematlva Methods (ECVAM)

•

•
•

•

The concentration used to evaluate sensitisation potential is the maximum
consistent with solubility and the need to avoid local and other systemic adverse
effects, and that this principle rather than strict adherence to the specificrecommended absolute concentrations as in OECD TO 429 should be used.
Negative test results associated with testing using concentrations of less than
10%, should undergo further evaluation.
Positive and negative (vehicle) control groups are used, as appropriate, perOECD TG 429.
The full LLNA should be perfonned when it is known that an assessment ofsensitisation potency is required.

ESAC recommends that further work should be undertaken to detennine if the 10%
concentration threshold referenced above is optimal.

Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra

27 April 2007

2

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ICCVAM Test Method Evaluation Report: Appendix D, Annex I

I.

The ESAC was established by the European Commission, and is composed ofnominees from the ED Members States, industry, academia and animal welfare,together with representatives of the relevant Commission services.
This statement was endorsed by the following members of the ESAC:
Ms Sonja Beken (Belgium)
Ms Dagmar Hrova (Czech Republic)
Mr Tonu PUssa (Estonia)
Mr Lionel Larue (France)
Mr Manfred Liebsch (Germany)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Constantin Mircioiu (Romania)
Mr Albert Breier (Slovakia)
Ms Argelia Castano (Spain)
Mr Patric Amcoff (Sweden)
Mr Jon Richmond (UK)Mr Carl Westmoreland (COLIPA)
Ms Vera Rogiers (ECOPA)
Ms Nathalie Alepee (EFPIA)
Mr Robert Combes (ESTIV)Mr Hasso Seibert (European Science Foundation)
The following Commission Services and Observer Organisations were involved in the
consultation process, but not in the endorsement process itself.
Mr Thomas Hartung (ECVAM; chairman)

Mr Jens Unge (ECVAM; ESAC secretary)
Ms EIke Anklam (Director of IHCP)
Ms Susanna Louhimies (DG Environment)
Ms Barbara Mentre (DG ENTR)
Ms Grace Patlewicz (ECB, DG JRC)Mr Christian Wimmer (DG Research)
Mr Hajime Kojima (JACYAM)
Ms Laurence Musset (OBCD)
Mr Barry Philips (Eurogroup for Animal Welfare)
Mr William Stokes (NICEATM, USA)

2. NIH (1999). The murine local lymph node assay. The results of an independent peer
review evaluation coordinated by the Interagency Coordinating Commictee on the
Validation of Altemative Methods (lCCVAM) aDd the National Toxicology Program
Center for the Evaluation of Altemative Tox icological Methods (NICEATM). NIH
Publication n.99-4494.
(http://iccvam.niehs.nih.gov/methods/immunotoxli mmunotox .htm)
3. EMEA (2000). Report from the ad-hoc expert meeting on testing for
immunohypersensitivity (11/01/2000). European Agency for the Evaluation of
Medicinal Products.

3

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ICCVAM Test Method Evaluation Report: Appendix D, Annex I

4.

SCCNFP (2000). Opinion adopted by the SCCNFP during the 11 lh plenary meeting,
17 February 2000.
(http://ec.europa.eu/healthlph_riskicommittees/sccp/docshtml/sccp_outII4_en.htm)

5. A Cockshott, P Evans, CA Ryan, GF Gerberick, CJ Betts, RJ Dearman, I Kimber and
DA Basketter (2006). The local lymph node assay in practice: a current regulatory
perspective. Human & Experimental Toxicology 25,387-394.

6. I Kimber, RJ Dearman, 0 Betts, GF Gerberick, CA Ryan, PS Kern, GY Patlewicz
and DA Basketter (2006.) The local lymph node assay and skin sensitisation: a cut­
down screen to reduce animal requirements? Contact Dermatitis 54, 181-185.

4

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ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Annex II
Physicochemical Properties of Substances Evaluated in the rLLNA

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ICCVAM Test Method Evaluation Report: Appendix D, Annex II[This Page Intentionally Left Blank]

D-72

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source(16-beta)-21-(Acetyloxy)-17hydroxy-16-methylpregna1,4,9(11)-triene-3,20-dione

17,21-Dihydroxy16beta-methylpregna1,4,9(11)-triene-3,20dione 21-acetate

910-99-6

398.50

3.56

Solid

Pharmaceuticalchemicals

GSK(1r)-1,2,3,4-Tetrahydro-6,7dimethoxy-2-methyl-1-[(3,4,5trimethoxyphenyl)methyl]isoqu
ino-line [r-(r*,r*)]-2,3bis(benzoyloxy)-butanedioate
(1:1)

–

104832-01-1

745.79

3.16

Solid

Pharmaceuticalchemicals

GSK

–

–

–

–

–

–

LLNA/EC3
Validation
Study

–

190965-45-8

365.23

5.26

Solid

Pharmaceuticalchemicals

GSK–

773059-57-7

250.21

0.83

Solid

Pharmaceuticalchemicals

GSK–

148776-18-5

502.40

7.51

Solid

Pharmaceuticalchemicals

GSK–

414910-30-8

712.73

5.63

Solid

Pharmaceuticalchemicals

GSK

Denagliptin

483369-58-0

373.38

2.31

Solid

Pharmaceuticalchemicals

GSK(1R,4R)-4-Isopropenyl-1methyl-2methylenecyclohexane(2-Bromo-5-propoxyphenyl)(2hydroxy-4-methoxyphenyl)methadone(2e)-2-[(2-Formyl-4hydroxyphenyl)-methylidene]butanedioic acid
(2-Oxo-1-phenyl-pyrrolidin-3yl)(triphenyl)-phosphonium
bromide(2R,4S)-4-(4-Acetyl-1piperazinyl)-n-{(1r)-1-[3,5bis(trifluoro-methyl)phenyl]ethyl}-2-(4-fluoro-2methylphenyl)-n-methyl-1piperidine-carboxamide
monomethane-sulfonate(2S,4S)-1-[(2s)-2-Amino-3,3bis(4-fluorophenyl)-1oxopropyl]-4-fluoro-2-pyrrolidine carbonitrile

D-73

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source(3as,4r,5s,6s,8r,9r,9ar,10r)-6Ethenyldeca-hydro-5-hydroxy4,6,9,10-tetramethyl-1-oxo3a,9-propano-3ahcyclopentacyclo-octen-8-yl[[(3-exo)-8-methyl-8azabicyclo-[3.2.1]oct-3yl]thio]-acetate

Retapamulin

224452-66-8

517.78

5.21

Solid

Pharmaceuticalchemicals

GSK(3as,4r,5s,6s,8r,9r,9ar,10r)-6Ethenyldeca-hydro-5-hydroxy4,6,9,10-tetra-methyl-1-oxo3a,9-propano-3ahcyclopentacycloocten-8-ylhydroxyacetate

Pleuromulin

125-65-5

378.51

3.98

Solid

Pharmaceuticalchemicals

GSK(3-Endo)-8-methyl-8azabicyclo[3.2.1]-octan-3-ol

Tropine120-29-6

141.22

-0.39

Solid

Pharmaceuticalchemicals

GSK

156928-09-5

130.14

-1.19

Solid

Pharmaceuticalchemicals

GSK

313682-08-5

703.84

4.32

Solid

Pharmaceuticalchemicals

GSK

820957-38-8

494.60

2.89

Solid

Pharmaceuticalchemicals

GSK(3r,3as,6ar)-Hexahydrofuro[2,3-b]furan-3-ol(3r,3as,6ar)-Hexahydrofuro[2,3-b]furan-3-yl [(1s,2r)-3[(1,3-benzodioxol-5ylsulfonyl)(2-methylpropyl)amino]-2-hydroxy-1-[[4-[(2methyl-4thiazolyl)methoxy]phenyl]methyl]-propyl]carbamate(3R6R)-3-(2,3-Dihydro-1hinden-2-yl)-1-[(1r)-1-(2methyl-1,3-oxazol-4-yl)-2-(4morpholinyl)-2-oxoethyl]-6[(1s)-1-methylpropyl]-2,5piperazinedione–

Brecanavir

–

D-74

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source(+)-trans-p-Menth-2ene

5113-93-9

138.25

4.70

Liquid–LLNA/EC3
Validation
Study(4r,5s)-(-)-1,5-Dimethyl-4phenyl-2-imidazolidinone–

92841-65-1

190.25

1.38

Solid

Pharmaceuticalchemicals

GSK(4r,5s)-1,5-Dimethyl-3-(1-oxo2-propenyl)-4-phenyl-2imidazo-lidinone–

139109-23-2

244.30

3.33

Solid

Pharmaceuticalchemicals

GSK(4S)-1-(tert-Butoxycarbonyl)-4fluoro-l-prolinamide–

426844-22-6

232.26

0.98

Solid

Pharmaceuticalchemicals

GSK(4S)-1-(tert-Butoxycarbonyl)-4fluoro-l-proline–

203866-13-1

233.24

1.75

Solid

Pharmaceuticalchemicals

GSK(4S,5R)-1-[(1R,2R,3S)-3-(1,3Benzodioxol-5-yl)-1-(2benzyloxy-4-methoxyphenyl)1-hydroxy-6-propoxy-2indanoyl]-3,4-dimethyl-5phenyl-2-imidazolidinone–

190965-47-0

740.86

9.58

Solid

Pharmaceuticalchemicals

GSK(4Z)-2-Methyl-6-methyleneoct4-ene

–

–

–

–

–

–

(5R)-5-Isopropenyl-2-methyl1-methylene-2-cyclohexene

–

–

–

–

Liquid–(Alpha-r)-n-alpha-dimethyl3,5-bis(trifluoro-methyl–

334477-60-0

409.30

3.59

Solid

Pharmaceuticalchemicals

GSK(R,S)-3-Amino-2,3,4,5tetrahydro-n-(1-methylethyl)2,4-dioxo-n,5-diphenyl-1h-1,5benzodiazepine-1-acetamide–

184944-86-3

442.52

3.21

Solid

Pharmaceuticalchemicals

GSK(s)-(-)-1-Phenylpropyl-amine–

3789-59-1

135.21

1.93

LiquidPharmaceuticalchemicals

GSK

Substance Name
(3S,6R)-3-isopropyl-6methylcyclohexene

D-75

LLNA/EC3
Validation
Study
LLNA/EC3
Validation
StudyICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
(S)-2-(4-Fluoro-2methylphenyl)4-piperidinone(s)-alpha-hydroxybenzeneacetic acid salt[3aS(3aAlpha,4beta,5alpha,6alpha,8beta,9alpha,9abeta,10S*)]-6Ethenyldecahydro-5-hydroxy4,6,9,10-tetramethyl-1-oxo3a,9-propano-3aHcyclopentacycloocten-8-yl[(methylsulfonyl)-oxy]acetate[4-(Ethoxymethyl)-2,6dimethoxyphenyl]-boronic acid
[4S-[1(E),4Alpha,5alpha]]-1[3-[2-[4-Methoxy-2(phenylmethoxy)-benzoyl]-4propoxyphenyl]-1-oxo-2propenyl]-3,4-dimethyl-5phenyl-2-imidazoli-dinone
1-(2',3',4',5'Tetramethylphenyl)-3-(4'tetrabutylphenyl)-propane-1,3dione
1-(2',3',4',5'Tetramethylphenyl)butane-1,3dione
1-(2,3-epoxypropoxy)-2,2-bis[(2,3epoxypropoxy)methylbutane
1-(2',5' Dimethylphenyl)butane1,3-dione
1-(2',5'-diethylphenyl)butane1,3,-dione

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

414910-13-7

359.40

1.68

Solid

Pharmaceuticalchemicals

GSK–

60924-38-1

456.60

4.11

Solid

Pharmaceuticalchemicals

GSK–

591249-50-2

240.07

1.79

Solid

Pharmaceuticalchemicals

GSK–

190965-46-9

618.74

9.34

Solid

Pharmaceuticalchemicals

GSK

–

–

336.47

5.35––

Gerberick–

167998-73-4

221.32

3.14––

Gerberick

–

–

–

–

–

–

BGIA

–

56290-55-2

193.27

2.65––

Gerberick–

167998-76-7

221.32

3.14––

Gerberick

D-76

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
1-(3',4',5'Tetramethoxyphenyl)-4dimethylpentane-1,3-dione
1-(4-Ethoxy-phenyl)-2-[4(methyl-sulfonyl)phenyl]ethanone
1-(p-methoxyphenyl)-1-penten3-one
1,1,3-Trimethyl-2formyylcyclohexa-2,4-dione
1,1-Dimethylethyl [(1s)-1[bis(4-fluorophenyl)-methyl]-2[(2s,4s)-2-cyano-4-fluoro-1pyrrolidinyl]-2-oxoethyl]carbamate
1,1-Dimethylethyl [(1s)-2-[4[(2-methyl-4thiazolyl)methoxy]phenyl]-1(2s)-oxiranylethyl]-carbamate
1,1-Dimethylethyl 3-[[[[(3s)2,3,4,5-tetrahydro-1-[2-[(1methylethyl)phenylamino]-2oxoethyl]-2,4-dioxo-5-phenyl1h-1,5-benzodiazepin-3yl]amino]carbonyl]amino]
benzoate
1,2,3,5,6,7-Hexahydro-2thioxo-4h-cyclopentapyrimidin-4-one
1,2-Benzisothiazolin-3-one

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

135099-98-8

297.37

2.47––

Gerberick–346413-00-1

318.40

2.46

Solid

Pharmaceuticalchemicals

GSK

Powdery ketone104-27-8

190.24

2.65

Solid

–

GerberickSafranal

116-26-7

150.22

2.54

LiquidHydrocarbons,
CyclicGerberick–483368-24-7

473.50

4.14

Solid

Pharmaceuticalchemicals

GSK–

313680-92-1

390.51

3.32

Solid

Pharmaceuticalchemicals

GSK–

305366-94-3

661.76

6.74

Solid

Pharmaceuticalchemicals

GSK–

35563-27-0

168.22

0.65

Solid

Pharmaceuticalchemicals

GSK

Solid

SulfurCompounds;
Heterocyclic
Compounds

GerberickProxan; Proxel active

2634-33-5

151.19D-77

1.42

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

cis-1,2Cyclohexanediamine

1436-59-5

114.190.09

LiquidAmines

BGIA

1,2-Dibromo-2,4dicyanobutane–

35691-65-7

265.93

1.91

Solid

NitrilesGerberick

1,3-Benzodioxazole-5sulphonyl chloride–

115010-10-1

220.63

0.14

Solid

Pharmaceuticalchemicals–
Digylidyl hexanediol;
1,6-Hexanediol
diglycidyl ether

123-31-9

110.11

1.17

Solid

Phenols

Gerberick16096-31-4

230.300.84

LiquidEthers

BGIA

Substance Name
1,2-Diaminocyclohexane

1,4-dihydroquinone
1,6-Bis(2,3epoxypropoxy)hexane

Data SourceGSK

1-[3-(Cyclopentyl-oxy)-4methoxy-phenyl]-4oxocyclohexane carbonitrile–

152630-47-2

313.40

2.23

Solid

Pharmaceuticalchemicals

GSK

1-[5-[(4-Fluorophenyl)methyl]2-furanyl]ethanone–

280571-34-8

218.23

2.97

Solid

Pharmaceuticalchemicals

GSK

3344-77-2

265.23

3.40

Solid

Alcohols

Gerberick

12-Bromododecanoic acid

12-Bromolauryl
alcohol
12-Bromolauric acid73367-80-3

279.21

3.02

Solid

LipidsGerberick

14-Hydroxynor-morphinone–

84116-46-1

285.30NA

Solid

Pharmaceuticalchemicals

GSK

1-Bromobutane–

109-65-9

137.02

1.82

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromodocosane–

6938-66-5

389.51

6.25

Solid

Hydrocarbons,
Halogenated

Gerberick143-15-7

249.23

3.79

LiquidHydrocarbons,
Halogenated

Gerberick4276-49-7

361.45

5.76

Solid

Hydrocarbons,
Halogenated

Gerberick

12-Bromo-1-dodecanol

1-Bromododecane
1-Bromoeicosane

Lauryl bromide–

D-78

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

1-Bromoheptadecane–

3508-00-7

319.36

5.02

1-Bromohexadecane

n-Hexadecyl bromide;
Palmityl bromide;
Cetyl bromide

112-82-3

305.34

1-Bromohexane

n-Hexyl bromide

111-25-1

Substance Name

Chemical Class3

Data Source

Solid

Hydrocarbons,
Halogenated

Gerberick

4.77

LiquidHydrocarbons,
Halogenated

Gerberick165.07

2.31

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromononane–693-58-3

207.15

3.05

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromooctadecane–

112-89-0

333.39

5.26

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromopentadecane

n-Pentadecyl bromide

629-72-1

291.31

4.53

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromotetradecane–112-71-0277.28

4.28

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromotridecane–

765-09-3

263.26

4.03

LiquidHydrocarbons,
Halogenated

Gerberick

1-Bromoundecane–693-67-4

235.203.54

LiquidHydrocarbons,
Halogenated

Gerberick

1-Butanol–

71-36-3

74.12

1.06

LiquidDinitrochlorobenzene

97-00-7

202.55

-0.06

Solid

–

4860-03-1

260.894.65

Liquid1-Chloro-2-dinitrobenzene

1-Chlorohexadecane

D-79

Alcohols; Lipids
Hydrocarbon,
Halogenated;
Nitro
Compounds;
Hydrocarbons,
CyclicHydrocarbons,
Halogenated

Gerberick

NTP,
GerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

1086-00-6

250.72

4.89

Solid

Hydrocarbons,
Cyclic; Polycyclic
Compounds

Gerberick

1-Chloromethylpyrene
1-Chlorononane

n-Nonyl chloride

2473-01-0

162.702.93

LiquidHydrocarbons,
Halogenated

Gerberick

1-Chlorooctadecane

Stearyl chloride

3386-33-2

288.94

5.14

LiquidHydrocarbons,
Halogenated

Gerberick

1-Chlorotetradecane

Myristyl chloride

2425-54-9

232.83

4.16

LiquidHydrocarbons,
Halogenated

Gerberick4292-19-7

296.24

3.91

LiquidHydrocarbons,
Halogenated

Gerberick544-77-4

352.34

4.89

Liquid/Solid

Hydrocarbons,
Halogenated

Gerberick638-45-9212.07

2.43

LiquidHydrocarbons,
Halogenated

Gerberick4282-42-2

254.15

3.17

LiquidHydrocarbons,
Halogenated

Gerberick629-93-6

380.39

5.39

Solid

Hydrocarbons,
Halogenated

Gerberick

19218-94-1

324.294.40–

Hydrocarbons,
Halogenated

Gerberick70-25-7147.09

-2.13

Solid

Amidines; Nitroso
Compounds

Gerberick

1-Iododecane
1-Iodohexadecane–
Palmityl iodide;Hexadecyl iodide

1-Iodohexane
1-Iodononane–
n-Nonyl iodide

1-Iodooctadecane–

1-Iodotetradecane

Myristyl iodide, nTetradecyl iodide

1-Methyl-3nitronitrosoguanidine

MNNG

1-Napthol–

90-15-3

144.17

2.54

Solid

1-Phenyl-1,2-propanedione–579-07-7

148.16

1.91

LiquidD-80

Hydrocarbons,
CyclicKetones;
Heterocyclic
Compounds

GerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

6668-24-2

179.24

2.40––

Gerberick–55846-68-1

221.32

3.14––

Gerberick–

18048-64-1

202.26

2.28

Solid

Pharmaceuticalchemicals

GSK

HC Red No. 3

2871-01-4

197.19

0.12

Solid

Amines

GW 406381

221148-46-5

393.47

3.86

Solid

Pharmaceuticalchemicals

GSK–

3197-25-9

231.25

1.57

Solid

Pharmaceuticalchemicals

GSK

2-(4-tert-Amylcyclohexyl)
acetaldehyde

QRM 2113620159-84-4

196.33

3.28––

Gerberick2-(Benzyl)tert-butyl)amino)-1(alpha,4-dihydroxy-mtolyl)ethanealpha-((Benzyl-tertbutylamino)methyl)m-xylene4,alpha,alpha'-triol

24085-03-8

329.44

2.51

Solid

Pharmaceuticalchemicals

GSK

2,2,6,6-Tetramethyl-heptane3,5-dione–1118-71-4

186.302.40

LiquidKetonesGerberick
LLNA/EC3
Validation
Study

GSK

Substance Name
1-Phenyl-2-methylbutane-1,3dione
1-Phenyloctane-1,3-dione
2-(3,4-Dimethyl-phenyl)-5methyl-2,4-dihydropyrazol-3one
2-(4-Amino-2nitrophenylamino)-ethanol
2-(4-Ethoxyphenyl)-3-[4(methyl-sulfonyl)phenyl]pyrazolo[1,5-b]-pyridazine
2-(4-Oxopentyl)-1h-isoindole1,3(2h)-dione

2,2-bis-[4-(2-hydroxy-3methacryloxypropoxy)phenyl)]propane
2,3,4,5-Tetrahydro-n-(1methylethyl)-2,4-dioxo-n,5diphenyl-3-[(phenylmethoxy)imino]-1h-1,5-benzodiazepine1-acetamide
2,3-Butanedione

Gerberick

1565-94-2

512.59

4.94

LiquidCarboxylic Acids;
Phenols;
Macromolecular
Substances–

305366-97-6

546.63

7.64

Solid

Pharmaceuticalchemicals

Erythritol anhydride;
Butadiene diepoxide

431-03-8

86.09

0.68

LiquidKetones

Bis-GMA

D-81

GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

444731-72-0

161.21

1.01

Solid

Pharmaceuticalchemicals

GSK

108-77-0

184.41

0.78

Solid

Heterocyclic
Compounds

GerberickAmines

LLNA/EC3
ValidationStudy

2,3-Dimethyl-2h-indazol-6amine
2,4,6-Trichloro-1,3,5-triazine

Cyanuric chloride

2,4-DiaminophenoxyethanolHCl–66422-95-5

168.19-1.28–

2,4-Dichloro-pyrimidine–

3934-20-1

148.98

1.17

Solid

2,4-Heptadienal–

5910-85-0

110.16

1.80–

2,4-Hexadienal–

142-83-6

96.13

1.37

Liquid

2,5-Diaminotoluene–

95-70-5

122.08(sulfate
156.25)

1.42

2,6-Dimethoxy-4-methyl-5-[3(trifluoromethyl)-phenoxy]-8quinolinamine–

106635-86-3

378.352,6-Dimethoxy-4-methyl-8nitro-5-[3-(trifluoromethyl)phenoxy]quinoline–

189746-15-4

2.4-Dinitrobenzene sulfonicacid
2-[(Benzyloxy)-imino]malonic
acid

DNBS
–

Pharmaceuticalchemicals

GSK

Aldehydes;
Hydrocarbons,
Acyclic
Aldehydes;
Hydrocarbons,
Acyclic

LLNA/EC3
Validation
Study

Solid
(sulfate)

AminesGerberick

5.73

Solid

Pharmaceuticalchemicals

GSK

408.34

6.09

Solid

Pharmaceuticalchemicals

GSK

89-02-1

248.17-1.53

LiquidHydrocarbons,
Cyclic; Sulfur
Compounds

Ryan

305366-96-5

223.19

1.36

Solid

Pharmaceuticalchemicals

GSK

D-82

GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
2-[1-(4-Bromophenyl)-1phenylethoxy]-n,ndimethylethanaminehydrochloride

Synonyms

Bromadryl

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

13977-28-1

384.75

4.71

Solid

Pharmaceuticalchemicals

GSK

2-Acetylcyclohexanone–

874-23-7

143.21

1.66

Solid

Hydrocarbons,
Cyclic; Ketones

Gerberick

2-Amino-6-chloro-4nitrophenol–

6358-09-4

188.57

0.26

Solid

AminesGerberick

2-Amino-di-phenylamine–

534-85-0

184.24

2.39

Solid

Pharmaceuticalchemicals

GSK

2-Aminoethyl-methylsulfone–

49773-20-8

159.63

-1.64

Solid

Pharmaceuticalchemicals

GSK

95-55-6

109.13

1.17

Solid

Amines; Phenols

Gerberick24085-08-3

363.89

3.34

Solid

Pharmaceuticalchemicals

GSK

2-Aminophenol
2-Benzyl-tert-butylamino-3'hydroxymethyl-4'hydroxyaceto-phenonehydrochlorideo-Aminophenol; 2Hydroxyaniline
Ethanone, 2-((1,1dimethylethyl)(phenymethyl)amino)1-(4-hydroxy-3(hydroxymethyl)
phenyl)-,hydrochloride

2-Bromo-5-hydroxybenzaldehyde–

2973-80-0

201.02

2.45

Solid

Pharmaceuticalchemicals

GSK

2-Bromo-5-propoxybenzoic
acid–

190965-43-6

259.10

3.33

Solid

Pharmaceuticalchemicals

GSK

2-Bromotetradecanoic acid

2-Bromomyristic acid

10520-81-7

307.27

3.51

Solid

Carboxylic Acids

Gerberick–

6340-55-2

207.66

3.57

Solid

Pharmaceuticalchemicals

GSK

2-Chloro-6-methoxy-4methylquinoline
2-Hydroxyethyl acrylate

HEA818-61-1

116.120.54

LiquidCarboxylic Acids

Gerberick

2-Hydroxypropyl methacrylate

2-HPMA

923-26-2

144.17

1.03

Solid

Carboxylic Acids

Gerberick

D-83

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3
Heterocyclic
Compounds
Phenols
SulfurCompounds;
Heterocyclic
Compounds

Data Source

2-Mercaptobenzothiazole

Captax149-30-4

167.25

1.80

Solid

2-Methoxy-4-methylphenol

Cresol

93-51-6

138.16

1.66

Liquid–2682-20-4

115.15

0.68

Solid

Product 240

525-76-8

161.16

1.52

Solid

–

Gerberick–55302-96-0

167.21

1.32––

Gerberick–

110-41-8

184.32

3.03

LiquidAldehydes

Gerberick

2-Morpholinoethyl isocyanide–

443882-99-3

281.67

4.55

Solid

Pharmaceuticalchemicals

GSK

2-Nitro-4-(propylthio)benzenamine–

54393-89-4

212.27

3.45

LiquidPharmaceuticalchemicals

GSK–

5307-14-2

153.13

0.01

Solid

Lyral

31906-04-4

210.32

2.89

Liquid

3,5-Dichloro-N-(3,4dichlorophenyl)-2hydroxybenzamide;TCS

1154-59-2

351.01

3.49

3,4-Dichloroanilinehydrochloride–

95-76-1

162.02

3,4-DihydrocoumarinHydroxydihydrocinnamic acid lactone

119-84-6

3,4-epoxyclohexylethylcyclopolymethylsiloxane

Tet-sil–

2-Methyl-2H-isothiazol-3-one
2-Methyl-4H,3,1-benzoxazin-4one
2-Methyl-5hydroxyethylaminophenol
2-Methylundecanal

2-Nitro-p-phenylenediamine
3 and 4-(4-Hydroxy-4methylpentyl)-3-cyclohexane1-carboxaldehyde
3, 3', 4', 5Tetrachlorosalicylanilide

GerberickGerberickGerberickAmines
Aldehydes;
Hydrocarbons,
CyclicGerberick

Solid

Amides; Amines

Gerberick

2.60

Solid

Pharmaceuticalchemicals

GSK

148.16

1.91

LiquidHeterocyclic
Compounds

Gerberick

–

–

–

D-84

–

Gerberick

LLNA/EC3
Validation
StudyICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
3,5,5-Trimethylhexanoyl
chloride3-[(2r)-3-[[2-(2,3-Dihydro-1hinden-2-yl)-1,1-dimethylethyl]amino]-2hydroxypropoxy]-4,5-difluorobenzene propanoic acid3'-[(2z)-[1-(3,4Dimethylphenyl)-1,5-dihydro3-methyl)-5-oxo-4h-pyrazol-4ylidene]hydrazino]-2'-hydroxy[1,1'-biphenyl]-3-carboxylicacid, compound with 2aminoethanol (2:1)
3-[4-[(6-Bromohexyl)oxy]butyl]benzene-sulfonamide
3-Aminomethyl-3,5,5trimethylcyclohexylamine
3-Aminophenol

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

36727-29-4

176.68

2.54

LiquidCarboxylic Acids

Gerberick–753449-67-1

447.53

2.13

Solid

Pharmaceuticalchemicals

GSK

Eltrombopag Olamine

496775-62-3

564.65

5.25

Solid

Pharmaceuticalchemicals

GSK–

452342-04-0

392.36

3.48

Solid

Pharmaceuticalchemicals

GSK

2855-13-2

170.301.90

LiquidAmines

BGIA

591-27-5

109.13

1.17

Solid

Amines; Phenols

Gerberick

5-Amino-1,3,3trimethylcyclohexanemethylamine; IPDA;
Isophorone diamine
m-Aminophenol; 3Hydroxyaniline

3-Bromomethyl-5, 5'-dimethyldihydro-2(3H)-furanone–

154750-20-6

207.07

1.79––

Gerberick

3-Chloro-4-fluorobenzoyl
chloride–

65055-17-6

193.01

2.42

Solid

Pharmaceuticalchemicals

GSK

109-55-7

102.180.92

LiquidAmines170928-69-5

248.32

3.00–

3-Dimethylaminopropylamine
3-Ethoxy-1-(2',3',4',5'tetramethylphenyl)propane-1,3dione

N,N-Dimethyl-1,3propanediamine;
DMAPA
–

D-85

Gerberick–GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

181633-36-3

188.21

1.80

Solid

Pharmaceuticalchemicals

GSK

3-Fluoro-5-(3pyridinyl)benzen-amine
3-Hydroxy-2-phenyl-4quinolinecarboxylic acid

Oxycinchophen

485-89-2

265.27

4.95

Solid

Pharmaceuticalchemicals

GSK

3-Hydroxy-4methoxybenzaldehyde

Isovanillin

621-59-0

152.15

1.28

Solid

Pharmaceuticalchemicals

GSK

3-Methyl-4-phenyl-1,2,5thiadiazole-1,1-dioxide

MPT3775-21-1

208.24

1.14––

Gerberick

3-Methyleugenol–

186743-26-0

178.23

2.40–

Ethers; Phenols

Gerberick

3-Methylisoeugenol–

186743-29-3

178.23

2.40–

Carboxylic Acids

Gerberick

3-Phenylenediaminem-Phenylenediamine

108-45-2

108.14

1.17

Solid

AminesGerberick

3-Propoxybenzoic acid–

190965-42-5

180.21

3.08

Solid

Pharmaceuticalchemicals

GSK

3-Propylidenphthalide–

17369-59-4

174.202.40

Liquid–Gerberick

4-(Bromomethyl)-benzoic acid
ethyl ester–

26496-94-6

243.103.42–

Pharmaceuticalchemicals

GSK

25646-71-3

836.97

-2.12

Solid

Amides; Sulfur
Compounds

Gerberick

90035-34-0

250.22

4.31

Solid

Pharmaceuticalchemicals

GSK

362-06-7

219.18

2.52––

Gerberick35578-47-3

368.02

5.34

Solid

–

LLNA/EC3
Validation
Study

4-(N-Ethyl-N-2-methansulfamido-ethyl)-2-methyl-1,4,phenylenediamine

CD-4 developer

4'-(Trifluoro-methyl)-[1,1'biphenyl]-4-carboxaldehyde
4,4,4-Trifluoro-1-phenylbutane1,3-dione
4,4-Dibromobenzil

–
BFA
–

D-86

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
4-[4-[[(3R)-1-Butyl-3-[(r)cyclohexyl-hydroxymethyl]2,5-dioxo-1,4,9triazaspiro[5.5]-undec-9yl]methyl]phenoxy]benzoicacid
4-Allylanisole

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Aplaviroc

461443-59-4

577.73

3.92

Solid

Pharmaceuticalchemicals

GSK

Estragole

140-67-0

148.202.54

LiquidEthers; Phenols

GerberickSynonyms

4-Amino-3-nitrophenyl
thiocyanate–

54029-45-7

195.20

2.21

Solid

Pharmaceuticalchemicals

GSK

4-Bromo-1-phthalimidopentane–

59353-62-7

296.17

3.48

LiquidPharmaceuticalchemicals

GSK

4-Chloro-6-iodoquinazoline–

98556-31-1

290.49

2.96

Solid

Pharmaceuticalchemicals

GSK

4-Fluoro-2-pyrrolidinecarboxamide–

748165-40-4

132.14

-1.01

Solid

Pharmaceuticalchemicals

GSK

4-Hydroxybenzoic acid–

99-96-7

138.12

1.03

Solid

Phenols;
Carboxylic Acids

Gerberick

4-Iodo-1-phthalimido-pentane–

63460-47-9

343.17

3.87

Solid

Pharmaceuticalchemicals

GSK

4-Isopropyl-1methylenecyclohexane

–

–

–

–

–

–

–
Metol; Paramethylaminophenol sulfate

100-06-1

150.18

1.91

Solid

Ethers

LLNA/EC3
Validation
Study
Gerberick

55-55-0344.38

-0.13

Solid

Amines; Phenols

Gerberick

4'-Methoxyacetophenone
4-Methylaminophenol sulfate
4-Nitrobenzyl bromide

1-(Bromomethyl)-4nitrobenzene

100-11-8

216.03

1.40

Solid

Hydrocarbons,
Cyclic; Nitro
Compounds

Gerberick

4-Phenylenediamine

p-PDA, pPhenylenediamine

106-50-3

108.14

1.17

Solid

AminesGerberick

D-87

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Gerberick29043-97-8

126.16

1.42–

Heterocyclic
Compounds;
SulfurCompounds;
Lactones

444731-52-6

437.53

3.65

Solid

Pharmaceuticalchemicals

GSK–

69733-09-7

186.23

-0.07

Solid

Pharmaceuticalchemicals

GSK

2-Hydroxy-p-toluidine

2835-95-2

123.15

0.79

Solid

–

NTP

5-Chloro-2,6-dimethoxy-4methyl-8-nitroquinoline–

189746-21-2

282.69

3.95

Solid

Pharmaceuticalchemicals

GSK

5-Chloro-2,6-dimethoxy-4methylquinoline–

189746-19-8

237.69

4.13

Solid

Pharmaceuticalchemicals

GSK

5'-Chloro-2'-hydroxy-3'-nitro[1,1'-biphenyl]-3-carboxylicacid–376592-58-4

293.67

4.03

Solid

Pharmaceuticalchemicals

GSK

Gerberick

5,5-Dimethyl-3methylenedihydro-2(3H)furanone
5-[[4-[(2,3-Dimethyl-2hindazol-6-yl)-methylamino]-2pyrimidinyl]amino]-2methylbenzene-sulfonamide
5-Amino-2-methylbenzenesulfonamide
5-Amino-O-Cresol–

Pazopanib

5-Chloro-2-methyl-4isothiazolin-3-one–

26172-55-4

149.600.92

LiquidSulfurCompounds;
Heterocyclic
Compounds

5-Chloro-6-methoxy-4-methyl8-nitro-2(1h)quinolinone–

189746-23-4

268.66

1.99

Solid

Pharmaceuticalchemicals

GSK

5-Methoxy-2-nitro-4(trifluoromethyl)benzene
acetonitrile–

178896-77-0

260.17

2.42

Solid

Pharmaceuticalchemicals

GSK

5-Methoxy-6-(trifluoromethyl)2,3-dihydro-1h-indole–

178896-79-2

217.19

3.25

Solid

Pharmaceuticalchemicals

GSK

13706-86-0

128.17

1.42

Liquid–Gerberick

5-Methyl-2,3-hexanedione

Acetyl isovaleryl

D-88

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

5-Methyl-2-phenyl-2-hexenal–

21834-92-4

188.27

3.77

Liquid–5-Methyleugenol–

186743-25-9

178.23

2.40–

Ethers; Phenols

6-(Diethylamino)-1-hexanol–

06947-12-2

173.301.73

LiquidPharmaceuticalchemicals

GSK

6-(Trifluoro-methyl)-2,3dihydro-5-methyl-1h-indole,hydrochloride–

280121-24-6

237.65

3.69

Solid

Pharmaceuticalchemicals

GSK

6-[(2-Methyl-3-pyridinyl)oxy]3-pyridinamine–

181633-42-1

201.23

1.42

Solid

Pharmaceuticalchemicals

GSK

6-Chloro-1-hexanol–

2009-83-8

136.62

1.59

LiquidPharmaceuticalchemicals

GSK

6-Diethylaminohexyl bromidehydrobromide–

64993-14-2

317.11

3.57

Solid

Pharmaceuticalchemicals

GSK

6-Iodo-quinazolin-4-ol–

16064-08-7

272.05

1.49

Solid

Pharmaceuticalchemicals

GSK

6-Methoxy-4-methyl-2(1H)quinolinone–

5342-23-4

189.22

1.51

Solid

Pharmaceuticalchemicals

GSK

92-48-8

160.17

2.15

Solid

186743-24-8

178.232.40–13041-12-8

178.23

2.40–

57-97-6

256.34

5.39

Solid

6-Methylcoumarin
6-Methyleugenol
6-Methylisoeugenol
7,12Dimethylbenz[a]anthracene

6-MC
–
–
DMBA; 9,10Dimethyl-1,2benzanthracene

D-89

Heterocyclic
Compounds
Ethers; Phenols
Carboxylic Acids
Hydrocarbons,
Cyclic; Polycyclic
Compounds

Data Source
LLNA/EC3
Validation
Study
GerberickGerberickGerberickGerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source7-[(4z)-3-(Aminomethyl)-4(methoxyimino)-1pyrrolidinyl]-1-cyclopropyl-6fluoro-1,4-dihydro-4-oxo-1,8naphthyridine-3-carboxylicacid, monomethane-sulfonate

Gemifloxacin
mesylate

210353-53-0

485.50

-1.25

Solid

Pharmaceuticalchemicals

GSK

7-Bromotetradecane

7-Tetradecyl bromide;
7-Myristyl bromide

74036-97-8

277.294.28–

Hydrocarbons,
Halogenated

Gerberick8-[(4-Phthalimido-1methylbutyl)amino]-2,6dimethoxy-4-methyl-5-(3trifluoromethylphenoxy)quinoline–

106635-87-4

593.61

8.70

Solid

Pharmaceuticalchemicals

GSK

8-Amino-6-methoxy-4methylquinoline–

57514-21-3

188.23

2.30

Solid

Pharmaceuticalchemicals

GSK

8-Chloro-3-pentyl-3,7-dihydro1h-purine-2,6-dione–

862892-90-8

256.69

2.27

Solid

Pharmaceuticalchemicals

GSK

8-Hydroxy-5-[(1r)-1-hydroxy2-[[2-[4-[(6-methoxy[1,1'biphenyl]-3-yl)amino]phenyl]ethyl]amino]ethyl]-2(1h)quinolinone–

530084-87-8

521.62

3.98

Solid

Pharmaceuticalchemicals

GSK

A SC600

–

–

–

–

–

514-10-3

302.46

4.61

Solid

124-04-9

146.14

-0.02

Solid

Pharmaceuticalchemicals

GSK

–

–

–

–

Formulation

Bayer

Abietic acid
Adipic acid
AE F016382 00 TK71 A101

Sylvic acid
1,4Butanedicarboxylic
acid
–

D-90

Formulation
Hydrocarbons,
Cyclic; Polycyclic
Compounds

Bayer
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Alpha-(p-toluenesulfonyl)-4fluorobenzyliso-nitrile–

165806-95-1

289.33

2.04

Solid

Pharmaceuticalchemicals

GSKalpha-Amyl cinnamic aldehyde–

122-40-7

202.30

3.52

Solid

Aldehydes

Gerberickalpha-Butyl cinnamic aldehydealpha-Methyl cinnamic
aldehydealphaMethylphenylacetaldehyde–

7492-44-6

188.27

3.28

LiquidAldehydes

Gerberick–

101-39-3

146.19

2.54

LiquidAldehydes

Gerberick

93-53-8

134.182.29

LiquidAldehydes

Gerberick
LLNA/EC3
ValidationStudyalpha-Phellandrenealpha-Terpinene
Aniline2-Phenyl
propionaldehyde
Menthadiene;
Dihydro-p-cymene; pMentha-1,5-diene; 2methyl-5-(1methylethyl)- 1,3cyclohexadiene
1-Isopropyl-4-methyl1,3-cyclohexadiene;
p-Mentha-1,3-diene
Benzenamine

99-83-2

136.23

4.62

Solid

Hydrocarbons,
Cyclic;Hydrocarbons,
Other

99-86-5

136.23

4.75

Solid

Hydrocarbons,
Other

62-53-3

93.13

1.56

LiquidAminesPharmaceuticalchemicals

LLNA/EC3
Validation
Study
Gerberick

Anthranilic acid–

118-92-3

131.14

1.21

Solid

Atrazine SC–

1912-24-9

215.68

2.82

Solid

Azithromycin–

83905-01-5

748.99

3.24

Solid

Bakelite EPR 161–

9012-45-7

–

–

Solid

Macromolecular
substances

BGIA

Bakelite EPR 162–

9012-45-7

–

–

Solid

Macromolecular
substances

BGIA

D-91

Heterocyclic
Compounds
Polycyclic
Compounds;
Carbohydrates,
Lactones

GSK
ECPA, NTPNTPICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

9012-45-7

–

–

Solid

Macromolecular
substances

BGIA

20048-27-5

318.38

0.74

Solid

Amines

LLNA/EC3
Validation
Study

8015-73-4––

LiquidLipidsLalko & Api

100-52-7

106.12

1.80

LiquidGerberick–

8001-54-5–––

Aldehydes
Onium
Compounds

Trimellitic anhydride552-30-7

192.13

0.75

Solid

–

50-32-8

252.31

5.39

Solid

94-09-7

165.19

1.52

Solid

106-51-4

108.10

1.17

Solid

Quinones

Gerberick

Benzyl benzoate–
p-Quinone; 1,4Cyclohexadienedione–

Hydrocarbons,
Cyclic; Polycyclic
Compounds
Carboxylic Acids

120-51-4

212.25

3.14

LiquidGerberick

Benzyl bromide

alpha-Bromotoluene

100-39-0

171.03

2.56

LiquidCarboxylic Acids
Hydrocarbons,
Cyclic122-57-6

146.19

2.54

Solid

KetonesGerberickHydrocarbons,
Cyclic;Hydrocarbons,
Other

LLNA/EC3
Validation
Study

Substance Name
Bakelite EPR 164

Bandrowski’s base

Basil oil
Benzaldehyde
Benzalkonium chloride
Benzene-1,3,4-tricarboxylic
anhydride
Benzo[a]pyrene
Benzocaine
Benzoquinone

Benzylidene acetone
beta-Phellandrene

1,4-Cyclohexadiene1,4-diamine; 1,4Benzenediamine;
N,N''-(2,5-diamino2,5-cyclohexadiene1,4-diylidene)bis(9CI)
Ocimum basilicum
herb oil–

4-phenyl-3-buten-2one
3-methylene-6-(1methylethyl)
cyclohexene; pMentha-1(7),2-diene555-10-2

136.23

D-92

4.70

–

Anhydrides;
Carboxylic Acids

CESIO
GerberickGerberickGerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3–

1210-39-5

208.23

2.78

Liquid–57-57-8

72.06

0.43

LiquidLactones

99-84-3

136.23

4.83––

282.38

4.37

–

–

–

–

–

–

–

–
n-butyl acrylate; nButyl propenoate; 2Propenoic acid; Butyl
ester–

1675-54-3

340.42

4.09

LiquidEthers

141-32-2

128.17

2.20

LiquidCarboxylic Acids2426-08-6

130.191.42

LiquidEthersC11-azlactone–

176665-06-8

267.41

3.24––

Gerberick

C15-azlactone–

176665-09-1

323.52

4.23––

GerberickC17-azlactone–

176665-11-5

351.58

4.72––

Gerberick

C19-azlactone––

379.63

5.21––

GerberickC4-azlactone–

176664-99-6

169.22

1.52––

GerberickC6-azlactone–

176665-02-4

197.282.01––

GerberickC9-azlactone–

176665-04-6

239.362.75–

465-29-2

166.22

2.15

Solid

Substance Namebeta-Phenylcinnamaldehyde
beta-Propiolactone

beta-Terpinene–
p-Mentha-1(7),3diene; (1methylethyl)-4methylene-1cyclohexene

bis-1,3-(2',5'-dimethylphenyl)propane-1,3-dione
Bis-3,4-epoxycyclohexyl-ethylphenyl-methylsilane
Bisphenol A-diglycidyl ether
Butyl acrylate
Butyl glycidyl ether

Camphorquinone–
Ph-SilCamphoroquinone

D-93

Hydrocarbons,
Other–
Hydrocarbons,
Other

Data Source
LLNA/EC3
Validation
Study
Gerberick
LLNA/EC3
Validation
Study
Gerberick
LLNA/EC3
Validation
Study
Gerberick
NTP,
LLNA/EC3
Validation
Study
GerberickGerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Gerberick

108-90-7

112.56

2.19

LiquidHydrocarbons,
Cyclic;Hydrocarbons,
Halogenated

1897-45-6

265.91

3.66

Solid

Nitriles104-54-1

134.18

2.29

Solid

Alcohols

LLNA/EC3
Validation
Study
Gerberick

104-55-2

132.16

2.29

LiquidAldehydes

Gerberick

Cilomilast

153259-65-5

343.23

3.20

Solid

Pharmaceuticalchemicals

2277-19-2

140.23

2.29

LiquidAldehydes5392-40-5

152.232.54/
3.45

LiquidHydrocarbons,
Other

Lalko & Api,
Gerberick

Citronella oil–
3,7-Dimethyl-2,6octadienal; GeranialNeral mixture–

8000-29-1––

LiquidLalko & Api

Clarithromycin–

81103-11-9

747.95

3.18

Solid

Clotrimazole–

23593-75-1

344.84

5.35

Solid

LipidsPolycyclic
Compounds;
Carbohydrates;
Lactones
Heterocyclic
Compounds

8000-34-8––

LiquidLipidsLalko & Api

8015-97-2––

LiquidLipidsLalko & Api

LipidsHeterocyclic
Compounds
Carboxylic Acids

Lalko & Api

Chlorobenzene

Chlorothalonil–
Tetrachloroisophthalodinitrile

Cinnamic alcohol
Cinnamic aldehyde
cis-4-Cyano-4-[3(cyclopentyloxy)-4methoxyphenyl]cyclohexanecarboxylic acidcis-6-Nonenal
CitralClove bud oil
Clove leaf oil–
Cinnamaldehyde

Clove oil; Oil ofcloves–Clove stem oil–

8015-98-3––

LiquidCoumarin–

91-64-5

146.15

1.91

Solid

Cyclamen aldehyde–

103-95-7

190.293.28

Liquid

D-94

GSK
Gerberick

NTP
GerberickGerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
Cytosine

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

4-Amino-2(1H)pyrimidinone

71-30-7

120.11

-1.85

Solid

Pharmaceuticalchemicals

GSK

–

–

–

–

FormulationBayer

D EC25

–D EW 15

–

–

–

–

–

Formulation

Bayer

Dicyclohexylcarbodiimide–

538-75-0

206.33

6.83

Solid

IminesNTP

Diethyl maleate–

141-05-9

172.180.89

LiquidGerberick

Diethyl sulfate–

64-67-5

154.19-0.09

LiquidDiethylacetaldehyde–

97-96-1

100.16

1.56

LiquidCarboxylic Acids
SulfurCompounds
Aldehydes

Diethylenetriamine–

111-40-0103.170.29

LiquidAminesGerberick

84-66-2

222.24

1.87

LiquidCarboxylic Acids

Gerberick2785-87-7

166.22

2.15

LiquidEthers; Phenols

Gerberick

Diethylpthalate
Dihydroeugenol–
2-Methoxy-4propylphenol; 4Propylguaicol

GerberickGerberick

Dimethyl 4-cyano-4-(3cyclopentyloxy-4methoxyphenyl)-pimelate–

152630-48-3

403.48

3.31

Solid

Pharmaceuticalchemicals

GSK

Dimethyl carbonate–616-38-6

90.08

0.10

LiquidPharmaceuticalchemicals

GSK

Dimethyl sulfate–

77-78-1

126.13-0.59

LiquidDimethylsulfoxide–

67-68-5

78.13

0.57

LiquidDinocap EC–

39300-45-3

364.39

5.76

LiquidDipropylene triamineBis(3aminopropyl)amine

56-18-8

131.22-1.15

LiquidDodecyl methanesulfonate

Laurylmethanesulfonate

51323-71-8

264.43

2.51

–

D-95

SulfurCompounds
SulfurCompounds
Hydrocarbons,
CyclicAmines
Esters; Sulfur
Compounds

GerberickGerberick
ECPA
BGIA
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

35130-97-3

219.31

-0.11

Solid

Pharmaceuticalchemicals

GSK–

56880-11-6

211.31

1.53

LiquidPharmaceuticalchemicals

GSK–

68672-65-1

599.76

7.75

Solid

Pharmaceuticalchemicals

GSK

Ethyl 1h-1,2,4-triazole-3carboxylate–

64922-04-9

141.13

-0.02

Solid

Pharmaceuticalchemicals

GSK

Ethyl 2,6-dichloro-5-fluorobeta-oxo-3-pyridinepropanoate–

96568-04-6

280.09

2.15

Solid

Pharmaceuticalchemicals

GSK

Ethyl 4-iodobenzoate–

51934-41-9

276.08

3.76

LiquidPharmaceuticalchemicals

GSK

Ethyl benzoylacetate–

94-02-0

192.21

2.01

LiquidEsters; Ethers

Gerberick

Ethyl vanillin–

121-32-4

166.18

1.52

Solid

Aldehydes

Gerberick

Ethyl-2-(Hydroxymethyl)-1,3Propanediol Triacrylate

–

–

–

–

–

–

Ethylacrylate–

140-88-5

100.12LiquidCarboxylic Acids

LiquidCarboxylic Acids

NTP,
GerberickGerberick

Substance Name
Endo-tropine-3-mesylate
Ethyl (3-endo)-8-methyl-8azabicyclo[3.2.1]-octane-3acetate
Ethyl (z)-alpha-[[2-(1,1dimethylethoxy)-1,1-dimethyl2-oxoethoxy]imino]-2[(triphenylmethyl)amino]- 4thiazoleacetate

NTP

Ethylene glycol dimethacrylate

EGDMA

97-90-5

198.22

0.92/
1.22
1.38

Ethylenediamine free base–
Octyl acrylate; 2Ethylhexyl 2propenoate; Acrylicacid; 2-ethylhexyl
ester

107-15-3

60.10

0.19

LiquidAminesGerberick103-11-7

184.28

4.09

LiquidCarboxylic Acids

LLNA/EC3
Validation
Study

Ethylhexyl acrylate

D-96

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

97-53-0

164.202.15/
2.73

LiquidCarboxylic Acids

Lalko & Api,
Gerberick

EXP 10810 A

2-Methoxy-4-(2propenyl)phenol; 4Allyl-2methoxyphenol; 4Allylguaiacol
–

–

–

–

–

Formulation

Bayer

EXP 11120 A

–

–

–

–

–

Formulation

Bayer

F & Fo WG 50 + 25

–

–

–

–

–

Formulation

Bayer

FAR01042-00

–

–

–

–

–

Formulation

Bayer

FAR01060-00

–

–

–

–

–

Bayer

Farnesal–

502-67-0

220.36

3.77

LiquidFatty acid glutamate

–

–

–

–

–

27072-45-3

389.38

3.32

Solid

Formulation
Alcohols;
Hydrocarbons,other; Lipids–
Polycyclic
Compounds;
Isocyanates;
SulfurCompounds

50-00-0

30.03

0.33/
0.35

LiquidAldehydes

110-17-8

116.07

0.05

Solid

Carboxylic Acids

EugenolFluorescein isothiocyanate

FITC

Formaldehyde
Fumaric acid–
2-Butenedioic acid;
Butenedioic acid;
Fumarate

Furil–

492-94-4

190.15

1.38

Solid

Fx + Me EW 69

–

–

–

–
LiquidLiquidHeterocyclic
Compounds
Formulation
Hydrocarbons,
Other–

LiquidAldehydes

Geraniol

Rhodinol106-24-1

154.25

Geranium oil

Pelargonium oil

8000-46-2

–

–
2.54/
3.47
–

111-30-8

100.12

0.92

Glutaraldehyde

–

D-97

Gerberick
CESIO

Gerberick
Ryan,
Gerberick
EFfCI
GerberickBayer
Lalko & Api,
Gerberick
Lalko & Api
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
Glycerol
Glyceryl thioglycolate
GlyoxalHexaneHexyl cinnamic aldehyde
Hydroxycitronellal
Hydroxyethylethylenediamine
Imidazolidinyl urea
Iodopropynyl butylcarbamate
Isoeugenol
Isononanoyl chloride
Isopropanol

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

56-81-5

92.09

0.05

LiquidAlcohols;
Carbohydrates

Gerberick30618-84-9

166.19-1.29–

LipidsCESIO

107-22-2

58.04

0.19

LiquidAldehydes

Gerberick–

110-54-3

86.18

1.94

LiquidHydrocarbons,
Acyclic

Gerberick

HCA; alpha-Hexylcinnamaldehyde; 2(Phenylmethylene)
octanal101-86-0

216.32

3.77/
4.82

LiquidAldehydes

BGIA,
Gerberick–

107-75-5

172.26

2.15

LiquidHydrocarbons,
Other

Gerberick

111-41-1

104.15-2.13

LiquidAlcohols; Amines

BGIA

39236-46-9

388.29

-3.00

Solid

Urea

87977-28-4

281.09

2.45

Solid

Carboxylic Acids

97-54-1

164.202.15

LiquidCarboxylic Acids

Gerberick

57077-36-8

176.69

2.54–

Carboxylic Acids

Gerberick

67-63-0

60.10

0.82

LiquidAlcohols

Gerberick35695-36-4

126.16

0.51

Solid

Pharmaceuticalchemicals

Acetic acid, mercapto, monoester with
1,2,3-propanetriol
Oxaldehyde;
Ethanedial; Biformyl

N-(2-Hydroxyethyl)
ethylenediamine
Germall 115, Imidurea
3-iodo-2propynylbutylcarbamate
2-Methoxy-4propenylphenol; 4Propenylguaiacol–
Isopropyl alcohol, 2Propanol

Gerberick
LLNA/EC3
Validation
Study

Isopropyl dicyandiamide–

Isopropyl myristate–110-27-0

270.46

3.88

LiquidLipidsGerberick

Isopropyleugenol–

51474-90-9

206.292.89–

Ethers; Phenols

Gerberick

Isopropylisoeugenol–

2953-00-7

206.292.89–

EthersGerberick

D-98

GSK

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
Jasmine absolute(Grandiflorum)
Jasmine absolute (Sambac)
Kanamycin
Lactic acid
Lauryl gallate
Laurylglycerin derivitive

Lemongrass oil

Linalool alcohol

Linalool aldehyde
Linoleic acid
Linolenic acid
Litsea cubeba oil
Maleic acid

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Jasmine oil

8022-96-6––

LiquidLipidsLalko & Api

Jasmine oil

8022-96-6
59-01-8;8063-07-8––

LiquidLipidsLalko & Api

484.50

-0.90

Solid

Carbohydrates

Gerberick598-82-3

90.08

0.05

LiquidCarboxylic Acids

Gerberick

1166-52-5

338.44

3.21

Solid

Carboxylic Acids

Gerberick

–

–

–

–

–

CESIO

8007-02-1––

Liquid78-70-6

154.25

2.54/
3.38

Liquid

–

–

–

–

–

Grape seed oil
9,12,15Octadecatrienoic acid–cis-Butenedioic acid;
Toxilic acid60-33-3

280.45

7.51

LiquidLipidsGerberick,
LLNA/EC3
Validation
Study
LLNA/EC3
Validation
Study
EFfCI

463-40-1

278.43

7.30

LiquidLipids

EFfCI

68855-99-2––

Liquid–Lalko & Api

110-16-7

116.07

0.05

Solid

Carboxylic Acids

EFfCI

Synonyms–
2-Hydroxypropanoic
acid––
Citral terpenes; Indian
melissa oil; Indian oilof verbena;
Cymbopogon citratus
oil
Linalool; Linalol;
Linalyl alcohol

Lipids;Hydrocarbons,otherHydrocarbons–

Lalko & Api

m-Chloropropio-phenone

3'Chloropropiophenone

34841-35-5

168.62

2.90

Solid

Pharmaceuticalchemicals

GSK

Methyl 4(bromomethyl)benzoate–

2417-72-3

229.08

2.89

Solid

Pharmaceuticalchemicals

GSK

D-99

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source–

2417-72-3

229.08

2.89

Solid

Pharmaceuticalchemicals

GSK

Methyl propenoate;
Acrylic acid methyl
ester; Methoxycarbonylethylene

96-33-3

86.09

0.73

LiquidCarboxylic Acids

LLNA/EC3
Validation
Study

Methyl dodecanesulfonate–

2374-65-4

264.43

2.51–

Esters; Sulfur
Compounds

GerberickMethyl hexacecyl sulfonate–

4230-15-3

320.53

3.49–

Hydrocarbons,
Acyclic; Sulfur
Compounds

GerberickMethyl hexadecenesulfonate–

26452-48-2

318.52

3.49–

Ethers; Sulfur
Compounds

GerberickMethyl methanesulfonate–

66-27-3

110.13-0.20

LiquidHydrocarbons,
Acyclic; Sulfur
Compounds

GerberickMethyl pyruvate–

600-22-6

102.09-0.96

LiquidCarboxylic Acids

LLNA/EC3
Validation
Study

Methyl salicylate

Oil of wintergreen, 2Hydroxybenzoic acidmethyl ester

119-36-8

152.15

1.28/2.6
0LiquidPhenols;
Carboxylic Acids

NTP,
GerberickMethyl(2-sulfomethyl)octadecanoate––

454.67

4.89–

Ethers; Sulfur
Compounds

GerberickMethyl-2-nonynoate–

111-80-8

168.24

2.15

LiquidLipidsGerberick

Substance Name
Methyl 4(bromomethyl)benzoate
Methyl acrylate

Methyl-4-hydroxybenzoate

Methylparaben

99-76-3

152.15

1.28

Solid

Carboxylic Acids

GerberickMethylmethacrylate

Pegalan

80-62-6

100.12

1.28

LiquidCarboxylic Acids;
Macromolecular
Substances

LLNA/EC3
Validation
Study

D-100

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

m-Phenylenebis(methylamine)

1,3-xylenediamine; mXylylenediamine; 1,3Bis(aminomethyl)benzene

1477-55-0

136.190.15

LiquidHydrocarbons,
Cyclic

BGIA–

444731-74-2

273.73

3.02

Solid

Pharmaceuticalchemicals

GSK–

444731-75-3

287.75

2.88

Solid

Pharmaceuticalchemicals

GSK

15537-76-5

324.64

3.22

Solid

Pharmaceuticalchemicals

GSKn-(2-Chloro-4-pyrimidinyl)2,3-drimethyl-2h-indazol-6aminen-(2-Chloro-4-pyrimidinyl)n,2,3-trimethyl-2h-indazol-6aminen-(3,4-Dichlorophenyl)-n'-(1methylethyl)imidodicarbonimidic diamide
monohydrochloride

Chlorproguanilhydrochloride

Data Sourcen-(4-Methoxyphenyl)-3oxobutanamide–

5437-98-9

207.23

0.88

Solid

Pharmaceuticalchemicals

GSKn-[(1,1-Dimethylethoxy)carbonyl]-l-tyrosine, ethyl ester–

72594-77-5

309.37

2.66

Solid

Pharmaceuticalchemicals

GSK

152811-62-6

369.51

4.01

Solid

Pharmaceuticalchemicals

GSK–

356057-34-6

666.79

8.33

Solid

Pharmaceuticalchemicals

GSK–201677-59-0

350.22

2.51

Solid

Pharmaceuticalchemicals

GSKn-[(1-Butyl-4piperidinyl)methyl]-3,4dihydro-2h-[1,3]oxazino[3,2a]indole-10-carboxamide
n-[2-(Diethylamino)ethyl]-2[[(4-fluorophenyl)methyl]thio]-4,5,6,7-tetrahydro4-oxo-n-[[4'-(trifluoromethyl)[1,1'-biphenyl]-4-yl]methyl]1h-cyclopentapyrim-idine-1acetamide
n-[2-Benzyloxy-5-(2-bromo-1hydroxy-ethyl)-phenyl]formamide

Piboserod

D-101

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Sourcen-{[(1,1Dimethylethyl)oxy]carbonyl}4-fluoro-beta-(4-fluorophenyl)l-phenylalanine–

481055-29-2

377.39

4.31

Solid

Pharmaceuticalchemicals

GSK

n-Amino-pyridinium–

35073-04-2

223.02

0.35

Solid

Pharmaceuticalchemicals

GSK

759-73-9

117.11

-0.73

Solid

N-Ethyl-N-nitrosourea

ENU

Nitroso
Compounds; Urea
Inorganic
Chemical, Metals;
Inorganic
Chemical,Elements

GerberickNickel Sulfate–

7786-81-4

154.76

-0.17

–

Ryan

n-Isopropyl-n-phenyl-2-(2phenylamino-phenylamino)acetamide–

161455-90-9

359.48

4.91

Solid

Pharmaceuticalchemicals

GSK

N-Methyl-N-nitrosourea

MNU

684-93-5

103.08

-0.97

Solid

Nitroso
Compounds; Urea

GerberickNonanoyl chloride

Pelargonoyl chloride

764-85-2

176.68

2.54

LiquidCarboxylic Acids

Gerberick

Non-ionic surfactant 1

–

–

–

–

–

–

CESIO

Non-ionic surfactant 2

–

–

–

–

–

–

CESIO

Non-ionic surfactant 3

–

–

–

–

–

–

CESIO

Non-ionic surfactant 4

–

–

–

–

–

–

CESIO

Non-ionic surfactant 5

–

–

–

–

–

–

CESIO

Non-ionic surfactant 6

–

–

–

–

–

–

CESIO

Non-ionic surfactant 7

–

–

–

–

–

–

CESIO

Non-ionic surfactant 8

–

–

–

–

–

–

CESIO

Non-ionic surfactant 9

–

–

–

–

–

–

CESIO

D-102

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Norbornene fluoroalcohol

Synonyms

CASRN

Mol.
Weight(g/mol)2[(bicyclo[2.2.1]hept5-ene-2yloxy)methyl]1,1,1,3,3,3-hexafluoro2-propanol

305815-63-8

290.20–124-07-2

144.21111-87-5

Octanoic acid
Octinol
Oleic acid

Capryl alcohol; Octyl
alcohol
cis-9-Octadecenoic
acid; Elainic acid

Oleyl methane sulfonate
Oripavine–
Oripavine

Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Liquid–LLNA/EC3
Validation
Study

1.66

LiquidCarboxylic Acids;
LipidsGerberick130.23

2.81

LiquidAlcohols; Lipids

EFfCI

112-80-1

282.46

7.73

LiquidLipidsEFfCI35709-09-2

346.57

3.98

LiquidHydrocarbons,
Acyclic; Sulfur
Compounds

Gerberick467-04-9

297.36

1.21

Solid

Pharmaceuticalchemicals

GSK

Oxalic acid–144-62-7

90.03

-0.59

Solid

Carboxylic Acids

GerberickOxazolone

4-Ethoxymethylene-2phenyloxazol-5-one

15646-46-5

217.22

1.87

Solid

Heterocyclic
Compounds

Gerberick–68609-97-2––

Liquid–Cymbopogon martini
oil; Geranium oil, east
indian–42874-03-3

361.70

5.21

Solid

8014-19-5––

Liquid–Lalko & Api

112-67-4

274.88

4.26

LiquidGerberick–

61-33-6

334.39

2.09

Solid

Penta; PCP

87-86-5

266.34

2.79

Solid

LipidsAmides; Sulfur
Compounds;
Heterocyclic
Compounds
Phenols

Oxirane, mono((C12-14alkyloxy)methyl) derivs
Oxyfluorfen EC
Palmarosa oil
Palmitoyl chloride
Penicillin G
Pentachlorophenol

D-103

–
EthersBGIA
ECPAGerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

Pentaerythritol Triacrylate–3524-68-3

298.290.91–

Carboxylic Acids,
Alcohols

NTP

Perillaldehyde–

2111-75-3

150.22

2.54

LiquidPhenyl benzoate–

93-99-2

198.22

2.89

Solid

Hydrocarbons,
Other
Carboxylic Acids

Phenylacetaldehyde
Phenylmethyl 2-(4-fluoro-2methylphenyl)-4-oxo-3,4dihydro-1(2h)-pyridinecarboxylate
Pluronic L92
p-Methylhydrocinnamic
aldehyde–127-78-1

120.15

2.05

LiquidAldehydes–

414909-98-1

339.37

3.94

Solid

Pharmaceuticalchemicals

GSK

–

–

–

–

–

Ryan

5406-12-2

148.21

2.54

Liquid–Gerberick

Substance Name

Potassium dichromate–
p-Cresyl
propionaldehyde

PDC

Precursor surfactant 1
Precursor surfactant 2
Propylene glycol––
1,2Dihydroxypropane;
1,2-Propanediol

7778-50-9294.18

0.62

Solid

–

–

–

–

–

–

–

–

57-55-6

76.09

0.43

Inorganic
Chemical,
Chromium
Compounds;
Inorganic
Chemical,Potassium
Compounds–

GerberickGerberickGerberick

NTP, Ryan,
Gerberick

CESIO

–

CESIO

LiquidAlcohols

Gerberick

Propylparaben

Propyl 4hydroxybenzoate

94-13-3

180.20

1.77

Solid

Phenols;
Carboxylic Acids

Gerberickp-tert-Butyl-a-ethylhydrocinnamal

Lilial80-54-6

204.31

3.52

LiquidAldehydes

Gerberickp-tert-Butylphenylglycidylether

4-tert-Butylphenyl
2,3-epoxypropyl ether

3101-60-8

206.28

3.52

Liquid

–

D-104

BGIA

ICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Pyridine–

110-86-1

79.10

1.31

LiquidQuinoxyfen SC–

124495-18-7

308.13

5.69

LiquidQuinoxyfen/cyproconazole–

124495-187/ 11309699-4

308.134/29
1.776

5.69/
3.25

LiquidHeterocyclic
Compounds

ECPA

R(+)-Limonene–

5989-27-5

136.24

2.93

LiquidHydrocarbons;
Hydrocarbons,
CyclicGerberick

R-Carvone–

2244-16-8

150.22

3.07

LiquidHydrocarbons,
Other

R-Carvoxime–2051-55-0

165.23

3.57

Solid

–

–

252873-35-1

295.25

0.83

Solid

Pharmaceuticalchemicals

1,3-Dihydroxybenzene

108-46-3

110.11

1.17

Solid

Rifamycin SV–

14897-39-3

697.77

5.04

Solid

Saccharin–

81-07-2

183.18

0.64

Solid

69-72-7

138.12

1.03

Solid

Phenols;
Carboxylic Acids

Gerberick

–

–

–

–

–

CESIO

140-90-9

144.19

2.11

Solid

Pharmaceuticalchemicals

GSK

Substance Namerel-(3r,3as,6ar)Hexahydrofuro[2,3-b]furan-3yl 4-nitrophenyl carbonate
Resorcinol

Salicylic acid
Saturated diglycerin
Sodium ethyl xanthate

2-Hydroxybenzoic
acid–
Carbonodithioic acid,O-ethyl ester, sodium
salt

D-105

Chemical Class3
Heterocyclic
Compounds
Heterocyclic
Compounds

Phenols
Heterocyclic
Compounds,
Polycyclic
Compounds
SulfurCompounds;
Heterocyclic
Compounds

Data Source
Gerberick
ECPA

LLNA/EC3
Validation
Study
LLNA/EC3
Validation
Study
GSK
Basketter
NTP

GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name

Synonyms

CASRN

Sodium lauroyl lactylate

Pationic 138C
Sodium dodecyl
sulfate, SLS, SDS,
Irium

13557-75-0

Mol.
Weight(g/mol)366.43151-21-3––

Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

2.58

–

Gerberick288.38

1.87/
1.69

Solid

–
Alcohols; Sulfur
Compounds;
Lipids6834-92-0

122.06-5.65–

Minerals, SiliconCompounds

NTP

94612-91-6

336.38

2.23––

Gerberick–
2,6,10,15,19,23Hexamethyl2,6,10,14,18,22tetracosahexaene–

68917-46-4––

Liquid–Lalko & Api

111-02-4

410.72

14.12

LiquidHydrocarbons,
Acyclic

EFfCI

3810-74-01457.39

-8.50

Solid

Carbohydrates

Gerberick

Butanedioic acid
4-Aminobenzenesulfonamide; pAnilinesulfonamide;p-Sulfamidoaniline
p-Aminobenzenesulfonic acid; pAnilinesulfonic acid
[R-(R*,R*)]-2,3Dihydroxybutanedioic
acid; L-Tartaric acid110-15-6

118.09

-0.75

Solid

Carboxylic Acids

EFfCI

63-74-1

172.21

0.40

Solid

Amides; Sulfur
Compounds;
AminesGerberick

121-57-3

173.19

0.40

Solid

Hydrocarbons,
Cyclic; Sulfur
Compounds

Gerberick

87-69-4

150.09

0.87

Solid

Alcohols;
Carboxylic Acids

Gerbericktert-Butyl-3-aminobenzoate–

92146-82-2

193.25

2.63

Solid

Pharmaceuticalchemicals

GSK

Tetramethyl thiuram disulfide

Thiram; Bis(dimethylthiocarbamoyl) disulfide

137-26-8

240.44

1.17

Solid

Carboxylic Acids;
SulfurCompounds

GerberickSodium lauryl sulfate
Sodium metasilicate
Sodium-3,3,5trimethylhexanoyloxybenzenesulfonate
Spearmint oil
Squalene
Streptomycin sulfate
Succinic acidSulfanilamide

Sulfanilic acid
Tartaric acid

D-106

BGIA,
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Synonyms

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalFormtrans-2-Decenal–

3913-71-1

154.25

2.54

Liquidtrans-2-Hexenal–

6728-26-3

98.15

1.56

LiquidTrans-2-methyl-2-butenal–497-03-0

84.12

1.15

LiquidAldehydes–
17,21-Dihydroxy16beta-methylpregna1,4,9(11)-triene-3,20dione104-46-1

148.21

2.54

LiquidEthers; Phenols

13504-15-9

356.47

3.02

Solid

Pharmaceuticalchemicals

GSK

1582-09-8

335.28

5.31–

Hydrocarbons,
Cyclic; Amine

ECPA

Substance Nametrans-Anethol
TrienolChemical Class3
Aldehydes;
Hydrocarbons,
Other
Heterocyclic
Compounds

Data Source
GerberickGerberick
LLNA/EC3
Validation
Study
Gerberick

Trifluralin EC–

Trimethylhexamine diamine

–

–

–

–

–

–

BGIA

Trimethylolpropane Triacrylate–

15625-89-5

296.32

2.86

LiquidCarboxylic Acids

NTP

Undec-10-enal–

112-45-8

168.28

2.79

LiquidAldehydes

Undecylenic acid

10-Undecenoic acid112-38-9

184.28

4.37

LiquidLipidsUnsaturated fatty acid

–

–

–

–

–

–

CESIO

Unsaturated fatty acid ester

–

–

–

–

–

–

CESIO

Vanillin–

121-33-5

152.15

1.28

Solid

Aldehydes

GerberickVeratraldehyde–

120-14-9

166.18

1.45

Solid

Pharmaceuticalchemicals

Vinylidene dichloride
Vinylpyridine–
Ethylenepyridine

75-35-4

96.94

1.45

Liquid1337-81-1

105.141.80

LiquidD-107

Gerberick
EFfCI

Hydrocarbons,
Acyclic;
Hydrocarbons,
HalogenatedHeterocyclic
Compounds

GSK

GerberickGerberickICCVAM Test Method Evaluation Report: Appendix D, Annex II

Substance Name
Ylang Ylang (Extra)
Ylang Ylang (III)Synonyms
Cananga oil;
Canangium odoratum
genuina oil
Cananga oil;
Canangium odoratum
genuina oil

CASRN

Mol.
Weight(g/mol)Log
Kow1,2

PhysicalForm

Chemical Class3

Data Source

8006-81-3––

Liquid–Lalko & Api

8006-81-3––

Liquid–Lalko & Api

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; g/mol = grams per mole.
1
Kow represents the octanol-water partition coefficient (expressed on log scale).
2
When two numbers are shown for Kow, the first number is the value calculated by the method of Moriguchi et al. (1994) and provided in Gerberick et al. (2005). The second
number was calculated by the method of Meylan and Howard (1995) and obtained from the website: http://www.srcinc.com/what-we-do/databaseforms.aspx?id=385. LogP (log
Kow) values for GSK chemicals were calculated using the method provided by Daylight Chemical Information Systems (see:http://www.daylight.com/dayhtml/doc/clogp/index.html).
3
Chemical classifications based on the Medical Subject Headings classification for chemicals and drugs, as developed by the National Library of Medicine (available at
http://www.nlm.nih.gov/mesh/meshhome.html). Chemical classification of "pharmaceutical chemicals" for the GSK chemicals was suggested by Dr. Michael Olson of GSK,
which in spirit captures three types of pharmaceutical active substances (actives, intermediates, and starting materials).
4
Basketter = Basketter et al. 2007; Bayer = Bayer CropScience SA Studies, submitted by E. Debruyne; BGIA = Berufsgenossenschaftliches Institut fur Arbeitsschutz (German
Institute for Occupational Safety and Health) Study Report, submitted by H.W. Vohr; CESIO = Comite Europeen des Agents de Surface et de Leurs Intermediaires Organiques(European Committee of Surfactants and Their Organic Intermediates) Report, submitted by K. Skirda; ECPA = European Crop Protection Association LLNA Project Report,
submitted by P. Botham; EFfCI = European Federation for Cosmetic Ingredients study, submitted by P. Ungeheuer; Gerberick = Gerberick et al. 2005; GSK = Glaxo
SmithKline, submitted by M.J. Olson; Lalko & Api = Lalko & Api (2006), submitted by A. Api (Research Institute for Fragrance Materials [RIFM]); LLNA/EC3 Validation
Study, submitted by D. Basketter, I. Kimber, and F. Gerberick; NTP = NTP Study, submitted by D. Germolec; Ryan = Ryan et al. (2002).

D-108

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Annex III
Traditional LLNA Data Used for the Performance Analysis of the rLLNA

D-109

ICCVAM Test Method Evaluation Report: Appendix D, Annex III[This Page Intentionally Left Blank]

D-110

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Resorcinol

108-46-3

AOO

+

+

6

1

1.80

2.5

2.30

5.0

2.60

10

6.30

25

10.10

50

12.50

A SC600

PA/H2O

-

-

NC

10

1.40

25

1.80

50

2.30

100

1.60

AE F016382 00
TK71 A101

PA/H2O

-

-

NC

3.6

1.00

7.1

0.80

17.9

1.00

35.7

1.10

D EC25

PA/H2O

-

-

NC

0.5

0.56

1

0.63

2.5

0.59

D EW 15

PA/H2O

-

-

NC

2.5

1.90

5

1.50

10

2.50

25

2.50

EXP 10810 A

PA/H2O

+

+

2.1

10

6.40

25

8.40

50

9.20

EXP 11120 A

PA/H2O

+

+

64.9

10

0.96

25

0.66

50

1.60

100

6.30

F & Fo WG 50 + 25

PA/H2O

+

+

0.003
1

2.5

11.70

5

12.60

10

14.10

25

15.20

FAR01042-00

PA/H2O

-

-

NC

10

1.40

25

2.10

50

1.40

100

2.50

FAR01060-00

PA/H2O

+

+

88.5

10

0.40

25

0.80

50

1.00

100

3.60

Fx + Me EW 69

PA/H2O

+

+

25.2

5

0.83

10

1.55

25

2.95

50

8.55

1-(2,3epoxypropoxy)-2,2bis [(2,3epoxypropoxy)methylbutane
1,2-Diaminocyclohexane

ACE

+

+

1.4

1

2.06

3

6.52

10

12.00

Basketter et al.
(2007)
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
BGIA Study Report,
Submitted by H.W.
Vohr

ACE

+

+

0.4

0.1

1.19

0.3

1.81

1

8.39

1436-595

D-111

BGIA Study Report,
Submitted by H.W.
Vohr

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

1,6-Bis(2,3epoxypropoxy)hexane
3-Aminomethyl3,5,5trimethylcyclohexyl
amine
Bakelite EPR 161

1609631-4

ACE

+

+

1.9

0.3

0.94

1

1.67

3

4.65

2855-132

ACE

+

+

1.0

0.3

1.17

1

2.68

3

20.16

9012-457

ACE

+

+

0.7

0.1

1.02

0.3

2.37

1

3.49

Bakelite EPR 162

9012-457

ACE

+

+

0.1

0.3

10.53

1

19.94

3

39.89

Bakelite EPR 164

9012-457

ACE

+

+

0.2

0.3

5.58

1

16.11

3

28.13

Dipropylene
triamine

56-18-8

ACE

+

+

0.9

0.3

2.16

1

3.17

3

12.45

Hexyl cinnamic
aldehyde101-86-0

AOO

-

-

NC

2.5

1.12

5

1.19

10

2.84

Hexyl cinnamic
aldehyde101-86-0

ACE

+

+

1.2

3

4.56

10

6.63

30

9.86

Hydroxyethylethylenediamine

111-41-1

ACE

+

+

IDR3

3

2.00

10

1.72

30

6.60

m-Phenylenebis(methylamine)

1477-550

ACE

+

+

0.4

0.3

1.92

1

9.09

3

44.20

Oxirane,mono((C12-14alkyloxy)methyl)
derivsp-tert-Butylphenylglycidylether

6860997-2

ACE

+

+

0.6

0.3

2.35

1

4.16

3

22.74

3101-608

ACE

+

+

0.4

0.1

1.36

0.3

1.68

1

14.22

151-21-3

Pluronic
L92

+

+

4.9

5

3.05

10

4.78

25

8.46

ACE

+

+

1.9

1

2.15

3

4.00

10

8.86

ACE

+

+

0.1

0.5

9.00

1

11.10

2

7.60

Sodium laurylsulfateTrimethylhexamine
diamineBenzalkonium
chloride

8001-545

D-112

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
BGIA Study Report,
Submitted by H.W.
Vohr
CESIO Report,
Submitted by K.
SkirdaICCVAM Test Method Evaluation Report: Appendix D, Annex III

rLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

+

+

IDR3

5

1.50

25

1.80

50

1.20

100

4.80

AOO

+

+

4.7

10

8.00

25

14.00

50

31.00

Laurylglycerinderivative

DMF

+

+

24.3

5

1.62

10

2.36

25

3.03

Non-ionic
surfactant 1

AOO

+

+

27.5

25

2.80

50

4.80

100

6.50

Non-ionic
surfactant 2

AOO

-

+

47.1

25

1.50

50

3.20

100

2.90

Non-ionic
surfactant 3

AOO

+

+

19.8

25

4.70

50

9.80

100

13.30

Non-ionic
surfactant 4

AOO

+

+

0.012

25

36.00

50

39.00

100

162.0
0

Non-ionic
surfactant 5

AOO

+

+

37.5

25

2.70

50

3.30

100

3.20

Non-ionic
surfactant 6

AOO

+

+

34.4

25

2.70

50

3.50

100

6.50

Non-ionic
surfactant 7

AOO

+

+

IDR3

25

6.30

50

50.80

100

7.40

Non-ionic
surfactant 8

AOO

+

+

IDR3

25

4.20

50

3.30

100

5.60

Non-ionic
surfactant 9

AOO

+

+

10.5

25

3.50

50

3.90

100

7.70

Precursor surfactant
1

AOO

+

+

60.7

25

2.20

50

2.70

100

4.10

Precursor surfactant
2

AOO

+

+

24.0

25

3.10

50

4.80

100

4.40

Saturated diglycerin

EtOH/
H 2O

-

-

NC

25

1.40

50

2.10

100

1.90

Unsaturated fatty
acid

AOO

+

+

22.2

25

3.40

50

5.70

100

6.50

Substance Name

CASRN

VehicleFatty acidglutamate
Glyceryl
thioglycolate

3061884-9

D-113

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

CESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

Unsaturated fatty
acid ester

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

AOO

+

+

27.1

25

2.80

50

5.20

100

4.70

Atrazine SC

1912-249

Pluronic
L92
(1%)

+

+

31.3

12.5

1.80

25

2.80

50

3.60

Dinocap EC

3930045-3

Pluronic
L92
(1%)

+

+

1.1

0.8

2.00

4

14.20

21

26.70

Oxyfluorfen EC

4287403-3

Pluronic
L92
(1%)

-

-

NC

1

0.30

7

0.90

33

2.30

Quinoxyfen SC

12449518-7

Pluronic
L92
(1%)

-

-

NC

7

1.10

33

1.70

100

0.80

Quinoxyfen/
cyproconazole

12449518-7/
11309699-4
1582-098

Pluronic
L92
(1%)

+

+

27.8

12.5

2.00

25

2.30

50

8.60

Pluronic
L92
(1%)

+

+

7.0

7

3.10

33

26.30

100

61.50

Fumaric acid110-17-8

DMSO

-

-

NC

5

1.30

10

2.30

25

1.40

Linoleic acid60-33-3

AOO

+

+

14.1

10

1.50

25

7.00

50

9.10

Linolenic acid463-40-1

AOO

+

+

9.9

10

3.10

25

9.30

50

10.30

Maleic acid110-16-7

DMSO

+

+

7.0

10

6.70

25

16.10

50

16.10

Octinol

111-87-5

AOO

+

+

4.7

10

5.60

25

8.80

50

11.20

Oleic acid112-80-1

AOO

+

+

10.5

10

2.60

25

14.90

50

6.90

Squalene

111-02-4

AOO

+

+

7.9

10

3.80

25

6.90

50

8.20

Trifluralin EC

D-114

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

75

7.10

100

7.30

75

15.80

100

30.10

6
Conc.
(%)

6 SI

Data Source

CESIO Report,
Submitted by K.
SkirdaECPA LLNA
Project Report,
Submitted by P.
BothamECPA LLNA
Project Report,
Submitted by P.
BothamECPA LLNA
Project Report,
Submitted by P.
BothamECPA LLNA
Project Report,
Submitted by P.
BothamECPA LLNA
Project Report,
Submitted by P.
BothamECPA LLNA
Project Report,
Submitted by P.
BothamEFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

Succinic acid110-15-6

DMSO

-

-

NC

5

1.20

10

1.20

25

1.30

Undecylenic acid112-38-9

AOO

+

+

19.4

10

2.50

25

3.30

50

4.40

ACE

-

-

NC

10

1.60

20

1.20

40

1.60

16799873-4

ACE

+

+

8.3

10

7.00

20

22.10

40

22.40

Gerberick et al.
(2005)

5629055-2

ACE

+

+

12.5

10

2.3

20

5.1

40

9.5

Gerberick et al.
(2005)

16799876-7

ACE

+

+

9.6

10

3.9

20

19.2

40

18.7

Gerberick et al.
(2005)

13509998-8

ACE

-

-

NC

10

2.80

20

1.10

40

0.70

Gerberick et al.
(2005)

104-27-8

AOO

+

+

9.3

10

3.5

25

10

50

26.1

Gerberick et al.
(2005)

116-26-7

AOO

+

+

7.5

0.5

0.70

1

1.10

2.5

1.10

2634-335

DMF

+

+

2.3

10

3.80

30

4.40

50

4.90

3569165-7
123-31-9

AOO

+

+

0.9

0.5

1.40

1

3.40

2.5

3.50

5

5.40

AOO

+

+

0.1

0.1

2.80

0.25

5.80

0.5

13.70

1

15.20

3344-772
7336780-3

AOO

+

+

6.9

5

2.20

10

4.3

25

9.8

AOO

+

+

17.9

5

1.30

10

2.00

25

3.9

109-65-9

AOO

-

-

NC

5

1.1

10

1.2

25

1

6938-665
143-15-7

AOO

+

+

8.3

2.5

1.2

5

1.6

10

3.7

AOO

+

+

17.7

5

1.1

10

1.4

25

4.5

1-(2',3',4',5'Tetramethylphenyl)-3-(4'tetrabutylphenyl)propane-1,3-dione
1-(2',3',4',5'Tetramethylphenyl)
butane-1,3-dione
1-(2',5'Dimethylphenyl)
butane-1,3-dione
1-(2',5'diethylphenyl)
butane-1,3,-dione
1-(3',4',5'Tetramethoxyphenyl)-4dimethylpentane1,3-dione
1-(pmethoxyphenyl)-1penten-3-one
1,1,3-Trimethyl-2formyylcyclohexa2,4-dione
1,2Benzisothiazolin-3one
1,2-Dibromo-2,4dicyanobutane
1,4-dihydroquinone
12-Bromo-1dodecanol
12Bromododecanoic
acid
1-Bromobutane
1-Bromodocosane
1-Bromododecane

D-115

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

EFfCI study,
Submitted by P.
Ungeheuer
EFfCI study,
Submitted by P.
Ungeheuer
Gerberick et al.
(2005)

5

2.70

10

3.30

Gerberick et al.
(2005)
Gerberick et al.
(2005)

2.5

13.10

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

1-Bromoeicosane

4276-497
3508-007
112-82-3

AOO

+

+

6.1

5

2.1

10

6.2

25

8.4

AOO

+

+

4.8

5

3.2

10

6

25

9.6

AOO

+

+

2.3

1

1.1

2.5

3.3

5

7.9

111-25-1

AOO

+

+

10.3

1

1.7

10

2.9

50

18.6

1-Bromononane693-58-3

AOO

-

-

NC

5

1.2

10

1.4

25

2.8

1-Bromooctadecane

112-89-0

AOO

+

+

15.2

5

1.8

10

2.2

25

4.5

1Bromopentadecane
1Bromotetradecane
1-Bromotridecane629-72-1

AOO

+

+

5.1

5

2.9

10

7.8

25

19.6

112-71-0

AOO

+

+

9.2

5

1.5

10

3.3

25

11.3

765-09-3

AOO

+

+

10.2

5

1.6

10

2.9

25

10.4

1-Bromoundecane693-67-4

AOO

+

+

19.6

5

1.3

10

1.4

25

3.9

1-Butanol

71-36-3

dH2O

-

-

NC

5

1.6

10

1.2

20

1.4

1-Chloro-2,4dinitrobenzene
1Chlorohexadecane
1-Chloromethylpyrene
1-Chlorononane

97-00-7

AOO

+

+

0.05

0.01

1.50

0.025

1.8

0.05

2.4

AOO

+

+

9.1

5

1.6

10

3.3

25

5.7

AOO

+

+

0.005

0.025

11.6

0.05

15.4

0.1

18.6

AOO

-

-

NC

10

1

25

1.6

50

2.3

AOO

+

+

16.3

10

1.7

25

4.8

50

7.3

AOO

+

+

20.2

10

1.1

25

3.9

50

6.3

AOO

+

+

13.1

5

1.70

10

2.30

25

5.70

1-Iodohexadecane

4860-031
1086-006
2473-010
3386-3322425-549
4292-197
544-77-4

AOO

+

+

19.1

10

1.60

25

3.90

50

6.40

1-Iodohexane

638-45-9

AOO

-

-

NC

10

0.90

25

1.20

50

2.50

1-Iodononane

4282-422
629-93-6

AOO

+

+

24.2

10

1.30

25

3.10

50

4.60

AOO

-

-

NC

5

1

10

1.4

25

1.9

1921894-1
70-25-7

AOO

+

+

13.8

10

1.70

25

6.90

50

9.70

AOO

+

+

0.03

0.05

27.5

0.1

60.4

0.25

78.3

1Bromoheptadecane
1Bromohexadecane
1-Bromohexane

1-Chlorooctadecane
1Chlorotetradecane
1-Iododecane

1-Iodooctadecane
1-Iodotetradecane
1-Methyl-3-nitronitrosoguanidine

D-116

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

10

11.1

25

13.5

0.1

8.9

0.25

38

6
Conc.
(%)

50

6 SI

16.8

Data Source

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

1-Napthol

90-15-3

AOO

+

+

1.3

0.1

1.40

0.25

1.00

0.5

1.20

1

1.50

2.5

8.50

1-Phenyl-1,2propanedione
1-Phenyl-2methylbutane-1,3dione
1-Phenyloctane-1,3-dione
2-(4-Amino-2nitrophenylamino)ethanol
2-(4-tertAmylcyclohexyl)
acetaldehyde
2,2,6,6Tetramethylheptane-3,5-dione
2,3-Butanedione579-07-7

AOO

+

+

1.3

5

12.80

10

17.70

25

20.10

6668-242

ACE

+

+

29.1

10

1.70

20

2.00

40

4.20

5584668-1
2871-014

ACE

+

+

10.4

10

2.80

20

6.60

40

8.70

AOO

+

+

2.2

0.1

0.50

0.25

1.20

0.5

1.90

62015984-4

AOO

+

+

36.8

25

2.1

50

4.00

100

9.10

Gerberick et al.
(2005)

1118-714

ACE

+

+

26.7

10

2.10

20

2.80

40

3.40

Gerberick et al.
(2005)

431-03-8

AOO

+

+

11.3

5

1.4

10

2.8

25

5.2

2,4,6-Trichloro1,3,5-triazine
2,4-Heptadienal

108-77-0

AOO

+

+

0.09

1

21.80

2.5

28.90

5

34.00

5910-850
95-70-5

AOO

+

+

4.0

0.5

1.1

1

1.4

2.5

1.9

5

3.7

DMSO

+

+

0.17

0.125

2.6

0.25

3.5

0.5

4.1

1

5.5

2-Acetylcyclohexanone
2-Amino-6-chloro4-nitrophenol
2-Aminophenol874-23-7

ACE

-

-

NC

10

0.8

20

0.7

40

0.8

6358-094
95-55-6

AOO

+

+

2.2

0.1

1.7

0.25

1.4

0.5

2.1

1

AOO

+

+

0.4

0.5

3.5

1

5

2.5

7.4

2-Bromotetradecanoic acid
2-Hydroxyethyl
acrylate
2-Hydroxypropyl
methacrylate
2-Mercaptobenzothiazole
2-Methoxy-4methylphenol
2-Methyl-2Hisothiazol-3-one
2-Methyl-4H,3,1benzoxazin-4-one

1052081-7
818-61-1

AOO

+

+

3.4

5

4.7

10

7.7

25

10.1

AOO

+

+

1.4

5

10.70

10

14.80

25

18.10

923-26-2

AOO

-

-

NC

10

1.1

25

1.2

50

1.3

149-30-4

DMF

+

+

1.7

1

2.3

3

4.4

10

8.6

93-51-6

AOO

+

+

5.8

4.2

1.80

8.4

5.00

21

8.50

2682-204525-76-8

AOO

-

+

1.9

0.25

1.50

0.5

1.50

1

1.8

2.5

DMSO

+

+

0.7

5

7.60

10

9.20

25

10.80

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)

2,5-Diaminotoluene

D-117

1

1.8

2.5

3.3

10

8.10

1.5

2.5

3.4

3.8

5

2.5

6
Conc.
(%)

6 SI

Data Source

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

2-Methyl-5hydroxyethylaminophenol
2-Methylundecanal

5530296-0

AOO

+

+

0.4

0.1

1.20

0.25

0.80

0.5

3.60

1

2.6

2.5

7.4

Gerberick et al.
(2005)

110-41-8

AOO

+

+

10.0

0.5

1.40

1

1.30

2.5

1.30

5

2.40

10

3.00

5307-142
3190604-4

AOO

+

+

0.4

0.1

1.80

0.25

2.20

0.5

3.30

1

7.90

2.5

11.90

AOO

+

+

17.1

1

0.60

2.5

0.70

5

0.60

10

1.30

25

4.90

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)

1154-592

ACE

+

+

0.04

0.25

11.20

0.5

14.40

1

18.00

Gerberick et al.
(2005)

119-84-6

AOO

+

+

5.6

2.5

1.6

5

2.5

10

6.6

3672729-4
591-27-5

AOO

+

+

2.7

5

7.20

10

12.00

25

19.00

AOO

+

+

3.2

2.5

2.8

5

3.5

10

5.7

15475020-6

AOO

+

+

3.5

3.19

2.7

6.37

5.1

12.74

7.1

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)

109-55-7

AOO

+

+

2.2

0.5

1.30

1

1.10

2.5

3.50

17092869-5

ACE

+

+

33

10

1.1

20

1.7

40

3.7

3775-211

AOO

+

+

1.4

0.1

1.3

0.25

1.1

0.5

2.1

18674326-0
18674329-3
108-45-2

AOO

+

+

32

11

1.5

27

2.3

54

6.4

AOO

+

+

3.6

2.5

2.20

5.5

4.30

11

6.00

AOO

+

+

0.5

2.5

11.70

5

15.50

10

19.20

1736959-4
2564671-3

AOO

+

+

3.7

5

4.90

10

9.10

25

15.10

DMSO

+

+

0.6

0.1

1.2

1

4.5

5

5.9

2-Nitro-pphenylenediamine
3 and 4-(4Hydroxy-4methylpentyl)-3cyclohexane-1carboxaldehyde
3, 3', 4', 5Tetrachlorosalicylanilide
3,4Dihydrocoumarin
3,5,5-Trimethylhexanoyl chloride
3-Aminophenol
3-Bromomethyl-5,5'-dimethyldihydro-2(3H)furanone
3-Dimethylaminopropylamine
3-Ethoxy-1(2',3',4',5'tetramethylphenyl)propane-1,3-dione
3-Methyl-4-phenyl1,2,5-thiadiazole1,1-dioxide
3-Methyleugenol
3-Methylisoeugenol
3-Phenylenediamine
3-Propylidenphthalide4-(N-Ethyl-N-2methan-sulfamidoethyl)-2-methyl1,4,-phenylenediamine

D-118

6
Conc.
(%)

6 SI

Data Source

5

7.00

10

13.90

Gerberick et al.
(2005)
Gerberick et al.
(2005)

1

1.9

2.5

5.6

Gerberick et al.
(2005)

10

6.3

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4,4,4-Trifluoro-1phenylbutane-1,3dione
4-Allylanisole362-06-7

ACE

+

+

20

10

2.10

20

3.00

40

4.60

140-67-0

AOO

+

+

18

10

1.20

25

4.7

50

4.5

4-Hydroxybenzoicacid
4'-Methoxyacetophenone
4-Methylaminophenol sulfate
4-Nitrobenzyl
bromide4-Phenylenediamine
5,5-Dimethyl-3methylenedihydro2(3H)-furanone

99-96-7

DMSO

-

-

NC

5

1.4

10

1.5

25

1.3

100-06-1

AOO

-

-

NC

10

1.3

25

1

50

1

55-55-0

DMF

+

+

0.8

0.5

2.50

1

3.40

2.5

6.70

100-11-8

AOO

+

+

0.05

0.01

0.90

0.03

1.30

0.05

106-50-3

AOO

+

+

0.16

0.05

1.90

0.1

2.30

2904397-8

AOO

+

+

2.0

2

3

4

2617255-4
1370686-0
18674325-9
92-48-8

DMF

+

+

0.009

0.01

3.50

AOO

+

+

26

25

AOO

+

+

13

ACE

-

-

18674324-8
1304112-8
57-97-6

AOO

+

AOO

5-Chloro-2-methyl4-isothiazolin-3-one
5-Methyl-2,3hexanedione
5-Methyleugenol
6-Methylcoumarin
6-Methyleugenol
6-Methylisoeugenol
7,12-Dimethylbenz[a]anthracene
7Bromotetradecane
Abietic acidalpha-Amyl
cinnamic aldehydealpha-Butyl
cinnamic aldehydealpha-Methyl
cinnamic aldehydealphaMethylphenylacetaldehyde
Aniline

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6 SI

Data Source

Gerberick et al.
(2005)
100

8

3.50

0.1

11.50

0.25

4.00

0.5

5.70

7.4

8

9.2

0.03

12.30

0.1

22.70

2.90

50

6.00

100

14.30

11

2.7

27

4.9

54

4.3

NC

5

1

10

1

25

1.1

+

17

11

1.9

27

4.9

54

8.3

+

+

1.6

2.5

5.90

5.5

11.10

11

15.7

DMF

+

+

0.006

0.025

7.60

0.5

17.70

1

15.60

7403697-8
514-10-3

AOO

+

+

21

5

0.9

10

1.2

25

3.6

AOO

+

+

15

5

1.5

10

2

25

5.2

122-40-7

AOO

+

+

10.6

1

1.5

2.5

1.7

5

2.2

10

2.8

25

8.2

7492-446
101-39-3

AOO

+

+

11.2

1

1.4

2.5

1.7

5

1.7

10

2.1

25

13

AOO

+

+

4.5

1

1.80

2.5

1.50

5

3.40

10

3.3

25

15.3

93-53-8

AOO

+

+

6.3

0.5

2

1

2.2

2.5

1

5

2.2

10

5.2

62-53-3

AOO

+

+

89

5

1.1

10

0.9

25

2

50

1.9

100

3.3

D-119

6
Conc.
(%)

1.0

6.60

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

Benzaldehyde

100-52-7

AOO

-

-

NC

1

2.1

2.5

1.7

5

2.2

10

1.8

25

2

Benzene-1,3,4tricarboxylic
anhydride
Benzo[a]pyrene552-30-7

AOO

+

+

9.2

1

1.10

2.5

2.00

5

2.00

10

3.20

25

4.60

50-32-8

AOO

+

+

0.5

17.6

1

19.2

2.5

27

Benzocaine

94-09-7

AOO

-

-

0.000
9
NC

2.5

2.1

5

1.8

10

2.7

Benzoquinone

106-51-4

AOO

+

+

0.5

36.4

1

42.3

2.5

52.3

Benzyl benzoate

120-51-4

AOO

+

+

0.009
9
17

5

2.3

25

3.5

Benzyl bromide

100-39-0

AOO

+

+

0.2

0.25

3.5

0.5

11.5

1

16.1

Benzylidene
acetonebeta-Propiolactone

122-57-6

AOO

+

+

3.7

10

8.5

25

13.6

50

12.8

57-57-8

AOO

+

+

0.15

0.025

1.50

1.0

13.00

2.5

19.90

ACE

-

-

NC

10

1.8

20

1.6

40

2.1

AOO

+

+

1.5

1

2

3

6

10

17.4

AOO

+

+

30.9

10

1.40

25

2.20

50

5.60

AOO

+

+

16

8.3

1.30

20.7

4.00

41.3

8.50

AOO

+

+

18

10

1.80

25

4.10

50

7.50

AOO

+

+

19

10.87

1.70

27.17

4.30

54.33

4.60

AOO

-

+

26

11.73

2.50

29.33

3.10

58.67

2.50

AOO

+

+

1.4

0.52

1.10

1.31

2.30

2.62

4.10

AOO

+

+

1.3

0.61

1.20

1.52

3.50

3.05

7.60

AOO

+

+

2.8

1.85

1.40

3.7

4.60

7.4

10.10

Camphorquinone

17666499-6
17666502-4
17666504-6
465-29-2

AOO

-

+

10

5

2.8

10

3

25

1.7

Chlorobenzene

108-90-7

AOO

-

-

NC

5

1.1

10

1.7

25

1.6

Cinnamic alcohol

104-54-1

AOO

+

+

21

10

1.8

25

3.5

50

Cinnamic aldehyde

104-55-2

AOO

+

+

3.0

0.5

1.40

1.0

0.90

2.5

bis-1,3-(2',5'dimethylphenyl)propane-1,3-dione
Bisphenol Adiglycidyl ether
Butyl glycidyl ether
C11-azlactone
C15-azlactone
C17-azlactone

1675-543
2426-086
17666506-8
17666509-1
17666511-5

C19-azlactone
C4-azlactone
C6-azlactone
C9-azlactone

D-120

25

1.8

50

1.2

2.5

16.4

5

25.1

5.23

11.70

3.9

90

5.7

1.90

5.0

7.10

10.0

15.80

6
Conc.
(%)

6 SI

Data Source

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

cis-6-Nonenal

AOO

+

+

22

10

1.60

25.0

3.30

50.0

4.50

100.0

13.70

AOO

+

+

13

5

1.20

10

2.10

25

6.30

AOO

+

+

4.8

2.5

1.6

5

3.1

10

3

Coumarin

2277-192
5392-405
2359375-1
91-64-5

AOO

-

-

NC

5

2.70

10

2.90

25

2.30

Cyclamen aldehyde

103-95-7

AOO

+

+

22.0

1

1.40

2.5

1.30

10

1.80

25

3.3

Diethyl maleate

141-05-9

AOO

+

+

5.8

25

16.30

50

22.60

100

13.10

Diethyl sulfate

64-67-5

AOO

+

+

3.3

1

0.8

2.5

1.9

10

12

Diethylacetaldehyde
Diethylenetriamine

97-96-1

AOO

+

+

76

25

1.2

50

0.8

75

2.4

100

16.3

111-40-0

AOO

+

+

5.8

10

6.40

25

12.10

Diethylpthalate

84-66-2

AOO

-

-

NC

25

1.00

50

1.30

100

1.50

Dihydroeugenol

AOO

+

+

6.8

5.1

2.70

10.1

3.60

25.3

7.80

Dimethyl sulfate

2785-877
77-78-1

AOO

+

+

0.19

0.25

3.80

0.5

6.00

1

5.70

Dimethyl sulfoxide

67-68-5

AOO

+

+

72

25

2.70

50

2.30

100

3.90

Dodecylmethanesulfonate
Ethylbenzoylacetate
Ethyl vanillin

5132371-8
94-02-0

AOO

+

+

8.8

5

2.10

10

3.30

25

9.00

ACE

-

-

NC

10

0.9

20

0.9

40

1.2

121-32-4

AOO

-

-

NC

2.5

0.65

5

1.05

10

0.74

25

Ethyl acrylate

140-88-5

AOO

+

+

28

10

1.2

25

2.7

50

5

Ethylene glycol
dimethacrylate
Ethylenediamine
free base
Eugenol

97-90-5

MEK

+

+

28

10

1.20

25

2.40

50

7.00

107-15-3

AOO

+

+

2.2

0.1

1.10

0.25

1.20

0.5

1.60

97-53-0

AOO

+

+

13

2.5

1.60

5

1.50

10

Farnesal

502-67-0

AOO

+

+

12

1

0.60

2.5

1.10

Fluorescein
isothiocyanate

2707245-3

+

+

0.143

0.5

8.60

1

Formaldehyde

50-00-0

ACE/
DBP(50:50)
ACE

+

+

0.61

0.093

1.10

Furil

492-94-4

AOO

-

-

NC

5

1.20

CitralClotrimazole

5
Conc.
(%)

5 SI

50

5.2

0.36

50

0.29

1

1.90

2.5

3.30

2.40

25

5.50

5

1.70

10

2.50

25

7.00

11.70

2.5

16.60

0.185

2.30

0.37

2.30

0.925

3.90

1.85

4.00

10

1.70

25

2.20

D-121

6
Conc.
(%)

5

6 SI

6.10

Data Source

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

Geraniol

106-24-1

+

+

26

1

1.00

3

1.00

10

1.30

30

3.40

50

3.90

Glutaraldehyde

111-30-8

EtOH/
DEP(75:25)
ACE

+

+

0.1

0.05

1.30

0.125

4.30

0.25

7.60

0.5

11.60

1.25

17.70

Glycerol

56-81-5

DMF

-

-

NC

25

1.10

50

0.70

100

0.50

Glyoxal107-22-2

AOO

+

+

1.4

1

2.50

2.5

4.20

5

5.20

10

10.30

25

15.80

Hexane110-54-3

AOO

-

-

NC

25

0.8

50

0.8

100

2.2

Hexyl cinnamic
aldehyde
Hydroxycitronellal

101-86-0

AOO

+

+

11

2.5

1.30

5

1.10

10

2.50

25

10.00

50

17.00

107-75-5

AOO

+

+

33

2.5

2.20

5

1.00

10

0.80

25

1.10

50

7.10

Imidazolidinyl urea

DMF

+

+

24

10

1.70

25

3.10

50

5.50

Isoeugenol

3923646-9
97-54-1

AOO

+

+

1.7

0.5

1.00

1

1.10

5

12.40

Isononanoyl
chloride
Isopropanol

5707736-8
67-63-0

AOO

+

+

2.7

5

6.60

10

10.60

25

12.60

AOO

-

-

NC

10

1.7

25

1.1

50

1

Isopropyl myristate110-27-0

AOO

+

+

44

25

2.10

50

3.30

100

3.40

Isopropyleugenol

5147490-92953-007
59-01-8;8063-078
598-82-3

AOO

-

-

NC

12

1.8

29.0

1.8

59.0

2.2

AOO

+

+

0.6

0.6

3.00

1.2

5.70

3.0

10.70

AOO

-

-

NC

5

2.2

10

0.8

25

1

DMSO

-

-

NC

5

1

10

1.4

25

2.2

1166-525
78-70-6

DMSO

+

+

0.3

1

12.10

10

29.70

25

29.30

AOO

+

+

30

25

2.5

50

4.8

100

8.3

2374-654
4230-153
2645248-2

AOO

+

+

0.4

1

21.6

2.5

39.9

5

48.6

AOO

-

-

NC

5

1

10

1.3

25

1.5

AOO

+

+

0.8

5

26.7

10

35.4

25

32.9

66-27-3

AOO

+

+

2.7

0.25

0.7

1

0.7

10

3.6

119-36-8

AOO

-

-

NC

1.0

1

2.5

1.1

5.0

1.6

Isopropylisoeugenol
Kanamycin
Lactic acid
Lauryl gallate
Linalool alcohol
Methyldodecanesulfonate
Methyl hexacecyl
sulfonate
Methylhexadecanesulfonate
Methylmethanesulfonate
Methyl salicylate

D-122

50

10

6 SI

Data Source

Gerberick et al.
(2005)
2.5

18.00

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)

36

1.4

6
Conc.
(%)

20

0.9

Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

AOO

+

+

2.0

2.5

5.10

5.0

11.60

10.0

25.60

Gerberick et al.
(2005)

+

+

2.5

5

10.4

10

17.7

20

24.4

99-76-3

EtOH
(80%)
DMF

-

-

NC

10

0.80

25

0.90

50

0.80

759-73-9

AOO

+

+

1.1

0.25

1.00

1

2.70

10

22.30

684-93-5

AOO

+

+

0.05

0.05

2.7

0.1

7.1

0.25

15.4

764-85-2

AOO

+

+

1.8

5

12.70

10

19.40

25

20.90

Octanoic acid124-07-2

AOO

-

-

NC

10

0.70

25.0

1.00

50.0

1.60

Oleyl methane
sulfonateOxalic acid

3570909-2
144-62-7

AOO

+

+

25

5

1.00

10.0

1.30

25.0

3.00

DMF

+

+

15

5

2.40

10

2.80

25

3.40

Oxazolone

AOO

+

+

0.003

0.003

2.90

0.005

4.90

0.01

12.00

Palmitoyl chloride

1564646-5
112-67-4

AOO

+

+

8.8

5

2.10

10

3.30

25

4.50

Penicillin G

61-33-6

DMSO

+

+

30

2.5

1.00

5.0

1.00

10

1.40

Pentachlorophenol

87-86-5

DMSO

+

+

20

10

2.10

25.0

3.50

50.0

5.40

Perillaldehyde

AOO

+

+

4.0

0.5

1.20

1.0

1.10

2.5

0.90

Phenyl benzoate

2111-753
93-99-2

AOO

+

+

20

5

2.30

10

2.10

25

3.50

Phenylacetaldehyde

127-78-1

AOO

+

+

3.0

1

0.70

2.5

1.80

5.0

p-Methylhydrocinnamic aldehyde
Potassium
dichromate
Propylene glycol

5406-122
7778-509
57-55-6

AOO

+

+

14

2.5

1.20

5

1.40

DMSO

+

+

0.08

0.025

1.60

0.05

1.40

dH2O

-

-

NC

50

1.20

100.0

1.60

Propylparaben

94-13-3

AOO

-

-

NC

5

1.40

10

p-tert-Butyl-aethylhydrocinnamal
Pyridine

80-54-6

AOO

+

+

19

1

1.3

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)

110-86-1

AOO

+

+

72

25

5989-275

AOO

+

+

69

25

Substance Name

Methyl(2sulfomethyl)octadecanoate
Methyl-2nonynoate
Methyl-4hydroxybenzoate
N-Ethyl-Nnitrosourea
N-Methyl-Nnitrosourea
Nonanoyl chloride

R(+)-Limonene

CASRN

111-80-8

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

0.025

22.00

0.05

33.00

25.0

2.10

50.0

6.60

5.0

4.30

7.80

10

8.80

25.0

19.00

10

2.60

25

4.2

50

10.7

0.1

3.80

0.25

5.30

0.5

16.10

1.00

25.0

1.30

2.5

2.5

10

2

25

3.7

50

9.3

1.10

50.0

2.30

100.0

3.90

1.8

50

2.4

100

4

D-123

6
Conc.
(%)

6 SI

Data Source

Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

Saccharin

81-07-2

DMSO

-

-

NC

25

1.30

50.0

1.30

75.0

1.50

Salicylic acid

69-72-7

AOO

-

-

NC

5

0.80

10

1.50

25.0

2.50

Sodium lauroyl
lactylate
Sodium laurylsulfateSodium-3,3,5trimethylhexanoyloxybenzenesulfonate
Streptomycin
sulfate1355775-0
151-21-3

AOO

+

+

15

5

1.40

10.0

2.50

25.0

3.90

DMF

+

+

14

1

0.90

2.5

1.10

5.0

1.70

9461291-6

DMSO

+

+

6.4

5

2.30

10.0

4.80

25.0

7.80

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

10.0

2.60

20.0

3.50

25.0

2.00

50.0

1.90

DMF

-

-

NC

2.5

1.20

5.0

1.40

10

1.30

Sulfanilamide

3810-740
63-74-1

DMF

-

-

NC

10.0

1.00

25.0

1.00

50.0

0.90

Sulfanilic acid121-57-3

DMF

-

-

NC

5.0

1.50

10

1.90

25.0

2.20

Tartaric acid

87-69-4

DMF

-

-

NC

5

1.00

10

0.90

25

1.50

Tetramethyl
thiuram disulfide
trans-2-Decenal

137-26-8

AOO

+

+

5.2

2.5

2.40

5.0

2.90

10

5.10

AOO

+

+

2.5

0.5

1.30

1

1.10

2.5

3.00

5

6

10

9.5

AOO

+

+

5.5

0.5

1.2

1

1.2

2.5

2.3

5

2.6

10

6.4

trans-Anethol

3913-711
6728-263
104-46-1

AOO

+

+

2.3

4.5

13.50

9

24.7

22.6

37.3

Undec-10-enal

112-45-8

AOO

+

+

6.8

5.0

1.70

10

5.30

25.0

7.50

50.0

8.70

75.0

8.80

Vanillin

121-33-5

AOO

-

-

NC

2.5

0.90

5.0

1.40

10

1.50

25.0

1.20

50.0

1.40

Vinylidene
dichloride
Vinylpyridine

75-35-4

AOO

-

-

NC

10

0.80

25.0

0.80

50.0

0.90

1337-811
910-99-6

AOO

+

+

1.6

2.5

7.40

5.0

14.20

10

14.80

DMF

-

-

NC

2.5

1.30

5

1.27

10

0.89

trans-2-Hexenal(16-beta)-21(Acetyloxy)-17hydroxy-16methylpregna1,4,9(11)-triene3,20-dione

D-124

6
Conc.
(%)

6 SI

Data Source

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

(1r)-1,2,3,4Tetrahydro-6,7dimethoxy-2methyl-1-[(3,4,5trimethoxyphenyl)
methyl]isoquinoline [r-(r*,r*)]-2,3bis(benzoyloxy)butanedioate (1:1)(2-Bromo-5propoxyphenyl)(2hydroxy-4methoxyphenyl)methadone(2e)-2-[(2-Formyl4-hydroxyphenyl)methylidene]butanedioic acid

10483201-1

DMF

-

-

NC

10

0.92

25

1.12

50

1.27

Glaxo SmithKline,
Submitted by M.J.
Olson

19096545-8

ACE

-

-

NC

0.5

1.10

5

0.90

50

1.70

Glaxo SmithKline,
Submitted by M.J.
Olson

77305957-7

DMF

+

+

48

0.5

0.80

5

1.45

50

3.08

Glaxo SmithKline,
Submitted by M.J.
Olson

(2-Oxo-1-phenylpyrrolidin-3yl)(triphenyl)phosphonium
bromide(2R,4S)-4-(4Acetyl-1piperazinyl)-n{(1r)-1-[3,5bis(trifluoromethyl)phenyl]ethyl}-2-(4-fluoro2-methylphenyl)-nmethyl-1piperidinecarboxamide
monomethanesulfonate(2S,4S)-1-[(2s)-2Amino-3,3-bis(4fluorophenyl)-1oxopropyl]-4fluoro-2-pyrrolidine carbonitrile

14877618-5

DMSO

-

-

NC

2.5

1.64

5

2.45

10

1.40

Glaxo SmithKline,
Submitted by M.J.
Olson

41491030-8

DMF

-

-

NC

2.5

1.07

5

0.90

10

1.47

Glaxo SmithKline,
Submitted by M.J.
Olson

48336958-0

DMSO

-

-

NC

5

0.99

10

1.60

25

2.44

Glaxo SmithKline,
Submitted by M.J.
Olson

D-125

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

(3as,4r,5s,6s,8r,9r,9ar,10r)-6Ethenyldeca-hydro5-hydroxy-4,6,9,10tetramethyl-1-oxo3a,9-propano-3ahcyclopentacycloocten-8-yl [[(3exo)-8-methyl-8azabicyclo[3.2.1]oct-3yl]thio]-acetate(3as,4r,5s,6s,8r,9r,9ar,10r)-6Ethenyldeca-hydro5-hydroxy-4,6,9,10tetra-methyl-1-oxo3a,9-propano-3ahcyclopentacyclooct
en-8-ylhydroxyacetate(3-Endo)-8-methyl8-azabicyclo[3.2.1]octan-3-ol

22445266-8

AOO

-

-

NC

0.5

1.00

2

1.40

5

1.20

Glaxo SmithKline,
Submitted by M.J.
Olson

125-65-5

DMF

-

-

NC

10

1.38

25

1.42

50

1.56

Glaxo SmithKline,
Submitted by M.J.
Olson

120-29-6

DMF

+

+

8.9

10

4.11

25

12.85

50

26.14

Glaxo SmithKline,
Submitted by M.J.
Olson

15692809-5

DMF

-

-

NC

1

0.78

3

0.91

10

0.95

31368208-5

DMF

-

-

NC

2.5

1.46

7.5

0.92

25

1.04

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

82095738-8

DMF

-

-

NC

5

0.74

10

1.41

25

1.62

(3r,3as,6ar)Hexahydrofuro[2,3-b]furan-3-ol(3r,3as,6ar)Hexahydrofuro[2,3-b]furan-3-yl[(1s,2r)-3-[(1,3benzodioxol-5ylsulfonyl)(2methylpropyl)amino]-2-hydroxy1-[[4-[(2-methyl-4thiazolyl)methoxy]
phenyl]methyl]propyl]carbamate(3R6R)-3-(2,3Dihydro-1h-inden2-yl)-1-[(1r)-1-(2methyl-1,3-oxazol4-yl)-2-(4morpholinyl)-2oxoethyl]-6-[(1s)-1methylpropyl]-2,5piperazinedione

D-126

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

(4r,5s)-(-)-1,5Dimethyl-4-phenyl2-imidazolidinone(4r,5s)-1,5Dimethyl-3-(1-oxo2-propenyl)-4phenyl-2-imidazolidinone(4S)-1-(tertButoxycarbonyl)-4fluoro-lprolinamide(4S)-1-(tertButoxycarbonyl)-4fluoro-l-proline

9284165-1

DMSO

-

-

NC

10

1.80

25

2.10

50

1.90

13910923-2

ACE

+

+

0.004

0.5

14.10

5

19.50

50

16.80

42684422-6

DMF

-

-

NC

2.5

1.12

5

0.99

10

1.60

Glaxo SmithKline,
Submitted by M.J.
Olson

20386613-1

DMF

-

-

NC

5

0.88

10

0.82

25

1.29

Glaxo SmithKline,
Submitted by M.J.
Olson

19096547-0

DMF

-

-

NC

0.5

0.78

5

1.14

10

1.39

Glaxo SmithKline,
Submitted by M.J.
Olson

33447760-0

DMF

-

-

NC

10

1.32

25

1.41

50

1.63

18494486-3

PG

-

-

NC

5

1.06

10

1.00

25

1.19

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

3789-591

AOO

-

-

NC

0.5

0.81

5

0.69

50

0.99

41491013-7

DMF

-

-

NC

10

1.79

25

1.85

50

2.10

(4S,5R)-1[(1R,2R,3S)-3-(1,3Benzodioxol-5-yl)1-(2-benzyloxy-4methoxyphenyl)-1hydroxy-6propoxy-2indanoyl]-3,4dimethyl-5-phenyl2-imidazolidinone(Alpha-r)-n-alphadimethyl-3,5bis(trifluoro-methyl(R,S)-3-Amino2,3,4,5-tetrahydron-(1-methylethyl)2,4-dioxo-n,5diphenyl-1h-1,5benzodiazepine-1acetamide(s)-(-)-1Phenylpropylamine(S)-2-(4-Fluoro-2methylphenyl)4piperidinone (s)alphahydroxybenzeneacetic acid salt

D-127

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

[3aS-(3aAlpha,4beta,5alpha,6alpha,8beta,
9alpha,9abeta,
10S*)]-6Ethenyldecahydro5-hydroxy-4,6,9,10tetramethyl-1-oxo3a,9-propano-3aHcyclopentacyclooct
en-8-yl[(methylsulfonyl)oxy]acetate[4-(Ethoxymethyl)2,6dimethoxyphenyl]boronic acid
[4S-[1(E),4Alpha,5alpha]]-1-[3-[2-[4Methoxy-2(phenylmethoxy)benzoyl]-4propoxyphenyl]-1oxo-2-propenyl]3,4-dimethyl-5phenyl-2-imidazolidinone
1-(4-Ethoxyphenyl)-2-[4(methylsulfonyl)phenyl]ethanone
1,1-Dimethylethyl[(1s)-1-[bis(4fluorophenyl)methyl]-2-[(2s,4s)2-cyano-4-fluoro-1pyrrolidinyl]-2oxoethyl]carbamate
1,1-Dimethylethyl[(1s)-2-[4-[(2methyl-4thiazolyl)methoxy]
phenyl]-1-(2s)oxiranylethyl]carbamate

6092438-1

DMF

-

-

NC

10

2.46

25

2.40

50

1.54

Glaxo SmithKline,
Submitted by M.J.
Olson

59124950-2

DMF

-

-

NC

10

0.87

25

0.58

50

1.00

Glaxo SmithKline,
Submitted by M.J.
Olson

19096546-9

DMF

-

-

NC

0.05

0.88

0.5

0.59

5

0.56

Glaxo SmithKline,
Submitted by M.J.
Olson

34641300-1

DMSO

-

-

NC

1

1.82

2.5

2.34

5

1.89

Glaxo SmithKline,
Submitted by M.J.
Olson

48336824-7

AOO

-

-

NC

10

0.97

25

0.81

50

0.99

Glaxo SmithKline,
Submitted by M.J.
Olson

31368092-1

DMF

-

-

NC

5

1.04

10

0.84

25

1.16

Glaxo SmithKline,
Submitted by M.J.
Olson

D-128

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

1,1-Dimethylethyl
3-[[[[(3s)-2,3,4,5tetrahydro-1-[2-[(1methylethyl)phenyl
amino]-2oxoethyl]-2,4dioxo-5-phenyl-1h1,5-benzodiazepin3-yl]amino]
carbonyl]amino]
benzoate
1,2,3,5,6,7Hexahydro-2thioxo-4hcyclopentapyrimidin-4-one
1,3Benzodioxazole-5sulphonyl chloride
1-[3-(Cyclopentyloxy)-4-methoxyphenyl]-4oxocyclohexane
carbonitrile
1-[5-[(4Fluorophenyl)methyl]-2furanyl]ethanone
14-Hydroxynormorphinone

30536694-3

DMF

+

+

38

10

2.10

25

2.20

50

3.80

Glaxo SmithKline,
Submitted by M.J.
Olson

3556327-0

PG

-

-

NC

5

0.63

10

1.71

25

1.37

Glaxo SmithKline,
Submitted by M.J.
Olson

11501010-1

AOO

+

+

0.4

10

13.54

25

16.56

50

16.76

15263047-2

DMSO

-

-

NC

10

2.70

25

2.80

50

2.20

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

28057134-8

AOO

-

-

NC

0.5

1.00

5

1.00

50

1.20

Glaxo SmithKline,
Submitted by M.J.
Olson

8411646-1

PG

+

+

8.4

5

1.20

10

3.88

25

6.24

2-(3,4-Dimethylphenyl)-5--methyl2,4-dihydropyrazol3-one
2-(4Ethoxyphenyl)-3[4-(methylsulfonyl)phenyl]pyrazolo[1,5-b]pyridazine
2-(4-Oxopentyl)1h-isoindole1,3(2h)-dione
2-(Benzyl)tertbutyl)amino)-1(alpha,4-dihydroxym-tolyl)ethane

1804864-1

DMF

+

+

IDR3

2.5

4.41

7.5

4.82

25

8.46

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

22114846-5

DMF

-

-

NC

5

0.98

10

0.97

25

0.94

Glaxo SmithKline,
Submitted by M.J.
Olson

3197-259

AOO

-

-

NC

0.25

0.58

2.5

1.54

25

0.67

2408503-8

DMF

-

-

NC

10

0.67

25

1.10

50

1.28

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

D-129

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

2,3,4,5-Tetrahydron-(1-methylethyl)2,4-dioxo-n,5diphenyl-3[(phenylmethoxy)imino]-1h-1,5benzodiazepine-1acetamide
2,3-Dimethyl-2hindazol-6-amine

30536697-6

DMF

-

-

NC

5

1.18

10

1.76

25

1.67

Glaxo SmithKline,
Submitted by M.J.
Olson

44473172-0

DMF

-

-

NC

5

0.74

10

0.95

25

1.16

2,4-Dichloropyrimidine

3934-201

DMF

+

+

0.7

0.25

0.76

0.75

3.46

2.5

8.64

2,6-Dimethoxy-4methyl-5-[3(trifluoromethyl)phenoxy]-8quinolinamine
2,6-Dimethoxy-4methyl-8-nitro-5[3(trifluoromethyl)phenoxy]quinoline
2-[(Benzyloxy)imino]malonic acid10663586-3

AOO

-

-

NC

0.5

2.30

5

2.10

50

1.80

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

18974615-4

PG

+

+

3.5

3

2.90

10

4.29

30

5.34

Glaxo SmithKline,
Submitted by M.J.
Olson

30536696-5

AOO

-

-

NC

10

1.17

25

1.88

50

2.40

2-[1-(4Bromophenyl)-1phenylethoxy]-n,ndimethylethanaminehydrochloride
2-Amino-diphenylamine

1397728-1

DMF

+

+

5.5

0.5

2.38

5

2.88

15

5.08

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

534-85-0

AOO

+

+

0.5

10

10.20

25

12.40

50

7.70

2-Aminoethylmethylsulfone

4977320-8

-

-

NC

10

0.40

25

0.30

50

0.30

2-Benzyl-tertbutylamino-3'hydroxymethyl-4'hydroxyacetophenonehydrochloride
2-Bromo-5hydroxybenzaldehyde

2408508-3

0.5%
Tween
80 in
H 2O
DMF

+

+

22

0.5

0.96

5

1.54

50

5.44

Glaxo SmithKline,
Submitted by M.J.
Olson

AOO

+

+

2.6

0.5

1.25

5

4.93

50

21.40

Glaxo SmithKline,
Submitted by M.J.
Olson

2973-800

D-130

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

2-Bromo-5propoxybenzoic
acid
2-Chloro-6methoxy-4methylquinoline
2-chloro-1-[(3fluorophenyl)methoxy]-4nitrobenzene
2-Nitro-4(propylthio)benzenamine

19096543-6

ACE

-

-

NC

0.5

0.60

5

0.70

50

1.10

6340-552

DMF

-

-

NC

0.1

1.07

1

0.98

10

1.11

44388299-3

AOO

+

+

IDR3

5

3.96

10

2.62

25

3.22

5439389-4

AOO

Equiv

+

IDR3

0.5

1.974

5

1.344

15

8.005

Glaxo SmithKline,
Submitted by M.J.
Olson

3,4-Dichloroanilinehydrochloride

95-76-1

DMF

+

+

18

0.25

1.02

2.5

1.75

25

3.53

3-[(2r)-3-[[2-(2,3Dihydro-1h-inden2-yl)-1,1-dimethylethyl]amino]-2hydroxypropoxy]4,5-difluorobenzene propanoicacid
3'-[(2z)-[1-(3,4Dimethylphenyl)1,5-dihydro-3methyl)-5-oxo-4hpyrazol-4ylidene]hydrazino]2'-hydroxy-[1,1'biphenyl]-3carboxylic acid,compound with 2aminoethanol (2:1)
3-[4-[(6Bromohexyl)oxy]butyl]benzenesulfonamide
3-Chloro-4fluorobenzoyl
chloride
3-Fluoro-5-(3pyridinyl)benzenamine
3-Hydroxy-2phenyl-4quinolinecarboxylic
acid

75344967-1

DMF

-

-

NC

5

0.71

15

1.02

50

1.28

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

49677562-3

AOO

-

-

NC

5

1.38

15

1.05

50

0.84

Glaxo SmithKline,
Submitted by M.J.
Olson

45234204-0

AOO

-

-

NC

10

1.02

25

0.82

50

0.68

Glaxo SmithKline,
Submitted by M.J.
Olson

6505517-6

PG

+

+

7.8

3

2.24

10

3.36

30

8.99

18163336-3

DMSO

+

+

15

0.5

1.90

5

2.20

50

5.90

485-89-2

DMSO

-

-

NC

0.05

0.56

0.5

0.79

5

1.04

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

D-131

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

3-Hydroxy-4methoxybenzaldehyde
3-Propoxybenzoic
acid621-59-0

DMF

-

-

NC

0.25

0.75

2.5

1.15

25

1.35

19096542-5

ACE

-

-

NC

0.5

1.10

5

1.20

50

1.10

4-(Bromomethyl)benzoic acid ethyl
ester
4'-(Trifluoromethyl)-[1,1'biphenyl]-4carboxaldehyde
4-[4-[[(3R)-1Butyl-3-[(r)cyclohexylhydroxymethyl]2,5-dioxo-1,4,9triazaspiro[5.5]undec-9yl]methyl]phenoxy]
benzoic acid
4-Amino-3nitrophenyl
thiocyanate
4-Bromo-1phthalimidopentane

2649694-6

AOO

+

+

IDR3

0.5

11.73

5

12.87

50

ND6

9003534-0

DMF

-

-

NC

1

1.36

3

1.55

10

2.58

46144359-4

DMSO

-

-

NC

2.5

1.07

5

1.27

10

1.63

Glaxo SmithKline,
Submitted by M.J.
Olson

5402945-7

DMSO

+

+

0.8

0.5

2.30

1

3.32

5

3.55

5935362-7

ACE

+

+

27

0.5

1.00

5

1.10

50

4.20

4-Chloro-6iodoquinazoline

9855631-1

AOO

+

+

IDR3

5

11.30

10

9.30

25

17.30

4-Fluoro-2pyrrolidinecarboxamide
4-Iodo-1phthalimidopentane
5-[[4-[(2,3Dimethyl-2hindazol-6-yl)methylamino]-2pyrimidinyl]amino]-2-methylbenzenesulfonamide
5-Amino-2methylbenzenesulfonamide

74816540-4

DMF

-

-

NC

10

1.22

25

1.15

50

1.03

6346047-9

ACE

+

+

5.0

0.5

1.70

5

3.00

50

9.50

44473152-6

AOO

-

-

NC

5

1.13

10

0.91

25

0.91

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

6973-097

DMF

-

-

NC

5

1.36

10

1.12

25

1.42

D-132

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

5-Chloro-2,6dimethoxy-4methyl-8nitroquinoline
5-Chloro-2,6dimethoxy-4methylquinoline
5'-Chloro-2'hydroxy-3'-nitro[1,1'-biphenyl]-3carboxylic acid
5-Chloro-6methoxy-4-methyl8-nitro2(1h)quinolinone
5-Methoxy-2-nitro4-(trifluoromethyl)
benzene acetonitrile

18974621-2

DMSO

+

+

IDR3

0.5

8.00

5

7.00

10

7.50

Glaxo SmithKline,
Submitted by M.J.
Olson

18974619-8

DMSO

-

-

NC

0.5

0.90

5

0.70

25

1.00

37659258-4

DMF

+

+

20

5

1.37

15

2.83

50

3.96

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

18974623-4

PG

+

+

IDR3

2.5

10.82

5

9.86

10

10.72

Glaxo SmithKline,
Submitted by M.J.
Olson

17889677-0

DMSO

-

-

NC

0.5

1.30

5

1.50

50

1.60

Glaxo SmithKline,
Submitted by M.J.
Olson

5-Methoxy-6(trifluoromethyl)2,3-dihydro-1hindole
6-(Diethylamino)1-hexanol

17889679-2

DMSO

+

+

37

0.5

1.10

5

1.30

50

3.70

Glaxo SmithKline,
Submitted by M.J.
Olson

0694712-2

PG

+

+

10

3

0.79

10

2.92

30

25.50

6-(Trifluoromethyl)-2,3dihydro-5-methyl1h-indole,hydrochloride
6-[(2-Methyl-3pyridinyl)oxy]-3pyridinamine
6-Chloro-1-hexanol

28012124-6

ETOH(100%)

-

-

NC

0.5

1.10

5

1.00

50

1.20

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

18163342-1

DMSO

+

+

45

0.5

1.00

5

1.40

50

3.20

2009-838

AOO

-

-

NC

5

2.35

15

1.66

50

1.92

6Diethylaminohexyl
bromidehydrobromide
6-Iodo-quinazolin4-ol

6499314-2

PG

+

+

5.3

3

1.76

10

5.46

30

14.69

1606408-7

DMF

-

-

NC

1

0.72

2.5

1.16

5

0.93

6-Methoxy-4methyl-2(1H)quinolinone

5342-234

PG

-

-

NC

3

1.21

10

1.49

30

1.32

D-133

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

7-[(4z)-3(Aminomethyl)-4(methoxyimino)-1pyrrolidinyl]-1cyclopropyl-6fluoro-1,4-dihydro4-oxo-1,8naphthyridine-3carboxylic acid,monomethanesulfonate
8-[(4-Phthalimido1-methylbutyl)
amino]-2,6dimethoxy-4methyl-5-(3trifluoromethylphenoxy)quinoline
8-Amino-6methoxy-4methylquinoline
8-Chloro-3-pentyl3,7-dihydro-1hpurine-2,6-dione21035353-0

DMSO

+

+

8.6

0.1

0.75

1

1.38

10

3.30

Glaxo SmithKline,
Submitted by M.J.
Olson

10663587-4

PG

-

-

NC

3

2.22

10

1.50

30

1.49

Glaxo SmithKline,
Submitted by M.J.
Olson

5751421-3

PG

-

-

NC

3

1.23

10

2.83

30

2.50

86289290-8

DMF

+

+

32

5

2.00

15

0.85

50

5.29

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

8-Hydroxy-5-[(1r)1-hydroxy-2-[[2-[4[(6-methoxy[1,1'biphenyl]-3yl)amino]phenyl]ethyl]amino]ethyl]2(1h)-quinolinone
Adipic acid

53008487-8

DMF

-

-

NC

5

0.96

10

2.34

25

1.58

Glaxo SmithKline,
Submitted by M.J.
Olson

124-04-9

DMSO

-

-

NC

10

1.01

25

0.93

50

0.79

16580695-1

DMF

+

+

45

0.5

4.72

5

2.78

50

3.03

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

118-92-3

AOO

-

-

NC

10

0.90

25

1.10

50

1.40

15325965-5

DMSO

-

-

NC

0.5

1.16

5

1.27

10

1.28

71-30-7

PG

-

-

NC

5

0.40

10

0.90

25

0.70

Alpha-(ptoluenesulfonyl)-4fluorobenzylisonitrile
Anthranilic acidcis-4-Cyano-4-[3(cyclopentyloxy)-4methoxyphenyl]
cyclohexanecarboxylic
acid
Cytosinehemihydrate

D-134

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

Dimethyl 4-cyano4-(3cyclopentyloxy-4methoxyphenyl)pimelate
Dimethyl carbonate

15263048-3

AOO

-

-

NC

0.5

1.66

5

1.59

50

1.76

Glaxo SmithKline,
Submitted by M.J.
Olson

616-38-6

AOO

-

-

NC

0.5

0.64

5

0.69

50

1.71

Endo-tropine-3mesylate

3513097-3

DMF

+

+

4.4

5

3.45

10

5.98

25

25.06

Ethyl (3-endo)-8methyl-8azabicyclo[3.2.1]octane-3-acetate
Ethyl (z)-alpha-[[2(1,1dimethylethoxy)1,1-dimethyl-2oxoethoxy]imino]2[(triphenylmethyl)amino]- 4thiazoleacetate
Ethyl 1h-1,2,4triazole-3carboxylate
Ethyl 2,6-dichloro5-fluoro-beta-oxo3pyridinepropanoate
Ethyl 4iodobenzoate

5688011-6

DMF

+

+

5.5

10

6.77

25

12.58

50

ND6

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

6867265-1

Butanone

-

-

NC

2.5

0.80

5

1.32

10

0.92

Glaxo SmithKline,
Submitted by M.J.
Olson

6492204-9

DMF

-

-

NC

0.25

1.00

2.5

1.20

25

1.00

9656804-6

AOO

-

-

NC

0.25

0.59

2.5

1.33

25

1.97

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

5193441-9

AOO

+

+

8.0

0.5

1.10

5

2.20

50

14.30

Isopropyl
dicyandiamide

3569536-4

DMF

-

-

NC

0.25

1.36

2.5

1.35

25

1.24

m-Chloropropiophenone

3484135-5

AOO

-

-

NC

10

0.86

25

0.73

50

1.25

Methyl 4(bromomethyl)benzoaten-(2-Chloro-4pyrimidinyl)-2,3drimethyl-2hindazol-6-amine

2417-723

AOO

+

+

IDR3

0.5

26.83

5

18.47

50

ND6

44473174-2

DMF

-

-

NC

1

0.80

2.5

0.74

5

1.07

D-135

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

n-(2-Chloro-4pyrimidinyl)-n,2,3trimethyl-2hindazol-6-aminen-(3,4Dichlorophenyl)-n'(1-methylethyl)imidodicarbonimidic diamide
monohydrochloriden-(4Methoxyphenyl)-3oxobutanamiden-[(1,1Dimethylethoxy)carbonyl]-ltyrosine, ethyl estern-[(1-Butyl-4piperidinyl)methyl]-3,4-dihydro-2h[1,3]oxazino[3,2a]indole-10carboxamiden-[2(Diethylamino)ethyl]-2-[[(4fluorophenyl)methyl]thio]4,5,6,7-tetrahydro4-oxo-n-[[4'(trifluoromethyl)[1,1'-biphenyl]-4yl]methyl]-1hcyclopentapyrimidine-1-acetamide
n-[2-Benzyloxy-5(2-bromo-1hydroxy-ethyl)phenyl]-formamiden-{[(1,1Dimethylethyl)oxy]
carbonyl}-4-fluorobeta-(4fluorophenyl)-lphenylalanine
1-Aminopyridazinium iodide

44473175-3

DMF

-

-

NC

1

1.28

2.5

1.47

5

2.15

Glaxo SmithKline,
Submitted by M.J.
Olson

1553776-5

DMF

+

+

1.3

0.25

1.64

2.5

4.61

25

ND6

Glaxo SmithKline,
Submitted by M.J.
Olson

5437-989

PG

+

+

2.2

3

3.10

10

3.49

30

10.33

7259477-5

AOO

-

-

NC

10

1.18

25

1.09

50

0.64

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

15281162-6

AOO

-

-

NC

0.5

0.80

5

1.20

25

1.30

Glaxo SmithKline,
Submitted by M.J.
Olson

35605734-6

EtOH/
dH2O
(4:1)

+

+

11

5

1.11

10

2.43

25

12.71

Glaxo SmithKline,
Submitted by M.J.
Olson

20167759-0

DMF

-

-

NC

10

0.98

25

0.68

50

0.97

Glaxo SmithKline,
Submitted by M.J.
Olson

48105529-2

DMF

-

-

NC

10

1.47

25

2.41

50

2.28

Glaxo SmithKline,
Submitted by M.J.
Olson

3507304-2

DMF

-

-

NC

10

1.17

25

1.43

50

1.25

Glaxo SmithKline,
Submitted by M.J.
Olson

D-136

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

n-Isopropyl-nphenyl-2-(2phenylaminophenylamino)acetamide
Oripavine

16145590-9

DMF

+

+

2.1

2.5

3.80

5

7.30

10

6.60

Glaxo SmithKline,
Submitted by M.J.
Olson

467-04-9

DMF

+

+

8.6

2.5

1.90

5

2.50

10

3.20

41490998-1

DMF

-

-

NC

10

1.67

25

1.85

50

1.47

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson

25287335-1

DMF

+

+

IDR3

2.5

3.48

7.5

3.40

25

3.54

Glaxo SmithKline,
Submitted by M.J.
Olson

140-90-9

PG

+

+

7.3

5

1.57

10

4.70

25

9.42

tert-Butyl-3aminobenzoate

9214682-2

DMF

-

-

NC

10

1.24

25

1.02

50

1.08

Trienol

1350415-9

DMF

-

-

NC

5

1.40

10

1.10

25

1.00

Veratraldehyde

120-14-9

AOO

+

+

3.2

0.5

2.63

5

3.24

50

3.47

Basil oil8015-734

EtOH/
DEP
(1:3)

+

+

IDR3

2.5

3.00

5.0

3.00

10.0

8.00

25.0

17.60

50.0

25.20

Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Glaxo SmithKline,
Submitted by M.J.
Olson
Lalko & Api(2006), Submitted
by A. Api (RIFM)

Citral5392-405

+

+

6.3

2.5

2.80

5.0

2.30

10.0

5.10

25.0

11.40

50.0

22.10

Citronella oil

8000-291

EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)

-

-

NC

2.5

1.40

5.0

0.90

10.0

1.20

25.0

1.20

50.0

2.70

Clove bud oil

8000-348

EtOH/
DEP
(1:3)

+

+

7.1

1.0

1.10

2.5

1.80

5.0

2.50

10.0

3.70

25.0

5.90

Lalko & Api(2006), Submitted
by A. Api (RIFM)

Clove leaf oil

8015-972

EtOH/
DEP
(1:3)

+

+

8.0

2.5

1.60

5.0

1.50

10.0

4.00

25.0

9.50

50.0

11.40

Lalko & Api(2006), Submitted
by A. Api (RIFM)

Phenylmethyl 2-(4fluoro-2methylphenyl)-4oxo-3,4-dihydro1(2h)-pyridinecarboxylaterel-(3r,3as,6ar)Hexahydrofuro[2,3b]furan-3-yl 4nitrophenyl
carbonate
Sodium ethyl
xanthate

D-137

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

Clove stem oil

8015-983

+

+

7.0

1.0

1.60

2.5

1.70

5.0

2.20

10.0

4.20

25.0

8.90

Eugenol

97-53-0

EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)

+

+

5.5

2.5

1.20

5.0

2.70

10.0

6.00

25.0

14.30

50.0

19.40

Geraniol

106-24-1

+

+

12

2.5

1.70

5.0

2.40

10.0

2.80

25.0

4.80

50.0

6.00

Geranium oil

8000-462

EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)

-

-

NC

2.5

1.20

5.0

0.70

10.0

1.70

25.0

1.80

50.0

2.80

Jasmine absolute(Grandiflorum)8022-966

EtOH/
DEP
(1:3)

+

+

5.9

1.0

1.20

2.5

1.80

5.0

2.00

10.0

7.40

25.0

11.80

Lalko & Api(2006), Submitted
by A. Api (RIFM)

Jasmine absolute(Sambac)8022-966

+

+

36

10

1.70

25.0

2.50

50.0

3.60

75.0

10.80

100.0

16.20

Lemongrass oil

8007-021

EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)

+

+

6.5

2.5

0.90

5.0

2.10

10.0

5.10

25.0

10.30

50.0

13.10

Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)

Litsea cubeba oil

6885599-2

+

+

8.5

2.5

2.00

5.0

2.30

10.0

3.30

25.0

7.90

50.0

16.00

Palmarosa oil

8014-195

+

+

9.5

2.5

1.10

5.0

2.10

10.0

3.10

25.0

3.60

50.0

5.00

Spearmint oil

6891746-4

EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)

+

+

8.2

0.5

1.20

1.0

1.10

2.5

1.20

5.0

1.90

10.0

3.60

Ylang Ylang(Extra)8006-813

+

+

6.8

0.5

1.50

1.0

1.40

2.5

2.10

5.0

2.50

10.0

3.90

Ylang Ylang (III)8006-813

EtOH/
DEP
(1:3)
EtOH/
DEP
(1:3)

-

-

NC

0.5

1.30

1.0

1.70

2.5

2.10

5.0

2.60

10.0

2.60

AOO

-

-

NC

1

1.30

5

1.80

10

1.20

15

2.30

25

2.90

(1R,4R)-4Isopropenyl-1methyl-2methylenecyclohexane

D-138

6
Conc.
(%)

6 SI

Data Source

Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)

Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)
Lalko & Api(2006), Submitted
by A. Api (RIFM)
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

(3S,6R)-3isopropyl-6methylcyclohexene

5113-939

AOO

-

-

NC

1

0.84

10

1.00

25

2.90

-

-

NC

1

1.10

5

0.87

10

0.78

AOO

+

+

7.3

0.5

0.94

5

1.90

15

6.60

(4Z)-2-Methyl-6methyleneoct-4-ene(5R)-5-Isopropenyl2-methyl-1methylene-2cyclohexene

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

15

0.89

25

2.10

2,2-bis-[4-(2hydroxy-3methacryloxypropoxy)phenyl)]propane

1565-942

AOO

+

+

45

35

2.00

75

5.90

2,4-DiaminophenoxyethanolHCl6642295-5

AOO

+

+

5.5

1

1.60

2,5

1.60

5

2.70

10

5.70

25

8.30

2,4-Hexadienal

142-83-6

AOO

+

+

3.5

0.5

0.90

1

1.50

2.5

2.20

5

4.20

10

14.80

AOO

-

-

NC

50

1.20

100

1.20

AOO

+

+

21

5

1.50

10

1.60

25

3.60

50

5.70

3,4epoxyclohexylethyl-cyclopolymethylsiloxane
4,4-Dibromobenzil

3557847-3

D-139

6
Conc.
(%)

6 SI

Data Source

LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

4-Isopropyl-1methylenecyclohexane

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

AOO

-

-

NC

1

1.20

10

0.71

25

1.40

5-Methyl-2-phenyl2-hexenal

2183492-4

AOO

+

+

4.4

0.5

1.00

1.0

1.30

2.5

0.50

a-Phellandrene

99-83-2

AOO

+

+

5.4

1.0

1.10

10

5.00

25

28.00

a-Terpinene

99-86-5

AOO

+

+

8.9

1

1.1

5

1.5

10

Bandrowski’s base2004827-5

AOO

+

+

0.02

0.01

1.10

0.025

3.10

beta-Phenylcinnamaldehyde

1210-395

AOO

+

+

0.6

0.1

2.00

0.25

AOO

+

+

16

25

3.70

AOO

+

+

NC

1.0

1.10

Bis-3,4epoxycyclohexylethyl-phenylmethylsilane (PhSil)b-Phellandrene

555-10-2

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

5

3.80

10

17.70

3.40

15

8.90

25

23.00

0.05

5.70

0.1

6.50

0.25

5.60

2.30

0.5

1.90

1

5.90

2.5

10.60

35

4.20

50

7.90

10

4.80

20

23.00

D-140

6
Conc.
(%)

6 SI

Data Source

LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

b-Terpinene

99-84-3

AOO

-

-

NC

1

1.4

10

1.30

25

2.1

Butyl acrylate141-32-2

AOO

+

+

11

1

0.70

2.5

1.30

5

1.50

10

2.50

25

8.70

Chlorothalonil

1897-456

DMF

+

+

0.004

0.003

2.10

0.01

9.40

0.03

13.80

0.1

18.40

0.3

27.20

Ethyl hexyl acrylate

103-11-7

AOO

+

+

9.7

0.5

1.10

1

1.20

2.5

0.90

5

1.20

10

3.10

Iodopropynyl
butylcarbamate

8797728-4

AOO

+

+

0.9

0.1

0.70

1

3.40

5

4.20

10

12.00

Linalool alcohol

78-70-6

AOO

-

-

NC

1

1.00

10

1.30

30

1.30

AOO

+

+

9.5

1

1.20

5

2.00

15

4.20

AOO

+

+

20

1

0.80

2.5

0.80

5

1.30

10

1.60

25

3.80

Linalool aldehyde

Methyl acrylate

96-33-3

D-141

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
GerberickICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

Methyl pyruvate

600-22-6

AOO

+

+

2.4

1.0

1.20

2.5

3.10

5.0

4.70

10

8.00

Methylmethacrylate

80-62-6

AOO

+

+

90

10

1.40

30

1.50

50

1.50

75

2.10

100

3.60

Norbornene
fluoroalcohol

30581563-8

AOO

+

+

46

5.0

0.70

10

0.80

25.0

1.90

50

3.20

100

3.70

R-Carvone

2244-168

AOO

+

+

13

6

1.30

12

2.60

20

6.20

R-Carvoxime2051-550

AOO

+

+

0.6

0.1

2.10

1

3.70

5

8.10

Trans-2-methyl-2butenal497-03-0

AOO

-

-

NC

10

1.50

25

1.00

50

1.80

1-Chloro-2dinitrobenzene

97-00-7

AOO

+

+

0.1

0.01

1.17

0.025

1.12

0.05

1.93

0.10

1.95

0.25

7.10

5-Amino-O-Cresol

2835-952

AOO

+

+

7.7

2.5

1.45

5.00

2.77

10.00

3.19

Atrazine

1912-249

ACE

-

-

NC

10

1.29

20.00

1.38

30.00

0.76

Azithromycin

8390501-5

ACE

-

+

IDR3

10

3.72

20.00

1.54

40.00

2.10

D-142

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I.
Kimber, and F.
Gerberick
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
GermolecICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

Butyl acrylate141-32-2

ACE

+

+

24

10

1.00

20.00

2.18

30.00

4.07

Clarithromycin

8110311-9

ACE

-

-

NC

1

1.78

2.00

1.03

4.00

1.18

Dicyclohexylcarbod
iimide538-75-0

ACE

+

+

0.057

0.006

1.03

0.03

1.71

0.06

3.16

Ethyl acrylate

140-885

ACE

-

-

NC

10

0.89

20.00

1.19

30.00

0.91

ACE

+

+

0.13

0.3

1.00

0.10

1.52

0.15

4.13

0.30

4.59

119-368

AOO

-

-

NC

1

0.86

2.50

1.19

5.00

1.16

10.00

1.41

Pentaerythritol
Triacrylate3524-683

ACE

-

-

NC

0.005

1.19

0.01

0.92

0.05

1.68

0.10

2.43

Potassium
dichromate

7778-509

DMSO

+

+

0.2

0.025

1.21

0.05

1.84

0.10

2.22

0.25

3.39

Rifamycin SV

1489739-3

AOO

-

-

NC

3

0.94

10.00

1.02

30.00

1.33

Sodiummetasilicate

6834-920

EtOH
(15%)

-

-

NC

2

0.87

4.00

1.40

6.00

1.29

Trimethylolpropane
Triacrylate

1562589-5

ACE

-

-

NC

0.05

0.96

0.10

0.87

0.25

1.62

2.4-Dinitrobenzene
sulfonic acid
2.4-Dinitrobenzene
sulfonic acid
2.4-Dinitrobenzene
sulfonic acid
2.4-Dinitrobenzene
sulfonic acid
Formaldehyde

89-02-1

H 2O

+

+

15

1

1.70

10

1.50

20

4.40

NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
Ryan et al. (2002)

89-02-1

+

+

6.4

1

0.90

10

4.40

20

11.60

Ryan et al. (2002)

89-02-1

Pluronic
L92
DMF

+

+

0.8

1

4.00

10

16.30

20

18.50

Ryan et al. (2002)

89-02-1

DMSO

+

+

2.0

1

1.70

10

13.70

20

16.10

Ryan et al. (2002)

50-00-0

H 2O

+

+

15

1

1.20

10

2.50

20

3.60

Ryan et al. (2002)

Formaldehyde

50-00-0

+

+

4.2

1

2.00

10

4.80

20

8.80

Ryan et al. (2002)

Formaldehyde
Formaldehyde

50-00-0
50-00-0

Pluronic
L92
DMF
DMSO

+
+

+
+

0.3
0.3

1
1

6.70
7.50

10
10

13.20
16.00

20
20

17.70
17.60

Ryan et al. (2002)
Ryan et al. (2002)

Ethyl-2(Hydroxymethyl)1,3- Propanediol
Triacrylate
Methyl salicylate

D-143

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

6
Conc.
(%)

6 SI

Data Source

NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
NTP Study,
Submitted by D.
Germolec
20.00

1.72

ICCVAM Test Method Evaluation Report: Appendix D, Annex III

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1
SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

Nickel sulfate

7786-814

-

+

2.5

0.25

2.00

0.5

2.40

1

2.80

2.5

3.00

5

2.30

Ryan et al. (2002)

Nickel sulfate

7786-814
7786-814

Pluronic
L92
(1%)
DMF

-

-

NC

0.25

0.90

0.5

1.10

1

1.60

2.5

1.60

5

2.20

Ryan et al. (2002)

DMSO

+

+

4.8

0.25

1.30

0.5

1.40

1

1.40

2.5

1.80

5

3.10

Ryan et al. (2002)

Nickel sulfate
Pluronic L92
Potassium
dichromate

7778-509

Potassium
dichromate
Potassium
dichromate

7778-509
7778-509

6
Conc.
(%)

50

6 SI

2

Data Source

H 2O

-

-

NC

1

1.30

2.5

1.00

5

1.00

10

0.80

25

0.80

Pluronic
L92
(1%)
DMF

+

+

0.2

0.025

1.10

0.05

1.10

0.1

1.40

0.25

4.90

0.5

5.40

Ryan et al. (2002)
Ryan et al. (2002)

+

+

0.03

0.025

2.90

0.05

4.30

0.1

9.10

0.25

15.10

0.5

22.60

Ryan et al. (2002)

DMSO

+

+

0.05

0.025

1.40

0.05

2.50

0.1

9.50

0.25

25.90

0.5

10.10

Ryan et al. (2002)

Abbreviations: ACE = acetone; AOO = acetone: olive oil (4:1 by volume); BGIA = Berufsgenossenschaftliches Institut fur Arbeitsschutz (German Institute for Occupational Safety and Health); CASRN = Chemical Abstract Service
Registry Number; CESIO = Comite Europeen des Agents de Surface et de Leurs Intermediaires Organiques (European Committee of Surfactants and their Organic Intermediates; Conc. = concentration; DBP = dibutyl phosphate; DEP= diethyl phthalate; dH2O = distilled water; DMF = dimethylformamide; DMSO = dimethyl sulfoxide; ECPA = European Crop Protection Association; EFfCI = European Federation for Cosmetic Ingredients; ECPA = European Crop
Protection Association; EtOH = ethanol; H2O = water; LLNA = Local Lymph Node Assay; MEK = methyl ethyl ketone; NC = not calculated because no SI value was ≥ 3 (i.e., substance was a nonsensitizer); ND = no data; NTP =National Toxicology Program; PA/ H2O = pluronic acid/ H2O (1%); PG = propylene glycol; rLLNA = Reduced Local Lymph Node Assay; RIFM = Research Institute for Fragrance Materials; SI = stimulation index; Trad. = traditional
1"+" = Sensitizer; "-" = Non-sensitizer
EC3 represents the estimated concentration needed to produce a stimulation index of three (i.e., a three fold increase in lymphocyte proliferation is observed for the test substance versus the vehicle control substance) and was
calculated using the methods described in Ryan et al. (2007).
3
IDR indicates an insufficient dose response to calculate an EC3 value using the methods in Ryan et al. (2007)
4
Result of initial study
5
Result of second study
6
Data not obtained due to toxicity
2

D-144

ICCVAM Test Method Evaluation Report: Appendix D, Annex IV

Annex IV
Substances in the NICEATM LLNA Database for which an Initial Dose of
10% or Greater Elicited a Negative Result but a Subsequent Higher Dose
Elicited a Positive Response

D-145

ICCVAM Test Method Evaluation Report: Appendix D, Annex 1V[This Page Intentionally Left Blank]

D-146

ICCVAM Test Method Evaluation Report: Appendix D, Annex IV

Substance Name

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1 SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

1-(2',5'Dimethylphenyl)butane-1,3dione

56290-55-2

ACE

+

+

12.5

10

2.30

20

5.10

40

9.50

305366-943

DMF

+

+

37.5

10

2.10

25

2.20

50

3.80

3386-33-2

AOO

+

+

16.3

10

1.70

25

4.80

50

7.30

1-Chlorotetradecane

2425-54-9

AOO

+

+

20.2

10

1.10

25

3.90

50

6.30

1-Iodohexadecane544-77-4

AOO

+

+

19.1

10

1.60

25

3.90

50

6.40

1-Iodononane

4282-42-2

AOO

+

+

24.2

10

1.30

25

3.10

50

4.60

1-Iodotetradecane

19218-94-1

AOO

+

+

13.8

10

1.70

25

6.90

50

9.70

1-Phenyl-2-methylbutane-1,3dione
1-Phenyloctane--1,3-dione

6668-24-2

ACE

+

+

29.1

10

1.70

20

2.00

40

4.20

55846-68-1

ACE

+

+

10.5

10

2.80

20

6.60

40

8.70

2-(4-tertAmylcyclohexyl)acetaldehyde

620159-844

AOO

+

+

36.8

25

2.10

50

4.00

100

9.10

2,2,6,6-Tetramethyl-heptane3,5-dione
2,2-bis-[4-(2-hydroxy-3methacryloxypropoxy)phenyl)]-propane

1118-71-4

ACE

+

+

26.7

10

2.10

20

2.80

40

3.40

1565-94-2

AOO

+

+

45.3

35

2.00

75

5.90

3-Ethoxy-1-(2',3',4',5'tetramethylphenyl)propane-1,3dione
3-Methyleugenol

170928-695

ACE

+

+

33.0

10

1.10

20

1.70

40

3.70

186743-260
362-06-7

AOO

+

+

31.6

11

1.50

27

2.30

54

6.40

ACE

+

+

20.0

10

2.10

20

3.00

40

4.60

140-67-0

AOO

+

+

17.7

10

1.20

25

4.70

50

4.50

5-Methyl-2,3-hexanedione

13706-86-0

AOO

+

+

25.8

25

2.90

50

6.00

100

14.30

5-Methyleugenol

186743-259

AOO

+

+

13.2

11

2.70

27

4.90

54

4.30

1,1-Dimethylethyl 3-[[[[(3s)2,3,4,5-tetrahydro-1-[2-[(1methylethyl)phenylamino]-2oxoethyl]-2,4-dioxo-5-phenyl1h-1,5-benzodiazepin-3yl]amino]carbonyl]amino]benzoate
1-Chlorooctadecane

4,4,4-Trifluoro-1phenylbutane-1,3-dione
4-Allylanisole

D-147

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

Data Source

Gerberick et al.
(2005)

Glaxo SmithKline,
Submitted by M.J.
Olson

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I. Kimber,and F. Gerberick
Gerberick et al.
(2005)

100

8.00

Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)ICCVAM Test Method Evaluation Report: Appendix D, Annex 1V

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1 SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

AOO

+

+

16.9

11

1.90

27

4.90

54

8.30

Atrazine SC

186743-248
1912-24-9

Pluronic
L92 (1%)

+

+

31.3

13

1.80

25

2.80

50

3.60

Butyl acrylate141-32-2

ACE

+

+

24.4

10

1.00

20

2.18

30

4.07

Butyl glycidyl ether

2426-08-6

AOO

+

+

30.9

10

1.40

25

2.20

50

5.60

C15-azlactone

176665-091
176665-115

AOO

+

+

17.8

10

1.80

25

4.10

50

7.50

AOO

+

+

19.0

11

1.70

27

4.30

54

4.60

AOO

-

+

26.4

12

2.50

29

3.10

59

2.50

Substance Name

6-Methyleugenol

C17-azlactone
C19-azlactone

4
Conc.
(%)

4 SI

75

7.10

Cinnamic alcohol

104-54-1

AOO

+

+

20.6

10

1.80

25

3.50

50

3.90

90

5.70

cis-6-Nonenal

2277-19-2

AOO

+

+

22.4

10

1.60

25

3.30

50

4.50

100

13.70

Diethylacetaldehyde

97-96-1

AOO

+

+

76.1

25

1.20

50

0.80

75

2.40

100

16.30

Dimethyl sulfoxide

67-68-5

AOO

+

+

71.9

25

2.70

50

2.30

100

3.90

Ethyl acrylate

140-88-5

AOO

+

+

28.3

10

1.20

25

2.70

50

5.00

Ethylene glycol dimethacrylate

97-90-5

MEK

+

+

28.3

10

1.20

25

2.40

50

7.00

EXP 11120 A

Pluronic
acid/H2O
(1%)

+

+

64.9

10

0.96

25

0.66

50

1.60

100

6.30

FAR01060-00

Pluronic
acid/H2O
(1%)

+

+

88.5

10

0.40

25

0.80

50

1.00

100

3.60

75

10.80

Imidazolidinyl urea

39236-46-9

DMF

+

+

23.9

10

1.70

25

3.10

50

5.50

Isopropyl myristate110-27-0

AOO

+

+

43.8

25

2.10

50

3.30

100

3.40

Jasmine absolute (Sambac)

8022-96-6

EtOH/DEP
(1:3)

+

+

36.4

10

1.70

25

2.50

50

3.60

Linalool alcohol

78-70-6

AOO

+

+

30.4

25

2.50

50

4.80

100

8.30

Linoleic acid60-33-3

AOO

+

+

14.1

10

1.50

25

7.00

50

9.10

D-148

5
Conc.
(%)

5 SI

100

7.30

100

16.20

Data Source

Gerberick et al.
(2005)
ECPA LLNA
Project Report,
Submitted by P.
BothamNTP Study,
Submitted by D.
Germolec
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Bayer CropScience
SA Studies,
Submitted by E.
Debruyne
Gerberick et al.
(2005)
Gerberick et al.
(2005)
Lalko & Api (2006),
Submitted by A. Api(RIFM)
Gerberick et al.
(2005)
EFfCI study,
Submitted by P.
Ungeheuer

ICCVAM Test Method Evaluation Report: Appendix D, Annex IV

CASRN

VehiclerLLNA1

Trad.
LLNA1

EC32

1
Conc.
(%)

1 SI

2
Conc.
(%)

2 SI

3
Conc.
(%)

3 SI

4
Conc.
(%)

4 SI

5
Conc.
(%)

5 SI

Data Source

80-62-6

AOO

+

+

90.0

10

1.40

30

1.50

50

1.50

75

2.10

100

3.60

Non-ionic surfactant 1

AOO

+

+

27.5

25

2.80

50

4.80

100

6.50

Non-ionic surfactant 2

AOO

-

+

47.1

25

1.50

50

3.20

100

2.90

Non-ionic surfactant 5

AOO

+

+

37.5

25

2.70

50

3.30

100

3.20

Non-ionic surfactant 6

AOO

+

+

34.4

25

2.70

50

3.50

100

6.50

75

15.80

100

30.10

LLNA/EC3
Validation Study,
Submitted by D.
Basketter, I. Kimber,and F. Gerberick
CESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaCESIO Report,
Submitted by K.
SkirdaEFfCI study,
Submitted by P.
Ungeheuer
Gerberick et al.
(2005)CESIO Report,
Submitted by K.
SkirdaGerberick et al.
(2005)
ECPA LLNA
Project Report,
Submitted by P.
BothamGerberick et al.
(2005)
EFfCI study,
Submitted by P.
Ungeheuer
CESIO Report,
Submitted by K.
SkirdaSubstance Name

Methyl methacrylate

Oleic acid112-80-1

AOO

+

+

10.5

10

2.60

25

14.90

50

6.90

Pentachlorophenol

87-86-5

DMSO

+

+

19.6

10

2.10

25

3.50

50

5.40

AOO

+

+

60.7

25

2.20

50

2.70

100

4.10

Precursor surfactant 1
Pyridine

110-86-1

AOO

+

+

71.9

25

1.10

50

2.30

100

3.90

Quinoxyfen/cyproconazole

Pluronic
L92 (1%)

+

+

27.8

13

2.00

25

2.30

50

8.60

R(+)-Limonene

124495-187/
113096-994
5989-27-5

AOO

+

+

68.8

25

1.80

50

2.40

100

4.00

Undecylenic acid112-38-9

AOO

+

+

19.4

10

2.50

25

3.30

50

4.40

AOO

+

+

27.1

25

2.80

50

5.20

100

4.70

Unsaturated fatty acid ester

Abbreviations: ACE = acetone; AOO = Acetone: olive oil (4:1 by volume); CASRN = Chemical Abstract Services Registry Number; CESIO = Comite Europeen des Agents de Surface et de Leurs Intermediaires Organiques (European
Committee of Surfactants and their Organic Intermediates; Conc. = concentration; DEP = diethyl phthalate; DMF = dimethylformamide; DMSO = dimethyl sulfoxide; EFfCI = European Federation for Cosmetic Ingredients; ECPA =European Crop Protection Association; EtOH = ethanol; H2O = water; LLNA = Local Lymph Node Assay; MEK = methyl ethyl ketone; NTP = National Toxicology Program; rLLNA = Reduced Local Lymph Node Assay; RIFM =Research Institute for Fragrance Materials; SI = stimulation Index; Trad. = traditional
12"+" = Sensitizer; "-" = Non-sensitizer
EC3 represents the estimated concentration needed to produce a stimulation index of three (i.e., a three-fold increase in lymphocyte proliferation is observed for the test substance versus the vehicle control substance).

D-149

ICCVAM Test Method Evaluation Report: Appendix D, Annex IV[This Page Intentionally Left Blank]

D-150

ICCVAM Test Method Evaluation Report: Appendix E

Appendix E
ECVAM Scientific Advisory Committee Statement on the Validity of the rLLNA

E-1

ICCVAM Test Method Evaluation Report: Appendix E[This Page Intentionally Left Blank]

E-2

ICCVAM Test Method Evaluation Report: Appendix E
EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE
Instltute tor Health and Consume r Protection
European Centre for the Validation of Attemative Methods (ECVAM)ESAC Statement on the Reduced LocaJ Lymph Node Assay (rLLNA)th

At its 26 Meeting, held on 26-27 April 2007 at the European Centre for the Validationof Alternative Methods (BCVAM), Ispra, Italy, the non-Commission members of the
BCVAM Scientific Advisory Committee (ESAC)1 unanimously endorsed the following
statement
Skin sensitisation is an important toxicological endpoint with respect to human safety.
Having reviewed the final report of the independent peer review evaluation co-ordinatedby ICCVAM and NlCEATM2 , the report by the EMEA3, the pre-report of the
sccNFP", and evidence made available since the original submissions to ICCVAM, in
March 2000 the 14th meeting ofESAC stated:"Following a review of the scientific report and pUblications on the local lymph
node assay (LLNA) it is concluded that the LLNA is a scientifically validated
test which can be used to assess the skin sensitisation potential of chemicals. The
LLNA should be the preferred method, as it uses fewer animals and causes lesspain and distress than the conventional guinea-pig methods. In some instancesand for scientific reasons, the conventional methods can be used."
Since its acceptance for regulatory purposes, the LLNA has proved suitable for the
purposes of satisfying a range of ED and other regulatory requirements s.
The developers of the LLNA have now undertaken a retrospective analysis of publisheddata obtained with the LLNA 6.
They conclude that within a tiered testing strategy in the context of REACH a "reduced"
version of the LLNA (rLLNA), using only a negative control group and the equivalentof the high-dose group from the full LLNA, can be used as a screening test to
distinguish between sensitisers and non-sensitisers.
ESAC established a peer review panel to evaluate if there was the potential to minimise
animal use by employing the rLLNA as a screening test as part of a tiered-testing
strategy for chemicals.
Mindful that with the rLLNA:•
When compared with the full LLNA the rLLNA cannot and will not result in
additional false positives.
•
When compared with the full LLNA the rLLNA may produce a few false
negati ves (3: 169 in the reference document, reducing to 2: 169 when negative
results obtained with concentrations of <10% are considered invalid)•
The test results provided by the rLLNA do not allow the determination of thepotency of a sensitising chemical.
ESAC states that the peer reviewed and pubJished information is of a quality and nature
to support the use of the rLLNA within tiered-testing strategies to reliably distinguishbetween chemicals that are skin sensitisers and non-sensitisers, and that animal use canbe minimised providing:

E-3

ICCVAM Test Method Evaluation Report: Appendix E
EUROPEAN COMMISSION
DIRECTORATE GENERAL JRC
JOINT RESEARCH CENTRE

Instltuie lor Health and Consu mer Protection
European Centre for the Validation of Akematlva Methods (ECVAM)

•

•
•

•

The concentration used to evaluate sensitisation potential is the maximum
consistent with solubility and the need to avoid local and other systemic adverse
effects, and that this principle rather than strict adherence to the specificrecommended absolute concentrations as in OECD TO 429 should be used.
Negative test results associated with testing using concentrations of less than
10%, should undergo further evaluation.
Positive and negative (vehicle) control groups are used, as appropriate, perOECD TG 429.
The full LLNA should be perfonned when it is known that an assessment ofsensitisation potency is required.

ESAC recommends that further work should be undertaken to detennine if the 10%
concentration threshold referenced above is optimal.

Thomas Hartung
Head of Unit
ECVAM
Institute for Health & Consumer Protection
Joint Research Centre
European Commission
Ispra

27 April 2007

2

E-4

ICCVAM Test Method Evaluation Report: Appendix E

I.

The ESAC was established by the European Commission, and is composed ofnominees from the ED Members States, industry, academia and animal welfare,together with representatives of the relevant Commission services.
This statement was endorsed by the following members of the ESAC:
Ms Sonja Beken (Belgium)
Ms Dagmar Hrova (Czech Republic)
Mr Tonu PUssa (Estonia)
Mr Lionel Larue (France)
Mr Manfred Liebsch (Germany)
Ms Annalaura Stammati (Italy)
Mr Jan van der Valk (The Netherlands)
Mr Constantin Mircioiu (Romania)
Mr Albert Breier (Slovakia)
Ms Argelia Castano (Spain)
Mr Patric Amcoff (Sweden)
Mr Jon Richmond (UK)Mr Carl Westmoreland (COLIPA)
Ms Vera Rogiers (ECOPA)
Ms Nathalie Alepee (EFPIA)
Mr Robert Combes (ESTIV)Mr Hasso Seibert (European Science Foundation)
The following Commission Services and Observer Organisations were involved in the
consultation process, but not in the endorsement process itself.
Mr Thomas Hartung (ECVAM; chairman)

Mr Jens Unge (ECVAM; ESAC secretary)
Ms EIke Anklam (Director of IHCP)
Ms Susanna Louhimies (DG Environment)
Ms Barbara Mentre (DG ENTR)
Ms Grace Patlewicz (ECB, DG JRC)Mr Christian Wimmer (DG Research)
Mr Hajime Kojima (JACYAM)
Ms Laurence Musset (OBCD)
Mr Barry Philips (Eurogroup for Animal Welfare)
Mr William Stokes (NICEATM, USA)

2. NIH (1999). The murine local lymph node assay. The results of an independent peer
review evaluation coordinated by the Interagency Coordinating Commictee on the
Validation of Altemative Methods (lCCVAM) aDd the National Toxicology Program
Center for the Evaluation of Altemative Tox icological Methods (NICEATM). NIH
Publication n.99-4494.
(http://iccvam.niehs.nih.gov/methods/immunotoxli mmunotox .htm)
3. EMEA (2000). Report from the ad-hoc expert meeting on testing for
immunohypersensitivity (11/01/2000). European Agency for the Evaluation of
Medicinal Products.

3

E-5

ICCVAM Test Method Evaluation Report: Appendix E

4.

SCCNFP (2000). Opinion adopted by the SCCNFP during the 11 lh plenary meeting,
17 February 2000.
(http://ec.europa.eu/healthlph_riskicommittees/sccp/docshtml/sccp_outII4_en.htm)

5. A Cockshott, P Evans, CA Ryan, GF Gerberick, CJ Betts, RJ Dearman, I Kimber and
DA Basketter (2006). The local lymph node assay in practice: a current regulatory
perspective. Human & Experimental Toxicology 25,387-394.

6. I Kimber, RJ Dearman, 0 Betts, GF Gerberick, CA Ryan, PS Kern, GY Patlewicz
and DA Basketter (2006.) The local lymph node assay and skin sensitisation: a cut­
down screen to reduce animal requirements? Contact Dermatitis 54, 181-185.

4E-6

ICCVAM Test Method Evaluation Report: Appendix F

Appendix F
Independent Scientific Peer Review Panel Assessment
F1

Independent Scientific Peer Review Panel Report: Validation Statusof New Versions and Applications of the Murine Local Lymph Node
Assay: A Test Method for Assessing the Allergic Contact Dermatitis
Potential of Chemicals and Products ........................................................... F-3

F2

Summary Minutes from Peer Review Panel Meeting on
March 4-6, 2008 ........................................................................................ F-161

F-1

ICCVAM Test Method Evaluation Report: Appendix F1[This Page Intentionally Left Blank]

F-2

ICCVAM Test Method Evaluation Report: Appendix F1Appendix F1
Independent Scientific Peer Review Panel Report: Validation Statusof New Versions and Applications of the Murine Local Lymph Node
Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential ofChemicals and Products

F-3

ICCVAM Test Method Evaluation Report: Appendix F1[This Page Intentionally Left Blank]

F-4

ICCVAM Test Method Evaluation Report: Appendix F1

Independent Scientific Peer Review Panel Report:
Validation Status of New Versions and Applications of the
Murine Local Lymph Node Assay: A Test Method for Assessing
the Allergic Contact Dermatitis Potential of Chemicals andProducts
May 2008

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

National Toxicology Program
P.O. Box 12233
Research Triangle Park, NC 27709
F-5

ICCVAM Test Method Evaluation Report: Appendix F1This document is available electronically at
http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf

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

F-6

ICCVAM Test Method Evaluation Report: Appendix F1Table of Contents
List of Tables ................................................................................................................... F-9
List of Abbreviations and Acronyms............................................................................ F-10
Members of the Independent Scientific Peer Review Panel ........................................ F-12
Preface ........................................................................................................................... F-15
Executive Summary ...................................................................................................... F-19
1.0

2.0

3.0

Murine Local Lymph Node Assay (LLNA) Limit Dose Procedure ................. F-33
1.1 Comments on the Draft Background Review Document (BRD) for
Completeness, Errors, and Omissions.......................................................... F-33
1.2

Comments on the Validation Status of the LLNA Limit Dose Procedure..... F-36

1.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA Limit Dose Procedure ...................................................................... F-41

LLNA for Testing Aqueous Solutions, Metals, and Mixtures.......................... F-45
2.1 Comments on the Draft Addendum for Completeness, Errors, andOmissions ................................................................................................... F-45
2.2

Comments on the Validation Status of the Traditional LLNA for Testing
Aqueous Solutions, Metals and Mixtures .................................................... F-45

2.3

Comments on the Draft ICCVAM Test Method Recommendations on the
Traditional LLNA for Testing Aqueous Solutions, Metals, and Mixtures .... F-47

Non-Radioactive LLNA Protocol - The LLNA: Daicel Adenosine Triphosphate(LLNA: DA) Test Method ................................................................................. F-51
3.1 Comments on the Draft BRD for Completeness, Errors, and Omissions ...... F-51
3.2

Comments on the Validation Status of the LLNA: DA ................................ F-52

3.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: DA ................................................................................................. F-57

F-7

ICCVAM Test Method Evaluation Report: Appendix F1

4.0

5.0

6.0

7.0

8.0

Non-Radioactive LLNA Protocol - The LLNA: Bromodeoxyuridine Detected by
Flow Cytometry (BrdU-FC) Test Method ........................................................ F-61
4.1 Comments on the Draft BRD for Completeness, Errors, and Omissions.......F-61
4.2

Comments on the Validation Status of the LLNA: BrdU-FC........................F-63

4.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-FC .........................................................................................F-66

Non-Radioactive LLNA Protocol - The LLNA: Bromodeoxyuridine Detected by
ELISA (BrdU-ELISA) Test Method ................................................................. F-71
5.1

Comments on the Draft BRD for Completeness, Errors, and Omissions.......F-71

5.2

Comments on the Validation Status of the LLNA: BrdU-ELISA..................F-73

5.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-ELISA ...................................................................................F-77

Draft ICCVAM LLNA Performance Standards.............................................. F-81
6.1 Comments on the Proposed Purpose and Applicability.................................F-81
6.2

Comments on the Essential Test Method Components .................................F-82

6.3

Comments on the Proposed Reference Substances .......................................F-83

6.4

Comments on the Test Method Accuracy Standards.....................................F-85

6.5

Comments on the Test Method Reliability Standards ...................................F-87

6.6

Summary .....................................................................................................F-88

6.7

Additional Statistical Comments..................................................................F-89

Use of the LLNA for Potency Determinations.................................................. F-91
7.1 Comments on the Draft BRD for Completeness, Errors, and Omissions.......F-91
7.2

Comments on the Validation Status of the Traditional LLNA to Determine
Skin-Sensitization Potency...........................................................................F-91

7.3

Comments on the Draft ICCVAM Test Method Recommendations for the
Use of the LLNA for Potency Determination ...............................................F-95

References.......................................................................................................... F-97

Annex A: Peer Review Panel Member Biosketches .................................................. F-101
Annex B: Questions for the Peer Review Panel ......................................................... F-111

F-8

ICCVAM Test Method Evaluation Report: Appendix F1

List of Tables
Table F-1

Power Calculations for the Traditional LLNA ..................................... F-37

Table F-2

Power Calculations for the LLNA: DA ................................................. F-53

Table F-3

Power Calculations for the LLNA: BrdU-FC ....................................... F-68

Table F-4

Power Calculations for the LLNA: BrdU-ELISA................................. F-72

F-9

ICCVAM Test Method Evaluation Report: Appendix F1

List of Abbreviations and Acronyms
ACD
ANOVA
AOO
BRD
BrdU
BT
CD4
CPSC
CRO
CV
DMF
DMSO
DNCB
EC3
ECt
ECVAM
ELISA
eLLNA: BrdU-FC
EPA
FC
FR
GLP
GPMT
GSK
HCA
HMT
HRIPT
HTdR
ICCVAMISO
IWG
JaCVAM
LLNA
LLNA: BrdU-ELISA
LLNA: BrdU-FC
LLNA: DA
LNC
LOEL
MEK
NICEATM

Allergic contact dermatitis
Analysis of varianceAcetone: olive oil (4:1)
Background review document
Bromodeoxyuridine
Buehler Test
Cluster of differentiation 4
U.S. Consumer Product Safety Commission
Clinical research organization
Coefficient of variation
Dimethylformamide
Dimethylsulfoxide
Dinitrochlorobenzene
Estimated concentration needed to produce a stimulation index
of 3
Estimated concentration needed to produce a stimulation index
that is indicative of a positive response
European Centre for the Validation of Alternative Methods
Enzyme Linked Immunosorbent Assay
Enhanced LLNA with BrdU detected by flow cytometry
U.S. Environmental Protection Agency
Flow cytometry
Federal Register
Good Laboratory PracticeGuinea Pig Maximization Test
GlaxoSmithKline
Hexyl cinnamic aldehyde
Human Maximization Test
Human Repeat Insult Patch Test
3
H-Methyl Thymidine
Interagency Coordinating Committee on the Validation of
Alternative Methods
International Organization for Standardization
Immunotoxicity Working Group
Japanese Center for Validation of Alternative Methods
Murine local lymph node assay
LLNA with BrdU detected by ELISALLNA with BrdU detected by FC
LLNA: Daicel adenosine triphosphate
Lymph node cells
Lowest observed effect level
Methyl ethyl ketone
NTP Interagency Center for the Evaluation of Alternative
Toxicological Methods
F-10

ICCVAM Test Method Evaluation Report: Appendix F1

NIEHS
NIH
NOEL
NTP
OD
OECD
QSAR
REACH
rLLNA
SAR
SD
SI
SDS
SLS
TG
Th
vs.

National Institute of Environmental Health Sciences
National Institutes of Health
No observed effect level
National Toxicology Program
Optical density
Organisation for Economic Co-operation and Development
Quantitative structure-activity relationship
Registration, Evaluation, and Authorisation of Chemicals
Reduced local lymph node assay
Structure-activity relationshipStandard deviation
Stimulation index
Sodium dodecyl sulfate
Sodium lauryl sulfate
Test guideline
T-helperVersus

F-11

ICCVAM Test Method Evaluation Report: Appendix F1

Members of the Independent Scientific Peer Review Panel
Michael Luster, Ph.D. (Panel Chair), Senior Consultant to the National Institute ofOccupational Safety and Health (NIOSH) Health Effects Laboratory, Morgantown, WV
Nathalie Alépée, Ph.D., Associate Research Fellow, Pfizer PDRD MCT Laboratory, France
Anne Marie Api, Ph.D., Vice President, Human Health Sciences, Research Institute for
Fragrance Materials, Woodcliff Lake, NJ
Nancy Flournoy, M.S., Ph.D. (Biostatistician), Professor and Chair, Dept. of Mathematics &Statistics, University of Missouri-Columbia, Columbia, MO
Thomas Gebel, Ph.D., Regulatory Toxicologist, Federal Institute for Occupational Safety &Health, Dortmund, Germany
Sidney Green, Ph.D.1, Graduate Professor, Howard University, Washington, DC
Kim Headrick, B. Admin., B.Sc., International Harmonization Senior Policy Advisor, Health
Canada, Ottawa, Ontario, Canada
Dagmar Jírová, M.D., Ph.D., Toxicologist, Research Manager, Head of Reference Center for
Cosmetics, Head of Reference Laboratory for Experimental Immunotoxicology, National
Institute of Public Health, Czech RepublicDavid Lovell, Ph.D. (Biostatistician), Reader in Medical Statistics, Postgraduate Medical
School, University of Surrey, Guildford, Surrey, U.K.
Howard Maibach, M.D., Professor, Department of Dermatology, University of California-San
Francisco, San Francisco, CA
James McDougal, Ph.D., Professor and Director of Toxicology Research, Department ofPharmacology and Toxicology, Boonshoft School of Medicine, Wright State University,
Dayton, OH
Michael Olson, Ph.D., Director of Occupational Toxicology, Corporate Environment Healthand Safety, GlaxoSmithKline, Research Triangle Park, NC
Raymond Pieters, Ph.D., Associate Professor, Immunotoxicology Group Leader, Institute for
Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands
Jean Regal, Ph.D., Professor, Department of Pharmacology, University of Minnesota Medical
School, Duluth, MN
Jonathan Richmond, MB ChB, FRCSEd1, Head of Division, Animals Scientific Procedures
Division, Home Office, London, U.K.

1

Drs. Green and Richmond were unable to attend the public meeting on March 4-6, 2008. However, they wereinvolved in the review of the background review documents and concur with the conclusions andrecommendations included in this report.
F-12

ICCVAM Test Method Evaluation Report: Appendix F1Peter Theran, V.M.D., Massachusetts Society for the Prevention of Cruelty to Animals,
Novato, CA
Stephen Ullrich, Ph.D., Dallas/Ft. Worth Living Legends Professor & Professor of
Immunology, Post-graduate School of Biomedical Science, University of Texas M.D. Anderson
Cancer Center, Houston, TX
Michael Woolhiser, Ph.D., Technical Leader - Immunotoxicology, Toxicology &Environmental Research & Consulting Immunology, Dow Chemical, Midland, MI
Takahiko Yoshida, M.D., Ph.D., Professor, Department of Health Science, Asahikawa
Medical College, Hokkaido, Japan

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ICCVAM Test Method Evaluation Report: Appendix F1

Preface
In 1999, the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) recommended the murine local lymph node assay (LLNA) to U.S. Federal agencies
as a valid substitute for currently accepted guinea pig test methods to assess the allergic contact
dermatitis potential of many, but not all, types of substances. The recommendation was basedon a comprehensive evaluation of the validation status of the LLNA that included an assessment
by an international independent scientific peer review panel (Panel). The Panel report and the
ICCVAM LLNA test method recommendations (ICCVAM 1999) are available at the National
Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM)-ICCVAM website.2 The LLNA was subsequently incorporated into
national and international test guidelines for the assessment of skin sensitization (OECD 2002;
ISO 2002; EPA 2003). For this Panel report, this LLNA will be referred to as the “traditional”
LLNA.
On January 10, 2007, the U.S. Consumer Product Safety Commission (CPSC) formallyrequested through NICEATM that ICCVAM assess the validation status of:3•

The traditional LLNA as a stand-alone assay for potency determinations (including
severity) for the purpose of hazard classification•

Three modifications of the traditional LLNA not requiring the use of radioactive
materials•

The LLNA limit dose procedure (also referred to as the “reduced” LLNA)•

The ability of the traditional LLNA to test mixtures, metals, and aqueous solutions(i.e., to re-evaluate the applicability domain for the traditional LLNA)NICEATM, in coordination with ICCVAM and the ICCVAM Immunotoxicity Working Group,
prepared a comprehensive draft background review document (BRD) for each modified versionof the traditional LLNA test method being evaluated, as well as a draft applicability domain
addendum to the final BRD published previously on the traditional LLNA. Each draft BRD andthe draft addendum detailed the available data and information from the published literature andsubmissions received in response to a 2007 Federal Register (FR) notice that had requested
data related to CPSC’s nomination (FR notice Vol. 72, No. 95, p. 27815-27817, May 17, 2007).
In addition, ICCVAM developed draft LLNA Performance Standards intended for use invalidating alternative test methods that are functionally and mechanistically similar to the
traditional LLNA. Finally, ICCVAM, based on the information contained in each of the draft
BRDs and the draft addendum, developed draft test method recommendations.
The various supporting documents and the draft ICCVAM recommendations were provided toa new international Panel for an independent scientific review. In addition, NICEATM
2

The 1999 ICCVAM Panel report and recommendations can be obtained at:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
3
The CPSC nomination can be obtained at:
http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
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ICCVAM Test Method Evaluation Report: Appendix F1announced the availability of these documents on the NICEATM-ICCVAM website(http://iccvam.niehs.gov) for public comment in a FR notice (Vol. 73, No. 5, p. 1360-1362,
January 8, 2008) and via the ICCVAM listserv. The FR notice also announced the public Panelmeeting, to be convened at the CPSC Headquarters in Bethesda, MD on March 4–6, 2008.
The Panel was charged with:•

Reviewing each ICCVAM draft BRD and the draft addendum for completeness andidentifying any errors or omissions of existing relevant data or information•

Evaluating the information in each draft BRD and the draft addendum to determine
the extent to which each of the applicable criteria for validation and acceptance of
toxicological test methods (ICCVAM 2003) had been appropriately addressed forthe recommended use of the new versions and applications of the traditional LLNA•

Considering the ICCVAM draft test method recommendations for the following andcommenting on the extent to which they are supported by the information provided
in the draft BRDs and the draft addendum:– proposed test method uses
– proposed recommended standardized protocols– proposed test method performance standards– proposed additional studies•

Evaluating the draft ICCVAM LLNA Performance Standards and considering
whether they were adequate for assessing the accuracy and reliability of alternative
test methods that are functionally and mechanistically similar to the traditional
LLNA

During our public meeting in March 2008, the Panel discussed each charge, listened to publiccomments, and developed conclusions and recommendations for ICCVAM on each of thenominated activities. The Panel wished to emphasize that they were to consider two overall
questions. They were to consider: (1) whether the validation status of the each of the above
proposed modifications or alternative uses of the LLNA had been adequately characterized for
its intended purpose according to established ICCVAM validation criteria (available on the
NICEATM-ICCVAM website, http://iccvam.niehs.gov), and (2) whether proposed
modifications or alternative uses of the LLNA are sufficiently accurate and reliable to be used
for the identification of sensitizing substances and non-sensitizing substances in place of the
traditional LLNA procedure.
This report details the Panel's independent conclusions and recommendations. ICCVAM willconsider this report, along with all relevant public comments, as it develops final test method
recommendations. The final ICCVAM test method recommendations will be forwarded to U.S.
Federal agencies for their consideration in accordance with the ICCVAM Authorization Act of2000 (Public Law 106-545).

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ICCVAM Test Method Evaluation Report: Appendix F1

The Panel gratefully acknowledges the efforts of NICEATM staff in coordinating the logisticsof the peer review Panel meeting and in preparing materials for their review. The Panel also
thanks each of the test method developers, Drs. George DeGeorge (LLNA: BrdU-FC), Kenji
Idehara (LLNA: DA), and Masahiro Takeyoshi, (LLNA: BrdU-ELISA) for providing
summaries and additional clarifications of the non-radioactive test methods under review.
Finally, as Panel Chair, I want to thank each Panel member for her or his thoughtful andobjective review of these LLNA-related activities.
Michael Luster, Ph.D.
Chair, LLNA Peer Review PanelMay 2008

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ICCVAM Test Method Evaluation Report: Appendix F1

Executive Summary
This report describes the conclusions and recommendations of an international independentscientific peer review panel (Panel). This Panel was charged by the Interagency Coordinating
Committee on the Validation of Alternative Methods (ICCVAM) with evaluating the validation
status of new versions and applications of the murine local lymph node assay (LLNA) for
assessing the allergic contact dermatitis (ACD) potential of chemicals and products. The
LLNA, which was first evaluated in 1999 by ICCVAM, is hereafter referred to as the“traditional LLNA” to distinguish it from other versions considered by the Panel. The newversions and applications considered include:•

The LLNA limit dose procedure (also referred to as the “reduced” LLNA4)•

The ability of the traditional LLNA to test mixtures, metals, and aqueous solutions(i.e., a re-evaluation of the applicability domain for the traditional LLNA)•

Three modifications of the traditional LLNA not requiring the use of radioactive
materials:– LLNA: DA (Local Lymph Node Assay: Daicel Adenosine Triphosphate)– LLNA: BrdU-FC (Local Lymph Node Assay: Bromodeoxyuridine detected byflow cytometry)– LLNA: BrdU-ELISA (Local Lymph Node Assay: Bromodeoxyuridine detectedby ELISA)•The traditional LLNA as a stand-alone assay for potency determinations (including
severity) for the purpose of hazard classification

The Panel also evaluated the draft ICCVAM LLNA Performance Standards and considered
whether they were adequate for assessing the accuracy and reliability of alternative test methods
that are functionally and mechanistically similar to the traditional LLNA.
LLNA Limit Dose Procedure
The Panel agreed that the LLNA limit dose procedure, which normally allows for testing at one
dose level, should be routinely recommended for hazard identification when used for testing
purposes which do not require dose response information, because it would offer time, cost,throughput and logistical benefits as well as using fewer animals. In instances when a necessity
to measure relative skin-sensitization potency for the purpose of risk assessment was present,
then the traditional LLNA should be used in order to generate dose response information. Still,
4

As described in this report, the Panel agreed that consideration should be given to applying the same term to
the LLNA limit dose procedure since in various places throughout the draft BRD it was referred to differently
as either the “cut-down,” the “limit dose,” or the “reduced LLNA” (i.e., “rLLNA”). Since the European
Centre for the Validation of Alternative Methods (ECVAM) has already established a naming convention of“rLLNA,” the Panel recommended adopting the ECVAM terminology to harmonize the terminology usedamong the international validation agencies. However, because the ICCVAM documents that were reviewed
use "LLNA limit dose procedure" that term is retained in this report.
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ICCVAM Test Method Evaluation Report: Appendix F1

the Panel recommended use of the LLNA limit dose procedure as the initial testing procedure to
identify sensitizers and non-sensitizers before conducting the traditional LLNA even when dose
response information is required since if the test substance were negative in the limit dose
procedure, it would not be necessary to conduct a multiple-dose LLNA test.
The draft background review document (BRD) for the LLNA limit dose procedure provides acomprehensive review of available data and information for assessing the usefulness andlimitations of this modified version of the LLNA for the purpose of skin-sensitization hazard
classification. The Panel evaluated the draft BRD for completeness, errors, and omissions andrecommended that it be updated to reflect their suggestions/corrections relating to general,
statistical, and specific editorial issues. In particular, the Panel noted that the differences interminology used for this procedure caused confusion and recommended that an internationallyharmonized term be adopted. They suggested referring to the procedure as the “reduced LLNA”(i.e., “rLLNA”) since that is being used by the European Centre for the Validation of
Alternative Methods (ECVAM).
The Panel concluded that the stimulation index (SI) based on the ratio of 3.0 as the cutoff value
was indicative of a response that was sufficiently greater than the control and would beconsidered an immunologically relevant response, but recommended that statistical analyses beused to definitively establish that a response induced by a test substance is significantly
different from the vehicle control. The Panel agreed that the LLNA protocol recommended by
ICCVAM (ICCVAM 1999; Dean et al. 2001) should be the standard protocol for all future
LLNA limit dose studies using the traditional LLNA protocol. Specifically, prospective LLNA
limit dose procedure studies should require that lymph nodes be collected from individual
animals instead of pooling them with other animals in a treatment group, which is also currently
permitted by the Organisation for Economic Co-operation and Development (OECD) Test
Guideline 429 (OECD 2002). Individual animal response data are necessary in order tostatistically analyze for differences between treated and control data. In addition, having datafrom individual animals also allows for identification of technical problems and outlier animals
within a dose group. Based on power calculations provided as supplemental information, thePanel agreed that five animals per dose group is an appropriate number to recommend for
LLNA limit dose studies following the traditional LLNA protocol. It should be noted that thePanel’s analysis of the LLNA limit dose dataset was not restricted to studies with confirmed
individual animal data, and that the Panel considered data known to have been generated using
pooled group data. The Panel stated that, internationally, both individual and pooled animal data
have likely been used both for regulatory decisions and for in-house decisions relating to
product development and risk management. In addition, the fact that the retrospective data
analysis set out in the draft LLNA limit dose procedure BRD did not distinguish between
individual or pooled animal data suggested that both met the quality standards for inclusion in
the draft BRD.
Although they did not reach consensus, the Panel suggested that for laboratories in which the
LLNA is “routinely” performed and have demonstrated the ability to consistently obtain
positive results, hexyl cinnamic aldehyde (HCA) or another positive control (e.g., a substance
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ICCVAM Test Method Evaluation Report: Appendix F1that matches the chemical class of the test substances) could be run at intervals for quality
control purposes rather than concurrent with each experiment. The Panel cited Kimber et al.
(2006), which describes “routine” use of the “rLLNA” utilizing only a vehicle and a high-dosegroup, as a rationale for this suggestion. However, the Panel does not recommend omitting the
concurrent positive control in laboratories that perform the LLNA only “occasionally.”
Based on the analyses presented in the draft BRD, the Panel considered the accuracy of the
LLNA limit dose procedure to have been adequately evaluated and compared to the traditional
LLNA, mindful of the limitations associated with a retrospective evaluation. For instance, itcannot be assumed that the compounds tested in the retrospective studies were always tested at
the highest possible dose unless such information was explicitly indicated. In this regard, thePanel recommended that a more detailed description of what is considered “avoidance ofexcessive irritation” and “evidence of systemic toxicity” be included in any LLNA protocol inorder to aid in choosing the most appropriate high (i.e., limit) dose, although specific indicatorsof “systemic toxicity or excessive irritation” were not formally discussed.
The Panel agreed that it was appropriate to assume that the intra- and inter-laboratory
reproducibility of the LLNA limit dose procedure and the traditional LLNA would be similar,
because reproducibility is more dependent on the method than on the number of dose groups.
However, reducing the number of test substances dose groups from three to one might reduce thesensitivity of the assay. The traditional LLNA may have a greater chance of correctly identifying
a sensitizer even in the presence of one or more technical errors since data from three dosegroups are being considered and an SI ≥3.0 at any dose group would result in the substance beingclassified as a sensitizer. However, for the purpose of adopting an assay that uses fewer animals
and provides increased throughput for testing purposes, these hypothetical considerations are nota sufficient reason to argue against use of the limit dose LLNA procedure.
LLNA for Testing Aqueous Solutions, Metals, and Mixtures
The draft ICCVAM recommendations state that, although more data are needed to assess the useof the LLNA for testing for mixtures and aqueous solutions before a recommendation can bemade, the traditional LLNA appears to be useful for the testing of metal compounds, with the
exception of nickel. The Panel agreed with these draft ICCVAM recommendations. Regarding
the use of the LLNA for testing mixtures, the Panel acknowledged that the ability of ICCVAM todevelop draft test method recommendations was limited not only by the amount of data
available, but the relatively poor concordance of traditional LLNA outcomes in comparison to
those obtained in guinea pig tests, and recommended that this be noted in the final ICCVAM
recommendations. The term “mixtures” can represent an infinite number of materials and it
would be more beneficial to specify types or formulations of mixtures that are being examined.
Regarding metals, the Panel concluded that the accuracy statistics for the traditional LLNA whencompared to results obtained from evaluation in humans supported use of the traditional LLNA
as a hazard identification tool for metals, with the exception of nickel, which produces variable
responses. One minority opinion stated that the results for nickel compounds were not entirely
questionable and that the traditional LLNA might also be suitable for testing nickel compounds.
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ICCVAM Test Method Evaluation Report: Appendix F1Thus, the Panel recommended further evaluation of the variable results obtained for nickel in thecontext of the available literature on allergic contact dermatitis to nickel in humans.
Regarding substances tested in aqueous solutions, the Panel suggested expanding the brief
section of the draft test method recommendations discussing the test method protocol for the
traditional LLNA to specifically point out how the conclusions of the applicability domain
evaluation may affect the standard traditional LLNA protocol. For instance, it could besuggested that aqueous test solutions be avoided due to problems associated with skinapplication. It would be preferable for a hierarchy of organic solvents to be considered as
dosing vehicles, with emphasis on using a vehicle to which humans may actually be exposed incircumstances linked to occupational sensitization.
The Panel agreed with the draft ICCVAM recommendation for continued accrual of
information from traditional LLNA evaluations of mixtures, metals, and aqueous solutions withcomparative data for guinea pig (i.e., guinea pig maximization test [GPMT] or Buehler test[BT]) and human (i.e., human maximization test [HMT] or human repeat insult patch test
[HRIPT]) tests. However, the Panel suggested that, given resource limitations, it would be
important to organize the recommendations based on relative priority.
The draft Addendum to the original validation report for the traditional LLNA (ICCVAM 1999)
provided a comprehensive review of currently available data and information for evaluating theusefulness and limitations of the traditional LLNA for assessing the skin-sensitization potentialof mixtures, metal compounds, and substances tested in aqueous solutions. The Panel evaluatedthe draft Addendum for completeness, errors, and omissions and concluded that there were noapparent errors or omissions, although they did state that the term “mixtures” was used too
broadly (i.e., can represent an infinite number of materials) and it would be more beneficial tospecify types or formulations of mixtures that are being examined.
The Panel did not identify any classes of chemicals missing from the dataset used to review theutility of the traditional LLNA for testing aqueous solutions. However, while they did notpropose an alternative, the Panel expressed concern over the most appropriate definition for anaqueous solution (defined in the draft Addendum as any solution containing ≥20% water). For
the mixtures included in the analysis, the Panel noted that quantitative compositions had notbeen provided and therefore they could not comment on whether these mixtures wererepresentative of the types of mixtures typically tested in the traditional LLNA. With respect tometals (none of which are mixtures), there was a paucity of important representatives ofcommercially useful metals such as platinum, palladium, iron, zinc, manganese and silver in thedata set. The Panel suggested that to enlarge the group of metal non-sensitizers, substances used
as cosmetic ingredients (e.g., titanium dioxide) and aluminum compounds currently used in
antiperspirants might be considered.
The Panel agreed that, although it was important to identify data obtained according to GLP
guidelines, data obtained from non-GLP studies should not be excluded automatically from thisretrospective analysis. The Panel concluded that other factors could be used to identify highquality data. Examples include data published in peer-reviewed journals or obtained from astudy conducted in a laboratory that has GLP capabilities.
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ICCVAM Test Method Evaluation Report: Appendix F1

The Panel concluded that, considering the limited comparative data that were available,
particularly for mixtures and aqueous solutions, the accuracy assessment of the traditional
LLNA for testing mixtures, metals, and aqueous solutions when compared to available human
and/or guinea pig test results was as comprehensive as was possible. The limited amount ofcomparative data made it unfeasible to draw definitive conclusions for mixtures and aqueous
solutions from the available accuracy statistics.
Non-Radioactive LLNA Protocol - The LLNA: DA Test Method
The Panel concluded that the available data and test method performance support the ICCVAM
draft recommendations for the LLNA: Daicel Adenosine Triphosphate test method (LLNA:DA), and that the test method may be useful for identifying substances as potential skinsensitizers and non-sensitizers, but that this recommendation is contingent upon receipt, review,
and analyses of additional existing data and information from the test method developer.
Therefore, this non-radioactive version of the traditional LLNA cannot currently berecommended for the hazard identification of skin sensitizing substances, regardless of whether
or not there are restrictions on the use of radioactive materials, until such time as this existingdata has been received and confirmed.
The draft LLNA: DA BRD was compiled to provide a comprehensive review of available dataand information evaluating the usefulness and limitations of the LLNA: DA test method toassess the allergic contact dermatitis potential of chemicals and other substances. The Panelevaluated the draft BRD for completeness, errors, and omissions and recommended that theirsuggestions/corrections relating to general, statistical, and specific editorial issues be
incorporated into future revisions.
The Panel agreed that five animals per dose group should be recommended for validation ofmodified LLNA test methods. The Panel, however, noted that supplemental power calculations
for the LLNA: DA test method indicated that the power for detecting a three-fold increase in
the treatment group was estimated to be 95% for a sample size of three mice per dose group.
Thus, the Panel identified the use of three animals per dose group as a potential opportunity to
reduce animal number when using modified assays in the future, assuming all essential
validation requirements can be successfully met. A minority opinion expressed by five Panelmembers was that if laboratories were operating under OECD guidance (OECD 2002) and a
reliable validation dataset had been generated, then pooled data from at least four animals perdose group could be considered.
Generally, the Panel viewed the difference in treatment schedule between the LLNA: DA andthe traditional LLNA to be potentially significant if the LLNA: DA induced the elicitation
phase of skin sensitization. The Panel was concerned that the 1% sodium lauryl sulfate (SLS)
pretreatment step in the LLNA: DA might modify the inherent sensitivity of the LLNA. They
recommended that the test method developer (Daicel Chemical Industries, Ltd.) justify the useof 1% SLS or consider an alternative decision criterion (i.e., an SI threshold other than 3.0)
such that the 1% SLS pretreatment is no longer necessary.

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ICCVAM Test Method Evaluation Report: Appendix F1

The Panel considered the database of substances tested in the LLNA: DA to be representative ofa sufficient range of chemicals expected to be tested for skin-sensitization potential, andconcluded that the accuracy analysis had made appropriate comparisons to the traditional
LLNA, guinea pig tests, and human data/experience. The Panel could not identify specificcharacteristics associated with the one false negative (i.e., 2-mercaptobenzothiazole) or the one
false positive (i.e., benzalkonium chloride), but reemphasized that the potential impact ofpretreatment with 1% SLS in this context needed to be considered.
With regard to test method reliability, the Panel concluded the intralaboratory reproducibility of
the LLNA: DA had not been adequately evaluated. They noted that the two sensitizers tested
had similar chemical structures (i.e., eugenol and isoeugenol) and that it was unclear if the tests
were truly independent. The Panel also noted that the interlaboratory reproducibility of theassay could not be adequately evaluated given the lack of original laboratory data andlimitations in the study design. In particular, they cited the use of pooled lymph nodes from themice in each treatment group and the testing of each substance at predetermined dose levels
established by the lead laboratory as study design limitations. Still, a Panel minority consideredpooled data acceptable and the setting of dose levels for all laboratories based on results fromthe lead laboratory to be reasonable.
The Panel also commented that ideally, test substances should be coded during the validation ofa new assay, although they did not feel that a lack of coding constituted a reason for rejecting
the current LLNA: DA dataset. The Panel also commented that although GLP compliance ishighly recommended for validation studies, the current studies should not be rejected solely on
the basis of a lack of GLP compliance. However, the Panel considered it important to obtain theoriginal records for all validation studies (which have been requested by NICEATM) in order toconfirm that the reported data were the same as the data recorded in the laboratory notebooks.
With regard to the 5% (1/19) false negative and 10% (1/10) false positive rates obtained with
the LLNA: DA, the Panel commented that it was important to identify reasons why thesubstances gave “false” results, taking into consideration factors such as intended use of thesubstances and the target population. They agreed that it might be useful to follow a suspectedinaccuracy with an investigation of the mechanistic basis for the discordance since it may help
to establish a biologically-based rationale for the discordance.
The Panel noted that the available LLNA: DA data did not support all of the ICCVAM draft
recommendations in the proposed test method standardized LLNA: DA protocol. First, although
the Panel agreed with the ICCVAM protocol that recommends five animals per dose group,
they noted that supplemental statistical information provided for the LLNA: DA test method
implied that using less than five animals per dose group was acceptable (e.g., a 3.0-fold increasein the SI value would likely be detected with 99% confidence when using four animals per dosegroup). In addition, the Panel considered it important to adequately characterize the effect of the
1% SLS pretreatment step in the LLNA: DA, and it should be demonstrated that the day 8
applications do not induce a skin reaction that could be indicative of the onset of the elicitation
phase of skin sensitization. Keeping these points in mind, the Panel agreed that if the limit dose
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ICCVAM Test Method Evaluation Report: Appendix F1

procedure was applicable to the traditional LLNA, then it would also be applicable to the
LLNA: DA in order to further reduce the number of animals used.
The Panel also stated that the available data supported the ICCVAM draft recommendations for
the LLNA: DA in terms of future studies, which included performing a more comprehensive
evaluation using more non-sensitizers within and across laboratories. A minority opinion statedby one Panel member was that although testing more sensitizers might be warranted forinterlaboratory validation studies, a sufficient number of non-sensitizers (n=11) had alreadybeen tested within the same laboratory.
The Panel also commented that the protocol differences between the LLNA: DA and the
traditional LLNA could not clearly be constituted as “major” or “minor” changes. However,they considered this issue largely irrelevant if a test method was able to correctly predict thedermal sensitization potential of a test substance. Consequently, the Panel concluded that thecurrent draft ICCVAM Performance Standards could be applicable to the LLNA: DA as amechanistically and functionally similar test method.
Non-Radioactive LLNA Protocol - The LLNA: BrdU-FC Test Method
Overall, the Panel concluded that the available data and test method performance of the LLNA
with bromodeoxyuridine (BrdU) detected by flow cytometry (LLNA: BrdU-FC) supported thedraft ICCVAM recommendations that it may be useful for identifying substances as potential
skin sensitizers and non-sensitizers, but that more information and existing data must be madeavailable before the LLNA: BrdU-FC can be recommended for routine use. The Panelconcluded that the test method usefulness and limitations identified in the draft ICCVAM
recommendations accurately summarized the limits of the information supplied and the
additional information that would need to be generated or provided for further consideration of
the test method. As a result, the Panel concluded that the LLNA: BrdU-FC could not currently
be considered as a scientifically valid replacement alternative to the traditional LLNA. Still, thePanel suggested that the test method recommendation should clearly state that the test method
was not “invalid,” but simply that there was currently not sufficient evidence and information
to state that it had been adequately validated.
The draft LLNA: BrdU-FC BRD was compiled to provide a comprehensive review of available
data and information evaluating the usefulness and limitations of the LLNA: BrdU-FC test
method to assess the ACD-inducing potential of substances. The Panel evaluated the draft BRD
for completeness, errors, and omissions and recommended that theirrecommendations/corrections relating to general, statistical, and specific editorial issues be
incorporated into future revisions.
The LLNA: BrdU-FC included routine measurements of ear swelling as an indicator ofexcessive skin irritation. The Panel viewed that this, or any other quantitative measurement ofskin irritation, should be carefully considered for inclusion in all LLNA protocols. The Panelconsidered inclusion of optional quantification of immunophenotypic markers as an additional
mechanism for distinguishing irritants from sensitizers to be useful, as it might reduce thefrequency of false positives (i.e., substances which are actually skin irritants) and improve
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ICCVAM Test Method Evaluation Report: Appendix F1comparisons with human data. However, they considered application of immunological markers
too detailed and costly for routine LLNA use (i.e., for hazard classification purposes) and moresuited for research purposes.
The Panel noted that the substances tested in the LLNA: BrdU-FC seemed representative of a
sufficient range of chemical classes and physical chemical properties, and thus that the test
method appeared applicable to many of the types of chemicals and products that are typicallytested for skin-sensitization potential. However, the Panel considered the total databaseavailable for evaluation of the validation status of the LLNA: BrdU-FC to be relatively small
compared to the large number of substances assessed in the traditional LLNA. Therefore, thePanel recommended caution when making conclusions related to its concordance with thetraditional LLNA. Still, the accuracy of the LLNA: BrdU-FC was considered adequately
evaluated and comparable to the traditional LLNA.
The Panel concluded that intralaboratory reproducibility was not adequately assessed and it
should be better evaluated in order to support the validation of this test method. The Panelsuggested that although the studies evaluated in the draft BRD were not GLP-compliant, thisshould not affect acceptance of the data for an evaluation of the validation status of this test
method. However, some sources of variability in the intralaboratory data, such as failure toappreciate differences in composition of dosing solutions between experiments caused by test
article instability or other phenomena, might be obscured if not in complete compliance with
GLP guidelines. Thus, the Panel suggested that any additional studies undertaken to validate thetest method should ideally be GLP-compliant.
The Panel agreed that the available data supported the ICCVAM draft recommendations for the
LLNA: BrdU-FC procedure in terms of the proposed test method standardized protocol. Theysuggested that the utility of ear swelling or other methods to detect inflammation appeared
warranted in every variation of the LLNA (including the traditional LLNA), but should befurther investigated before routine inclusion in the protocol is recommended. The Panel also
concluded that the traditional LLNA limit dose procedure could be applied to the LLNA: BrdUFC, keeping in mind the limitations associated with a “limit dose” procedure.
The Panel further agreed that the ICCVAM draft recommendations for future studies
highlighted the unanswered questions raised by the available data set. Specifically, conducting
interlaboratory studies as a part of the validation process is important. The Panel considered theimmunological markers suggested for the LLNA: BrdU-FC to be acceptable, but that additional
immunological markers for discrimination of irritant versus sensitization phenomena were also
possible. In general, for any future work, efforts should be made to decrease the variability andto thereby increase the power of the test in order to ensure that more animals were not needed
relative to the traditional LLNA or other alternative LLNA protocols.
The Panel considered the protocol differences between the LLNA: BrdU-FC and the traditional
LLNA to be “minor” changes, and therefore concluded that assessment of the validity of thistest method could be based on the draft ICCVAM LLNA Performance Standards. The Panelalso cautioned, however, that a clear definition of what constituted a “major” versus a “minor”
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ICCVAM Test Method Evaluation Report: Appendix F1change, or a different protocol altogether could be better addressed once the recommendations
for the current draft ICCVAM LLNA Performance Standards were finalized.
Non-Radioactive LLNA Protocol - The LLNA: BrdU-ELISA Test Method
The Panel concluded that the available data and test method performance for the LLNA with
BrdU detected by enzyme-linked immunosorbent assay (LLNA: BrdU-ELISA) support the
ICCVAM draft recommendations that it may be useful for identifying substances as potential
skin sensitizers and non-sensitizers, but that more information and existing data must be madeavailable before the LLNA: BrdU-ELISA can be recommended for use. The Panel also statedthat a detailed protocol was needed, in addition to sufficient quantitative data for broader
analysis on a larger set of balanced reference substances that take into account physicochemical
properties and sensitization potency, as well as an appropriate evaluation of interlaboratory
reproducibility.
The draft LLNA: BrdU-ELISA BRD was compiled to provide a comprehensive review ofavailable data and information evaluating the usefulness and limitations of the LLNA: BrdUELISA test method to assess the ACD-inducing potential of chemicals and other substances.
The Panel evaluated the draft BRD for completeness, errors, and omissions and recommended
that their suggestions/corrections relating to general, statistical and specific editorial issues be
incorporated into the final document.
The Panel’s main concern with the test method was that the accuracy of the LLNA: BrdUELISA at SI ≥3.0 was inadequate and not equivalent to the traditional LLNA. Furthermore,
although using a decision criterion of SI ≥1.3 improved the test’s performance in identifying
sensitizers from non-sensitizers, it did not resolve concerns about the test method. Based on a
power analysis for the LLNA: BrdU-ELISA, which was provided to the Panel as supplemental
information, the Panel concluded that it was difficult to justify using a SI ≥1.3 as the cutoff
value, given the much larger number of animals that would be required to detect a 1.3-foldincrease above vehicle controls with similar power to the traditional LLNA when five animals
per dose group are used. For a three-fold increase, the supplemental statistical analyses
indicated that a sample size of four was sufficient. Still, the Panel agreed with the ICCVAM
recommendation to use five animals per dose group and to collect individual animal data. They
concluded that this would allow for more robust calculations in the event that an outlier
prevented some of the data from being included in the analysis. A minority opinion by five
Panel members was stated that if laboratories were operating under OECD guidance (OECD2002) and a reliable validation dataset had been generated, then pooled data from a least fouranimals could be considered.
The Panel noted that in organizations where the use or disposal of radioactive materials was
restricted, the potential to use the LLNA: BrdU-ELISA could reduce the number of animals
needed per test compared to the traditional LLNA and would result in less pain and suffering
compared to using traditional guinea pig test methods. However, if the SI ≥1.3 was chosen as
the decision criterion because of its improved accuracy compared to SI ≥3.0, the Panel statedthat the number of mice needed to perform the LLNA: BrdU-ELISA test should be compared to
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ICCVAM Test Method Evaluation Report: Appendix F1

the number of guinea pigs that would be needed for skin-sensitization tests in order to assess if
the LLNA: BrdU-ELISA actually reduced overall animal use for skin-sensitization testing.
In general, the Panel considered the number of substances tested in the LLNA: BrdU-ELISA
too few, and that data from more substances tested using the traditional LLNA, guinea pig tests,
and human tests should have been included. The Panel also did not consider the available datafrom the LLNA: BrdU-ELISA to be representative of a sufficient range of chemical classes andphysical chemical properties. The limited dataset prevents an evaluation of whether the test
method would be considered applicable to any of the types of chemicals and products typicallytested for skin-sensitization potential.
However, the Panel concluded that the appropriate comparisons between the traditional LLNA,
guinea pig test and human data had been made. The Panel agreed that the false negative rate forhazard identification using the SI ≥3.0 in the LLNA: BrdU-ELISA was excessive (i.e., using
this SI threshold value, the LLNA: BrdU-ELISA misclassified 29% and 39% of the substancesclassified as sensitizers in the traditional LLNA or in humans, respectively).
The Panel also considered that the intralaboratory reproducibility of the LLNA: BrdU-ELISA
was not adequately evaluated and compared to the traditional LLNA. The Panel indicated thatthe number of substances was too few, and in some cases there was a wide variation in repeat
tests of the same substance. The Panel recommended a more comprehensive evaluation of theintralaboratory reproducibility of the test method, using different SI values, and that the analysisof the variability of the estimated concentration needed to produce a positive SI value (ECtvalues) be conducted on a log scale.
The Panel also noted that interlaboratory reproducibility for the LLNA: BrdU-ELISA could not
be evaluated because neither the design of the study sponsored by the Japanese Center for
Validation of Alternative Methods nor any of the resulting data had been provided in advanceof their evaluation. The Panel agreed that a multi-laboratory validation study using a balanced
set of chemicals would adequately characterize the interlaboratory reproducibility of the LLNA:
BrdU-ELISA.
In general, the Panel agreed that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA procedure in terms of the proposed test method
standardized protocols. However, as noted above, a minority opinion by five Panel members
was that there could be circumstances in which pooled data from at least four animals could
also be acceptable. The Panel also stated that if the LLNA: BrdU-ELISA was found to beequivalent to the traditional LLNA in the future that it would be appropriate to apply the LLNA
limit dose procedure to the test. The Panel also agreed with ICCVAM’s test method
recommendations for future studies and emphasized that more data were needed in order todetermine the appropriate threshold value for the decision criterion. The Panel concluded that it
might be more appropriate to use a statistically-based decision criterion rather than astimulation index to classify substances as sensitizers, and that this should be further
investigated.

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ICCVAM Test Method Evaluation Report: Appendix F1

The Panel agreed that the LLNA: BrdU-ELISA protocol differed from the traditional LLNA
only in the method used to assess lymphocyte proliferation and as such concluded that thisrepresented a “minor” change (as defined in the current draft ICCVAM LLNA PerformanceStandards) and separate performance standards for the LLNA: BrdU-ELISA were not needed.
Draft ICCVAM LLNA Performance Standards
The draft ICCVAM LLNA Performance Standards are intended to evaluate the acceptability of
proposed test methods that are mechanistically and functionally similar to the traditional LLNA.
ICCVAM proposed that the applicability of the draft ICCVAM LLNA Performance Standards
be restricted to protocols that incorporate “minor” modifications to the traditional LLNA
procedure, defined as changes only to the method for measuring lymphocyte proliferation. The
Panel agreed that different methods of measuring lymphocyte proliferation represent “minor”
modifications, but recommended that, instead of trying to define “minor” modifications, a better
strategy might be to define criteria that would need to be satisfied in order to ensure that thealternative test method was mechanistically and functionally similar to the traditional LLNA
(e.g., only measure cell proliferation associated with the induction phase of a skin-sensitization
reaction). The Panel considered that the draft performance standards were also appropriate for
evaluating other modifications. Examples of acceptable modifications included test animal sex,
strain, the use of rats rather than mice, the number of animals per group, and timing of test
article treatment. One minority opinion considered the potential impact of changes to protocolcomponents other than the method of measuring lymphocyte proliferation to be significant andtherefore would require more extensive validation, which was not defined.
The Panel indicated that alternative LLNA protocols that are undergoing validation should
contain essential test method components that follow the ICCVAM-recommended protocol(ICCVAM 1999; Dean et al. 2001), unless adequate scientific rationale for deviating from thisprotocol was provided.
The Panel also identified aspects of the LLNA that should be required as part of the test method
validation process: (1) application of the test substance to the skin with sampling of the lymph
nodes draining that site, (2) measurement of cell proliferation in the draining lymph node, (3)absence of a skin reaction that could be indicative of the onset of the elicitation phase of skinsensitization, (4) data collected at the level of the individual animal to allow for an estimate of
the variance within control and treatment groups (using this variance, a power analysis needs tobe conducted to demonstrate that the modified method is utilizing a sufficient number ofanimals per treatment group to permit hazard identification with at least 95% power), and (5) if
dose response information is needed, there are an adequate number of dose groups (n ≥3) withwhich to accurately characterize the dose response for a given test substance.
The Panel noted that the list of substances included in the draft ICCVAM LLNA PerformanceStandards was sufficiently representative of the types of materials that are likely to be tested forskin sensitization. However, among the 13 sensitizers in the list of “required” substances, only
five were considered to have robust data (i.e., traditional LLNA data based on at least three
independent studies).
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ICCVAM Test Method Evaluation Report: Appendix F1

To evaluate performance for use in hazard identification, the Panel concluded that all 22
substances in the draft ICCVAM-recommended list should be tested and accuracy statistics
calculated (Note: this list of substances includes “required” substances as well as “optional”
false negative and false positive substances, of which only 8/22 have “robust” datasets [n ≥ 3 asdefined by the Panel]). To the extent possible, a rationale for any discordant results should be
provided. However, the most potent sensitizers (e.g., dinitrochlorobenzene [DNCB]) should
always be identifiable. Also, considerable weight should be given to the balance between
animal welfare and human safety when considering the adequacy of test method accuracy.
Based on the limited data available for the sensitizers on the list and the lack of standardizationof test methods from which the results were obtained, the current database does not support
inclusion of ECt values as a component of the accuracy evaluation.
The Panel agreed with the draft ICCVAM recommendations for evaluating test method
reliability. These recommendations included obtaining ECt values that are generally within 0.5x
to 2.0x of the mean historical EC3 (i.e., estimated concentrations needed to produce an SI of 3)
values for hexyl cinnamic aldehyde (HCA) (intralaboratory, n=4 experiments in one
laboratory), or HCA and DNCB (interlaboratory, n=1 experiment in three laboratories).
However, the Panel recommended that the criteria for independent tests should be specified(e.g., different animal shipment, different reagents, different operator). The Panel concluded
that the proposed criteria for acceptability appeared to be appropriate in this case, because only
one or two substances were being evaluated (i.e., a statistical multiple-comparisons5 problem
does not exist). The Panel also suggested that historical control data using HCA and DNCB in
the same vehicle could be used to demonstrate adequate intra- and/or inter-laboratory
reproducibility.
The Panel also recommended that statistical tests to analyze the data might allow for a moreaccurate interpretation. They recommended that a suitable variance-stabilizing transformation(e.g., log transformation, square root transformation) be applied in all statistical analyses and inreporting summary standard deviations. The Panel also recommended that a more rigorous
evaluation be conducted of what would be considered an appropriate range of ECt values toinclude as a requirement. This would be a statistical evaluation that considers the variability of
ECt values generated among the sensitizers included on the performance standards reference
substances list and the statistical multiple comparisons problem.
Use of the LLNA for Potency Determinations
The Panel agreed with the draft ICCVAM recommendation that the LLNA should not be used
as a stand-alone assay for categorizing skin sensitizers as strong vs. weak, but that it could be
used as part of a weight-of-evidence evaluation (e.g., along with quantitative structure-activity
5

When multiple experiments are conducted and multiple observations, comparisons, or hypothesis tests are
conducted, the chance of observing rare events increases. Suppose, for example, that an interval is establishedsuch that 5% of observations from a particular population of data are outside that interval. Then if kindependent experiments generate data from this population (e.g., a standard normal distribution), the chancesthat all 20 results will lie inside the interval is (1.0 - 0.05)k (N. Flournoy, personal communication).
F-30

ICCVAM Test Method Evaluation Report: Appendix F1

relationships, peptide reactivity, human evidence, historical data from other experimental
animal studies) for this purpose. The Panel also agreed with the draft ICCVAM
recommendation that any LLNA studies conducted for the purpose of evaluating skinsensitization potency should use the ICCVAM recommended LLNA protocol. In addition, thePanel viewed that the relevant testing guidelines for the traditional LLNA should be revised toinclude the procedure for calculating an EC3 value.
A draft BRD was compiled by ICCVAM that provided a comprehensive review of available
data and information and an evaluation of the usefulness and limitations of the traditional
LLNA for the categorization of substances with regard to skin-sensitization potency. The Panelevaluated the draft BRD for completeness, errors, and omissions and noted alternative analyses
that would allow for a more complete evaluation of the use of the traditional LLNA for skinsensitization potency categorizations (see below).
The Panel agreed that the database of substances evaluated for potency determinations was
sufficient and represented a range of chemical classes and physicochemical properties
applicable to products typically tested for skin-sensitization potential. The Panel also concluded
that since the database was compiled from existing data, the lack of substance coding likely had
no impact on the retrospective evaluation presented in the draft BRD. Still, the Panelrecommended the coding of test substances in any future validation studies. The Panel generally
agreed that potency determinations based on traditional LLNA results should ideally be limited
to data from studies that evaluated lymph node proliferation in individual animals so thatoutliers and technical errors could be identified. However, they also agreed that pooled animal
data should not be excluded automatically from a retrospective analysis.
The Panel indicated that the relevance of the LLNA for potency determinations had beenadequately compared and evaluated to human (i.e., HMT or HRIPT) and guinea pig (i.e.,
GPMT or BT) data. A minority opinion stated by one Panel member was that the relevance ofthe traditional LLNA to human clinical observations had not been sufficiently determined.
In general, the Panel agreed that the proposed two-level categorization scheme (weak vs. strong
sensitizers) for both human and guinea pig data was appropriate. However, a minority opinion
stated by two Panel members was that a moderate category should be included since certain
compounds might be on the border between weak and strong sensitizers. Thus, they suggested
that the five-category scheme proposed by Kimber et al. (2003), which includes non-sensitizers,
might be recommended.
The Panel concluded that the decision criteria providing the best overall performance was theuse of <250 µg/cm2 to distinguish between strong and weak sensitizers in humans and the use of
an LLNA EC3 ≤9.4% to distinguish between strong and weak sensitizers in the LLNA. The
Panel stated that more data would be needed to determine if values different from these twowould be more appropriate. The Panel also recommended that safety factors other than 10 for
the lowest observed effect level (LOEL) be evaluated to determine if improved results could beobtained. The Panel also suggested an analysis that directly compares the LOEL values without
using a safety factor (i.e., using LOEL data only) and an analysis that only uses no observed
effect level data. The Panel further stated that traditional LLNA tests based on pooled or
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ICCVAM Test Method Evaluation Report: Appendix F1

individual lymph nodes for a dose group should be evaluated independently to assess the impactof using pooled data on the accuracy analysis for skin-sensitization potency. Finally, the Panelstated that the effect of different vehicles should be recognized as a limitation in the current
data analysis and a likely contributor to the variability observed within and across laboratories.
The Panel stated that data from studies that could not be confirmed as being GLP-compliant,but that were from peer-reviewed literature or sources with high-quality laboratory management
practices, were still appropriate to include in the accuracy analysis. However, the Panel stated
that, ideally, GLP compliance should be the standard, as it is clearly the only objective way tojudge the credibility of the data.
The Panel recommended that more data should be collected to determine the optimal thresholdin humans for distinguishing between strong and weak sensitizers. In addition, the Panel
discouraged conducting additional animal studies unless such studies would be expected to leadto an overall reduction in animal use. The Panel recommended that the LOELs from Akkan etal. (2003) be used instead of the DSA05 (i.e., the dose per skin area leading to a sensitization
incidence of 5%) values from Schneider and Akkan (2004) in all of the potency analyses. A
minority opinion by one Panel member stated that it was acceptable to use the DSA05 values
from Akkan et al. (2003) as LOEL values in the evaluation. This panelist mentioned that the
DSA05 value is a LOEL value adjusted to 5% incidence of induction in order to correct forhuman studies leading to different inductions. Furthermore, the panelist stated that because the
DSA05 is corrected for an induction rate of 5%, it would be better to compare with thetraditional LLNA EC3 than to use the default uncorrected LOEL.

F-32

ICCVAM Test Method Evaluation Report: Appendix F1

1.0

Murine Local Lymph Node Assay (LLNA) Limit Dose Procedure6

1.1

Comments on the Draft Background Review Document (BRD) for
Completeness, Errors and Omissions

1.1.1 General Comments
The international independent scientific peer review panel (hereafter, Panel) was asked if there
were errors in the draft LLNA limit dose procedure BRD that should be corrected, if omissionsof existing relevant data had been identified, or if there was additional information that should
be included. The Panel agreed that consideration should be given to applying the same term to
the LLNA limit dose procedure since in various places throughout the draft BRD it was referred
to differently as either the “cut-down,” the “limit dose,” or the “reduced LLNA” (rLLNA).
Since the European Centre for the Validation of Alternative Methods (ECVAM) has alreadyestablished a naming convention of “rLLNA,” the Panel recommended adopting the ECVAM
terminology to harmonize the terminology used among the international validation agencies.
The Panel recommended that since the validation of the LLNA limit dose procedureencompassed data that was analyzed retrospectively, a discussion of the limitations of a
retrospective evaluation of previously published LLNA results should be included in the final
BRD. In particular, the assumption that the highest dose in the retrospective dose-response
study would be equivalent to the highest possible dose tested in the limit dose procedure should
be addressed. Discussing such a limitation would be important since it bears directly on thevalidation of the limit dose procedure.
Further, since determination of the appropriate “limit dose” is critical to the LLNA limit dose
procedure, the Panel suggested that a discussion of how to arrive at the maximal concentration
for test substance dosing should be included in the final BRD. The final BRD should also
specifically define what is meant by the terms “avoidance of excessive irritation” and “systemic
toxicity” to aid in choosing the most appropriate maximum dose. In this regard, the Panelsuggested that a systematic and quantitative measurement of ear thickness and systemic toxicity
be considered or evaluated for routine inclusion in the LLNA protocol.
The Panel discussed modifying the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) LLNA protocol requirement for testing concurrent positive
controls (ICCVAM 1999; Dean et al. 2001) as a means of further streamlining the LLNA limit
dose procedure (i.e., reducing animal number, cost, etc.). Although the Panel did not reachconsensus, a suggestion was made that for laboratories in which the LLNA is “routinely”
performed and which had demonstrated the ability to consistently obtain positive results, hexyl
cinnamic aldehyde (HCA) or another positive control (e.g., a substance that matches thechemical class of the test substances) could be run at intervals for quality control purposes
rather than concurrent with each experiment. The Panel noted that Kimber et al. (2006) havedescribed the “routine” use of the “rLLNA” utilizing only a vehicle and a high-dose group. The
Panel also recommended that for laboratories that perform the LLNA only “occasionally,” aconcurrent positive control should be used. However, in their discussions, the Panel was notable to conclude what would constitute “routine” or “occasional” LLNA use or what would be

6

Also referred to as the “reduced” LLNA
F-33

ICCVAM Test Method Evaluation Report: Appendix F1

an appropriate interval between positive control testing when a concurrent positive control is
not used.
The Panel also noted that including the following additional information in the final BRD mightprove informative if included:•

An indication of any procedural problems reported for the tests•

An indication of the range of historical values obtained with the negative andpositive controls (the positive control historical range might give insight into theneed for a concurrent positive control)•

Any discussion of global harmonization should expand on why the draft BRD did
not place more reliance upon Organisation for Economic Co-operation andDevelopment (OECD) Test Guideline (TG) 429 (OECD 2002) as a normativereference•

For two of the substances tested multiple times (HCA and linalool alcohol), different
doses were used and discordant results were obtained. It should be noted for which(if either) of these tests, the highest dose tested was consistent with the dose
selection principles set out in the draft BRD

1.1.2 General Statistical Comments
The Panel also had some statistical comments related to the LLNA limit dose procedure. First,
the Panel viewed that a reference to the supplemental statistical information in which Dr.
Joseph Haseman performed power calculations on the traditional LLNA would be useful indetermining if the sample size used in the LLNA limit dose procedure was adequate for
evaluating skin-sensitization potential. Also, the Panel concluded that although a stimulationindex (SI) based on the ratio of 3.0 as the decision criterion for a sensitizer is informative,
statistical analysis determining if the test substance is significantly different from the control
substance should be recommended.
1.1.3 Comments with Specific References to the Text
The Panel also identified the following minor formatting and grammatical errors, andinformation gaps in the draft BRD:•

The manner of notating numerical data in the draft BRD tables was not consistent(e.g., in some places the value “one” was shown as 1, elsewhere as 1.0; in a few
places the SI was shown over the percent concentration used).

•Lines 291-294: The statement was made that “chemical class information is
included to provide an indication of the variety of structural elements present in thesubstances that were evaluated in this analysis, but it is not intended to suggest an
impact of structure on sensitization potential.” The latter concept is not entirely
correct; the portion of the sentence stating “but it is not intended to suggest an
impact of structure on sensitization potential” should be omitted. Consideration
should be given to using the large database of chemicals to selectively modify
structure-activity relationship (SAR) software for improved predictivity. This
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ICCVAM Test Method Evaluation Report: Appendix F1

could likely be accomplished by communication with software developers to pointout availability of the newly expanded ICCVAM dataset developed for evaluationof the LLNA limit dose procedure.
•Lines 299-300: The sentence is incomplete; “non-sensitizers” should be inserted at
the end of the sentence.

•Line 358: The citation to Sailstad et al. (2001) was not listed in Section 12.0
(References) and should be included.

•Lines 365-384 (Section 1.1.2): Consideration should be given to expanding thebackground on the mechanism and natural history of allergic contact dermatitis(ACD). Some additional detail regarding the biochemistry and cell biology of
immune induction and elicitation would be useful as an orientation to how the
LLNA functions as an integrated method of detection for ACD.

•

Lines 366–368: The introductory sentence on the prevalence of ACD as an
occupational health issue would benefit from amplification to also indicate that ACD
is of variable severity with some potentially severe ramifications, and that ACD isrecurrent upon rechallenge possibly requiring workplace accommodation or changeof employment.

•Lines 366-368: There is no reference provided for the statistic from the U.S.
Department of Labor Bureau and Labor Statistics cited in Section 1.1.2 of the draft
BRD.

•

Line 433: The reason for further evaluation of negative results with concentrations
less than 10% should be clarified (Kimber et al. 2006).

•

Lines 435-436: This bullet point re-plays conclusions made in the summary of the“rLLNA” issued by the scientific advisory committee of the European Centre for the
Validation of Alternative Methods (ECVAM). However, in the draft BRD thestatement has been altered and should instead read “..., as appropriate, per OECD
TG 429 (OECD 2002)” rather than citing ICCVAM (1999) and Dean et al. (2001).

•

Lines 452-453: The intent of the sentence would be clarified by modifying to read“...to identify potential human skin sensitizers through quantification of lymphocyte
proliferation in the test method.”•Lines 496-500: Reading of the Kimber et al. (2006) citation does not indicate arecommendation for a concurrent positive control group. Thus, the sentence in thedraft BRD that reports use of vehicle and positive control groups in the limit dose
procedure based on the Kimber et al. (2006) paper is incorrect.

•

Lines 509-510: This sentence should also mention that the LLNA limit dose
procedure, as published by Kimber et al. (2006), is rationalized not only as a meansof bioresource economy but as a valid means of streamlining the LLNA for
regulatory screening assessment purposes under regulation such as Registration,
Evaluation and Authorisation of Chemicals (REACH).
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ICCVAM Test Method Evaluation Report: Appendix F1

1.2
1.2.1

•

Lines 520-523: A footnote might be needed to explain why OECD TG 429 (OECD2002) is not referenced here.

•

Lines 629-636: Data donated by GlaxoSmithKline (GSK) to the National
Toxicology Program Interagency Center for the Validation of Alternative
Toxicological Methods (NICEATM)-ICCVAM LLNA review were generated under
GLP conditions at a clinical research organization (CRO) repeatedly audited for
GLP compliance by GSK. This information should be added to the text.

•

Section 6.1: The presentation of data and associated discussion regarding limitationsin accepting a 10% dose concentration cut-off should be repositioned for emphasis.
This information is important in developing suggestions for a standard LLNA limit
dose procedure. Data from Appendix D could be reduced to a small table or figure
and be integrated into the body of the final BRD.

•

Lines 722-723: This appears to be an incomplete sentence.

•Lines 815-822: Data donated by GSK to the NICEATM-ICCVAM LLNA review
were generated under GLP conditions at a CRO repeatedly audited for GLP
compliance by GSK. This information should be added to the text.

•

Table following line 1126: There is a typographical error in the 2 x 2 table. It
appears that the cell for Negative (New Test) x Total (Reference Test) should read“c + d” rather than “a + d.”

Comments on the Validation Status of the LLNA Limit Dose Procedure
Test Method Protocol

For the proposed LLNA limit dose procedure, ICCVAM recommended that the number ofanimals used in each group should be the same as that recommended by ICCVAM for the
traditional LLNA based on its 1998 evaluation (i.e., at least five animals per group), and that
individual animal data should be collected and reported (ICCVAM 1999; Dean et al. 2001). The
Panel was asked whether they agreed that these are appropriate protocol requirements for thelimit dose procedure. The Panel agreed that, based on the supplemental power calculations forthe traditional LLNA performed by Dr. Joseph Haseman (see Table F-1), a minimum of fiveanimals per treatment group should be recommended for all future studies employing the limit
dose procedure. In addition, the collection of individual animal data, as recommended by
ICCVAM for the traditional LLNA (ICCVAM 1999; Dean et al. 2001), should also apply to all
future studies following the LLNA limit dose procedure. Similarly, application of the LLNA
limit dose procedure to a modified LLNA protocol would require adherence to a validated
protocol with the exception of omitting the middle and low dose groups. Respective power
calculations would indicate if application of the LLNA limit dose procedure to a validated
modified LLNA protocol would allow fewer animals per dose group.

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ICCVAM Test Method Evaluation Report: Appendix F1Table F-1 Power Calculations for the Traditional LLNA1
3.0-foldincrease2

2.5-foldincrease

2.0-foldincrease

1.5-foldincrease

1.3-foldincrease

Mean Rx response

1034.4

862.0

689.6

517.2

448.24Log (Mean Rx
response)

6.942

6.759

6.536

6.248

6.105

Difference (log scale)

1.099

0.916

0.693

0.405

0.262

Difference/SD

2.40

2.00

1.51

0.88

0.57

Power for N=595%

80-90%

50-80%<50%<50%

Power for N=4

90%

80%

50%

<50%

<50%

Power for N=350-80%

50-80%

<50%

<50%

<50%

Other power––

95% (N=11)95% (N=29)

95% (N=68)

Other power––

90% (N=9)90% (N=23)

90% (N=54)

Parameter

Abbreviations: N = number of animals; Rx = Treatment; SD = standard deviation.
1
The power calculations above are based on a one-sided p<0.05 Student’s t test applied to log-transformed
data from vehicle control LLNA tests.
2
Fold-increase = Required increase above the vehicle control for a positive response (i.e., the stimulationindex)The primary rationale for both provisions is to underpin robust statistical analysis of LLNA
results. Furthermore, the use of individual animal data would allow for the evaluation of dosing
errors or other anomalies that might be masked by the use of pooled animal data.
1.2.2 Substances Used for the Validation Studies
The Panel was asked whether they considered the traditional LLNA database representative of a
sufficient range of chemical classes and physical chemical properties such that it would beapplicable to any of the types of chemicals and products that are typically tested for skinsensitization potential. If not, the Panel was asked which relevant chemical classes/properties(other than those identified as limitations in the traditional LLNA) should be tested withcaution, or not evaluated using the limit dose procedure, and which chemicals or products
should be evaluated to fill this data gap. The Panel agreed that, in general, the traditional LLNA
database included in the LLNA limit dose procedure evaluation was representative of a
sufficient range of chemical classes and physical chemical properties and that it should beapplicable to any of the types of chemicals and products that are typically tested in the
traditional LLNA for skin-sensitization potential. It was notable that the substances included inthis evaluation provided a diverse chemical database. Since much is known about themechanism of sensitization, the LLNA should theoretically identify any chemical that works bymigration of haptens to the lymph node. However, the Panel noted that substance classes thatare sometimes problematic in the LLNA (i.e., metals) would also likely be problematic in the
LLNA limit dose procedure. There were also some substance classes that had limited or no
representation in the draft BRD (i.e., mixtures/formulations, higher molecular weight
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ICCVAM Test Method Evaluation Report: Appendix F1

biopharmaceuticals, and medical device materials). Thus, in general, the LLNA (and the LLNA
limit dose procedure) is best used as part of a weight-of-evidence appraisal in which attributes
such as physical chemical parameters, SAR evaluation, and indications of other biologicalactivity involving potential chemical-to-biological macromolecule interactions, are carefully
considered along with LLNA results to evaluate dermal sensitizing potential.
Because the LLNA limit dose procedure was based on a retrospective evaluation of existing
data, most of which was not generated using coded chemicals, the Panel was asked whether alack of coding of test substances adversely impacted or biased the current evaluation. The Panelconsidered that although coding of chemicals should be recommended for prospective
validation studies, this evaluation was likely not adversely impacted or biased because of a
retrospective evaluation of existing data. This is supported by the fact that many of the chemical
structures included in the analyses do not appear to contain known structural motifs associatedwith ACD or chemical hypersensitivity and therefore there was no a priori expectation that thechemical tested would be a sensitizer. The Panel viewed it important to consider the issue ofcoding or bias in prospective validation studies.
For some substances submitted using the traditional LLNA test method, it was not possible toconfirm whether the data were generated using pooled animal data for each dose group (asallowed in OECD TG 429 [OECD 2002]). ICCVAM (1999), Dean et al. (2001), and EPA(2003) recommend the use of statistical analyses to help interpret LLNA study results, which
necessitates data collected at the level of the individual animal. Furthermore, Cockshott et al.
(2006) reported that using individual animal data allowed for technical problems or other
outliers during an experiment to be identified. The Panel was asked what impact the inclusionof pooled animal data might have on the accuracy analysis of the LLNA limit dose procedure.
The Panel concluded that, although it would be important to note whether individual or pooled
animal data were reported, the retrospective analysis of the LLNA limit dose procedure versus
the traditional LLNA should not be limited to studies with confirmed individual animal data.
The Panel stated that internationally, both individual and pooled animal data have likely beenused both for regulatory decisions and for in-house decisions relating to product development
and risk management. Also, the fact that the retrospective data analysis presented in the draft
LLNA limit dose procedure BRD did not distinguish between individual or pooled animal datasuggested that both met the quality standards for inclusion in the draft BRD.
1.2.3 Test Method Accuracy
The Panel was asked whether the relevance (e.g., accuracy/concordance, sensitivity, specificity,
false positive and false negative rates) of the LLNA limit dose procedure had been adequately
evaluated and compared to the traditional LLNA (refer also to Table 6-1 of the draft ICCVAM
BRD). The Panel concluded that the relevance of the LLNA limit dose procedure had beenadequately evaluated and compared to the traditional LLNA. Comparisons resulting in an
accuracy of 98.9% (461/466), a sensitivity of 98.4% (308/313) and a specificity of 100%
(153/153) for the LLNA limit dose procedure when compared to the traditional LLNA were
sufficient to consider it adequately validated for use in the evaluation of skin sensitization,
mindful of its known limitations that are described elsewhere. Still, the Panel noted that it was
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ICCVAM Test Method Evaluation Report: Appendix F1

important to keep in mind that a prospective analysis may not have the same accuracy as thisretrospective analysis.
Furthermore, there were five substances for which the highest concentration tested produced an
SI < 3.0, while lower concentrations of these substances produced an SI >3.0 (see Table 6-2 ofthe draft ICCVAM BRD). These substances were classified as false negatives compared towhat was obtained in the traditional LLNA. The Panel was asked to identify any characteristics
associated with these or other substances that might signal that this type of abnormal dose
response might occur, and therefore, that using the LLNA limit dose procedure would not beappropriate. The Panel could not identify any common characteristics associated with the fivefalse negative substances that would explain the non-linear dose response obtained. It was notknown if any procedural problems were reported with these studies or what values werereturned by the negative/positive control groups (in relation to other historical positive control
values).
Thus, the Panel suggested that it might be worthwhile to examine whether LLNA results withthese five false negative substances should be repeated. If the difference turned out to berepeatable, there could be hypothetical reasons to explain why the higher doses did not pass the
SI threshold of 3.0. For example, under certain experimental conditions, the target lymphocytes
may be selectively induced to a highly sensitive state by some chemicals at higher doses andmay undergo either induction of apoptosis or inhibition of cell proliferation. Still, there was noevidence that these substances were immunomodulators that might have differentiallystimulated or depressed the immune response depending on the dose and exposure. In any case,understanding false negatives is encouraged to ensure adequate protection of public health.
The Panel was asked whether the draft BRD adequately characterized the usefulness andlimitations of the LLNA limit dose procedure based on the accuracy analyses. Overall, thePanel agreed that the draft BRD adequately characterized the usefulness and limitations of the
LLNA limit dose procedure based on the accuracy analyses. Since the LLNA limit dose
procedure and the traditional LLNA have close concordance, there was no need for detailed
discussion in the draft BRD. However, it was not explicitly stated in the draft BRD thatcompared to a fully conducted traditional LLNA, a false positive result in the LLNA limit dose
procedure is not possible (i.e., if the single dose used in the proposed limit dose procedure gives
an SI ≥3.0, then so would the top dose in the traditional LLNA). Furthermore, prospective
testing with the LLNA limit dose procedure to predict the sensitization potential of an unknown
chemical was not discussed.
1.2.4 Test Method Reliability
The Panel was asked if it was appropriate to assume that the intra- and inter-laboratory
reproducibility of the LLNA limit dose procedure and the traditional LLNA would be similar,
based on the fact that they use identical protocols with the exception of the number of doses
used (i.e., would reducing the number of test substance dose groups from three to one reduce
the reliability of the assay?). The Panel agreed that it was appropriate to assume that the intraand inter-laboratory reproducibility of the LLNA limit dose procedure and the traditional
LLNA would be similar, because reproducibility is more dependent on the method than on the
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ICCVAM Test Method Evaluation Report: Appendix F1

number of dose groups. However, reducing the number of test substances dose groups fromthree to one could reduce the sensitivity of the assay (i.e., the ability to correctly identify
sensitizers). The traditional LLNA may have a greater chance of correctly identifying asensitizer even in the presence of one or more technical errors since there are data from threedose groups for consideration and an SI ≥3.0 at any dose group would result in the substancebeing classified as a sensitizer. However, for the purpose of adopting an assay that uses fewer
animals and provides increased throughput for screening purposes, these hypotheticalconsiderations are not a sufficient reason to argue against use of the limit dose LLNA
procedure.
1.2.5 Data Quality
For some studies included in the draft BRD, it was not possible to determine whether or notthey had been conducted in accordance with GLP guidelines. Furthermore, original records forsome of the non-GLP studies included in this evaluation could not be obtained. As a result, an
independent audit could not be conducted to confirm that the reported data was the same as thedata recorded in laboratory notebooks. Neither was it possible to obtain the results of GLP
audits for all studies conducted in accordance with GLP guidelines. The Panel was asked
whether the results of such studies (all of which are currently included) should be excluded
from the performance analyses. The Panel concluded that it was important to note if the data
were obtained from studies conducted according to GLP guidelines, as ideally this should be thecase. However, the Panel concluded that the data resulting from the retrospective studies that
could not be confirmed as GLP-compliant should not be excluded from the performance
analysis. Since there was not an indication that the reliability of the data presented forconsideration may have been compromised, omitting any data would likely lessen the impact of
the analysis. Furthermore, data obtained from peer-reviewed literature or final reports werelikely of sufficient quality.
1.2.6 Consideration of All Available Data and Relevant Information
The Panel was asked if all the relevant data identified in published or unpublished studies
conducted using the traditional LLNA had been adequately considered in the draft BRD. If not,the Panel was asked what other traditional LLNA data needed to be considered and how suchdata could be obtained. The Panel considered that the draft BRD had taken into account a large
majority of the relevant data identified in published and unpublished traditional LLNA studies.
The data received as a result of the Federal Register (FR) notices and the key literature
citations seemed to be inclusive of the relevant data for this analysis. Although additional datathat could have been included might exist, it was deemed unlikely that the current outcome(which is based on 466 substances) would be altered given the very small change in accuracy
statistics relative to Kimber et al. (2006), which was based on 211 substances.

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1.3

Comments on the Draft ICCVAM Test Method Recommendations on the LLNA
Limit Dose Procedure

1.3.1 Test Method Usefulness and Limitations
The Panel was asked to comment on whether the available data supported the ICCVAM draft
recommendations for the LLNA limit dose procedure in terms of the proposed test method
usefulness and limitations (i.e., that the LLNA limit dose procedure should be routinely
recommended for hazard identification when dose response information is not required). The
Panel considered that, based on the available information, the draft recommendations appeared
valid, but made the following suggestions:•

Further emphasis should be given to using the LLNA limit dose procedure as a partof a comprehensive weight-of-evidence evaluation of dermal sensitizing potential(e.g., including physical chemical evaluation, SAR information, including likelihoodof dermal penetration, ability of materials to adduct biomacromolecules).

•Such information in addition to LLNA results might also be useful in confirming orquestioning LLNA outcomes terms of in human hazard identification, since it shouldbe emphasized that a major application of the method is to prospectively detectharmful chemicals.

•

Solubility or thermodynamic activity data, beyond visual assessment (e.g., use ofchemically-specific methods to document solubility), should be used to confirm theappropriateness of the maximum dose tested.

•

Vehicle selection for the LLNA can affect the results and may not allow accuratecomparisons between chemicals applied in different vehicles. In choosing the best
vehicle, consider measured solubility information for the potential vehicle. Then, it
would be important to take into account how the vehicle affects the amount of thechemical that can be applied to the ear. More importantly, the impact that vehicle
selection has on the amount of applied chemical that actually gets into the mouse to
induce the sensitization response should be evaluated. Some of the recommended
LLNA vehicles (e.g., 4:1 acetone:olive oil (AOO), dimethylsulfoxide (DMSO),
methyl ethyl ketone) could be expected to disrupt the barrier properties of the skin.
Additionally, although propylene glycol might allow an increased amount ofchemical to be applied, it might also inhibit the penetration of a chemical byenhancing partitioning in the vehicle relative to the skin.

The Panel was asked whether the LLNA limit dose procedure should be routinely
recommended for the hazard identification of skin sensitizing chemicals when dose response
information is not required. With the points noted above in mind, the Panel agreed that it should
be routinely recommended since the LLNA limit dose procedure offers time, cost, throughput,and logistical benefits as well as using fewer animals. Still, the investigator should keep in mind
what is known of the chemical regarding general toxicity and note scenarios where abnormal
dose-response relationships in the traditional LLNA might result in false negatives in the limit
dose procedure (see Table 6-2 of the draft ICCVAM BRD).
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ICCVAM Test Method Evaluation Report: Appendix F1

The Panel was then asked whether the LLNA limit dose procedure should be routinely
recommended as the initial test to identify sensitizers before conducting the traditional LLNA,
as a way to further reduce animal use, even if dose response information is required, sincenegative results would not require further testing. The Panel agreed that use of the LLNA limit
dose procedure, as the initial testing procedure to identify sensitizers and non-sensitizers before
conducting the traditional LLNA, is justifiable even when dose response information isrequired. This is applicable in the occupational and public health setting where obtaining hazard
information is of critical importance. There is a benefit since dose-response information
generated in subsequent testing in the traditional LLNA for substances that were positive in thelimit dose procedure then gives further assurance of detecting hazardous substances andallowing a potency estimate. The benefits of screening out the negatives (which do not require
dose response information) is clear; however the animal welfare gains will depend on the
proportion of test substances in any class that turn out to be non-sensitizers and there might bepossible consequences of the delays resulting from a further round of testing for those materials
that are identified as sensitizers.
Based on the existing database, there is a false negative rate of 1.6% (5/313 positive
compounds) for the LLNA limit dose approach compared to the results obtained in the
traditional LLNA. The Panel was asked whether they considered that this is adequately
addressed by the proposed cautionary language and weight-of-evidence consideration for
negative substances. The Panel agreed that the small rate of false negatives was adequately
addressed in the draft test method recommendations by giving cautionary and weight-ofevidence consideration to the negative substances (and any possible false positive results).
Furthermore, given that the dose responses for these five materials were rather unusual, it was
not known whether these studies were repeatable, whether any procedural problems werereported with these studies, or what values were returned by the negative/positive control
groups (in relation to other historical positive control values). In general, the Panel viewed thatthe false negative rate of 1.6% would likely be unimportant when the larger differences between
the animal model and humans are considered.
1.3.2 Test Method Protocol
The Panel was asked whether they agreed that the available data support the ICCVAM draft
recommendations for the LLNA limit dose procedure in terms of the proposed standardized test
method protocol. The Panel agreed and recommended adherence to the ICCVAM (1999) LLNA
protocol for future studies of the LLNA limit dose procedure with the exception of omitting the
middle and low dose groups. Similarly, application of the LLNA limit dose procedure to amodified LLNA protocol would require adherence to the modified LLNA protocol with the
exception of omitting the middle and low dose groups. Adhering to the use of individual
animals for future studies was specifically stressed because it would allow for an estimate ofinter-animal variability.
The recommended ICCVAM protocol (ICCVAM 1999; Dean et al. 2001; EPA 2003), as wellas OECD TG 429 (OECD 2002), specifies that the highest dose tested should be the highest
soluble concentration that does not induce systemic toxicity and/or excessive skin irritation.
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However, Kimber et al. (2006) concluded that negative results obtained from studies where thehighest concentration tested was below 10% should be considered invalid, and adopted a 10%
application concentration as a threshold of confidence for categorization of a chemical as being
negative while noting that the figure should not be considered as inviolable. The Panel was
asked whether the data presented in the draft BRD (i.e., 51/313 positive substances in the
NICEATM database were negative at concentrations equal to or above 10%, but were positiveat even higher concentrations) were adequate to conclude that this threshold concentration is not
appropriate. The Panel viewed that this point should be clarified. ICCVAM recommended thatno threshold should be used to determine the validity of conduct of the LLNA limit dose
procedure. Instead, formal attempts to maximize dose delivery including documentation ofsolubility of the test substance in the vehicle used should be undertaken.
The Panel was asked whether additional testing should be required if a negative result was
obtained for a test substance in a study where the highest concentration that could be tested(based on systemic toxicity or excessive local irritation, as described in ICCVAM [1999], Deanet al. [2001], and EPA [2003]) was <10%. The Panel considered that, if a negative result was
obtained for a test substance under these conditions, additional testing should not be required,because at that point it would likely be a toxic effect and not sensitization. In contrast, theimperative should be to minimize the number of false negatives. For this purpose, rigorous
examination of maximum solubility or other parameters to ensure testing at maximum
concentration should be employed. In addition, weight-of-evidence considerations such as SAR
and physicochemical characteristics should be documented. More animal testing to verify
negative results should only be undertaken if the weight-of-evidence suggests that it would beappropriate.
The Panel was asked if the current approach for selecting the “limit dose” was appropriate orwhether there is a threshold concentration for the LLNA at which a negative result could always
be considered as an acceptable result. The Panel agreed that the current recommendation to
select a maximum applied dose in the LLNA limit dose procedure is appropriate. However, thedata presented in the draft BRD implied that at present it is not possible to establish a uniform
concentration threshold for the “limit dose.” Thus, it seemed justifiable that preliminary
experimentation (as would be typically performed during a dose range finding study) should be
conducted on vehicle selection, test substance solubility, and stability in the vehicle.
1.3.3 Future Studies
The Panel was asked if they agreed that the available data support the ICCVAM draft
recommendations for the LLNA limit dose procedure in terms of the proposed future studies.
Although limited in scope, the Panel considered that the available data supported the ICCVAM
draft recommendations for additional studies. The Panel agreed that attempts be made toinvestigate if maximum solubility was achieved (e.g., use of chemically-specific methods todocument solubility). For hazard assessment, it was considered troublesome that there were somany vehicle choices, because the vehicle could have a significant effect on whether (and howmuch) a test substance penetrated the skin barrier. Observed vehicle effects may relate todermal penetration as well as to immunomodulation. The Panel considered it desirable to follow
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ICCVAM Test Method Evaluation Report: Appendix F1

the hierarchy of vehicles recommended in the ICCVAM (ICCVAM 1999; Dean et al. 2001)
protocol. In addition, dedicated attempts must be made to investigate solubility in AOO
mixtures before using other vehicles. Regardless of the vehicle used, it is important to ensurethat a vehicle does not promote lymph node cell proliferation. The Panel also suggested that it
might be informative to test both known mild and severe sensitizers concurrently in all
recommended vehicles to evaluate if a specific vehicle choice(s) might influence the results.
Although the false negative rate in the current analysis was small, a need exists to better
understand factors that could lead to false negative results with future use of the LLNA limit
dose procedure. Thus, consideration should also be given to formal statistical assessments toverify group size and use of individual animal data in routine performance of the LLNA limit
dose procedure. Criteria should be established to verify proficiency with the LLNA limit dose
procedure. Such criteria could be used to answer questions about the necessity to performconcurrent positive controls.
1.3.4 Comments with Specific References to the Text
The Panel also identified the following comments and/or corrections to the draft ICCVAM test
method recommendations document on the LLNA limit dose procedure that should beconsidered by ICCVAM:•

Lines 26-28: Conclusions given here regarding the relative potency ratings of thefive materials classified as false negative in the analysis in Section 6.2 of the draft
BRD were newly introduced. This assessment should also have been considered forinclusion in Section 6.2 of the draft BRD.

•

Line 28: The citation of Gerberick et al. (2004) was not accompanied by a reference.

•Lines 62-70: The listing of substances not amenable to test in the LLNA could havebeen expanded to also include agents with anticipated pharmacodynamic action as
immune suppressants.

•

Line 69: The citation of Gaspari et al. (2007) was not accompanied by a reference.

•Lines 75-79: Dependent upon other considerations, this portion of the text could
have been modified to (1) clarify recommendations regarding routine use of
concurrent positive control (i.e., possible exception for laboratories conducting ahigh volume of LLNA work in which periodic positive control for quality control
purposes might suffice), and (2) expand on the details regarding indications ofexcessive irritation and/or systemic toxicity to aid in choice of maximal test dose.

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ICCVAM Test Method Evaluation Report: Appendix F1

2.0

LLNA for Testing Aqueous Solutions, Metals, And Mixtures

2.1

Comments on the Draft Addendum for Completeness, Errors, and Omissions

In regard to the draft Addendum to the traditional LLNA BRD, the Panel was asked tocomment on any errors that should be corrected or omissions of relevant data/information thatshould have been included. The Panel concluded that there were no apparent errors oromissions to the draft Addendum.
2.2

Comments on the Validation Status of the Traditional LLNA for Testing
Aqueous Solutions, Metals, and Mixtures

2.2.1 Substances Used for the Validation Studies
The Panel was asked whether the database of substances evaluated was representative of a
sufficient range of mixtures, metal compounds, and substances in aqueous solutions that are
typically tested for skin-sensitization potential. While there were limited data available on theeffects of mixtures, metals, and aqueous solution on skin-sensitization potential, the Panelconsidered the database to be generally representative. The Panel indicated that there did notseem to be obvious classes of chemicals missing from the data set used to evaluate the utility ofthe traditional LLNA for testing aqueous solutions. However, quantitative compositions for the
mixtures included in the analysis had not been provided. Thus, it was difficult to determine if
those mixtures were representative of the types of mixtures typically tested in the traditional
LLNA. With respect to metals, there was a paucity of commercially useful metals such as
platinum, palladium, iron, zinc, manganese, and silver compounds. To enlarge the group of
metal non-sensitizers, substances used as cosmetic ingredients (e.g., titanium dioxide) andaluminum compounds currently used in antiperspirants might be considered. However, thePanel considered that the inclusion of an array of other metals and at least one zinc andmanganese salt likely weighted the data set appropriately and it appeared sufficiently broad tosupport conclusions about the utility of the traditional LLNA for testing the skin-sensitization
potential of metals.
Substances or mixtures that were tested in an aqueous or an organic:aqueous vehicle werelabeled as aqueous solutions. For the purpose of this evaluation, a substance or mixture
containing at least 20% water was defined as an aqueous solution. The Panel was asked whether
this criterion was appropriate for defining an aqueous solution. The Panel was uncertain about
the appropriateness of this definition of an aqueous solution, but did not offer an alternative
definition. However, the Panel indicated that an organic:aqueous solution that is not miscible
would likely produce varying results because of partitioning of the chemical into either phase.
The Panel was asked whether the lack of coding of test substances might adversely impact orbias the current evaluation. While coding of chemicals is recommended for prospective
validation studies, the retrospective evaluations in the draft Addendum were based on existing
data, most of which were not generated using coded chemicals. However, the Panel agreed thatthe lack of chemical coding was not likely to bias the evaluation since this study was
retrospective. This is supported by the fact that many of the chemical structures included in the
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ICCVAM Test Method Evaluation Report: Appendix F1

analyses did not contain known structural motifs associated with allergic contact
dermatitis/chemical hypersensitivity and therefore there was no a priori expectation that thechemical tested would be a sensitizer. Furthermore, many of the substances tested wereapparently evaluated for hazard assessment purposes rather than to test the predictive ability ofthe traditional LLNA. Thus, there does not appear to be any bias in chemical selection for the
expanded dataset considered in the study of applicability domain for the traditional LLNA.
For some substances submitted using the traditional LLNA test method, it was not possible toconfirm whether the data were generated based on pooled lymph nodes among animals within adose group, as allowed in OECD TG 429 (OECD 2002), or individual animal responses, as
recommended by ICCVAM (1999) and required by EPA (2003). ICCVAM (1999) and EPA(2003) both recommend the use of statistical analyses to aid in the interpretation of traditional
LLNA study results; such analyses necessitate data collected from individual animals.
Additionally, Cockshott et al. (2006) reported that using individual animal data allowed foroutlier animal results within a dose group to be identified. The Panel was asked whether the
analysis of the performance of the traditional LLNA for testing mixtures, metal compounds, andsubstances in aqueous solutions should be limited to data from studies that collected individual
animal data, and then to comment on the potential impact on the accuracy analysis of including
results from studies in which pooled animal data were collected. The Panel concluded that,
although individual animal data were preferred, pooled animal data should not be excluded
automatically from this retrospective analysis.
2.2.2 Test Method Accuracy
The Panel was asked whether the relevance (e.g., accuracy/concordance, sensitivity, specificity,
false positive and false negative rates) of the traditional LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions had been adequately evaluated and comparedto the human and guinea pig test results. The Panel agreed that the comparative assessment of
the relevance of the traditional LLNA for testing mixtures, metal compounds, and substances inaqueous solutions appeared to be as comprehensive as was feasible. However, because of thelimited number of comparisons available, the accuracy statistics probably do not give acomplete picture of the usefulness and limitations of the traditional LLNA for identifying skinsensitizers among these types of substances or when using an aqueous vehicle.
When multiple traditional LLNA studies were available for the same substance, the “majority
call” (among studies using the same vehicle and generally tested over the same concentration
range) was used by ICCVAM to assign an overall classification for the purposes of the accuracy
analysis. For example, if chemical X was tested five times and was positive in three studies andnegative in two, the overall classification was positive. The Panel was asked whether theyagreed with this approach. They expressed their concern about the approach in the following
way; if all nickel-containing compounds in the analysis were viewed as a group, there were four
positive calls and four negative calls (see Appendix C2 of the draft Addendum). Using the“majority call” approach, the overall call would be determined by the next available study,
which may not provide the correct call. More data would be needed to confirm whether theclassification was appropriate. For this dataset, most of the “negative calls” had SI values that
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ICCVAM Test Method Evaluation Report: Appendix F1

approached 3.0. Thus, a more suitable method might be to base the overall call on the SI data,
while giving greater positive call consideration/weight to SI values just below 3.0. It may also
be useful to perform a meta-analysis. It is important for the Addendum to mention the potential
impact of using the “majority call” decision, rather than relying on a weight-of-evidence
approach, on the accuracy analyses.
2.2.3 Data Quality
For some studies included in the draft Addendum, it was not possible to determine whether or
not they had been conducted in accordance with GLP guidelines. Original records for some of
the non-GLP studies included in this evaluation could not be obtained. As a result, an
independent audit could not be conducted to confirm that the reported data was the same as thedata recorded in laboratory notebooks. Neither was it possible to obtain the results of GLP
audits for all studies conducted in accordance with GLP guidelines. The Panel was asked to
discuss what impact this lack of information might have on the evaluation of the traditional
LLNA for testing mixtures, metal compounds, and substances in aqueous solutions and whether
such studies should be excluded from an analysis of test method accuracy. The Panelconsidered it important to note if the data were obtained from studies conducted according tointernational GLP guidelines, since ideally this should be the process followed. However, thePanel viewed that data from studies that could not be confirmed as being GLP-compliant werestill appropriate to include in the accuracy analysis, provided that the data were from the peerreviewed literature or from sources with high quality laboratory management practices. Muchof the value for this draft Addendum was the potential to supplement the data available at thetime of the ICCVAM (1999) analysis. Additional information on test substance identification
would clearly be useful in the continued evaluation of the applicability domain of the traditional
LLNA, but omitting data on mixtures, metals, or use of aqueous solutions based solely on thelack of GLP compliance would lessen the impact of the current retrospective analysis and did
not seem warranted. However, if the original data were not available, it would be appropriate tonote this in the final version of the Addendum.
2.2.4 Consideration of All Available Data and Relevant Information
The Panel was asked whether the draft Addendum included all of the relevant data for studies
conducted using the traditional LLNA for testing mixtures, metal compounds, and substances inaqueous solutions. The Panel considered that, although it was possible that there might be a few
studies in the literature to augment the analysis, it seemed that the relevant data had been
identified and the response to the FR notice and the literature citations examined had included
the most relevant studies.
2.3

Comments on the Draft ICCVAM Test Method Recommendations on the
Traditional LLNA for Testing Aqueous Solutions, Metals, and Mixtures

2.3.1 Test Method Usefulness and Limitations
ICCVAM stated that more data would be needed before a recommendation on the usefulness
and limitations of the traditional LLNA for testing mixtures could be made, due to thelimitations associated with the available mixtures database (i.e., unknown formulae, lack of
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human data). The Panel was asked whether they agreed that the available data supported the
ICCVAM draft recommendations for the traditional LLNA with regard to testing mixtures interms of the proposed test method usefulness and limitations. The Panel agreed that ICCVAM’s
draft recommendation with respect to the traditional LLNA testing of mixtures appeared valid
based on the limitations inherent in the available data set. Still, the Panel urged that the
ICCVAM recommendation indicate that the approach may be viable. The Panel further
recommended that the test method recommendations summary should indicate that thelimitations include relatively poor concordance of traditional LLNA outcomes for mixtures withto those obtained in guinea pig tests. Routine comparisons of accuracy according toclassification criteria may not be sufficient to evaluate the concordance for mixtures, andfurthermore, the guinea pig tests are not necessarily valid for mixtures. The Panel also indicatedthat the term “mixtures” was used too broadly (i.e., can represent an infinite number ofmaterials) and it would be more beneficial to specify types or formulations of mixtures that are
being examined.
ICCVAM recommended that, based on the available data for metals, the traditional LLNA wasuseful for the testing of metal compounds, with the exception of nickel. The Panel was asked
whether they agreed that the available data supported the ICCVAM draft recommendations forthe traditional LLNA with regard to testing metals in terms of the proposed test method
usefulness and limitations. Based on the available information, the Panel agreed that the draft
recommendations with regard to testing metals appeared to be valid. In particular, the evidence
for most metals (e.g., accuracy of 86% (12/14), sensitivity of 100% (9/9), specificity of 60%(3/5), 0% (0/9) false negatives) when comparing traditional LLNA results to those obtained
from evaluations in humans supported the use of the traditional LLNA as a hazard identification
tool for metals, excluding nickel. However, the Panel recommended that it would be
worthwhile to study further the variable results obtained for nickel since there is a wealth ofliterature on allergic contact dermatitis of nickel in humans.
In a minority opinion, Dr. Dagmar Jírová stated that it should not be concluded that thetraditional LLNA was not suitable for testing nickel compounds, because the different vehicles
used may have had a significant impact on the ability of nickel to penetrate the skin and bebioavailable. She noted that nickel chloride and nickel sulfate were both positive in aqueous
solutions, and negative only when non-aqueous vehicles were used. In human exposures, nickel
compounds were applied in aqueous solutions. Thus, this may serve as sufficient justification touse aqueous vehicles when nickel, and perhaps also other substances, are tested and evaluatedin the traditional LLNA. When DMSO was used as the vehicle, the SI value increased withincreasing nickel concentration. Unfortunately, no data were available for concentrations over
5% for either nickel compound in DMSO. Nickel chloride as 10% in aqueous solution reached
an SI of 6.6. Inconsistent test results due to the vehicle have also occurred in other in vitro
studies (e.g., phototoxicity). Thus, Dr. Jírová concluded that the traditional LLNA could be usedeven for testing nickel compounds when other vehicles (in particular aqueous) are used.
Due to the limited number of substances tested in aqueous solutions, ICCVAM recommended
that more data would be needed before a recommendation on the usefulness and limitations of
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ICCVAM Test Method Evaluation Report: Appendix F1

the traditional LLNA for testing substances in aqueous solutions could be made. The Panel was
asked whether they agreed that the available data supported this ICCVAM draft
recommendation for the traditional LLNA with regard to the testing of substances in aqueous
solutions. The Panel agreed that the draft ICCVAM recommendation was appropriate and that
more data were required before an adequate evaluation of the use of the traditional LLNA withaqueous solutions could be conducted.
2.3.2 Test Method Protocol
The Panel was asked whether they agreed that the available data supported the ICCVAM draft
recommendations for the traditional LLNA in terms of the proposed test method standardized
protocol. The Panel agreed that, in general, the results of the assessment in the draft Addendum
supported the proposals for standardized conduct of the traditional LLNA. However, thisconclusion depended on a side-by-side reading of the draft Addendum and the ICCVAM (1999)
protocol. The Panel suggested expanding the brief section of the draft test method
recommendations dealing with test method protocol for the traditional LLNA (Section 2.0) to
specifically point out how the conclusions of the applicability domain evaluation may affect thestandard traditional LLNA protocol. For example, the evaluation of aqueous solutions apparently
resulted in the methodological recommendation that aqueous test solutions be avoided and thefurther recommendation of a hierarchy of organic solvents to be considered as dosing vehicles.
The emphasis might be on using a vehicle to which humans may actually be exposed.
2.3.3 Future Studies
The Panel was asked whether they agreed that the available data support the ICCVAM draft
recommendations for the traditional LLNA in terms of the proposed future studies. The Panelagreed that the ICCVAM recommendation for continued accrual of information from traditional
LLNA evaluations of mixtures, metals, and aqueous solutions with comparative data for guinea
pig and human tests was appropriate. The traditional LLNA accuracy for metals of 86% andsensitivity of 100% (0% false negative) was excellent; a specificity of 60% (40% false positive)
was considered acceptable as over-classification maintains safe human use. The Panelencouraged the use of the traditional LLNA to acquire further information on mixtures, metals,
and aqueous solutions. However, the Panel suggested that, given resource limitations, it would
be important to prioritize the recommendations in order to focus on what is most important.

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3.0

Non-Radioactive LLNA Protocol - The LLNA: Daicel Adenosine
Triphosphate (LLNA: DA) Test Method

3.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

3.1.1 General Comments
The Panel was asked if there were any errors in the draft LLNA: DA BRD that should becorrected, if omissions of existing relevant data had been identified, or if there was additional
information that should be included. As a general comment, the Panel noted that the draft BRD
clearly and succinctly provided an overview of the LLNA: DA test method and the relevant
validation study data. The draft BRD indicated that the LLNA: DA differs from the traditional
LLNA in the method of measuring proliferation (measures levels of adenosine triphosphate[ATP] instead of radioactivity), substance treatment (pretreating the test site with 1% sodium
lauryl sulfate [SLS] prior to test substance application and an additional treatment on day 7),
and sampling time (draining auricular lymph nodes are collected on day 8 rather than on day 6).
Because the traditional LLNA evaluates the induction phase only, the relevance of results with
the LLNA: DA (and any other LLNA protocol) should always be considered in the context ofhuman experimental sensitization data, human epidemiologic data, and elicitation in the clinicalsetting.
3.1.2 Comments with Specific References to the Text
The Panel noted the following text that should be clarified or corrected in the final version of
the LLNA: DA BRD:•

Line 428: The text should read “1% SLS,” not “1% SDS”; the same terminology
should be used throughout rather than going back and forth between SLS andsodium dodecyl sulfate (SDS).

•

Line 449: The text and formula in lines 448-450 appear misplaced and instead seemto belong in Section 7.0. Additionally, X and Y should be defined and the “Var (lnSI)” formula should be clarified.

•

Table 3-1: There were 33 substances in the table, yet the discussion of the table in
the text referred to 31 substances. Although the reason for this apparent discrepancy
becomes more evident later on, this should be discussed up front.

•

Table 3-2 and 3-3: The interlaboratory distribution and testing of the sensitizers
versus non-sensitizers should be indicated here.

•

Table 6-1: Although the table clearly provided a comparison of the different
methods, it would be useful if the footer for this table also indicated the basis for thedifferences in substances included in each analysis (i.e., n=25, 26, or 29) as stated in
the text.

•

Table 7-1 was only moderately helpful because the standard deviations (SD) were
not calculated on a log scale. Given the skewness in the data, the ranges given weremisleading indicators of increases due to the vehicles. Without the samples sizes, an
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ICCVAM Test Method Evaluation Report: Appendix F1

analysis of variance was impossible to calculate, and that would have been theappropriate measure of differences between the experiments. In order to makerecommendations regarding needed reproducibility experiments, it would have been
helpful to have a power analysis for this situation. That is, for each vehicle with eachchemical, using two, three, four and five animals per dose group per experiment,
how many experiments need to be run to detect significant differences between theexperiments?
3.2

Comments on the Validation Status of the LLNA: DA

3.2.1 Test Method Protocol
Based on its 1998 evaluation of the traditional LLNA procedure, ICCVAM recommended thatat least five animals be used in each test group (ICCVAM 1999). The LLNA: DA validation
studies presented in the draft BRD were performed using four animals per dose group. Thus, thePanel was asked to comment on the potential impact of using fewer than five mice per dosegroup. The Panel noted that supplemental statistical information they were provided withindicated that the power for detecting a three-fold increase in the SI value in the treatmentgroup for the LLNA: DA dataset evaluated in the draft BRD was estimated to be 95% for asample size of three mice per dose group (see Table F-2). Since an increase of false negatives
may not be an issue, the potential opportunity exists for utilizing this smaller group size. The
Panel cautioned, however, that using less than five animals per group might result in a lessprecise estimate of the mean response, which, in turn, will impact accuracy. Also, if technicalerrors further reduce the sample size, accuracy is further reduced. Thus, the Panelrecommended that all initial validation studies adhere to the ICCVAM-recommended protocol(ICCVAM 1999; Dean et al. 2001) of five animals per dose group until sufficient information is
generated to indicate that the use of fewer animals per dose group is statistically valid.

F-52

ICCVAM Test Method Evaluation Report: Appendix F1Table F-2 Power Calculations for the LLNA: DA1
3.0-fold
Increase2

2.5-fold
Increase

2.0-fold
Increase

1.5-fold
Increase

1.3-fold
Increase

Mean Rx response

8835

7362.55890

4417.53828.5Log (mean Rx
response)

9.086

8.904

8.681

8.393

8.250

Difference fromcontrol (log scale)

1.098

0.916

0.693

0.405

0.262

Difference/SD

3.95

3.29

2.49

1.46

0.94

Power for N=5

99%

99%

95%

50-80%<50%

Power for N=499%

95-99%

90%

50%

<50%

Power for N=395%

90-95%

80%

<50%

<50%

Other power

–

–

–

95% (N=11)95% (N=25)

Other power

–

–

–

90% (N=9)90% (N=20)

Parameter

Abbreviations: N = number of animals; Rx = Treatment; SD = standard deviation.
1
The power calculations above are based on a one-sided p<0.05 Student’s t test applied to log-transformed
data from vehicle control LLNA:DA tests.
2
Fold-increase = Required increase above the vehicle control for a positive response

The data generated for the substances analyzed in the LLNA: DA interlaboratory validation
studies came from auricular lymph nodes that were pooled across animals in each treatmentgroup. The Panel was asked to comment on the potential impact of including pooled animal
data on the accuracy analysis of the LLNA: DA. The Panel noted that a statistical analysis ofdifferences between treatment-related and vehicle control ATP levels could not be determined
without measures of variability. Individual animal data highlights technical issues and allows
for consideration of dose-response information and statistical analyses.
The LLNA: DA differs from the traditional LLNA in the treatment schedule and by including a
pretreatment step with 1% SLS just prior to application of the test substance. The Panel was
asked to comment on the appropriateness of these protocol differences. The Panel did notconsider these differences to be significant, as long as it could be demonstrated that the 1% SLS
pretreatment step and the additional test substance treatment on day 7 did not induce a skinreaction indicative of the elicitation phase of skin sensitization. Although it was being used at alower concentration than the estimated concentration needed to produce a stimulation index of 3
(EC3), the Panel expressed concern about pretreating the mouse ear with 1% SLS since SLS isan irritant and positive in the traditional LLNA. Consequently, the inherent sensitivity of the
LLNA may be modified by the 1% SLS pretreatment step. To demonstrate that these concerns
are not justified, the Panel concluded that weak irritants and weak sensitizers needed to betested in the LLNA: DA assay with and without pretreatment with 1% SLS. The test method
developer might also consider using decision criteria other than SI ≥3.0 such that 1% SLS
pretreatment is no longer necessary.
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ICCVAM Test Method Evaluation Report: Appendix F1

3.2.2 Substances Used for the Validation Studies
The Panel was asked if they considered the substances tested in the LLNA: DA to be
representative of a sufficient range of chemical classes and physicochemical properties that thetest method would be applicable to any of the types of chemicals and products that are typicallytested for skin-sensitization potential. The Panel considered the database of test substancestested in the LLNA: DA representative of a sufficient range of chemicals. The selected
substances included solids and liquids and a range of solvents/vehicles. The database also
represented a range of sensitizing potency, a variety of different chemical classes andsubstances with differing requirements for metabolic activation. However, it might have been
useful to have also included substances with clearly different protein reaction mechanisms(protein binding), as well as dyes, natural extracts, and mixtures.
3.2.3 Test Method Accuracy
The accuracy analysis in the draft LLNA: DA BRD was based on overall concordance with thetraditional LLNA. Accuracy statistics compared to the guinea pig tests and humandata/experience were also provided. The Panel, when asked if they considered thesecomparisons appropriate for assessing the accuracy of the LLNA: DA, agreed that thecomparisons to the traditional LLNA performance and also to the guinea pig and humansensitization data were important. The Panel also stressed that, because the traditional LLNA
only evaluates the induction phase, the relevance of the LLNA: DA results should always be
considered in the context of human experimental sensitization data, human epidemiologic data,and elicitation in the clinical setting.
The Panel was asked if they considered the evaluation of the relevance of the LLNA: DA andthe comparison to the traditional LLNA to be adequate. The Panel noted that Table 6-1 of thedraft LLNA: DA BRD clearly provided a comparison of the different reference methods (i.e.,
traditional LLNA, human tests, and guinea pig tests). Thus, the Panel concluded that therelevance of the LLNA: DA had been adequately evaluated. However, including data on more
substances is likely to further strengthen confidence in the concordance data.
One substance, 2-mercaptobenzothiazole, produced a false negative response compared to the
traditional LLNA when tested using the LLNA: DA. The Panel was asked if they could identify
any characteristics associated with this or similar substances, compared to the correctly
identified sensitizers, that might signal that this type of discordant response would occur, andtherefore using the LLNA: DA to test such substances would not be appropriate (or thatnegative results for substances with such properties may warrant additional testing). The Panel
could not identify specific characteristics that might explain the false negative response using
the LLNA: DA. Although understanding the solubility and stability of the test substance indifferent vehicles is important, the differences in response did not seem to be explained by thevehicle differences (AOO and dimethylformamide [DMF]) between the two tests. In addition,
the impact of 1% SLS pretreatment on the negative response in the LLNA: DA is not known
but should be considered.

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ICCVAM Test Method Evaluation Report: Appendix F1

One substance, benzalkonium chloride, produced a false positive response compared to the
traditional LLNA and guinea pig test when tested using the LLNA: DA. The Panel was asked ifthey could identify any characteristics associated with this or similar substances, compared tothe correctly identified non-sensitizers that might signal that this type of discordant response
would occur, and therefore using the LLNA: DA to test such substances would not beappropriate (or that positive results for substances with such properties may warrant additional
testing). The Panel could not identify specific characteristics that might explain the false
positive response for this substance in the LLNA: DA. The Panel viewed that it was important
to note, however, that this chemical is a well-known skin irritant, and on occasion it had also
been considered a human sensitizer, typically on the basis of positive diagnostic patch test data.
Thus, the Panel reiterated that the relevance of LLNA: DA results should always be consideredin the context of human experimental sensitization data, human epidemiologic data, andelicitation in the clinical setting. The actual impact of the 1% SLS pretreatment step on the
LLNA: DA has not been well established, although van Och et al. (2000) and De Jong et al.
(2002) have reported that 1% SLS pretreatment enhances the response in the traditional LLNA.
3.2.4 Test Method Reliability
The Panel was asked if they considered the intralaboratory reproducibility of the LLNA: DA tohave been adequately evaluated and compared to the traditional LLNA (refer to Table 7-1 of thedraft LLNA: DA BRD). The Panel noted that only eugenol and isoeugenol, two sensitizers withsimilar chemical structures, were tested. The Panel recommended testing a positive control
commonly used in the traditional LLNA (e.g., HCA) for a more complete evaluation of
intralaboratory reproducibility. In addition, it was unclear if the tests were truly independent.
Factors that might indicate independence should have been documented (e.g., time interval
between experiments, different animal shipment, different reagents, different operator).
The Panel was also asked if they considered the interlaboratory reproducibility of the LLNA: DA
to have been adequately evaluated and compared to the traditional LLNA. The Panel noted thatthe interlaboratory reproducibility of the assay could not be adequately evaluated given the lackof original study data and limitations in the study design. Study design limitations included:•

Pooled lymph nodes were used from mice within a dose group. This precluded ananalysis of variation between laboratories.

•

The lead laboratory established the dose levels to be tested by the other laboratories
participating in the interlaboratory validation effort. In a minority opinion, Drs.
Nathalie Alépée and Michael Woolhiser asserted that for an effective and efficientinterlaboratory evaluation, it seemed reasonable to set dose levels for all laboratories
based on results from the lead laboratory.

In addition, the Panel considered that the interlaboratory studies could benefit by performing
more than one test on two commonly used positive controls (i.e., HCA and DNCB).
The draft LLNA: DA BRD contained an analysis of data from two interlaboratory
reproducibility validation studies that used coded substances, as well as an intralaboratory
accuracy validation study with 31 substances that were not coded. The Panel was asked if they
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ICCVAM Test Method Evaluation Report: Appendix F1

considered the lack of coding of the test substances to have adversely impacted or biased theintralaboratory accuracy evaluation. The Panel commented that, in the validation of a new
assay, it is better to avoid the potential for bias by testing coded substances. However, the Panelconcluded the data already generated for the LLNA: DA test method should be considered andnot be rejected in the current validation evaluation.
The lead laboratory established the dose levels tested by the participating laboratories in the twointerlaboratory validation studies. The Panel was asked if this adversely impacted or biased theevaluation. The Panel considered that the choice of the maximum test substance concentrationis crucial for the proper performance of the traditional LLNA as well as any modified LLNA.
Thus, predetermining the dose levels to be tested for each substance might have reduced
variability between the two interlaboratory studies. In a minority opinion, Drs. Nathalie Alépée
and Michael Woolhiser asserted that for an effective and efficient interlaboratory evaluation, it
seemed reasonable to set dose levels for all laboratories based on results from the leadlaboratory.
3.2.5 Data Quality
The studies evaluated in the draft BRD for the LLNA: DA were not conducted in accordancewith GLP guidelines although they were reportedly done in laboratories that conduct GLP
studies, and were conducted “in the spirit” of GLP (K. Idehara, personal communication).
Furthermore, the original records for the interlaboratory studies were requested but have notyet been obtained. As a result, an independent audit could not be conducted to confirm that thereported data was the same as the data recorded in laboratory notebooks. The Panel was asked
to comment on the potential impact this might have had on the evaluation of the LLNA: DA.
The Panel commented that, ideally, GLP compliance is recommended for validation studies,but the current studies should not be rejected based on the lack of GLP compliance alone.
However, all the raw data obtained through the validation process should be made available
and audited for accuracy. The Panel further commented that since the original records for theinterlaboratory studies have not yet been provided, recommendations from ICCVAM should becontingent upon receiving these data. Obtaining original laboratory records is a necessary step
to confirm that all data generated during the validation studies have been provided, and that thereported data are the same as the data recorded in laboratory notebooks.
3.2.6 Consideration of All Available Data and Relevant Information
The Panel was asked to comment on whether all of the relevant data identified in published or
unpublished studies that employed the LLNA: DA had been adequately compared. The Panelviewed that, generally, it seemed that all of the relevant results had been adequately identified
and considered. However, as mentioned above, all of the original data supporting these results
have not been provided. The Panel again expressed concern related to the effect of pretreating
the mouse ear with 1% SLS and the Panel therefore recommended that the results from van Och
et al. (2000) and De Jong et al. (2002) should be considered.

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ICCVAM Test Method Evaluation Report: Appendix F1

3.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: DA

3.3.1 Test Method Usefulness and Limitations
The Panel was asked to comment on whether the available data supported the ICCVAM draft
recommendations for the LLNA: DA procedure in terms of the proposed test method usefulness
and limitations. The Panel agreed with ICCVAM’s recommendation, which stated that the
LLNA: DA might be useful for identifying substances as potential skin sensitizers and nonsensitizers, but this recommendation was contingent upon the receipt of additional data andinformation. The Panel further added that information on the possibility of skin reactions
suggestive of the onset of the elicitation phase and the impact of the 1% SLS pretreatment stepon the performance of the LLNA: DA should be evaluated. The Panel also considered that the
ICCVAM proposed limitations needed to be more clearly defined, as it was not clear from thedraft recommendations what points were considered as limitations. For instance, limitations thatare known for the traditional LLNA would likely apply to this modified protocol as well andthese should be noted.
The Panel was asked whether restrictions on using radioactive materials would warrant that the
LLNA: DA be routinely recommended for hazard identification of skin sensitizing substancesin lieu of having to possibly use guinea pig tests. The Panel noted that, based on gaps in thecurrently available dataset and information described in this report, the LLNA: DA could not
yet be recommended for the routine use for hazard identification of skin sensitizing substances,regardless of whether restrictions on using radioactive materials were present or not. Generally,
non-radioactive LLNA test methods are preferred in lieu of using guinea pig tests because
fewer animals are used and animal pain and distress is reduced. However, policy issues
regarding restrictions on radioactivity should have no impact on this science-based conclusion.
The Panel was asked if, from a public health perspective, the recommended guidance for
evaluating negatives were sufficient to address concerns associated with the false negative rateof 5% (1/19 substances) calculated for the LLNA: DA. The Panel noted that this was not ascientific question, rather a risk characterization issue, and could not be answered without
considering other factors such as intended use, target population, etc. The Panel was also
asked if, from a testing strategy perspective, the ICCVAM guidance addressed concerns
associated with the false positive rate of 10% (1/10 substances) calculated for the LLNA: DA
and/or if they had other suggestions for additional guidance or limitations. The Panel again
commented that this was not a scientific question but a risk characterization issue and could
not be answered without considering other factors such as intended use, target population, etc.
Furthermore, the Panel noted that it would be difficult to generalize the finding of one test
substance being a “false” result. Instead, they considered it better to identify reasons why asubstance was a “false” result. Certainly, if a “false” result is suspected, confirmatory testingwith another mouse LLNA method was not recommended. It might be important to follow asuspected inaccuracy with an investigation of the mechanistic basis for the discordance.

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ICCVAM Test Method Evaluation Report: Appendix F1

3.3.2 Test Method Protocol
The Panel was asked if they agreed that the available data supported the ICCVAM draft
recommendations for the LLNA: DA procedure in terms of the proposed test method
standardized protocols or what recommendations they would make. The Panel noted thatavailable data did not support all of the ICCVAM draft recommendations in the LLNA: DA
standardized protocol. First, the ICCVAM protocol (ICCVAM 1999; Dean et al. 2001) for the
traditional LLNA recommends using at least five animals per dose group. Although the Panelagreed that five animals per dose group should be recommended for validation studies, theysuggested that power calculations would be useful in determining if subsequent use of themodified test method could use fewer animals per dose group. For the LLNA: DA test method,
the Panel noted that based on statistical power calculations that were provided as supplemental
information, using four animals per group instead of five did not appear to be a limitation (i.e.,
detecting a 3.0-fold increase in the SI with four animals per group was estimated to have a 99%
confidence level). In addition, the Panel generally agreed with the recommendation in the
ICCVAM protocol (ICCVAM 1999; Dean et al. 2001) that individual animal data should becollected. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová,
Raymond Pieters, and Michael Woolhiser stated that if laboratories were operating under
OECD guidance (OECD 2002) and a reliable validation dataset had been generated, thenpooled data from at least four animals could be considered acceptable.
Of greater importance, the Panel concluded that pretreatment with 1% SLS should not be
accepted until its impact on the performance of the LLNA: DA has been adequately
characterized. Although used at a concentration below its EC3, the Panel was concerned about
pretreating the mouse ear with an irritant reported as positive in the traditional LLNA. To
demonstrate that these concerns are not justified, the Panel recommended that substances thatare weak irritants and weak sensitizers be tested in the LLNA: DA with and without
pretreatment with 1% SLS. It also needed to be demonstrated that the 1% SLS pretreatments, aswell as the additional test substance treatment on day 7, did not induce a skin reaction that
could be indicative of the onset of the elicitation phase of skin sensitization.
The Panel was asked to comment on whether the limit dose procedure could be applied to the
LLNA: DA. The Panel concluded that if the limit dose procedure is considered applicable to the
traditional LLNA, then it should also be applicable to the LLNA: DA, in order to reduce thenumber of animals used. This would require adherence to the LLNA: DA test method protocol,
with the exception that the middle and low dose groups would be omitted in the limit doseversion.
3.3.3

Future Studies

The Panel was asked if they agreed that the available data support the ICCVAM draft
recommendations for the LLNA: DA in terms of the proposed future studies or, if not, what
recommendations they would make. The Panel stated that the available data supported the
ICCVAM draft recommendations for the LLNA: DA in terms of a more comprehensive
evaluation using more non-sensitizers within and across laboratories. In a minority opinion, Dr.
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ICCVAM Test Method Evaluation Report: Appendix F1

Thomas Gebel stated that although testing of more non-sensitizers might be warranted forinterlaboratory validation studies, a sufficient number of non-sensitizers had been tested within
the same laboratory (Table 6.3 in the draft BRD).
However, the Panel viewed that there were additional studies that ICCVAM might consider. As
previously mentioned, the Panel recommended that the 1% SLS pretreatment step should not be
accepted until its impact on the performance of the LLNA: DA had been adequately
characterized. Furthermore, it should be demonstrated that such pretreatments did not induce askin reaction that could be indicative of the onset of the elicitation phase of skin sensitization. It
might also be of interest to evaluate ATP as a marker of lymph node proliferation using the
traditional LLNA dosing scheme and lymph node collection schedule. Lastly, the Panelconsidered that studies on the reliability of outlier analysis in small sample sizes and the effects
of reduced sample size on the power of the LLNA: DA test method should be proposed.
3.3.4 Performance Standards
The draft LLNA: DA BRD indicated that the LLNA: DA protocol differed from the ICCVAMrecommended protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes. In addition, there
are differences between the two protocols that relate to how and when the test substance isapplied and when the lymph nodes are collected (Table 2-1 and Appendix A in the draft LLNA:
DA BRD). According to the proposed draft ICCVAM LLNA Performance Standards for the
traditional LLNA, any change to the LLNA protocol other than the method used to assess
lymphocyte proliferation would be considered a “major” change. The Panel was asked if theyagreed that these should be considered “major” changes and therefore the usefulness andlimitations of the LLNA: DA should not be assessed using the draft ICCVAM LLNA
Performance Standards. The Panel commented that answering this question depended on having
a clear definition of what constitutes a “major” versus a “minor” change, and what may
constitute a different protocol altogether. Depending on the goal of the assay, whether a change
is “major” versus “minor” may not be relevant. Ultimately, if a test method is able to make thecorrect prediction with regard to the dermal sensitization potential of a test substance, then theissue of “major” versus “minor” modifications might not apply. Considering the robust natureof the current draft ICCVAM LLNA Performance Standards, it is difficult to identify the needfor additional requirements for methods like the LLNA: DA. Thus, the draft ICCVAM LLNA
Performance Standards could be used to evaluate the LLNA: DA as a mechanistically andfunctionally similar test method.
The Panel was asked, even if the draft ICCVAM LLNA Performance Standards were not foundapplicable to the LLNA: DA, whether an analysis based on 13 of the 18 proposed requiredreference substances in the performance standards would impact the overall evaluation of thetest method accuracy. The Panel commented that the accuracy analysis based on 13 of the 18
proposed required reference substances in the performance standards (with one false negative
substance) should have no impact on the overall evaluation of test method accuracy as 31
substances have been tested. However, given the concern regarding pretreatment with 1% SLS,
the Panel stated that testing of substances with and without 1% SLS was needed to characterize
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ICCVAM Test Method Evaluation Report: Appendix F1

the effect of this pretreatment on the performance of the assay. The Panel concluded that as
described above, the idea of “major” versus “minor” changes might be reconsidered, thus thecurrent draft ICCVAM LLNA Performance Standards could be applicable to the LLNA: DA asa mechanistically and functionally similar test method.

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ICCVAM Test Method Evaluation Report: Appendix F1

4.0

Non-Radioactive LLNA Protocol - LLNA: Bromodeoxyuridine
Detected by Flow Cytometry (BrdU-FC) Test Method

4.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

4.1.1 General Comments
The Panel was asked if there were any errors in the draft LLNA: BrdU-FC BRD that should becorrected, if omissions of existing relevant data had been identified, or if there was additional
information that should be included. The Panel noted that overall, errors and omissions in thedraft LLNA: BrdU-FC BRD were few. The majority of omissions relating to the data records
were identified in the text, and all reasonable efforts to obtain additional information from MB
Research Labs, the developer of the LLNA: BrdU-FC, appear to have been made.
The following describes the identified errors, omissions, and/or information gaps in the draft
LLNA: BrdU-FC BRD that should be addressed:•

Data are available in the peer-reviewed literature on the application of BrdU in the
LLNA with histochemical or enzyme-linked immunosorbent assay (ELISA)
detection. This could be briefly mentioned in the final BRD for the LLNA: BrdU-FC
method, simply as a means of indicating the utility of non-radiolabeled tracer
methods in the LLNA.

•

It should be noted that a potential reason why nickel chloride was negative in the
LLNA: BrdU-FC may be due to oral tolerance in the mice that was induced bynickel-containing nipples of drinking bottles and nickel cages (Van Hoogstraten etal. 1993).

•

The vehicle(s) used with the test substances should be stated.

•

Information on experience of the inter-laboratory transferability of othertechnologies that depend upon flow cytometry technology as the key data read-out
should be included.

•

All raw data for the LLNA: BrdU-FC and the enhanced LLNA (eLLNA): BrdU-FC
should be made available.

4.1.2 Comments with Specific References to the Text
The Panel stated that the following comments and/or suggested corrections relevant to specificparts of the draft LLNA: BrdU-FC BRD text should be addressed:•

Line 226: Citation was made to a reference dated 2001 by MB Research Labs which
established their development of the LLNA: BrdU-FC; however, no reference wasincluded in Section 12.0 (References) of the draft BRD.

•

Lines 232-233: For a sensitizer, the SI should be greater than or equal to 3.

•

Line 246: “i.e., positive” should be explained.

•Line 254: 11% should be 17%.
F-61

ICCVAM Test Method Evaluation Report: Appendix F1•Line 263: For purposes of completeness, it may be worthwhile to add a brief
description of the comparative accuracy of the available traditional LLNA, LLNA:
BrdU-FC, and eLLNA: BrdU-FC results versus human maximization/patch test
data. This information is of importance and displayed in Tables 6-1 and 6-2.

•

Lines 286-288: The issue of the refinement/reduction in animal use that might follow
the availability of a scientifically validated non-radioactive variant of the LLNA was
mentioned in the draft BRD as a benefit but it was not quantified, and noauthoritative reference was cited in support.

•

Line 288: The final LLNA: BrdU-FC BRD should explain why the BrdU method
would result in less pain and distress to the animals (i.e., does the route of injectionof BrdU vs. 3H-methyl thymidine produce less discomfort?).

•Line 335: Reference was made to a citation dated 2001 by MB Research Labs which
established their development of the LLNA: BrdU-FC; however, no citation was
included in Section 12.0 (References) of the draft BRD.

•

Line 356: The sentence starting “To evaluate excessive skin…” implies that
evaluation of excessive skin irritation by measuring ear thickness is recommended
by the ICCVAM LLNA protocol although it is only recommended in the LLNA:
BrdU-FC protocol.

•

Lines 365-366: Consider supplementing the list of abbreviations for Figure 2-1 with
B220+, B:T, CD69+, and IAk+. Also, the figure shows I-Ak+ while all other text
uses IAk+. The MB Research Labs protocol shows I-Ak+.

•

Figure 2-1: Should be redrawn to show the SI decision point lines coming off of the“Analyze Proliferating LNC (lymph node cells)” box rather than the “Inject BrdU
and Excise…” box.

•

Lines 500-503: Classification of “equivocal results” was unclear without datacomparison (i.e., benzocaine produced divergent results in both tests). Were theseresults unlike what was expected from human data? What were the data for salicylic
acid and mercaptobenzothiazole?

•Lines 552-558: It may be useful for comparative purposes to add summary accuracy
data for the traditional LLNA versus human maximization/patch test data from thelarger data set reported in the 1999 ICCVAM LLNA report to the section of the final
BRD which discusses performance of the LLNA: BrdU-FC method.

•

Table 6-3: Benzocaine was missing and it seems that salicylic acid was the same inboth traditional LLNA and LLNA: BrdU-FC. Mercaptobenzothiazole was notreported for the LLNA: BrdU-FC. This needs to be corrected or explained.

•

Table 6-5: The human outcome for benzalkonium chloride and ethylene glycol
methacrylate should be negative. See also Table 6-6.

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ICCVAM Test Method Evaluation Report: Appendix F1

4.2

Comments on the Validation Status of the LLNA: BrdU-FC

4.2.1 Test Method Protocol
The LLNA: BrdU-FC protocol includes routine measurements of ear swelling as an indicator ofexcessive dermal irritation. The Panel was asked if they considered this procedure to be an
appropriate approach and if this measurement should be recommended for routine inclusion
into all LLNA protocols. The Panel stated that, as a quantitative parameter associated with
inflammatory cell influx and fluid retention near the site of test substance application, ear
swelling (or other quantitative measurements) should be carefully considered for inclusion into
all LLNA protocols. This might assist in differentiating between sensitizers and irritants, assist
in the interpretation of equivocal results, and possibly detect other procedure-related problems
that might require further exploration/consideration.
The LLNA: BrdU-FC protocol also includes optional quantification of immunophenotypicmarkers as an additional mechanism for distinguishing irritants from sensitizers. The Panel was
asked if they considered this to be an appropriate approach to reduce false positives, and if thecorrect markers were being considered. The Panel was also asked if these measurements should
be recommended for routine inclusion in the LLNA: BrdU-FC. The Panel agreed that the use of
immunological markers would be appropriate for detailed studies, as it might reduce thefrequency of false positives (irritants) and improve comparisons with human data. However,since the primary use of the LLNA is for discrimination of human hazard from direct chemical
contact, it could be argued that some false positives are acceptable (especially for methods
which have relatively lower rates of false negatives). Given this dominant use, application of
immunological markers would likely be too detailed and costly for routine LLNA use. Thus, thePanel suggested that results of ear swelling measurements be compared with the more
technically complex flow cytometry markers to determine if similar results might be obtained.
Furthermore, alternative immunological markers for discriminating between irritants andsensitizers may be available, although the draft LLNA: BrdU-FC BRD did not contain
information allowing any informed decision on whether other markers might be more predictive.
Thus, based on current knowledge, the current markers suggested in the draft LLNA: BrdU-FC
BRD seemed acceptable to the Panel. Two other possibilities suggested were a surface marker
relating to CD4 T-helper cells (Th) or Th1 cells (interferon-γ).
The Panel was also asked to comment on the appropriateness of the “sequential strategy” usedin the eLLNA: BrdU-FC (see Figure 2-1 of the draft LLNA: BrdU-FC BRD). Generally, thePanel viewed that incorporation of immune parameters improved the value of a predictive assay
and may also help explain mechanisms, which is important. Still, the “sequential strategy” usedin the eLLNA: BrdU-FC for discriminating irritation from sensitization might be more sensible
for research studies because of resource and cost considerations, and may not be appropriate forroutine use of the LLNA in hazard identification. For human hazard detection, more simplified
methods should be available for discrimination of irritants.

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ICCVAM Test Method Evaluation Report: Appendix F1

4.2.2 Substances Used for the Validation Studies
The Panel was asked to consider if the substances tested in the LLNA: BrdU-FC wererepresentative of a sufficient range of chemical classes and physicochemical properties suchthat the test method would be applicable to any of the types of chemicals and products that are
typically tested for skin-sensitization potential. The Panel agreed that if the proviso that theapplicability domain limitations published for the traditional LLNA remained in force, thesubstances tested in the LLNA: BrdU-FC seemed representative of a sufficient range ofchemical classes and physical chemical properties and it would likely be applicable to many of
the types of chemicals and products that are typically tested for skin-sensitization potential.
However, the available LLNA: BrdU-FC database was relatively small compared to the large
number of substances assessed in the traditional LLNA and this implied some caution inassuming that assay performance was concordant with the traditional LLNA.
4.2.3 Test Method Accuracy
The accuracy analysis presented in the draft LLNA: BrdU-FC BRD was based on overall
concordance with the traditional LLNA. Accuracy statistics compared to the guinea pig tests
and human data/experience were also provided. The Panel was asked if these comparisons wereappropriate for assessing the accuracy of the LLNA: BrdU-FC. The Panel viewed that since thetraditional LLNA is used to provide human hazard identification and information relevant tohuman health, the accuracy statistics compared to human data/experience were important. Since
the LLNA: BrdU-FC is fairly similar to the traditional LLNA, guinea pig comparisons might
not have been necessary. However, taken together, the availability of both humandata/experience and guinea pig data allowed additional insights that might have expanded theapplicability domain of the LLNA: BrdU-FC, or indicated improved performance with respect
to LLNA false negatives and positives.
The Panel was then asked if the relevance (e.g., accuracy/concordance, sensitivity, specificity,
false positive and false negative rates) of the LLNA: BrdU-FC had been adequately evaluatedand compared to the traditional LLNA. The Panel agreed that the relevance of the LLNA:
BrdU-FC was adequately evaluated and compared to the traditional LLNA, and supported theinclusion of accuracy analyses with and without equivocal materials.
Three substances (benzalkonium chloride, resorcinol, and Tween 80) produced a false positive
response compared to the traditional LLNA and guinea pig test when tested using the LLNA:
BrdU-FC (based on immunophenotyping, benzalkonium chloride was subsequently classified
as an irritant rather than a sensitizer). The Panel was asked if they could identify any
characteristics associated with these or similar substances that might suggest that using the
LLNA: BrdU-FC to test such substances would not be appropriate or that positive results for
substances with such properties may warrant additional testing. Overall, the Panel stated that
there were not any patterns or unifying concepts that explained the three false positive results inthe available data set. They noted that only a single laboratory is using the LLNA: BrdU-FC
method and recommended that the raw data on which the reports were prepared be madeavailable in order to allow further investigation. The Panel also suggested that additional studies
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ICCVAM Test Method Evaluation Report: Appendix F1

be conducted to determine whether LLNA: BrdU-FC results with these three substances arerepeatable.
Dr. Raymond Pieters stated that benzalkonium chloride and Tween 80 are consideredaggressive irritants, but both published data (Manetz and Meade. 1999; Varani et al. 2008) andunpublished data from his laboratory has shown that benzalkonium chloride (5%) is morepotent than SLS in the stimulation of lymph node cell proliferation and may therefore may
actually be considered a sensitizer. However, in the traditional LLNA these compounds did notincrease the SI above the threshold for a positive response (i.e., SI ≥3), so they were identified
as non-sensitizers.
4.2.4 Test Method Reliability
The Panel was asked if the intralaboratory reproducibility of the LLNA: BrdU-FC had beenadequately evaluated and compared to the traditional LLNA and if any limitations wereapparent based on this assessment. The draft LLNA: BrdU-FC BRD analyzed data from repeat
testing of HCA in six different vehicles and intralaboratory reproducibility was assessed by acoefficient of variation (CV) evaluation. The calculated CVs ranged from 30% to 53%. The
Panel agreed that the relatively large SD and associated CV values raised questions about theextent of experiment-to-experiment variability. There was less concern about vehicle choiceand effects on the range of group means than about the CVs greater than 50% for the groupmeans of HCA tested in DMSO and AOO. The large number of repeated experiments for thesetests would have been expected to dramatically reduce variability. The Panel concluded that theresults suggested that key elements of assay standardization were not yet developed. Further
evaluation using other positive control substances would have been valuable to more adequately
characterize reproducibility.
4.2.5 Data Quality
The studies evaluated in the draft BRD for the LLNA: BrdU-FC were not all conducted inaccordance with GLP guidelines although they were done in a laboratory that routinely
conducts GLP studies (G. DeGeorge, personal communication). The Panel was asked to discuss
what impact this might have on the evaluation of the LLNA: BrdU-FC. The Panel considered
that, even without formal GLP compliance, the current LLNA: BrdU-FC results appeared toreflect a sincere attempt to perform work of high quality. The only area in which a lack of full
GLP compliance may have been a source of assay variability was in the quantitative analysis of
dosing solutions. For instance, failure to appreciate differences in composition of dosing
solutions between experiments caused by test article instability or other phenomena may
account for the relative large variability in intralaboratory data and possibly of some of the
discordant results (i.e., false negatives and differences in LLNA: BrdU-FC results between
repeat studies for the same substance). Thus, the Panel viewed that any additional studies
undertaken to validate the test method should ideally be GLP-compliant.
Furthermore, the original records for these studies were requested but had not yet been obtainedat the time of the Panel review. As a result, an independent audit could not be conducted toconfirm that the reported data was the same as the data recorded in laboratory notebooks. The
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Panel was asked if they agreed that any recommendations from ICCVAM should be contingent
upon the completion of such an audit and findings that there were no significant errors in datatranscription. The Panel agreed that, although a request for original data had been made, it wasgood practice to hold final recommendations until an independent audit could be performed.
While it would be expected that no serious errors would be uncovered which would alter thecurrent findings, an audit would confirm assay performance to date and position ICCVAM forfurther consideration of the LLNA: BrdU-FC.
4.2.6 Consideration of All Available Data and Relevant Information
The Panel was also asked if, based on the draft LLNA: BrdU-FC BRD, all the relevant data
identified in published or unpublished studies that employ this test method had been adequately
considered. Furthermore, they were asked that if there were other comparative test method datathat were not considered in the draft BRD, how such data might be obtained. Overall, the Panelconsidered that all the relevant data identified in published or unpublished studies that
employed this test method had been adequately considered in the draft LLNA: BrdU-FC BRD.
However, some additional information was available in the peer-reviewed literature on
application of BrdU in the LLNA with other methods of detection (e.g., histochemistry,
ELISA). The Panel felt that these could have been briefly mentioned in the draft BRD for the
LLNA: BrdU-FC method, simply as a means of indicating the utility of non-radiolabeled tracer
methods in the LLNA. Furthermore, if an analysis of the CV for the traditional LLNA wasundertaken, a more direct comparison with the LLNA: BrdU-FC could have been performed.
4.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-FC

4.3.1 Test Method Usefulness and Limitations
The Panel was asked if they agreed that the available data and test method performance support
the ICCVAM draft recommendations for the LLNA: BrdU-FC in terms of the proposed test
method usefulness and limitations (i.e., that it may be useful for identifying substances aspotential skin sensitizers and non-sensitizers, but that more information and data are needed
before a recommended use of the LLNA: BrdU-FC can be made). The Panel agreed that theavailable data and test method performance of the LLNA: BrdU-FC support the draft ICCVAM
recommendations. They considered the proposed test method usefulness and limitations to havewell summarized the limits of the information supplied and the additional information thatwould need to be generated or provided for further consideration of this test method. As aresult, the LLNA: BrdU-FC could not at this stage be considered scientifically validated as areplacement alternative to the traditional LLNA. Still, the test method recommendation should
clearly state that the test method was not “invalid” but simply that there was currently not
sufficient evidence and information to affirm that it had been adequately validated by ICCVAM.
Instead, the Panel considered that the LLNA: BrdU-FC could be recommended in instanceswhere mechanistic information about a sensitizer is required.
The Panel was asked if restrictions on using radioactive materials were or were not present,
whether or not the LLNA: BrdU-FC should be routinely recommended for hazard identification
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ICCVAM Test Method Evaluation Report: Appendix F1of skin sensitizing substances in lieu of having to possibly use guinea pig tests. The Panelagreed that it is preferable to use alternative methods for the LLNA (i.e., ELISA detection of
BrdU or histochemical detection of BrdU-labeled cells), as opposed to application of guinea pig
test methods, if a limitation on radioisotope use exists (e.g., the lack of a radioactivity use
license). This rationale is based on avoidance of the less quantitative guinea pig test methods,
which may employ adjuvant treatment with associated animal stress and harm. Still, at thistime, the Panel considered that data gaps in the LLNA: BrdU-FC method precluded
recommending it for routine hazard identification of skin sensitizing substances in lieu of the
traditional LLNA, whether or not limitations on using radioactive materials exist. Policy issues
regarding restrictions on radioactivity should have no impact on this science-based conclusion.
The Panel was asked if the ICCVAM recommendations adequately addressed concerns
associated with the false positive rate of 17% (3/18 substances) calculated for the LLNA: BrdUFC and if there were other suggestions for additional guidance or limitations that should beconsidered. The Panel agreed that the relatively high false positive rate was adequately
identified and discussed, and that no mechanistic reason could be identified for these results
based on available information. The Panel noted that it might be worthwhile to point out in the
final BRD the impact on human health of false positive results versus false negative results inthe context of hazard screening and identification. Consideration of factors such as intended use
and target population of the false positive substances would further aid in characterizing humanrisk for these substances.
4.3.2 Test Method Protocol
The Panel was asked whether or not they agreed that the available data supported the ICCVAM
draft recommendations for the LLNA: BrdU-FC procedure in terms of the proposed test method
standardized protocol and if not, what recommendations would they make. The Panel noted thatthe draft ICCVAM recommendations for conduct of a standardized method for the LLNA:
BrdU-FC variant were relatively brief and stated only that all applicable portions of the 1999
ICCVAM procedure be carefully followed. The Panel agreed that the available data supported
the ICCVAM draft recommendations for the LLNA: BrdU-FC procedure in terms of adheringto the ICCVAM LLNA protocol (ICCVAM 1999; Dean et al. 2001). In particular, the Panelagreed that at least five animals per dose group should be used, particularly in light of Dr.
Haseman's power analysis (see Table F-3). The Panel did note however that power calculations
could be undertaken to determine if fewer animals per dose group might be adequate for postvalidation studies, though Dr. Haseman's power analyses suggest that this is unlikely. The
majority of the Panel also agreed with the ICCVAM-recommended protocol to use individual
animal data although a minority opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar
Jírová, Raymond Pieters, and Michael Woolhiser stated that if laboratories were operating
under OECD TG 429 guidance (OECD 2002) and a reliable validation dataset had been
generated, then pooled data from at least four animals per dose group could be considered
acceptable.
Further, the Panel considered the methodological description of the LLNA: BrdU-FC procedure
supplied by MB Research Labs (Appendix A to the draft BRD) to be comprehensive. The
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utility of ear swelling or other methods to detect inflammation/excessive local irritation appear
to be warranted in every variation of the LLNA (including the traditional LLNA), but should befurther investigated before routine inclusion in any protocol is recommended.
The Panel was asked whether the LLNA limit dose procedure could be applied to the LLNA:
BrdU-FC. The Panel agreed that the LLNA limit dose procedure could be applied to the LLNA:
BrdU-FC as long as the limitations associated with the limit dose procedure were appreciated.
Furthermore, application of the limit dose procedure to the LLNA: BrdU-FC would require
adherence to a validated LLNA: BrdU-FC test method protocol with the exception that the
middle and low dose groups would be excluded. Furthermore, it would need to be confirmedthat the number/pattern of sensitizers that would have been identified/missed from the “highdose” group would mirror that of the traditional LLNA.
Table F-3

Power Calculations for the LLNA: BrdU-FC1
3.0-fold
Increase2

2.5-fold
Increase

2.0-fold
Increase

1.5-fold
Increase

1.3-fold
Increase

Mean Rx response

30279

25232.5

20186

15139.5

13120.9

Log (Mean Rx response)

10.31810.136

9.913

9.625

9.482

Difference from control(log scale)

1.098

0.916

0.693

0.405

0.262

Difference/SD

1.75

1.46

1.10

0.65

0.42

Power for N=580%

50-80%

<50%

<50%

<50%

Power for N=450-80%

50%

<50%

<50%

<50%

Power for N=3

50%

<50%

<50%

<50%

<50%

Other Power

95% (N=9)95% (N=12)

95% (N=19)

95% (N=52)

95% (N>100)

Other Power

90% (N=7)

90% (N=10)

90% (N=15)

90% (N=42)

90% (N>100)

Parameter

Abbreviations: N = number of animals; Rx = Treatment; SD = standard deviation.
1
The power calculations above are based on a one-sided p<0.05 Student’s t test applied to log-transformed
data from vehicle control LLNA BrdU-FC tests.
2
Fold-increase = Required increase above the vehicle control for a positive response

4.3.3 Future Studies
Finally, the Panel was asked whether they agreed that the available data supported the
ICCVAM draft recommendations for the LLNA: BrdU-FC in terms of the proposed futurestudies. The Panel agreed that the ICCVAM draft recommendations for future studies
highlighted the unanswered questions raised by the available database. Specifically, conducting
interlaboratory studies as a part of the validation process was considered important. As
mentioned previously, the Panel viewed that the immunological markers suggested for the
LLNA: BrdU-FC in the draft BRD were acceptable but that additional immunological markers
for discrimination of irritant versus sensitization phenomena might also be identified. A
suggestion for a future study was to use the surface marker relating to CD4 Th cells or internalmarker relating to Th1 cells (interferon-γ).
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ICCVAM Test Method Evaluation Report: Appendix F1

In general, for any future work, the Panel considered that efforts should be made to decrease thevariability and thereby increase the power of the test in order to ensure that more animals were
not needed relative to the traditional LLNA or other alternative LLNA protocols. For instance,
further optimization of the LLNA: BrdU-FC method should include kinetic studies todemonstrate that the optimal protocol was being used.
4.3.4 Performance Standards
The draft BRD indicated that the LLNA: BrdU-FC protocol differs from the ICCVAMrecommended protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes. According to the
proposed draft ICCVAM LLNA Performance Standards for the traditional LLNA, any change
to the LLNA protocol other than the method used to assess lymphocyte proliferation wasconsidered a “major” change. According to this criterion, the Panel considered the protocol
differences between the LLNA: BrdU-FC and the traditional LLNA to be “minor” changes, andtherefore considered that the validity of this test method could be based on the draft ICCVAM
LLNA Performance Standards. However, the Panel also recognized that this depended on aclear definition of what constituted a “major” versus a “minor” change, or a different protocol
altogether. Thus, further consideration of this topic could be addressed once therecommendations for the current draft ICCVAM LLNA Performance Standards were finalized.
The Panel found it difficult to identify any additional requirements for methods like the LLNA:
BrdU-FC.
Even if the draft ICCVAM LLNA Performance Standards were not found to apply to the
LLNA: BrdU-FC, the Panel considered that the impact of the LLNA: BrdU-FC accuracy
analysis based on 13 of the 18 proposed required reference substances in the draft ICCVAM
LLNA Performance Standards should not have a major impact on the overall evaluation of test
method accuracy, as 45 substances, representative of an appropriate range, were tested.
However, based on consideration for development of LLNA performance standards, it would bedesirable for validation purposes that the substances missing from the range of 18 standard
materials be assessed in the LLNA: BrdU-FC protocol.
The draft LLNA: BrdU-FC BRD also indicated that three out of six sensitizers for which EC3data were available had EC3 values that were outside of the proposed 0.5x to 2.0x EC3
acceptability range, which was developed based on the traditional LLNA. The Panel viewedthat the primary concern seemed to have less to do with the variation in the response than with aconcern that the range of response would skew the interpretation of any LLNA: BrdU-FC
results used for sensitization potency estimates. Furthermore, it was not known if the samevehicle was used to derive both EC3 values/ranges. The proposed 0.5 x to 2.0 x range seemed to
be based upon empirical/goodness of fit rather than any biological constant. The
appropriateness of this range should be considered further when the finalized ICCVAM LLNA
Performance Standards document is considered. In general, if the vehicles were different thequestion is irrelevant.

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ICCVAM Test Method Evaluation Report: Appendix F1

4.3.5 Comments with Specific References to the Text
The Panel made the following comment with specific reference to the text in the draft ICCVAM
test method recommendations on the LLNA: BrdU-FC and suggested that it be addressed:•

Lines 39-41: For parallel construction of this sentence with the preceding sentence,suggest substituting the following “One of the other equivocal substances, salicylicacid, is one of the recommended reference standard materials used as a nonsensitizer in the draft ICCVAM LLNA Performance Standards….” This is based on
the assumption that salicylic acid was the substance intended for discussion and that
it was used in the draft ICCVAM LLNA Performance Standards as a model nonsensitizer.

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ICCVAM Test Method Evaluation Report: Appendix F1

5.0

Non-Radioactive LLNA Protocol - The LLNA:
Bromodeoxyuridine Detected by ELISA (BrdU-ELISA) Test Method

5.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

5.1.1 General Comments
The Panel was asked if there were any errors in the draft LLNA: BrdU-ELISA BRD thatshould be corrected, if omissions of existing relevant data had been identified, or if there wasadditional information that should be included. The Panel noted that, in general, all of the data
included were relevant, and that it was apparent that considerable effort had been involved in
carefully developing the comprehensive database. The Panel noted that they would havepreferred to have the original papers by Dr. Takeyoshi included in the review materials, butthey were easily retrieved from the journal websites. The Panel indicated that raw data (i.e.,
the actual optical density at 370 nm [OD370] readings for the triplicates and the SD of thetriplicates) are necessary for a thorough evaluation. Additionally, the Panel noted that only arelatively small number of substances had been tested in the LLNA: BrdU-ELISA.
When considering the animal welfare impact of implementing the LLNA: BrdU-ELISA, thePanel agreed that it would be less painful than guinea pig tests in those circumstances where theuse of radioactive materials are restricted. Thus, the Panel agreed that the test represents a
potential refinement. The Panel further stated that, if there is not an option to replace the guinea
pig test with a non-animal test, decreasing the extent of pain and distress should be the firstanimal welfare priority. The Panel cautioned that at some point, however, the numbers ofanimals being utilized must be considered. An eventual recommendation that the LLNA: BrdUELISA be routinely used instead of guinea pig test methods where the use of radioactive
substances are restricted would apparently require a significant increase in the number of micekilled per test (to increase the statistical power of the test method - see Section 5.1.2 below) if
an SI ≥1.3 is deemed the appropriate criterion to use for determining a positive response. The
Panel stated that it would be helpful to know how many guinea pigs are currently being usednationally and internationally for skin-sensitization tests, and how many mice would be used in
the LLNA: BrdU-ELISA with the SI ≥1.3 criterion. Even an order of magnitude estimate would
help the Panel judge whether the increase in numbers of mice needed is justified as the quest to
relieve pain in guinea pigs is pursued.
5.1.2 General Statistical Comments
The Panel was concerned about using an SI of ≥1.3 to optimize the performance of the LLNA:
BrdU-ELISA method. One Panel member’s extensive experience with ELISA protocols wascited as evidence that the difference between the OD370 of the vehicle and the positive test at 1.3
would not likely be statistically significant. The Panel recommended that the raw data must be
reviewed to evaluate this. In addition, based on Dr. Joseph Haseman’s power analysis (seeTable F-4), the Panel stated that it was difficult to justify using a SI ≥1.3 as the decision
criterion since it would result in a significant increase in the number of animals needed toobtain an acceptable confidence level. In this regard, the Panel recommended that power
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ICCVAM Test Method Evaluation Report: Appendix F1

calculations should be routinely recommended to ensure that the appropriate number of animals
per dose group is being analyzed.
Table F-4 Power Calculations for the LLNA: BrdU-ELISA1
3.0-fold
Increase2

2.0-fold
Increase

1.3-fold
Increase

Mean Rx response

0.399

0.266

0.173

Log (mean Rx
response)

-0.92

-1.32

-1.75

Difference fromcontrol (log scale)

1.10

0.70

0.27

Difference/SD

3.64

2.32

0.89

Power for N=499%

80-90%<50%

Other power

95% (N=3)

95% (N=5)

50% (N=8)

Other power–

50-80% (N=3)

80% (N=16)

Other power––

90% (N=22)

Parameter

Abbreviations: N = number of animals; Rx = Treatment; SD = standard deviation.
1
The power calculations above are based on a one-sided p<0.05 Student’s t test applied to log-transformed
data from vehicle control LLNA:BrdU-ELISA tests.
2
Fold-increase = Required increase above the vehicle control for a positive response

5.1.3 Comments with Specific References to the Text
The Panel also identified the following minor formatting and grammatical errors, as well as
information gaps, in the draft BRD:•

The Panel noted a discrepancy between the draft LLNA: BrdU-ELISA BRD and thedraft ICCVAM LLNA Performance Standards in the vehicle used for testing 2mercaptobenzothiazole. Table 6-2 of the draft LLNA: BrdU-ELISA BRD indicatedthat the vehicle was AOO but the revised draft ICCVAM LLNA PerformanceStandards indicated that the vehicle was DMF (see page C15, C22 of September 7,
2007, draft and page B-6 of January 7, 2008, revised draft). Additionally, Table 1 on
page C-7 of the revised draft ICCVAM LLNA Performance Standards listed AOO
as the vehicle for 2- mercaptobenzothiazole. For both vehicles, the revised draft
ICCVAM LLNA Performance Standards indicated that the EC3 value is 2.5%,
although the text on page 10 of the draft LLNA: BrdU-ELISA BRD stated “theNICEATM database of traditional LLNA studies indicates that 2mercaptobenzothiazole has a higher EC3 value when tested in AOO (meanEC3=9.8%) compared with DMF (mean EC3=2.5%)....”•The Panel noted that Table 6-1 of the draft LLNA: BrdU-ELISA BRD indicatedthat, when compared to the guinea pig and human test data, the sensitivity andspecificity of the LLNA: BrdU-ELISA is lower than that of the traditional LLNA. In
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ICCVAM Test Method Evaluation Report: Appendix F1fact, depending on the SI threshold value used, the sensitivity and specificity of the
LLNA: BrdU-ELISA can be higher than that of the traditional LLNA.
The Panel recommended that the draft LLNA: BrdU-ELISA BRD be updated to rectify theseerrors and omissions.
5.2

Comments on the Validation Status of the LLNA: BrdU-ELISA

5.2.1 Test Method Protocol
The data generated for the substances analyzed in the LLNA: BrdU-ELISA test method camefrom auricular lymph nodes from four individual mice in each dose group. The ICCVAMrecommended LLNA protocol (ICCVAM 1999; Dean et al. 2001) and OECD TG 429 (OECD2002) recommend a minimum of five animals per dose group when collecting individual animal
data. The Panel was asked what impact might the use of four animals per dose group have on
the accuracy and reliability of the LLNA: BrdU-ELISA, and if the Panel agreed with the
ICCVAM recommendation that future use of this test method protocol should include fiveanimals per dose group. The Panel majority agreed with the ICCVAM recommendation that
future use of this test method should use five animals per dose group and collect individual
animal data, as per the ICCVAM-recommended protocol. A minority opinion by Drs. Nathalie
Alépée, Thomas Gebel, Dagmar Jírová, Raymond Pieters, and Michael Woolhiser stated that if
laboratories were operating under OECD guidance (OECD 2002) and a reliable validation
dataset had been generated, then pooled data from at least four animals could be considered
acceptable. Based on the supplemental data provided by Dr. Haseman, the power to detect athree-fold increase with a sample size of four was determined to be 99%. These calculations,however, assume that a sample size of four is always obtained. If a sample size of four wasplanned and fewer usable data values were obtained, then the experiment might becompromised. Furthermore, the Panel concluded that testing for and eliminating “outliers” fromexperiments with small sample sizes is questionable. A reduction in sample size from five tofour was not recommended unless data was provided on the frequency with which “outliers”
occurred and an analysis is performed that establishes that a reduction in the nominal sample
size from five to four would not compromise the performance of the test method. The Panelstated that the handling of suspected “outliers” and the use of robust statistics are issues thatneed to be addressed in such an analysis. For example, robust procedures may compensate forapparent “outliers” and eliminate the impulse to discard data. An example is calculating themean values used in the SI on a log scale and then exponentiating the result to construct the SI.
The Panel also indicated that it was important to routinely include a positive control group in
test method validation experiments (e.g., HCA), which was likely not the case for most of the
LLNA: BrdU-ELISA validation experiments. Although the Panel did not reach consensus, they
did consider the suggestion that for laboratories in which the LLNA is “routinely” performed,
positive controls (e.g., HCA or a substance that matches the chemical class of the test
substances) could be run at intervals for quality control purposes rather than concurrent witheach experiment in which substances are tested. The Panel also discussed that omitting the
concurrent positive control should not be recommended for laboratories that perform the LLNA
only “occasionally.” In their discussions, the Panel was not able to conclude what should
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constitute “routine” or “occasional” LLNA use or what would be an appropriate interval
between positive control testing when a concurrent positive control is not used.
5.2.2 Substances Used for the Validation Studies
The Panel was asked whether the LLNA: BrdU-ELISA database was representative of a
sufficient range of chemical classes and physicochemical properties such that the test method
would be applicable to any of the types of chemicals and products typically tested for skinsensitization potential. The Panel indicated that the ratio of solids to liquids was notcomparable; more solids should be included. The Panel further indicated that more substances
for which traditional LLNA data are available should be tested, and that compounds including
metals (e.g., nickel, cobalt), mixtures, and substances in aqueous solutions should be included.
5.2.3 Test Method Accuracy
The current accuracy analysis using an SI ≥3.0 or SI ≥1.3 to identify sensitizers is based on
overall concordance with the traditional LLNA. Accuracy statistics compared to the guinea pig
tests and human data/experience were also provided to the Panel. The Panel was asked whether
these comparisons were appropriate for assessing the accuracy of the LLNA: BrdU-ELISA. The
Panel indicated that comparing the LLNA: BrdU-ELISA performance to the traditional LLNA
and the guinea pig tests were appropriate. Comparisons between the LLNA: BrdU-ELISA andhuman data were considered particularly valuable because the traditional LLNA doesn’t match
human data with 100% accuracy. For this reason, the Panel considered comparing theperformance of the LLNA: BrdU-ELISA with that of the traditional LLNA with respect topredicting the human outcomes to be the best method of comparing these two LLNA protocols.
The Panel concluded that in moving forward with any test method recommendation, key
importance should be placed on interpreting the test results and making them clinicallyapplicable to humans.
Takeyoshi et al. (2007) performed an accuracy analysis using decision criteria other than an SI≥3.0 to classify substances as sensitizers. Maximal accuracy for the LLNA: BrdU-ELISA
occurred when an SI ≥1.3 was used to distinguish between sensitizers and non-sensitizers.
Using this decision criterion, the LLNA: BrdU-ELISA achieved an accuracy of 91% (21/23),
with a sensitivity of 100% (16/16) and a specificity of 71% (5/7) (i.e., there were no false
negatives and two false positives). The Panel was asked whether this analysis supported arecommendation that the decision criteria be based on an SI ≥1.3, and if there were concernswith using such a small increase (i.e., 1.3-fold) above the vehicle control response as the basis
for identifying a positive response. The Panel did not support using an SI ≥1.3 as the criterion
for positive results. An SI=1.0 means there was no difference between the vehicle control andthe test substance. An SI=1.3 represents a 30% increase from the vehicle control. The
difference between the OD370 of the vehicle and the positive test at 1.3 may not be statisticallysignificant. An SI=3.0, which represents a three-fold difference between the vehicle and apositive test, would be a more believable positive difference. If the positive test criteria must be
reduced to 1.3, then the Panel questioned whether the protocol is useful in its current state. The
supplemental information that provided power calculations indicated that it would not be
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ICCVAM Test Method Evaluation Report: Appendix F1

realistic to expect to detect a 1.3 fold increase in the control response without a significant
addition of animals. Although using SI ≥1.3 increases the accuracy of the test, it comes at anincreased cost to animals, which merits consideration. Furthermore, the ICCVAM (1999) reportstated that an irritating chemical might induce proliferation, but that the response seldom
exceeds an SI ≥3.0 (page 6). The Panel concluded that this might provide further justification
against using a low SI (e.g., 1.3) as a threshold for a positive response.
The Panel was asked if the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: BrdU-ELISA, using the SI ≥3.0 criterion, hadbeen adequately evaluated and compared to the traditional LLNA (refer also to Table 6-1 of thedraft LLNA: BrdU-ELISA BRD). If not, the Panel was asked what other analyses should be
performed. The Panel agreed that the relevance of the LLNA: BrdU-ELISA, using the SI ≥3.0
criterion, had been adequately evaluated. The Panel further stated that a better evaluation could
be performed, however, if the database for the LLNA: BrdU-ELISA included more substanceswith traditional LLNA, guinea pig, and human data. The Panel considered the false negative
rate of the test method to be excessive when results are compared with that obtained in the
traditional LLNA (29/33/27% for the various datasets) or with human data (39%) – the results
should be at least comparable with the traditional LLNA.
Using the SI ≥3.0 criterion, there were four substances (aniline, 4-chloroaniline, 2mercaptothiazole, and hydroxycitronellal) that produced false negative responses when tested
using the LLNA: BrdU-ELISA compared to the traditional LLNA. The Panel was asked
whether it could identify any characteristics associated with these or similar substances,compared to the correctly identified sensitizers, that might indicate that such substances should
not be tested in the LLNA: BrdU-ELISA or that negative results for such substances should
indicate a need for confirmatory testing. The Panel could not identify any characteristics
associated with these substances that might allow the identification of these substances as false
negatives prior to testing. The Panel stated that the LLNA: BrdU-ELISA test, using the standard
SI ≥3.0 to indicate positive results, simply does not perform well for identifying sensitizers.
5.2.4 Test Method Reliability
The Panel was asked whether the intralaboratory reproducibility of the LLNA: BrdU-ELISA
had been adequately evaluated and compared to the traditional LLNA, and whether any
limitations were apparent based on this intralaboratory reproducibility assessment. The Panelindicated that the number of substances evaluated for intralaboratory reproducibility was too
few and, in some cases, there was a wide variation in repeat test results for the same substance.
Only six substances (five sensitizers and only one non-sensitizer) were tested multiple times.
The non-sensitizer, propylene glycol, was tested only twice and opposite results were obtained.
The Panel considered the results of an intralaboratory reproducibility evaluation that was basedon two discordant results only to be unacceptable. The numbers calculated in Table 7-1 of thedraft LLNA: BrdU-ELISA BRD are correct, but the Panel questioned the dependability of thedata since only two to three values were available for calculating the mean and CV. The Panelconsidered the CV values (over 30%) high, compared to the traditional LLNA (draft LLNA:
BrdU-ELISA BRD Tables 7-1 to 7-3). The Panel stated that at least four independent tests with
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three concentrations tested represent a solid basis for calculation. The Panel considered thenumber of tests for intralaboratory concordance analysis to be insufficient, and stated that more
intralaboratory testing is needed. The Panel recommended an evaluation of the intralaboratory
reproducibility of the EC ≥1.3 and that the analysis of the variability of the ECt be conducted
on a log scale.
The substances evaluated for intralaboratory reproducibility of the LLNA: BrdU-ELISA study
were not coded. The Panel was asked whether the lack of coding of test substances adversely
impacts or biases the current evaluation. The Panel stated that, although coding of substances ispreferred for independent testing and evaluation of test results, the current data should not berejected from consideration because the substances tested were not coded.
The Japanese Center for Validation of Alternative Methods (JaCVAM) has implemented amulti-laboratory validation study of the LLNA: BrdU-ELISA. The Panel was asked whether the
study design was appropriate to adequately determine the extent of interlaboratory
reproducibility for the LLNA: BrdU-ELISA. If not, the Panel was asked what other studies
should be performed. The Panel stated that they had insufficient time to evaluate the study
design and that they could not evaluate interlaboratory reproducibility because the study data
were not available at the time of their evaluation.
5.2.5 Data Quality
The studies evaluated in the draft BRD for the LLNA: BrdU-ELISA were not conducted instrict accordance with GLP guidelines, although there were reportedly performed inlaboratories that conduct GLP studies (M. Takeyoshi, personal communication). In otherwords, an audit report was not available. Also, the raw data were unavailable for an
independent audit. The Panel was asked to discuss what impact this might have on theevaluation of the LLNA: BrdU-ELISA. The Panel concluded that ideally, validation studies
should be performed in accordance with GLP guidelines. Although the systems employed fortests (i.e., test facilities, staff, reagents, etc.) were identical to those for GLP-compliant studies,
the data quality may be questioned and therefore should at least be available for a retrospective
independent audit. However, in this case, the Panel concluded that the lack of GLP compliance
was not likely the reason for the poor results obtained with the LLNA: BrdU-ELISA.
The original records for these studies were requested but had not been received by the time thePanel convened. As a result, an independent audit could not be conducted to confirm that thereported data in peer reviewed publications and a poster presentation is the same as the raw
data. The Panel was asked whether any recommendations from ICCVAM should be contingent
upon the completion of such an audit and findings that there were no significant errors in datatranscription. The Panel concluded that, to have confidence in data quality, ICCVAM
recommendations should be contingent upon the completion of an independent audit. Moreover,
if an SI ≥1.3 is used as the criterion for positive results, review of the raw data is necessary toconfirm statistically significant differences. The Panel concluded that this test, as described, hadpoor accuracy, poor sensitivity, and poor specificity. The Panel stated that changing the SI
decision criterion from 3.0 to improve test performance, especially to such a drastic change asSI ≥1.3, is a mistake and sets a dangerous precedent.
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5.2.6 Consideration of All Available Data and Relevant Information
Based on the draft LLNA: BrdU-ELISA BRD, the Panel was asked whether all the relevant
data identified in published or unpublished studies that employ this test method had beenadequately considered, and if other comparative test method data that were not considered wereavailable. If yes, the Panel was asked to suggest how to obtain such data. The Panel believedthat all of the relevant data, with the exception of the interlaboratory reproducibility study, were
presented and that they were not aware of any omissions.
5.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-ELISA
5.3.1 Test Method Usefulness and Limitations
The Panel was asked whether the available data support the ICCVAM draft recommendations
for the LLNA: BrdU-ELISA in terms of the proposed test method usefulness and limitations.
The Panel agreed with the ICCVAM recommendation that the LLNA: BrdU-ELISA may be
useful for identifying substances as potential skin sensitizers and non-sensitizers but that, at thistime, more information and data are needed before a recommended use of the LLNA: BrdUELISA can be made. The Panel also stated that a detailed protocol is needed, in addition to
sufficient quantitative data for a more comprehensive analysis based on a larger set of balanced
reference substances with regard to physicochemical properties and sensitization potency, as
well as an evaluation of interlaboratory reproducibility.
The Panel was asked whether the LLNA: BrdU-ELISA should be routinely recommended forhazard identification of skin sensitizing substances in lieu of using guinea pig tests if
restrictions on using radioactive materials are present, due to the fact that fewer animals might
be used and because pain and distress would be avoided. The Panel stated that if the accuracyof the test method was at least similar to the traditional LLNA, the LLNA: BrdU-ELISA
might be routinely recommended for hazard identification of skin-sensitizing substances interms of reduction of animals and refinement of the pain and distress associated with guinea
pig tests. Clearly, using the LLNA: BrdU-ELISA instead of the traditional LLNA or guinea
pig test methods would also offer advantages for the environment due to the use of a nonradioactive probe chemical. However, the Panel stated that the accuracy of the current LLNA:
BrdU-ELISA dataset at SI ≥3.0 was inadequate and not equivalent to the traditional LLNA.
The Panel also noted that if an SI ≥1.3 was used because of its apparent increased accuracy,
additional mice (over and above the number needed in the standard LLNA test) would
apparently be needed (see Table F-4). Thus, the Panel stated that reduction of animals would
not be achieved. In this regard, the Panel noted that some quantification of the total animal use
numbers would be useful as it is not clear whether the increased number of mice used would
outweigh the avoidance of pain and distress in guinea pigs.
The Panel was asked whether the LLNA: BrdU-ELISA procedure or other valid and accepted
non-radioactive method could be routinely recommended for hazard identification of skinsensitizing substances instead of the traditional LLNA if limitations in using radioactive
materials are not present. The Panel stated that the LLNA: BrdU-ELISA procedure could not beroutinely recommended for hazard identification of skin sensitizing substances instead of the
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traditional LLNA, because the accuracy of this test at SI ≥3.0 was inadequate. In other words,
the current dataset available for the LLNA: BrdU-ELISA did not predict the guinea pig or
human outcomes as accurately as the traditional LLNA. Thus, the Panel acknowledged that
there is the possibility that additional data might impact on the accuracy statistics and eliminate
this concern. The Panel stated that factors that weigh on a decision of replacement of the LLNA
with a non-radioactive method would include:•

Are more animals needed?

•

Is the replacement test safer and less complex?

•

Is the replacement test more efficient?

•

Is the replacement test less costly?

The Panel stated that additional factors to consider might exist, but overall recommended thatwhether or not restrictions on radioactivity exist, a test that causes the least pain and uses the
fewest number animals should be preferred, as long as adequate test method performance ismaintained. Clearly, policy issues regarding restrictions on radioactivity should have no impact
on this science-based conclusion.
The Panel was asked whether using a decision criterion of SI ≥1.3 instead of SI ≥3.0 resolved
any concerns with respect to potential false positives or false negatives that may occur in thistest method. The Panel was also asked for other suggestions for additional guidance orlimitations that should be considered. The Panel stated that using a decision criterion of SI ≥1.3
instead of SI ≥3.0 would not itself resolve any concerns; more raw data are needed for a broader
set of reference positive and negative sensitizers, including metals, mixtures, and aqueous
solutions. The Panel also stated that a detailed protocol is needed, as is an evaluation ofinterlaboratory reproducibility. The Panel considered the current database to be inadequate, but
based on the limited database, concluded that it might be more appropriate to use a statistically
based decision criteria than a stimulation index.
5.3.2 Test Method Protocol
The ICCVAM draft recommendations state that the LLNA: BrdU-ELISA protocol should
adhere to the ICCVAM LLNA protocol (ICCVAM 1999; Dean et al. 2001), except formeasurement of lymphocyte proliferation. The Panel was asked whether the available data
support the ICCVAM draft recommendations for the LLNA: BrdU-ELISA procedure in termsof the proposed test method standardized protocols. In general, the Panel agreed that theavailable data support the ICCVAM draft recommendations for this test method in terms of thestandardized protocol. As stated previously, the Panel majority agreed with the ICCVAM
recommendation that future studies should use five animals per dose group and collectindividual animal data, as per the ICCVAM-recommended protocol. A minority opinion by Drs.
Nathalie Alépée, Thomas Gebel, Dagmar Jírová, Raymond Pieters, and Michael Woolhiserstated that if laboratories were operating under OECD guidance (OECD 2002) and a reliable
validation dataset had been generated, then pooled data from at least four animals could beconsidered acceptable. The Panel further noted that using an SI <3.0 would require more
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animals to achieve adequate statistical power (Table F-4) and therefore any considerations of
reducing the SI to improve test method accuracy should include this point.
The Panel was asked whether the traditional LLNA limit dose procedure could be applied to the
LLNA: BrdU-ELISA. The Panel stated that, if the LLNA: BrdU-ELISA was considered
equivalent to the traditional LLNA, then it would be appropriate to apply the LLNA limit dose
procedure to this test method. The Panel explained that, as in the case of the traditional LLNA,
the protocol would be the same except for testing the maximum dose only, so applying the limit
dose procedure would appear to have the same opportunity to reduce the number of animals
needed to perform the test. However, using an SI ≥3.0 would not be appropriate because of theassociated low accuracy in identifying sensitizers.
5.3.3 Future Studies
The Panel was asked whether the available data support the ICCVAM draft recommendations
for the LLNA: BrdU-ELISA in terms of the proposed future studies. The Panel stated that the
proposed future studies were justified. The Panel concluded that it is important to consider nonradioactive methods because, in some laboratories, it is difficult or not permissible to use
radioactivity. The Panel also stated that, if more data were available and there was lessvariability in this test method, it might warrant re-evaluation. The Panel concluded that moredata are needed, especially for determination of the appropriate threshold value for the decision
criterion, and that interlaboratory reproducibility should be also evaluated (which presumably
will occur once the Japanese interlaboratory validation effort is complete).
5.3.4 Performance Standards
The LLNA: BrdU-ELISA protocol differs from the ICCVAM-recommended protocol for the
traditional LLNA (ICCVAM 1999; Dean et al. 2001) in the method used to assess lymphocyte
proliferation in the auricular lymph nodes. According to the proposed draft ICCVAM LLNA
Performance Standards for the traditional LLNA, any change to the LLNA protocol other thanthe method used to assess lymphocyte proliferation is considered a “major” change. The Panelwas asked whether protocol differences between the LLNA: BrdU-ELISA and the traditional
LLNA should be considered only “minor” changes and therefore if the validity of this test
method should be based on the draft ICCVAM LLNA Performance Standards. In general, thePanel agreed that the LLNA: BrdU-ELISA protocol differs only in the method used to assess
lymphocyte proliferation. Thus, based on the current draft ICCVAM LLNA PerformanceStandards, it should be considered as having only “minor” changes and therefore the validity ofthis test method could be based only on the draft ICCVAM LLNA Performance Standards.
However, the Panel concluded also that the answer to this question might differ depending on
what the draft ICCVAM LLNA Performance Standards ultimately constitutes as a “major”
change, a “minor” change, or a different protocol altogether. The Panel further stated that,
depending on the goal of the assay, these distinctions may not be relevant. Ultimately, if a test
method is able to make the correct prediction with regard to the sensitization potential of a test
substance, then the issue of “major” versus “minor” changes in the protocol should not apply.
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The Panel was asked, even if the draft ICCVAM LLNA Performance Standards do not apply to
the LLNA: BrdU-ELISA, what impact should the accuracy analysis based on eight of the 18
proposed required reference substances in the draft ICCVAM LLNA Performance Standards(only one false negative and no false positives) have on the overall evaluation of test method
accuracy. The Panel concluded that the accuracy analysis based only on eight of the 18
proposed required reference substances had a significant impact on the evaluation of test
method accuracy. The low number of experiments provided data that resulted in unacceptable
test method performance.
The Panel was asked whether there were concerns that 4/4 sensitizers, for which EC3 data wereavailable, had EC3 values that were outside of the proposed recommended 0.5x to 2.0x EC3
acceptability range developed based on the traditional LLNA. The Panel concluded that theEC3 values outside the recommended 0.5x to 2.0x EC3 acceptability range raised concerns
related to test reproducibility and reliability.7
The Panel was asked whether separate performance standards should be developed for the
LLNA: BrdU-ELISA. The Panel concluded that separate performance standards for the LLNA:
BrdU-ELISA were not needed because the test principles are identical to the traditional LLNA.

7

During their public meeting on March 4-6, 2008, the Panel’s discussion, conclusions and recommendations on
the LLNA: BrdU-ELISA took place prior to the discussion, conclusions and recommendations on the draft
ICCVAM LLNA performance standards. Following their discussion of the draft LLNA performancestandards, the Panel concluded that that an evaluation of test method accuracy should be based on overall
accuracy statistics when compared to the traditional LLNA, and not on a chemical-by-chemical match that isbased on obtaining an EC3 value within a specified range of EC3 values.
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6.0

Draft ICCVAM LLNA Performance Standards

6.1

Comments on the Proposed Purpose and Applicability

ICCVAM proposed that these performance standards should only be applicable to versions of
the LLNA that incorporate “minor” modifications to the traditional LLNA. Currently, this islimited to the use of non-radioactive reagents to measure lymphocyte proliferation. It isconsidered essential that the modified LLNA should otherwise adhere to all other aspects of the
traditional LLNA protocol, as defined by ICCVAM (1999) and Dean et al. (2001). This
includes aspects such as: the sex and strain of mouse used, the number of mice per dose group,
the timing and site of test article treatment, the duration between the last treatment and lymph
node collection, the inclusion of concurrent negative and positive control groups, the measured
endpoint (i.e., lymphocyte proliferation in the draining auricular lymph node), and thecollection of data at the level of the individual mouse. The Panel was asked if they agreed thatthe use of non-radioactive reagents for measuring cell proliferation in the lymph nodes
constitutes a “minor” modification to the traditional LLNA protocol.
The Panel noted that the draft ICCVAM LLNA Performance Standards are proposed forevaluating the acceptability of test methods that are functionally and mechanistically similar to
the traditional LLNA (i.e., measuring the same biological effect), and understood that ICCVAM
proposed that these performance standards should only be applicable to protocols thatincorporate “minor” modifications to the traditional LLNA, as defined above. The Panelunanimously agreed that based on ICCVAM’s definition, the use of non-radioactive reagentsfor measuring cell proliferation is a “minor” modification of the traditional LLNA protocol.
However, the Panel also agreed that other modifications may be considered “minor” and that abetter strategy for the performance standards might be to define criteria that need to be satisfied
to insure that the method is mechanistically and functionally similar (see criteria listed underessential test method components). Examples of potentially acceptable modifications identified
by the Panel include sex, strain, the use of rats rather than mice, number of animals per group,and timing of test article treatment.
Regardless of the modification, the Panel stated that the modified test method should bedesigned to measure only the induction phase of the immune response. This is crucial, since thetraditional LLNA is intended for hazard identification with the underlying principle that
stimulation of cell proliferation in the draining lymph node suggests that sensitization (i.e.,
induction) is occurring. Using only the induction phase as the method to identify hazardous
substances involves a short time frame, and reduces pain and distress in treated animals (i.e., nodermatitis response). Furthermore, the Panel stated that the performance standards should not
imply that the traditional LLNA, or any alternative LLNA protocol, is capable of specificallydistinguishing a type IV hypersensitivity reaction (as might be inferred from the text beginningwith line 342 of the draft ICCVAM LLNA Performance Standards document). Therefore,
reference to type IV hypersensitivity reaction should be removed from the document.
The Panel was asked if they considered it necessary that a modified LLNA keep the samedecision criteria for distinguishing between sensitizers and non-sensitizers as the traditional
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LLNA (i.e., an SI ≥3.0). The Panel considered it unnecessary for a modified LLNA to keep thissame decision criteria as a different method for measuring cellular proliferation might havebetter concordance with the human data at a SI different than 3.0. Thus, with any modified
LLNA, the SI threshold defining a sensitizer would need to be established (i.e., it is important
to consider if the results are biologically relevant to humans).
The Panel was asked if other procedural modifications could be identified as “minor,” based on
the description in the draft ICCVAM LLNA Performance Standards document, and therefore
could be evaluated for equivalence to the traditional LLNA using the proposed performancestandards. The Panel reiterated that sex, strain, the use of rats rather than mice, animals pergroup, and timing of test article treatment are also potentially “minor” modifications.
Furthermore, the proposed performance standards appear robust; therefore, regardless of themodification (i.e., “major” or “minor”), there is the same expectation for test method
performance. Dr. James McDougal offered a minority opinion to express his concern about thepotential impact that allowing alternative LLNA protocols with modifications other than themethod by which lymphocyte proliferation was measured would have.
The Panel was asked if they considered the draft ICCVAM LLNA Performance Standards
applicable to the LLNA limit dose procedure. The Panel noted that the current draft ICCVAM
LLNA Performance Standards could be applicable to the LLNA limit dose procedure as long as
it is recognized that this procedure can only be used for a yes/no hazard classification (i.e., an
ECt estimate is not feasible).
6.2

Comments on the Essential Test Method Components

The essential test method components are based on the ICCVAM-recommended protocol(ICCVAM 1999; Dean et al. 2001), which is the basis for the current EPA (2003) test guideline.
There are some notable differences between these protocols and OECD TG 429 (OECD 2002)
for the LLNA. The Panel was asked to comment on, when evaluations of non-radioactive
versions of the traditional LLNA are conducted using these performance standards, whether it isnecessary that the validation studies follow the ICCVAM-recommended protocol. The Panelindicated that ideally, there would be one globally recognized set of performance standards(ICCVAM, ECVAM, JaCVAM). However, when validating versions of the traditional LLNA
where the only difference is in the use of a non-radioactive method to measure cellproliferation, the ICCVAM-recommended protocol should be used. If more extensive changes
to the protocol are being considered, the following requirements should be considered during
modifications of the LLNA:•

Application of the test substance should be to the skin, with sampling of the lymph
nodes draining that site.

•

Cell proliferation should be measured in the draining lymph node.

•No skin reaction should be present, since presence of a skin reaction might indicate
the onset of the elicitation phase of skin sensitization.

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Data should be collected at the level of the individual animal to allow for an estimateof the variance within control and treatment groups. Using this variance, a power
analysis needs to be conducted to demonstrate that the modified method is utilizing asufficient number of animals per treatment group to permit hazard identification
with at least 95% power.

•

If dose response information is needed, there should be an adequate number of dosegroups (n ≥3) with which to adequately characterize the dose response for a given
test substance.

The Panel was asked to comment on whether validation studies should include a concurrent
positive control with each test substance and if so, whether the concurrent testing of the positive
control and test substance should be conducted in the same vehicle or if different vehicles were
acceptable. The Panel noted that a concurrent positive control should be included in eachvalidation study to ensure that the test system was operating as expected and technical errors
were not occurring. A concurrent positive control would be especially useful when an unknown
test material was being tested or when a laboratory was collecting a dataset to serve as historicalcontrol data. However, if a known sensitizer was being tested, a concurrent positive control
might not be needed, thus reducing animal use. Finally, the Panel concluded that the positive
control should be tested in the same vehicle as the test substance. Using a different vehicle forthe positive control would require an additional set of vehicle control animals.
The Panel was also asked whether the validation studies should use a minimum of fiveanimals per dose group and collect lymph node data from individual animals. The Panelcommented that until sufficient data were collected to enable a reliable power calculation to
be conducted to determine the optimal number of animals per dose group, at least five animals
per dose group should be used. The Panel also agreed that when validating a modified LLNA
protocol, lymph node proliferation should be evaluated at the level of the individual animal
within each dose group. Variance is only measurable if lymph nodes from individual animals
are assessed. If the variability within a dose group of a modified LLNA protocol was
substantially less than the traditional LLNA, reducing the number of animals per dose group
might yield similar results. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel,
Dagmar Jírová, Raymond Pieters, and Michael Woolhiser stated that if laboratories wereoperating under OECD guidance (OECD 2002) and a reliable validation dataset had been
generated, then pooled data from at least four animals could be considered acceptable.
6.3

Comments on the Proposed Reference Substances

The Panel was asked if they agreed with the selection and prioritization criteria used to selectthe performance standards reference substances. The Panel noted that the rationale for selectionof the reference substances included in the draft ICCVAM LLNA Performance Standards waswell documented (taking into account the physicochemical characteristics, the purity, thestability, the quality of the in vivo data, and the chemical classes covered). The substances also
appeared to be distributed over a wide range of EC3 values. However, the available database forsome of the substances was insufficient. Among the 13 sensitizers in the “required” list, only
five appear to have a robust database (i.e., have been tested in at least three independent
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studies). Thus, consideration should be given to revising the list of substances and/or making
the data for the substances on the current list more robust. Ideally, the reference list should bebased only on substances with robust data for LLNA, human, and guinea pig tests.
The rationale for the number of substances included on the “required” list of substances (n=18)
was provided in the draft ICCVAM LLNA Performance Standards. In addition, there were four
additional substances that were described as problematic in the traditional LLNA (i.e., false
negatives and false positives). The Panel was asked if they considered 18 required” substances
to be an adequate number upon which to evaluate the performance of non-radioactive LLNA
test methods, where the only protocol modification is the method for assessing cell proliferationin the auricular lymph nodes, and if not, how many reference chemicals should be tested. The
Panel commented that ideally, one would like to be able to demonstrate that an assay isequivalent to the traditional LLNA. However, with the small number of reference substances
available, establishing equivalence will be extremely difficult. Therefore, the Panelrecommended that, for use in hazard identification, a modified method should be evaluated withall 22 substances (including false negatives and false positives) and accuracy statistics
calculated. To the extent possible, rationale for any discordant results should be provided, but
the most potent sensitizers (e.g., DNCB) should always be identifiable. There also should beconsiderable weight given to the balance between animal welfare and human safety when
considering the adequacy of test method accuracy.
The panel considered it noteworthy that 19 of the 22 substances on the draft ICCVAM list arein common with the ECVAM performance standards list. The Panel also considered it
important that substances be coded during validation studies.
It is also relevant to note that the Panel discussed the value of GLP procedures on several
occasions during the meeting. In each instance, the Panel agreed that data collected under GLP
conditions would be greatly preferred, particularly for reasons of data quality and the associatedreliability of any interpretations. However, they noted that GLP compliance would not be
considered a requirement that would automatically exclude data from consideration. The Panelconcluded that other factors could be used to identify high quality data. Examples would
include published in a peer-reviewed journal or obtained from a study conducted in a laboratorythat routinely conducts GLP studies. Data generated under non-GLP conditions would be
subject to a critical quality review, and as such the Panel considered it important to obtain theoriginal records in order to confirm the reported data.
The Panel was asked if they considered the types of substances included in the reference
substance list, with regard to relative sensitization potency, physicochemical characteristics, andvehicles, to be representative of the overall diversity of substances that are likely to be tested forskin sensitization. The Panel concluded that although the list should not be considered allinclusive, it was sufficiently representative.
The Panel was also asked if there were other types of information relevant to skin sensitizationthat should be considered in order to demonstrate an adequately diverse reference list. The
Panel commented that identifying concentrations of each of the substances that are known tocause excessive local irritation or overt systemic toxicity would be useful.
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The Panel was asked if there were other substances that they considered to be more appropriate
for assessing the sensitivity (i.e., ability of the test method to correctly identify sensitizing
substances) and specificity (i.e., ability of the test method to correctly identify non-sensitizing
substances) of non-radioactive LLNA test methods, and for which there is available LLNA,
guinea pig, and human data. The Panel could not identify such substances given the time frame
for consideration but reiterated that substances in the reference list should have robust data.
A subset of “discordant chemicals” (i.e., false negative or two false positive compared to guinea
pig tests or human data) were included as “optional” substances that could be studied toevaluate if the proposed modifications might provide improved performance relative to the
traditional LLNA. The Panel was asked to comment on the appropriateness of including these
specific substances in the reference list, whether they should be required, whether different
substances should be included, and if more false negative/positive substances should be tested.
As mentioned previously, the Panel commented that it was appropriate to include such
substances in the reference list and that they should be required and evaluated during thevalidation of alternative LLNA assays that are functionally and mechanistically similar to the
traditional LLNA assay. The Panel noted that the substances that were considered discordant
depended on the species to which comparisons are made (i.e., LLNA vs. guinea pig or LLNA
vs. human). Still, since the “discordant compounds” were false negatives or positives in the
traditional LLNA, they would provide an opportunity to determine if modifications to the
traditional LLNA may even have increased accuracy.
Finally, the Panel was asked if “correct” results with these discordant chemicals would be
sufficient to consider the alternative test method to be more predictive of skin sensitization than
the traditional LLNA. The Panel concluded that correct results with the “discordant chemicals”
would not be sufficient to consider the alternative test method to be more predictive of skinsensitization, but it could provide supporting evidence to indicate further testing with additional
compounds would be of value.
6.4

Comments on the Test Method Accuracy Standards

The draft ICCVAM LLNA Performance Standards state that the non-radioactive proposed
LLNA test method should exactly match the accuracy of the traditional LLNA when evaluatedwith the minimum set of 18 reference substances. The Panel was asked if they agreed that test
method accuracy should be based on a chemical-by-chemical match with regard to identifying
the chemicals as sensitizers or non-sensitizers. The Panel commented that although an assaythat is able to predict the same hazard classification for the reference substances as the
traditional LLNA is desired, with the small number of reference substances available (n=18),
clearly establishing equivalence will be extremely difficult. Furthermore, even with this small
number, there is a statistical multiple-comparisons8 problem because more than one chemical is8

When multiple experiments are conducted and multiple observations, comparisons, or hypothesis tests are
conducted, the chance of observing rare events increases. Suppose, for example, that an interval is establishedsuch that 5% of observations from a particular population of data are outside that interval. Then if kindependent experiments generate data from this population (e.g., a standard normal distribution), the chancesthat all 20 results will lie inside the interval is (1.0 - 0.05)k (N. Flournoy, personal communication).
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being tested. The likelihood that a modified LLNA will fail to demonstrate equivalence to the
traditional LLNA will increase with the number of chemicals that must be identified correctly.
A statistical measure of concordance should be calculated so that accuracies can be compared
between methods.
The Panel reiterated their recommendation that, for use in hazard identification, a modified test
method should be evaluated with all 22 substances (including false negatives and false
positives) and accuracy statistics calculated. A statistical measure of concordance should be
calculated so that accuracies can be compared between methods. To the extent possible,
rationale for any discordant results should be provided. However, the most potent sensitizers(e.g., DNCB) should always be identifiable. Considerable weight should be given to the balance
between animal welfare and human safety when considering the adequacy of test method
accuracy.
The draft ICCVAM LLNA Performance Standards recommend that, for each sensitizer, thethreshold concentration that induces a positive SI response should be within 0.5x to 2.0x of theconcentration obtained for the EC3 in the traditional LLNA. As described in the draft ICCVAM
LLNA Performance Standards, statistical approaches have been used in an attempt to identify
an appropriate range, but these calculated ranges do not appear to be the most practical. The
Panel was asked to comment on the appropriateness of using this criterion to judge theequivalency of a non-radioactive version of the traditional LLNA and, if this approach was not
acceptable, to suggest an alternative along with the basis for this approach. The Panelcommented that the usefulness and limitations of the traditional LLNA for determining relative
potency have not been definitively established, and therefore equivalence should not be basedstrictly on potency. Furthermore, the current database does not support the inclusion of EC3
values as a component of the accuracy evaluation. The range of 0.5x to 2x EC3 value suggested
in the draft ICCVAM LLNA Performance Standards document are based on the experience
with a range of known skin sensitizers tested in the standard LLNA. However, based on theavailable data provided, the 0.5x to 2.0x EC3 range may be too restrictive if a strict
interpretation of equivalence is applied. The chances of a failure to achieve an EC3 within thisrange would vary from chemical to chemical depending upon the inherent underlying
variability and robustness in the estimation of the EC3. For those chemicals for which the EC3
can be accurately estimated, the failure rate may be close to zero. For other, more variablechemicals, perhaps with fewer data points, the failure rate for a single chemical will be muchhigher.
The Panel reiterated their concern with regard to EC3 values (i.e., the statistical multiple
comparisons problem). The likelihood that a modified LLNA will fail to demonstrateequivalence to the traditional LLNA will increase with the number of chemicals tested, theextent to which the new test must obtain the same EC3 value, and how independent the results
are for different chemicals in the same lab.
For five of the 13 sensitizers on the draft ICCVAM reference substances list, the reference EC3
value was based on a single LLNA study (Table C1 of the draft ICCVAM LLNA PerformanceStandards). The Panel was asked to comment on the appropriateness of including such
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ICCVAM Test Method Evaluation Report: Appendix F1chemicals in the list of recommended reference substances and whether or not the 0.5x to 2.0x
criteria should be applied to such substances. The Panel concluded that the appropriateness of
the 0.5x to 2.0x ECt range had not been adequately justified. It was inappropriate to include
chemicals represented by only one LLNA study on a list of recommended reference substances,
as there was insufficient data by which to calculate a robust mean ECt value. Thus, thosecompounds should either (1) be exchanged for compounds with sufficient EC3 data (i.e., havebeen tested in at least three independent studies using the same solvent), or (2) retained but notconsidered to be part of the ECt criterion until such data has been collected.
6.5

Comments on the Test Method Reliability Standards

The draft ICCVAM LLNA Performance Standards state that acceptable intralaboratory
reproducibility will be indicated by a laboratory obtaining, in each of four independent
experiments conducted with at least one week between each experiment, ECt values (theestimated concentration needed to produce an SI of a defined threshold [e.g., EC3]) for HCA
that are generally within 0.5x to 2.0x (i.e., 5% to 20%) of the historical mean EC3 concentration(10%) for this substance, based on existing available traditional LLNA data. The Panel was
asked if they considered four repeat experiments to be adequate. The Panel concluded that four
experiments would be adequate, as requiring four independent experiments is similar to theoriginal LLNA submission, as is a one-week interval between experiments. Therefore, theserequirements were appropriate for a comparison of modified methods to the traditional LLNA.
However, it would be useful to have this number evaluated statistically (see Section 6.7).
The Panel was asked if they considered testing HCA adequate for demonstrating intralaboratory
reproducibility and if not, which substance(s) should be tested. The Panel concluded that HCA
testing would be adequate for demonstrating intralaboratory reproducibility and would allow aneffective comparison to the traditional LLNA.
The Panel was asked to comment on whether the required one-week interval between
independent tests was adequate and/or appropriate. The Panel concluded that the minimum oneweek interval seemed logical and that the more important clarifying information might be theelements that define independent tests (e.g., different animal shipment, different reagents,
different operator, blind testing).
The Panel was asked to comment on the appropriateness of the criteria for acceptability(generally within 0.5x to 2.0x EC3 for HCA), or to describe another criteria and explain thebasis for their recommendation. The Panel concluded that the criteria for acceptability appeared
to be appropriate because the statistical multiple comparisons issue does not exist. However,given that there is so much data and experience with HCA and the fact that only one compoundis being tested (not 18), it is reasonable to evaluate reproducibility using the mean ± 3 standard
deviations rather than the 0.5x to 2.0x EC3 range to account for a single comparison (seeSection 6.7 regarding data transformation recommendations). The Panel noted that historicalcontrol data using HCA in the same vehicle could be used to demonstrate adequate
intralaboratory reproducibility.

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The draft ICCVAM LLNA Performance Standards state that acceptable interlaboratory
reproducibility will be indicated by each of three laboratories obtaining ECt values for HCA
and DNCB from a single experiment that are generally within 0.5x to 2.0x (5% to 20% and0.025 to 0.1%, respectively) of the mean historical EC3 concentration (10% and 0.05%,
respectively) obtained for these two substances in the traditional LLNA. The Panel was asked ifthey considered the single experiment per substance in each laboratory to be adequate. The
Panel concluded that, considering the overall validation plan for a given laboratory, multiple
experiments (n=3) within each laboratory should be conducted.
The Panel was asked if they considered testing HCA and DNCB to be adequate fordemonstrating interlaboratory reproducibility and if not, which substance(s) should be tested.
The Panel concluded that, since there is a great deal of data and experience with HCA and
DNCB, and many laboratories have successfully worked with them in the traditional LLNA,
they should be considered adequate for this purpose.
The Panel was asked if they considered the criteria for acceptability to be appropriate. The
Panel concluded that the criteria for acceptability (i.e., generally within 0.5x to 2.0x ECt for
HCA and DNCB) appeared to be appropriate because the statistical multiple comparisonsproblem was relatively minor given that only two substances are being tested. However, giventhat there is so much data and experience with HCA and DNCB and the fact that twocompounds are being tested (not 18), it is reasonable to evaluate reproducibility using the mean
± 4.5 standard deviations to account for statistical multiple comparisons (see Section 6.7
regarding data transformation recommendations). The Panel also noted that historical control
data using HCA and DNCB in the same respective vehicle could be used to demonstrateadequate interlaboratory reproducibility.
6.6

Summary

The Panel was asked what criteria should be used to evaluate the equivalence of a radioactive or
non-radioactive LLNA method to the traditional LLNA, if one were proposed with a “major”
change, as defined in the draft ICCVAM LLNA Performance Standards (e.g., different mousestrain or use of male mice, change in the schedule for test article administration, change inschedule for lymph node excision, etc.). The Panel commented that the idea of what is a“major” and a “minor” change should be re-considered (refer to Question 2 regarding essential
test components). The final version of the performance standards should be adequate toevaluate any protocol modifications.
The Panel was asked if a new set of performance standards would be required for a modified
version of the LLNA that incorporated one or more “major” protocol changes. Based on theabove response, the Panel concluded that a new set would not be required.
The Panel was asked to comment on how many reference substances might be consideredadequate for evaluating the validity of a modified version of the LLNA with a “major” protocol
change; specifically, if the 18 minimum reference substances in the draft ICCVAM LLNA
Performance Standards would be sufficient. The Panel concluded that additional substances
should not be considered necessary. However, since eight of the proposed sensitizers had
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ICCVAM Test Method Evaluation Report: Appendix F1

limited data (i.e., EC3 values based on ≤2 LLNA studies), other substances with more robust
data should be considered as replacements. Furthermore, if the goal is to evaluate a specificapplicability domain, additional test substances might be needed.
The Panel was asked to comment, regardless of the number of reference substances, whether
the alternative LLNA with a “major” change should be required to obtain the same “call” (andpotency for sensitizers) as the traditional LLNA for the 18 minimum reference substances in thedraft ICCVAM LLNA Performance Standards. The Panel reiterated that an assay that isequivalent to the traditional LLNA is desired, but with the small number of reference
substances available, clearly establishing equivalence will be extremely difficult. They also
reiterated their concern regarding the statistical multiple comparisons problem.
For use in hazard identification, a proposed modified LLNA should be evaluated with all
22 substances (including false negatives and false positives) and accuracy statistics calculated
so that accuracies can be compared between the modified test method and the traditional
LLNA. To the extent possible, rationale for any discordant results should be provided.
However, the most potent sensitizers (e.g., DNCB) should always be identifiable. Considerable
weight should be given to the balance between animal welfare and human safety when
considering the adequacy of test method accuracy.
The Panel was asked to identify any additional specific substances that should be used. The
Panel concluded that while additional substances should not be needed, it would be useful toidentify replacements for the eight proposed sensitizers with limited test data. If the goal is toevaluate a specific applicability domain, additional test substances might be needed.
6.7

Additional Statistical Comments

During the evaluation of the draft ICCVAM LLNA Performance Standards, the Panel noted anumber of statistical issues that should be addressed. They suggested that in order to achieve anormal distribution of the data and to reduce differences between groups, a suitable variancestabilizing transformation (e.g., log transformation, square root transformation) should beapplied in all statistical analyses and in reporting summary standard deviations. The Panel alsosuggested that there should be a more rigorous evaluation of what would be considered an
appropriate range of ECt values to include as a requirement. This would be a statisticalevaluation that takes into consideration the variability of ECt values generated among thesensitizers included on the performance standards reference substances list and the statisticalmultiple comparisons problem and the fact that sample sizes that are less than 30 invalidate
statistics based on the normal distribution (Young 2007).
Furthermore, bioequivalence models have been developed (Berger and Hsu 1996) and should
be applied to the LLNA. Probability values can be used as descriptive statistics and as suchprovide a summary measure of weight-of-evidence that would be useful for comparison of
performance standards across test methods. In this context, it would be informative to havestatistical tests of data generated for these purposes. A test of concordance for measuring theaccuracy of classification should be done.

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ICCVAM Test Method Evaluation Report: Appendix F1Intralaboratory tests should include analysis of variance (ANOVA)-like tests with a test for no
trend, with the null hypothesis being that there is a difference and the alternative being that thedifference is bioequivalent. Interlaboratory tests should include ANOVA-like tests with the null
hypothesis being that there is a difference and the alternative hypothesis being that thedifference is bioequivalent. The reliability tests require “bioequivalence” to be defined (i.e.,
what is acceptable to be considered equivalent).
It is not known whether these specific statistical tests can be identified in the literature or if theyneed to be developed. If they do need to be developed, this should be given a priority. Prior torunning reliability studies, these statistical methods should be used to determine the appropriate
number of substances and the number of times each substance needs to be tested within andamong laboratories in the study design (see also ISO 5725 [ISO 1994] and ASTM Standard
E691 [ASTM 2005]). The power for the traditional LLNA should be established for comparison
purposes.

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ICCVAM Test Method Evaluation Report: Appendix F1

7.0

Use of the LLNA for Potency Determinations

7.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

The Panel was asked if there were any errors in the draft BRD on the use of the LLNA for
potency determinations that should be corrected, if omissions of existing relevant data had been
identified, or if there was additional information that should be included. The Panel noted
alternative analyses that would better help evaluate the use of the traditional LLNA for skinsensitization potency (see the discussion of the categorization scheme in Section 7.2 and thediscussion of future studies in Section 7.3).
7.2

Comments on the Validation Status of the Traditional LLNA to Determine SkinSensitization Potency

7.2.1 Substances Used for the Validation Studies
The Panel was asked to consider whether the validation status of the traditional LLNA for
potency categorization (i.e., “strong” vs. “weak” sensitizers) has been adequately characterized,
and if the traditional LLNA is sufficiently accurate and reliable to be used as a stand-alone
assay for characterizing the potency of sensitizing substances, based on the comparison tohuman and guinea pig responses. The Panel agreed that the LLNA database of 170 substanceswith comparative guinea pig (i.e., Guinea Pig Maximization Test or Buehler Test) and/or
human data (i.e., Human Maximization Test [HMT] and/or Human Repeat Insult Patch Test
[HRIPT], but not human clinical observations) is sufficient in number and well balanced for thisevaluation. The database included 112 substances (97 sensitizers, 15 non-sensitizers) withcomparative human data and 105 substances (52 sensitizers, 53 non-sensitizers) withcomparative guinea pig data. Known contact sensitizers of public health concern from various
chemical groups are included. The Panel further agreed that these substances wererepresentative of a sufficient range of chemical classes and physical chemical properties so that
it would be applicable to the types of chemicals and products typically tested for skinsensitization potential.
While coding of chemicals to reduce bias is recommended for validation studies, this evaluation
was based on a retrospective evaluation of existing data, most of which were generated usingchemicals that were not coded. The Panel was asked whether the lack of coding of test
substances adversely impacted or biased the current evaluation. Given the nature of the studies(i.e., the testing was not conducted to demonstrate the ability of the LLNA to be used forpotency characterization), the Panel stated that the lack of coding likely had no impact on thecurrent evaluation.
For some substances tested for sensitization using the traditional LLNA, it was not possible todetermine whether the data were generated using pooled or individual animal lymph node
samples within a dose group (the former allowed in OECD TG 429 [OECD 2002]; the latter asrecommended in the ICCVAM 2001 protocol and required in the EPA 2003 skin sensitization
test guideline). Cockshott et al. (2006) reported that using individual animal data allowed fortechnical problems during an experiment and outlier animals within a dose group to be
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identified. Considering this, the Panel was asked whether the analysis of the performance of the
traditional LLNA for potency determinations should be limited to data from studies that can be
confirmed as using individual animal data collection procedures.
A majority of the Panel agreed that, ideally, future traditional LLNA potency determinations
should be based on data from studies that use individual data collection procedures, as thiswould allow for the identification of outliers that might skew the average group stimulationindex. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, RaymondPieters, and Michael Woolhiser stated that if laboratories were operating under OECD guidance(OECD 2002) and a reliable validation dataset had been generated, then pooled data from atleast four animals could be considered acceptable.
7.2.2 Test Method Accuracy
The Panel was further asked what impact the inclusion of pooled animal data might have on theaccuracy analysis included in Section 6.0 of the draft ICCVAM LLNA potency BRD. With
regard to this retrospective dataset, the Panel agreed that pooled data should not be excluded
from the current analysis to assess potency determinations for the traditional LLNA. The Panelstated that it is impossible to assess the impact of using pooled data without a separate analysisof the ability of the traditional LLNA to be used for characterizing skin-sensitization potency
using pooled vs. individual data, which the Panel recommended be done (see the discussion of
future studies in Section 7.3).
A minority opinion from Dr. Dagmar Jírová stated that, since OECD TG 429 (OECD 2002)
allows the use of both pooled and individual animal data, the analysis that includes both typesof data is appropriate. Even with the diversity of data sources (the vehicle is not known for 43%
of substances tested in the traditional LLNA; human data were obtained by different, even
undefined methods, etc.), the outcome of the evaluation was good, which documents thestrength and robustness of the traditional LLNA.
The Panel was asked whether the correct classification, as well as the over- and underclassification, rates of the traditional LLNA for sensitization potency determinations had beenadequately compared and appropriately evaluated based on the corresponding human andguinea pig data (refer also to Section 6.0 of the draft ICCVAM LLNA potency BRD). The
Panel agreed that the two approaches used in the draft BRD for analyzing the ability of the
traditional LLNA to discriminate between strong and weak skin sensitizers were appropriate
and correct. In these two approaches, the traditional LLNA was evaluated, after identifying the
optimal EC3 value, for its ability to correctly classify strong and weak sensitizers as defined byhuman or guinea pig threshold values based on: (1) sensitizers only, and (2) sensitizers
combined with false positives, false negatives, and non-sensitizers.
A minority opinion from Dr. Howard Maibach stated that the relevance of the traditional LLNA
to human clinical observations has not been sufficiently determined and should be.
The accuracy analysis (see Section 6.0 of draft ICCVAM BRD) focuses on a proposed twolevel categorization scheme (weak sensitizers vs. strong sensitizers) for both human and guinea
pig data. The Panel was asked whether this was an appropriate categorization scheme, or if
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ICCVAM Test Method Evaluation Report: Appendix F1other categorization schemes should be considered. The Panel agreed that the two-level
categorization scheme was appropriate, especially considering the fact that, for humansituations, risk assessment should be performed, and therefore more categories are not needed.
Even a weak sensitizer under heavy exposure and individual circumstances may reach acomparable risk level as a strong sensitizer under conditions of low exposure.
A minority opinion from Drs. Raymond Pieters and Michael Woolhiser recommended the
addition of at least a moderate category since certain compounds will always be on the border
between weak and strong. Dr. Pieters specifically recommended the categorization scheme ofKimber et al. (2003), which is based on five categories if non-sensitizers are included.
Of the two human threshold concentrations that are proposed in this two-category
categorization scheme (i.e., <250 µg/cm2 or <500 µg/cm2), the Panel was asked which threshold
was the most appropriate for categorizing sensitizing substances as strong vs. weak for humans,
or if another threshold was more appropriate for this purpose. The Panel noted that thisvalidation was based on comparison to guinea pig and HMT/HRIPT information. These data
relate only to induction and do not permit an assessment of risk in humans for elicitation.
For the data provided, the Panel concluded that the best results were obtained using the decision
criterion of 250 µg/cm2 and the corresponding optimal traditional LLNA EC3 value of 9.4%.
Using this cut-off when traditional LLNA false negative and false positive substances areincluded in the analysis, in addition to sensitizers in both the traditional LLNA and in humans
using the HRIPT and/or HMT, correct classification of strong sensitizers was 79% andunderclassification was 21%. Underclassification of substances in this context meansclassification as weak instead of strong sensitizers (i.e., they are not missed as sensitizers
regarding the labeling and safety of consumers). The Panel stated that more data are needed todetermine if another threshold is more appropriate.
When the potency categorization analysis was based on sensitizers only, the guinea pig tests
predicted weak sensitizers with higher accuracy than did the LLNA (89% vs. 75% for the 250µg/cm2 cutoff and 83% vs. 60% for the 500 µg/cm2 cutoff), which is logical because the guinea
pig test methodology involves all phases of the sensitization process and usually involves
adjuvants. However, the guinea pig tests were less accurate for the prediction of strong
sensitizers compared to LLNA (48% vs. 71% for the 250 µg/cm2 cutoff and 42% vs. 63% for
the 500 µg/cm2 cutoff), which represents a higher risk for consumers. For the protection of
public health, it is more important to correctly identify strong sensitizers than weak sensitizers.
The Panel was asked whether the draft BRD adequately characterized the usefulness andlimitations of the LLNA for potency categorizations. If not, the Panel was asked what additions
or changes should be made to the description of usefulness and limitations in the draft BRD.
The Panel stated that additional evaluations should be conducted to determine the impact on
potency categorization if the human threshold data are evaluated differently (e.g., alternative
lowest observed effect level [LOEL] safety factors other than 10, using LOEL data only, using
no observed effect level [NOEL] data only), and if this might improve the correlation between
the LLNA and the human results. According to the Panel, the approach of directly comparing
the LOEL values without using a safety factor compares values of similar significance in
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humans and in the LLNA. In other words, the LOEL in humans describes the thresholdinduction area dose in humans and the EC3 value in the traditional LLNA is the thresholdinduction area dose and thus could be the analogous value to the human LOEL. The Panelfurther stated that traditional LLNA tests based on pooled or individual lymph nodes for a dosegroup should be evaluated independently to assess the impact of using pooled data on theaccuracy analysis for skin-sensitization potency. Finally, the Panel stated that the effect ofdifferent vehicles should be recognized as a limitation in the data analysis given thedemonstrated variability of results.
7.2.3 Test Method Reliability
The Panel was asked whether the reliability (e.g., intralaboratory repeatability, intra- and interlaboratory reproducibility) of the traditional LLNA for potency determinations had beenadequately evaluated. If not, the Panel was asked what other analyses should be performed.
Similar to their recommendations for test method accuracy, the Panel stated that additional
evaluations of reliability should be conducted based on using different approaches for humanthreshold data (e.g., using alternative LOEL safety factors other than 10, using LOEL data only,
using NOEL data only). The Panel further stated that the reliability of LLNA based on using
pooled or individual animal data should be evaluated independently. Finally, the Panel statedthat the effect of different vehicles should be recognized as a limitation in the data analysis, as asource of increased variability.
7.2.4 Data Quality
It was not possible to determine whether or not all studies included in the draft LLNA potency
BRD had been conducted in accordance with GLP guidelines, nor was it possible to obtain the
results of GLP audits for all studies determined to be GLP-compliant. The Panel was asked to
discuss what impact this might have on the evaluation of the LLNA for potency determinations
and whether any of the non-GLP studies should be excluded from the analyses. The Panelconcluded that it was important to note if the data were obtained from studies conductedaccording to international GLP guidelines, as ideally this should be the case. However, thePanel concluded that data from studies that could not be confirmed as being GLP-compliant butthat were from peer-reviewed literature or other sources with high quality laboratory
management practices were still appropriate to include in this retrospective analysis.
As described in the draft BRD, original records for some of the non-GLP studies included inthis evaluation could not be obtained. As a result, an independent audit could not be conductedto confirm that the reported data is the same as the data recorded in laboratory notebooks.
Considering this, the Panel was asked whether the results of these studies (all of which are
currently included) be excluded from any of the performance analyses. The Panel considered
the data to have been generated by repeatedly published and reliable laboratories and therefore
did not question the adequacy/quality of the retrospective data analysis. Thus, although data
should be checked when available, exclusion of data was not deemed necessary, in this case.

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7.2.5 Consideration of All Available Data and Relevant Information
Based on the draft BRD, the Panel was asked whether all the relevant data identified inpublished or unpublished studies conducted using the traditional LLNA had been adequately
considered. If not, the Panel was asked what other studies should be considered. The Panelrecommended that the LOELs from Akkan et al. (2003) be used instead of the DSA05 values
from Schneider and Akkan (2004) in all of the potency analyses. A minority opinion by Dr.
Thomas Gebel stated that it was acceptable to use the DSA05 values from Akkan et al. (2003) as
LOEL values in the evaluation. Dr. Gebel mentioned that the DSA05 value is a LOEL value
adjusted to 5% incidence of induction. Akkan et al. (2003) used the DSA05 value to correct fordifferent human studies leading to different inductions. Dr. Gebel further stated that as the
DSA05 is corrected for an induction rate of 5%, it would be better to compare with thetraditional LLNA EC3 than to use the default uncorrected LOEL.
7.3

Comments on the Draft ICCVAM Test Method Recommendations for the Use of
the LLNA for Potency Determination

7.3.1 Test Method Usefulness and Limitations
With regard to the use of the LLNA for potency categorization (i.e., strong vs. weaksensitizers), the ICCVAM draft recommendation is that the traditional LLNA should not be
considered as a stand-alone test method for predicting sensitization potency, but must instead beused as part of a weight-of-evidence evaluation to discriminate between strong and weaksensitizers. This is based on the fact that, although there is a significant positive correlation
between traditional LLNA EC3 values and human sensitization threshold doses, this correlation
is not strong [see detailed discussion in the draft ICCVAM recommendations]. The Panelagreed that the traditional LLNA should not be considered a stand-alone assay for
categorization of skin-sensitization potency, but it could be used in a weight-of-evidence
evaluation (e.g., along with quantitative structure-activity relationship [QSAR], peptide
reactivity, human evidence) to discriminate between strong and weak sensitizers. The Panelfurther stated that there are additional studies proposed that may provide a better correlation andimprove prediction of potency categorization (see the discussion of future studies below).
A minority opinion from Drs. Thomas Gebel and Dagmar Jírová stated that there is a significant
positive correlation between EC3 values and human threshold values. It is likely that limitations
in estimating human threshold values and the inclusion of human NOEL values in the current
evaluation contributed negatively to the resulting R2 value of 0.405 (when LLNA EC3 data vs.
human threshold data were compared, see Table 6-2 of the draft ICCVAM BRD). Thus, the R2
value may improve when the additional analyses that have been suggested by the Panel are
conducted.
The Panel stated that the effect of different vehicles should be recognized as a limitation in thedata analysis and a likely source of within and between laboratory variability.

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ICCVAM Test Method Evaluation Report: Appendix F1

7.3.2 Test Method Protocol
The Panel was asked whether the ICCVAM-recommended LLNA protocol (ICCVAM 1999;
EPA 2003) should be used when generating data that will or might be considered forsensitization potency categorization decisions. The Panel agreed that this protocol should be
used. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, RaymondPieters, and Michael Woolhiser stated that if laboratories were operating under OECD guidance(OECD 2002) and a reliable validation dataset had been generated, then pooled data from atleast four animals could be considered acceptable.
The Panel was asked whether the relevant testing guidelines for the traditional LLNA should be
updated to include the calculation of an EC3 value. The Panel agreed with thisrecommendation. The calculation of an EC3 value is briefly described in the draft ICCVAM
LLNA Performance Standards for specific situations with references to Basketter et al. (2000)
and Ryan et al. (2007).
7.3.3 Future Studies
The Panel was asked whether the available data support the ICCVAM draft recommendations
for the traditional LLNA in terms of the proposed future studies. The Panel agreed andconcluded that more data are needed to determine the optimal threshold in humans for
distinguishing between strong and weak sensitizers. However, the Panel discouraged
conducting new animal studies unless it was likely that results from such studies would lead toan overall reduction in animal use. The Panel stated further that the traditional LLNA appears to
be a robust rodent assay for the quantification of the induction of cell-mediated immunity.
Thus, use of the traditional LLNA for potency determination can be used in conjunction withQSAR information, guinea pig assays, HRIPT/HMT, and the quantitative data of elicitation andfrequency of positive response in humans in a weight-of-evidence approach. The Panel further
stated that additional evaluations should be conducted to determine the impact on potency
categorization if the human threshold data are evaluated differently (e.g., alternative LOEL
safety factors other than 10, using LOEL data only, using NOEL data only). This might
improve the correlation between LLNA and human data. The Panel further stated that LLNA
tests based on pooled or individual animal data should be evaluated independently to assess theimpact of using pooled data on the accuracy for determining skin-sensitization potency.
The Panel recommended a statistical analysis to determine where an appropriate cutoff value
between weak or strong sensitizers might be best defined for traditional LLNA data. For
example, receiver operating characteristic curves could be used to identify the optimum cut-off
for determining the difference between weak and strong sensitizers.
Finally, the Panel stated that the effect of different vehicles should be recognized as a limitationin the current data analysis, that this was a source of variability within and between laboratories,
and that its impact should be considered in future analyses.

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8.0

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ASTM. 2005. ASTM Standard E691-05 Standard Practice for Conducting an Interlaboratory
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Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP. 2007.
Quantification of chemical peptide reactivity for screening contact allergens: a classification
tree model approach. Toxicol Sci 97:417-27.
ICCVAM. 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemicals/Compounds. The Results of an IndependentPeer Review Evaluation Coordinated by the Interagency Coordinating Committee on the
Validation of Alternative Toxicological Methods (ICCVAM) and the National Toxicology
Program Center for the Evaluation of Alternative Toxicological Methods (NICEATM). NIH
Publication No. 99-4494. Research Triangle Park, NC: National Institute of Environmental
Health Sciences.
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ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised,
and Alternative Test Methods. NIH Publication No: 03-4508. Research Triangle Park, NC:
National Institute of Environmental Health Sciences.
ISO. 1994. ISO 5725. Accuracy (trueness and precision) of measurement methods and results.
Geneva: International Organization for Standardization.
ISO. 2002. ISO 10993-10: Tests for Irritation and Sensitization. Geneva: International
Organization for Standardization.
Jowsey IR, Clapp CJ, Safford R, Gibbons BT, Basketter D. 2008. The impact of vehicle on therelative potency of skin sensitising chemicals in the local lymph node assay. In Press.
Kimber I, Basketter DA, Butler M, Gamer A, Garrigue JL, Newsome C, et al. 2003.
Classification of allergens according to potency: proposals. Food Chem Toxicol 41:1799-1809.
Kimber I, Dearman RJ, Betts CJ, Gerberick GF, Ryan CA, Kern PS, et al. 2006. The local
lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?
Contact Dermatitis 54:181-185.
Manetz TS, Meade BJ. 1999. Development of a flow cytometry assay for the identification anddifferentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cellmediated hypersensitivity responses. Toxicol Sci. 48:206-17.
OECD. 2002. Guideline for the Testing of Chemicals 429 - Skin Sensitisation: Local Lymph
Node Assay. Paris: OECD.
Ryan CA, Chaney JG, Gerberick GF, Kern PS, Dearman RJ, Kimber I, Basketter DA. 2007.
Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency.
Cutan Ocul Toxicol 26:135-145.
Schneider K, Akkan Z. 2004. Quantitative relationship between the local lymph node assay andhuman skin sensitization assays. Regul Toxicol Pharmacol 39:245-55.
Sailstad DM, Hattan D, Hill RN, Stokes WS. 2001. ICCVAM evaluation of the murine local
lymph node assay: I. The ICCVAM review process. Regul Toxicol Pharmacol 34:249-257.
Takeyoshi M. 2007. Promising endpoint and assay performance of non-radioisotopic local
lymph node assay based on BrdU incorporation. Sixth World Congress on Alternatives andAnimal Use in the Life Sciences. August 21-25, 2007. Tokyo, Japan.
Van Hoogstraten IM, Boos C, Boden D, Von Blomberg ME, Scheper RJ, Kraal G. 1993. Oral
induction of tolerance to nickel sensitization in mice. J Invest Dermatol 101:26-31.
Van Och FMM, Slob W, De Jong WH, Vandebriel RJ, Van Loveren H. 2000. A quantitative
method for assessing the sensitizing potency of low molecular weight chemicals using a local
lymph node assay: employment of a regression method that includes determination of theuncertainty margins. Toxicology 146:49-59.

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Varani J, Perone P, Spahlinger DM, Singer LM, Diegel KL, Bobrowski WF, et al. 2008.
Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer
culture for assessment of chemically induced skin damage. Toxicol Pathol 35:693-701.
Young SS. 2007. Gaming the system: chaos from multiple testing. Institute of Mathematical
Statistics Bulletin 36:13

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Annex I
Peer Review Panel Member Biosketches

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Panel Member Biosketches
Nathalie Alépée, Ph.D.
Dr. Alépée performed research leading to a Ph.D. in Medical Virology and Microbiology at
the Centre National de la Recherché Scientifique research institute, Gif sur Yvette, France.
She is currently the Global Pfizer Leader for photosafety, including the global portfolio
support and Associate Research Fellow in Investigative Toxicology, at Pfizer Global
Research and Development, Amboise, France. As a laboratory manager in the Molecular andCellular Toxicology Group with Pfizer, she implemented the local lymph node assay(LLNA) in the laboratory. She serves on the European Centre for the Validation of
Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC), representing theEuropean Federation of Pharmaceutical Industries Associations (EFPIA). She is also the
Pfizer representative to the European Partnership on Alternative to Animal Testing (EPAA),in two working groups; Identification of Opportunities, Including R&D (working group 2),
and Validation and Acceptance (working group 5). She served as a peer reviewer of the
reduced LLNA test protocol and prediction model for ESAC in 2007 and has been designatedas an ESAC peer reviewer for ECVAM's performance standards for the standard LLNA.
Anne Marie Api, Ph.D.
Dr. Api received a Ph.D. from Aston University in Birmingham, England and is currently
Vice President of Human Health Sciences at the Research Institute for Fragrance Materials(RIFM), as well as the Scientific Director. She is responsible for the human health scientificprogram, and the investigation and initiation of new research and testing projects for RIFM.
She is also Adjunct Assistant Professor at the University of Medicine and Dentistry of New
Jersey. She is a member of 10 professional organizations, including the American Contact
Dermatitis Society, the European Society of Contact Dermatitis, and the Society ofInvestigative Dermatology. She participated in the World Health Organization (WHO)International Workshop in Skin Sensitization in Chemical Risk Assessment held in Berlin,
Germany in 2006. She is author of over 100 publications and presentations relevant todermatology and dermatotoxicology.
Nancy Flournoy, M.S., Ph.D.
Dr. Flournoy received a M.S. degree in Biostatistics from the University of California at Los
Angeles, and a Ph.D. in Biomathematics from the University of Washington. She is Professor
and Chair of the Department of Statistics at the University of Missouri-Columbia. Her
research interests include adaptive designs, bioinformatics, chemometrics, clinical trials, andenvironmetrics. She has an extensive list of edited volumes and papers on statistical theory,
statistical genetics and immunology, epidemiology in immune suppressed subjects, clinical
trials for prevention and treatment of viral infection, transplantation biology and its effects ondigestion, lungs, eyes, mouth, and central nervous system, optimization of statisticalprocessing, and additional papers, interviews, and technical reports. She has editorial
responsibilities for numerous statistical journals, serves on numerous advisory boards, andnominating committees. She is a member and past Chair of the Council of Sections of theAmerican Statistical Association, and served in various other statistical, medical andtoxicological societies or programs as Chair or as a member of the Board of Directors. She isa former member of the Scientific Advisory Committee on Alternative Toxicological
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex I

Methods. She also served on the Expert Panels for the National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)
and the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) that evaluated the Revised Up-and-Down Procedure; the Current Validation
Status of In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants; andFive In Vitro Pyrogen Test Methods.
Thomas Gebel, Ph.D.
Dr. Gebel received a Ph.D. in Toxicology from the University of Mainz and is certified as a
toxicologist by the German Society of Toxicology. His scientific interests are in biomonitoring,
genetic toxicology, environmental hygiene, and occupational toxicology. He has publishedover 40 papers in peer-reviewed scientific journals. He is employed by the German Federal
Institute for Occupational Safety and Health, and is an Associate Professor at the University of
Goettingen. Dr. Gebel is currently a member of the Organisation for Economic Co-operation
and Development (OECD) Globally Harmonized System of Classification and Labeling ofChemicals (GHS) expert group on sensitization and head of the German advisory committee onclassification and labeling of existing substances and biocides. Dr. Gebel also is head of theGerman Delegations to the United Nations Economic and Social Council Sub-Committee ofExperts on the GHS, and to the OECD Task Force on Harmonisation of Classification andLabeling. He participated in the WHO International Workshop in Skin Sensitization in
Chemical Risk Assessment held in Berlin, Germany in 2006.
Sidney Green Ph.D., F.A.T.S.
Dr. Green received a Ph.D. in Biochemical Pharmacology from Howard University. His
research interests include toxicology, mutagenic assay systems, and alternatives to animals in
toxicology. He is currently Graduate Professor of Pharmacology at Howard University and afaculty member at the Centers for Alternatives to Animal Testing at the Johns Hopkins
University School of Public Health. Previously, he has been Director of the Department of
Toxicology at Covance Laboratories Inc. and the Director of the Division of ToxicologicalResearch at the U.S. Food and Drug Administration (FDA). Dr. Green is a Fellow of the
Academy of Toxicological Sciences (F.A.T.S.). He has served on numerous expert panels
and committees. He was a participant in an International Workshop organized by ICCVAM
and NICEATM on In Vitro Methods for Assessing Acute Systemic Toxicity in 2000. He
served on the ICCVAM/NICEATM Expert Panels that evaluated the Corrositex® Test
Method for Assessing Dermal Corrosivity Potential of Chemicals, and In Vitro Test Methods
for Identifying Ocular Corrosives and Severe Irritants. He is a former member of the
ICCVAM Advisory Committee on Alternative Toxicological Methods (ACATM) and of
SACATM. He has authored over 60 publications for peer-reviewed journals.
Kim Headrick, B.Admin., B.Sc.
Kim Headrick received Bachelor of Administration and B.Sc. degrees from the University ofOttawa, Canada. She is currently International Harmonization and Senior Policy Advisor for
Health Canada, and Chair of the UN Sub-Committee of Experts on GHS. She manages theoverall strategy for the implementation of the GHS in Canada. She was awarded the Queen
Elizabeth Commemorative Golden Jubilee Medal in 2002, which focuses on theachievements of people who, over the past 50 years, have created the Canada of today. She is
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a member of the OECD Task Force on Harmonization of Classification and Labelling and theOECD Expert Group Meeting on Sensitization Hazards.
Dagmar Jírová, M.D., Ph.D.
Dr. Jírová received a Ph.D. from the Medical Faculty of Hygiene at Charles University in
Prague. She is currently the Head of the Reference Center for Cosmetics, and Head of
National Reference Laboratory for Experimental Immunotoxicology at the National Instituteof Public Health in the Czech Republic. Her main responsibilities include safety assessmentof consumer products, particularly cosmetics and their ingredients, performance of
toxicological methods in vivo in animals, human patch testing for local toxicity assessment,
and introduction of in vitro techniques for screening of toxicological endpoints using cell andtissue cultures. She represents the Czech Republic in the Standing Committee on Cosmeticsof the European Commission. She is an ESAC-ECVAM member and was involved in Peer
Review Panel for Skin Irritation Validation Study and LLNA test protocol and prediction
model. She is author of more than 100 publications and presentations relevant todermatotoxicology including recent presentation at the 6th World Congress on Alternatives& Animal Use in the Life Sciences, held in Tokyo, 2007, titled “Comparison of Human Skin
Irritation and Photoirritation Patch Test Data with Cellular in vitro Assays and Animal invivo data.”
David Lovell, Ph.D., B.Sc. (Hons), F.S.S., FIBiol, CStat, CBiol
Dr. Lovell received a Ph.D. from the Department of Human Genetics and Biometry,
University College, London. He is currently Reader in Medical Statistics at the Postgraduate
Medical School at the University of Surrey. Previously, he was Associate Director and Headof Biostatistics support to Clinical Pharmacogenomics at Pfizer Global Research andDevelopment in Sandwich, Kent providing data management and statistical support topharmacogenetics and genomics. He joined Pfizer in 1999 as the Biometrics Head of ClinicalPharmacogenetics. Before joining Pfizer, Dr. Lovell was the Head of the Science Division at
BIBRA International, Carshalton, which included Molecular Biology, Genetic Toxicology,
Biostatistics and Computer Services. At BIBRA, Dr. Lovell managed the statistical andcomputing group providing specialized statistical support to BIBRA’s Clinical Unit andcontract research work. He conducted and managed research programs on genetics, statistics
and quantitative risk assessment for the European Union (EU) and U.K. Government
Departments. His research interests at BIBRA were in the use of mathematical and statisticalmethods together with genetic models in the understanding of toxicological mechanisms andrisk assessment problems. Dr. Lovell had previously been a Senior Research Officer with the
U.K. Medical Research Council (MRC) Experimental Embryology and Teratology Unit, avisiting Postdoctoral Fellow at the National Institute of Environmental Health Sciences(NIEHS) in North Carolina, U.S., a Geneticist at the MRC Laboratories, Carshalton, and a
Research Assistant in Cytogenetics at Birmingham University. He has acted as a consultant toa number of organizations, has considerable experience of working with Regulatory Authorities,
has many publications related to his work and has wide experience of making presentations to awide range of audiences. He is a member of the U.K. Government’s advisory Committee on
Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) andthe Independent Scientific Advisory Committee for Medicines and Healthcare Products
Regulatory Agency database research. He served on the NICEATM-ICCVAM Expert Panels
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex Ithat evaluated the Frog Embryo Teratogenesis Assay - Xenopus, In Vitro Test Methods for
Identifying Ocular Corrosives and Severe Irritants, and Five In Vitro Pyrogen Test Methods.
Michael Luster, Ph.D.
Dr. Luster received a Ph.D. in Immunology from Loyola University of Chicago. He wasformerly Chief, Toxicology and Molecular Biology Branch, Health Effects Laboratory
Division, National Institute for Occupational Safety and Health (NIOSH), and currently
serves as a senior advisor to the Director of the Health Effects Laboratories and the staff of
Toxicology and Molecular Biology Branch at NIOSH. Program areas include neuroscience,
dermatology, molecular carcinogenesis, molecular epidemiology, molecular toxicology,
molecular epidemiology, and inflammation/immunotoxicology. In addition, Dr. Luster
conducts basic and applied research in immunotoxicology including its application in risk
assessment. Current research activities include molecular epidemiology studies of genetic
polymorphism involved in workplace-related diseases and experimental studies involving
occupational allergic rhinitis. Dr. Luster is also working with various staff at the U.S.
Environmental Protection Agency (EPA) through the Risk Assessment Forum to develop
immunotoxicity testing guidelines. He also directed two studies for the NTP on the
Toxicology and the Carcinogenesis of Promethazine and Ortho-phenylphenol, in 1990 and
1986, respectively. He is a co-author of over 300 publications in peer-reviewed journals.
Howard Maibach, M.D.
Dr. Maibach received an M.D. from Tulane University. He is currently a professor in the
Department of Dermatology at the University of California, San Francisco (USCF), where he isalso Chief of the Occupational Dermatology Clinic. In his 35 years at UCSF, Dr. Maibach has
written and lectured extensively on dermatotoxicology and dermatopharmacology. His current
research programs include defining the chemical-biologic faces of irritant dermatitis and thestudy of percutaneous penetration. Dr. Maibach served on the 1998 ICCVAM Peer Panel that
evaluated the Murine LLNA. Dr. Maibach has been on the editorial boards of over 30 scientificjournals and is a member of 19 professional societies including the American Academy ofDermatology, San Francisco Dermatological Society, and the International Commission on
Occupational Health. He has co-authored over 1500 publications related to dermatology.
James McDougal, Ph.D., F.A.T.S.
Dr. McDougal earned a Ph.D. in Pharmacology/Toxicology at the University of Arizona. He
is currently Professor and Director of Toxicology Research in the Department ofPharmacology and Toxicology at Wright State University’s Boonshoft School of Medicine.
Prior to his appointment at Wright State, he worked in the Air Force toxicology researchorganization for about 17 years. He has active skin research programs related to dermal
pharmacokinetics, molecular biology of skin irritation, dermal risk assessment, andbiologically-based mathematical modeling. He has served on many national committees,
published more than 75 manuscripts, and consults for a wide variety of government andindustry organizations. Dr. McDougal is a member of the National Academy of Sciences(National Research Council) Committee on Toxicology and the American Congress ofGovernmental Industrial Hygienists Threshold Limit Value Committee for Chemical
substances. Dr. McDougal is also past president of the Dermal Toxicology Specialty Sectionof the Society of Toxicology.
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Michael Olson, Ph.D., A.T.S.
Dr. Olson received a Ph.D. in Toxicology from the University of Arkansas for Medical
Sciences, with dissertation research conducted at the FDA National Center for ToxicologicalResearch. Following graduate training, he served as NIEHS National Research Service
Award Post-doctoral Fellow in the Department of Pharmacology, School of Medicine University of North Carolina. Currently he is Director, Occupational Toxicology, Corporate
Environment Health and Safety for GlaxoSmithKline. Dr. Olson is a Fellow of the Academyof Toxicological Sciences (A.T.S.). His research interests include mechanisms of chemicallyinduced toxicity; genetic toxicity; xenobiotic metabolism; alternative methods in toxicology;
hazard evaluation, risk assessment, and communication. Dr. Olson has authored a number ofpeer-reviewed manuscripts and book chapters in these areas as well as preparing manyoccupational health effects reviews for pharmaceutical active ingredients, isolated
intermediates, and associated chemicals. He has served as an editorial board member and ad
hoc referee for numerous toxicology and biosciences journals. In addition, he has worked asa Visiting Scientist, EPA, as well as advisor to EPA Risk Assessment Forum, U.S. National
Institutes of Health (NIH) (Toxicology Study Section I), U.S. Air Force, Transportation
Research Board, and the National Research Council - National Academy of Sciences. A
member of several biomedical professional societies, Dr. Olson has served in elective andappointed positions in the Society of Toxicology, including Chairman of the Society of
Toxicology (SOT) Occupational Health Specialty Section.
Raymond Pieters, Ph.D.
Dr. Pieters received a Ph.D. at Utrecht University and is currently an Associate Professor at
the Institute for Risk Assessment Sciences, and Group Leader for Immunotoxicology at that
institution. In 2007, he presented a paper on Development of Strategies to Assess Drug
Hypersensitivity at the Congress of the European Societies of Toxicology. He was involvedin the development of the Reporter Antigen Popliteal Lymph Node Assay, an assay to assess
the immunomodulating potential of chemicals, which enables differentiation between
immunosensitizing chemicals (sensitizers), immunostimulating chemicals (irritants), andchemicals that have no apparent immunological effects. He has published over 70 papers on
sensitization and other subjects in immunotoxicology in peer-reviewed journals, including areview article, Murine Models of Drug Hypersensitivity, in 2005.
Jean Regal, Ph.D.
Dr. Regal received a Ph.D. in Pharmacology from the University of Minnesota. She iscurrently a Professor in the Department of Pharmacology, Department of Biochemistry &Molecular Biology and Associate Dean of Faculty Affairs, Medical School Duluth,
University of Minnesota. Her current research is focused on respiratory allergy, especiallyasthma. She has served on multiple NIH review panels regarding asthma, as an
immunotoxicologist in 2000 for an Institute of Medicine Committee on Health Effects
Associated with Exposures Experienced during the Persian Gulf War, as well as on the 1998
ICCVAM Peer Panel that evaluated the Murine LLNA. In 2007 she served as an ad hoc
reviewer for the NTP Board of Scientific Counselors for two nominations: Artificial Butter
Flavoring Mixture & O-phthalaldehyde, at NIEHS. Also in 2007, she served on an NIEHS
Center in Environmental Toxicology pilot project program for the University of Texas
Medical Branch at Galveston. She is currently Vice-President-elect of the Immunotoxicology
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex I

Specialty Section of SOT and Associate Editor of the Journal of Immunotoxicology. Dr.
Regal has authored over 50 research articles and reviews in peer-reviewed journals and holds
two patents on pulmonary administration of sCR1 and other complement inhibitory proteins.
Jonathan Richmond, B.Sc. (Hons) Med.Sci., MB ChB, FRCSEd, FRMS
Dr. Richmond received a Bachelor of Science in Medical Science with Honors (B.Sc. [Hons]Med.Sci.) and Bachelor of Medicine and Bachelor of Surgery (MB ChB) degrees with
Distinction in Medicine and Therapeutics from Edinburgh University. Presently, he is headof the Animals Scientific Procedures Division at the Home Office. He is a Fellow of theRoyal College of Surgeons of Edinburgh (FRCSEd) and a Fellow of the Royal Society of
Medicine (FRMS). Other appointments include convener of the U.K. interdepartmentalgroup on the 3Rs, board member U.K. National Centre for the 3Rs, convener of theInternational Standards Organization Technical Corrigendum 194/Working Group 3(Biocompatibility of Medical Device Materials), and member of related expert working
groups. He is a former member of the EU Committee on Scientific and Technical Progress
and past Chairman of the European Commission Technical Expert Working Group on ethicalreview. He served as chair of the peer review panel for the reduced LLNA test protocol andprediction model for ESAC in 2007 and has been designated as an ESAC peer reviewer for
ECVAM's performance standards for the standard LLNA. He served on the
ICCVAM/NICEATM Expert Panel that evaluated Five In Vitro Pyrogen Test Methods. He
has a variety of publications in peer-reviewed journals and national and international
meetings, on the principles and practice of surgery, regulation of biomedical research,principles of humane research, bioethics, and public policy.
Peter Theran, V.M.D.
Dr. Theran holds a Doctor of Veterinary Medicine degree from the University of Pennsylvania.
He has had many years of experience both as a veterinary internal medicine specialist at theMassachusetts Society for Prevention of Cruelty to Animals’ Angell Memorial Animal
Hospital in Boston, and as the director of Boston University Medical Center's Laboratory
Animal Science Center. He presently serves on a number of government committees as an
animal welfare member, and is a member of the Board of Directors of the Institute for In Vitro
Sciences in Gaithersburg, MD and Chimp Haven in Shreveport, Louisiana. He served on the
NICEATM-ICCVAM Expert Panels that evaluated the In Vitro Test Methods for Identifying
Ocular Corrosives and Severe Irritants, and Five In Vitro Pyrogen Test Methods. He is aformer member of ACATM and SACATM. He is presently working as a consultant.
Stephen Ullrich, Ph.D.
Dr. Ullrich received a Ph.D. in Microbiology from Georgetown University. He is currently
the Dallas/Fort Worth Living Legends Professor, and Professor of Immunology at the
University of Texas, M.D. Anderson Cancer Center, where he is also Associate Director, The
Center for Cancer Immunology Research. He is also a member of the Animal Research
Strategic Advisory Committee. He has served numerous national review committees andpanels, including the 1998 ICCVAM Peer Panel that evaluated the Murine LLNA. Dr.
Ullrich has authored over 75 peer-reviewed publications, over 30 invited articles, and heholds four patents in the U.S., E.U., and Australia for a UV-induced Immunosuppressive
Substance. He is the past President of the American Society for Photobiology.
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Michael Woolhiser, Ph.D.
Dr. Woolhiser received a Ph.D. in Pharmacology and Toxicology from the Medical College
of Virginia at Virginia Commonwealth University. He is a specialist in immunotoxicology
and is currently a toxicologist for the Dow Chemical Company where he serves as aTechnical Leader for Immunotoxicology, and Polyurethane Business Toxicology Consultant.
Dr. Woolhiser is also an Adjunct Professor at the Center for Integrative Toxicology,
Michigan State University. He is a member of the Program Committee of the Society of
Toxicology's Immunotoxicology Specialty Section. He has served on numerous working
groups, including an LLNA Expert Working Group under the European Crop Protection
Agency's Toxicology Expert Group, a European Centre for Ecotoxicology and Toxicology ofChemicals LLNA Task Force. He has authored 29 peer-reviewed publications.
Takahiko Yoshida, M.D., Ph.D.
Dr. Yoshida earned his M.D. and a Ph.D. in Medical Science from Tokai University. He iscurrently Professor in the Department of Health Science at Asahikawa Medical College.
Prior to this appointment, he held the posts of Instructor, Assistant Professor and Associate
Professor at the Tokai University School of Medicine. He has also been a Guest Researcher
at NIEHS. He has also worked as an occupational physician for major Japanese corporations,
including Toyota and Sony. Dr. Yoshida’s research interests include occupational health,public health, environmental health and preventative medicine. He is a member of theInternational Congress of Occupational Health, the Japanese Society of Hygiene, theJapanese Society of Immunotoxicology, the Japanese Society of Clinical Ecology, and the
SOT.

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Annex II
F1
F2
F3
F4
B5
F6
F7

Questions for the Peer Review Panel
LLNA Limit Dose Procedure .......................................................................... F-113
LLNA for Testing Aqueous Solutions, Metals, and Mixtures........................ F-119
Non-Radioactive LLNA Protocol - LLNA: DA Test Method ........................ F-125
Non-Radioactive LLNA Protocol - LLNA: BrdU-FC Test Method .............. F-133
Non-Radioactive LLNA Protocol - LLNA: BrdU-ELISA Test Method ........ F-141
Draft ICCVAM Performance Standards for the LLNA ................................ F-149
Use of the LLNA for Potency Determinations................................................ F-155

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Annex F1
Questions for the Peer Review Panel: LLNA Limit Dose Procedure

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Instructions for the Peer Review Panel: LLNA Limit Dose Procedure
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the murine local lymph node assay (LLNA) limit dose procedure as a substitute forthe traditional LLNA for assessing the allergic contact dermatitis potential of chemicals andother substances.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA limit dose procedure. You are first asked to review the information in the draft
ICCVAM LLNA limit dose procedure background review document (BRD) forcompleteness, and to identify any errors or omissions of existing relevant data or informationthat should be included. You are then asked to evaluate the information in this BRD todetermine the extent to which each of the applicable criteria for validation and acceptance of
toxicological test methods (ICCVAM 20039) have been appropriately addressed for the
proposed use of the LLNA limit dose procedure. Adequate validation10 is a prerequisite for atest method to be considered for use in regulatory decision-making by U.S. Federal agencies.
This validation process characterizes the usefulness and limitations of a test method for its
intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA limit dose procedure (i.e., the proposed test method use, the proposed recommended
standardized protocol, the proposed test method performance standards, and any proposed
additional studies) and comment on whether the recommendations are supported by the
information provided in the draft LLNA limit dose procedure BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA limit dose procedure.
These include questions prepared by the ICCVAM IWG to ensure that the assessment
provides adequate information to facilitate U.S. Federal agency decisions on the regulatory
acceptability of this test method, and/or adequate guidance for organizations that may be
involved in conducting or supporting further development, standardization, and/or validation.
The overall question to consider is whether the validation status of the LLNA limit dose
procedure has been adequately characterized for its intended purpose, and is it sufficiently
accurate and reliable to be used for the identification of sensitizing substances and nonsensitizing substances in place of the traditional LLNA procedure when there is not a needfor dose response information, in order to reduce the number of animals required for suchtesting.

9

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
10
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

I.

Questions to the Panel: Review for Errors and Omissions
1. Are there any errors or omissions of existing relevant data or information thatshould be included in the draft BRD?

II.

Questions to the Panel: LLNA Limit Dose Procedure Draft BRD
1. For the proposed LLNA limit dose procedure, ICCVAM recommends that the
number of animals used in each group should be the same as that recommended
by ICCVAM for the traditional LLNA based on its 1998 evaluation of the LLNA,
and that individual animal data should be collected and reported (ICCVAM,
1999). Do you agree that these are appropriate provisions for the limit dose
procedure? Please explain your answer.
2. Do you consider the traditional LLNA database representative of a sufficient
range of chemical classes and physical chemical properties that it would beapplicable to any of the types of chemicals and products that are typically tested
for skin-sensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using thelimit dose procedure? What chemicals or products should be evaluated to fill thisdata gap? Please explain your answer.
3. While coding of chemicals is recommended for prospective studies, thisevaluation is based on a retrospective evaluation of existing data, most of which
was not generated using coded chemicals to reduce the potential for bias. Does thelack of coding of test substances adversely impact or bias the current evaluation?
Please explain your answer.
4. For some substances submitted using the traditional LLNA test method, it was notpossible to confirm whether the data were generated using pooled animal data foreach dose group (as allowed in Test Guideline [TG] 429 of the Organisation for
Economic Co-operation and Development [OECD]). ICCVAM (1999), Dean etal. (2001), and EPA (2003) recommend the use of statistical analyses to help
interpret LLNA study results, which necessitates data collected at the level of theindividual animal, while Cockshott et al. (2006) reported that using individual
animal data allowed for technical problems during an experiment to be identified.
Considering this, should the analysis of the performance of the LLNA limit dose
procedure against the traditional LLNA be limited to data from studies that can be
confirmed as using individual animal data collection procedures? What impact
might the inclusion of pooled animal data have on the accuracy analysis of the
LLNA limit procedure? Please explain your answer.
5. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA limit dose procedure beenadequately evaluated and compared to the traditional LLNA (refer also to Table 61 of the draft ICCVAM BRD)? If not, what other analyses should be performed?
6. There were five substances for which the highest concentration tested produced
an SI of less than 3.0, while lower concentrations of these substances produced an
SI of greater than 3.0 (see Table 6-2 of the draft ICCVAM BRD). These
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substances are classified as “false negatives” compared to what was obtained in
the traditional LLNA. Can you identify any characteristics associated with theseor other substances that might signal that this type of abnormal dose response
might occur, and therefore using the LLNA limit dose procedure would not beappropriate? Please explain your answer.
7. Does the BRD adequately characterize the usefulness and limitations of the
LLNA limit dose procedure based on the accuracy analyses? If not, what
additions or changes should be made to the current usefulness and limitations?
Please explain your answer.
8. Is it appropriate to assume that the intra- and inter-laboratory reproducibility of
the LLNA limit dose procedure and the traditional LLNA will be similar, basedon the fact that they use identical protocols with the exception of the number ofdoses used? Do you agree? Does reducing the number of test substances dosegroups from three to one reduce the reliability of the assay? Please explain youranswer.
9. For some studies included in the draft BRD, it was not possible to determine
whether or not they had been conducted in accordance with Good Laboratory
Practice (GLP) guidelines. Original records for some of the non-GLP studies
included in this evaluation could not be obtained. As a result, an independent
audit could not be conducted to confirm that the reported data is the same as thedata recorded in laboratory notebooks. Neither was it possible to obtain the resultsof GLP audits for all studies conducted in accordance with GLP guidelines.
Considering this, should the results of these studies (all of which are currently
included) be excluded from any of the performance analyses? Please explain youranswer.
10. Based on the draft BRD, have all the relevant data identified in published or
unpublished studies conducted using the traditional LLNA been adequately
considered? If not, what other traditional LLNA data needs to be considered andhow can it be obtained?

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III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA Limit Dose Procedure
1. Do the available data support the ICCVAM draft recommendations for the LLNA
limit dose procedure in terms of the proposed test method usefulness and limitations?
If not, what recommendations would you make? Please explain your answer.
• Should the LLNA limit dose procedure be routinely recommended for thehazard identification of skin sensitizing chemicals when potency information is
not required? Please explain your answer.
• If potency information is required, should the LLNA limit dose procedure be
routinely recommended as the initial test to identify sensitizers before
conducting the traditional LLNA as a way to further reduce animal use, sincenegative results would not require further testing? Please explain your answer.
• Based on the existing database, there is a false negative rate of 1.6% (5/313
positive compounds) for the LLNA limit dose approach compared to the results
obtained in the traditional LLNA. Do you consider that this is adequately
addressed by the proposed cautionary language and weight of evidence
consideration for negative substances? Please explain your answer.
2. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA limit dose procedure in terms of the proposed test method
standardized protocol? If not, then what recommendations would you make?
Please explain your answer.
• The recommended ICCVAM protocol (ICCVAM 1999; Dean et al. 2001; EPA
2003), as well as OECD TG 429, specifies that the highest dose tested should be
the highest soluble concentration that does not induce systemic toxicity and/or
excessive skin irritation. However, Kimber et al. (2006) concluded that negative
results obtained from studies where the highest concentration tested was below
10% should be considered invalid, and adopted a 10% application concentration
as a threshold of confidence for categorization of a chemical as being negative
while noting that the figure should not be considered as inviolable. Are the data
presented in the draft BRD (i.e., 5/313 positive substances in the NICEATM
database were negative at concentrations ≤10%, but were positive at higher
concentrations) adequate to conclude that this threshold concentration is not
appropriate? If a negative result was obtained for a test substance in a study
where the highest concentration that could be tested (based on systemic toxicity
or excessive local irritation, as described in ICCVAM [1999], Dean et al.
[2001], and EPA [2003]) was <10%, should additional testing be required? Do
you agree that the current approach for selecting the “limit” dose is appropriate
or do you conclude that there is a threshold concentration for the LLNA atwhich a negative result could always be considered as an acceptable result? If
so, what is that concentration? Please explain your answer.
3. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA limit dose procedure in terms of the proposed future studies? If not,then what recommendations would you make? Please explain your answer.
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Annex F2
Questions for the Peer Review Panel:
LLNA for Testing Aqueous Solutions, Metals, and Mixtures

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Instructions for the Peer Review Panel: LLNA for Testing Aqueous Solutions, Metals,
and Mixtures
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is currently updating theoriginal validation report of the LLNA (ICCVAM 1999) based on a comprehensive review ofavailable data and information regarding the current validity of the LLNA for assessing theskin sensitizing potential of mixtures, metal compounds, and substances tested in aqueous
solutions. The information is based on a retrospective review of LLNA data derived from a
database of over 500 substances (including mixtures) tested in the LLNA and builds on theprevious ICCVAM evaluation of the LLNA, which was based on 209 substances (ICCVAM
1999). In the original ICCVAM report, the performance of the LLNA was compared to 1) the
results from guinea pig tests and 2) information about sensitizers in humans (e.g., humanmaximization test [HMT] results, substances used in human repeat insult patch test [HRIPT],
clinical data), where available. This addendum updates the LLNA performance analyses for
mixtures, metal compounds, and substances tested in aqueous solutions when compared tohuman and guinea pig results.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA. You are first asked to review the information in the draft Addendum to the
ICCVAM (1999) report for completeness, and to identify any errors or omissions of existing
relevant data or information that should be included. You are then asked to evaluate theinformation in this Addendum to determine the extent to which each of the applicable criteria
for validation and acceptance of toxicological test methods (ICCVAM 200311) have been
appropriately addressed for the proposed use of the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions. Adequate validation12 is a prerequisite for atest method to be considered for use in regulatory decision-making by U.S. Federal agencies.
This validation process characterizes the usefulness and limitations of a test method for its
intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA (i.e., the proposed test method use, the proposed recommended standardized protocol,
the proposed test method performance standards, and any proposed additional studies) andcomment on whether the recommendations are supported by the information provided in thedraft Addendum.
The questions relating to the draft Addendum that must be addressed are provided in
Sections I and II of this guidance, while Section III contains questions relating to the draft
ICCVAM test method recommendations on the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions.

11

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
12
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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These include questions prepared by the ICCVAM IWG to ensure that the assessment
provides adequate information to facilitate U.S. Federal agency decisions on the regulatory
acceptability of this test method, and/or adequate guidance for organizations that may be
involved in conducting or supporting further development, standardization, and/or validation.
The overall question to consider is whether the validation status of the LLNA for testing
mixtures, metal compounds, and substances in aqueous solutions has been adequately
characterized, and is it sufficiently accurate and reliable to be used for the identification ofsensitizing substances based on a comparison to either human or guinea pig responses.
I.

Questions to the Panel: Review for Errors and Omissions
1. In the draft Addendum, are there any errors that need to be corrected or omissionsof existing relevant data or information that should be included?

II.

Questions to the Panel: Updated LLNA Applicability Domain Addendum
1. Do you consider the database of substances evaluated representative of a
sufficient range of mixtures, metal compounds, and substances in aqueous
solutions that are typically tested for skin-sensitization potential? Please explain
your answer.
2. For the purpose of this evaluation, aqueous solutions were defined by the
proportion of water (at least 20%) (i.e., substances or mixtures that were tested in
an aqueous or an organic:aqueous vehicle were labeled as aqueous solutions). Do
you consider this to be an appropriate criterion for defining aqueous solutions? If
not, what would be more appropriate? Please explain your answer.
3. While coding of chemicals is recommended for prospective studies, thisevaluation is based on a retrospective evaluation of existing data, most of which
was not generated using coded chemicals to reduce bias. Does the lack of codingof test substances adversely impact or bias the current evaluation? Please provide
a rationale for your answer.
4. For some substances submitted using the LLNA, it was not possible to confirm
whether the data were generated using pooled animal data for each dose group (asallowed in Test Guideline [TG] 429 of the Organisation for Economic Cooperation and Development [OECD]) rather than individual animal data (asrecommended in the ICCVAM 2001 protocol)? Cockshott et al. (2006) reported
that using individual animal data allowed for technical problems during anexperiment to be identified. Considering this, should the analysis of theperformance of the LLNA for testing mixtures, metal compounds, and substancesin aqueous solutions be limited to data from studies that can be confirmed asusing individual animal data collection procedures? What impact might theinclusion of pooled animal data have on the accuracy analysis included in
Section 5.0 of the draft Addendum? Please explain your answer.
5. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions been adequately evaluated andcompared to the human and guinea pig (refer also to Section 5.0 of the draft
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Addendum)? If not, what other analyses should be performed? Please explain
your answer.
6. When multiple LLNA studies were available for the same substance, the majority
call (where all studies used the same vehicle and the same concentration range)
was used to assign an overall classification for the purposes of the accuracy
analysis. For example, if chemical X was tested 5 times and was positive in 3
studies and negative in two, the overall classification was positive. Do you agreewith the approach to assigning overall classifications? If not, how would you
propose that this be accomplished? Please explain your answer.
7. Does the Addendum adequately characterize the usefulness and limitations of the
LLNA for testing mixtures, metal compounds, and substances in aqueous
solutions based on the accuracy analyses? If not, what additions or changes
should be made to the current usefulness and limitations? Please explain youranswer.
8. For some studies included in the draft Addendum, it was not possible to determine
whether or not they had been conducted in accordance with Good Laboratory
Practice (GLP) guidelines. Neither was it possible to obtain the results of GLP
audits for all studies conducted in accordance with GLP guidelines. Please discuss
what impact this lack might have on the evaluation of the LLNA for testing
mixtures, metal compounds, and substances in aqueous solutions and whether
such studies should be excluded from any analysis.
9. As described in the draft Addendum, original records for some of the non-GLP
studies included in this evaluation could not be obtained. As a result, an
independent audit could not be conducted to confirm that the reported data is thesame as the data recorded in laboratory notebooks. Considering this, should theresults of these studies (all of which are currently included) be excluded from anyof the performance analyses? Please explain your answer.
10. Based on the draft Addendum, have all the relevant data identified in published or
unpublished studies conducted using the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions been adequately considered? If
not, what other studies should to be considered?
III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA for Testing Aqueous Solutions, Metals, and Mixtures
1. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA with regard to testing mixtures, metal compounds, and substances inaqueous solutions in terms of the proposed test method usefulness and limitations?
Please explain your answer. If not, what recommendations would you make?
2. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed test method standardized protocol? Please
explain your answer. If not, then what recommendations would the Panel make?

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3. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed future studies? Please explain youranswer. If not, then what recommendations would you make?

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Annex F3
Questions for the Peer Review Panel:
Non-Radioactive LLNA Protocol - LLNA: DA Test Method

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Instructions for the Peer Review Panel: Non-Radioactive LLNA Protocol: LLNA: DA
Test Method
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program (NTP) Interagency Center for
the Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating thevalidation status of the LLNA: DA (Local Lymph Node Assay-Daicel adenosine triphosphate[ATP]) for assessing the allergic contact dermatitis (ACD) potential of chemicals and othersubstances. This test method, developed by Daicel Chemical Industries, Ltd. (Tokyo, Japan),is a non-radiolabeled version of the traditional LLNA, and is based on measuring levels of
ATP in the auricular lymph nodes as an indicator of increased cell proliferation.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA: DA. You are first asked to review the information in the draft ICCVAM
LLNA: DA background review document (BRD) for completeness, and to identify any errors
or omissions of existing relevant data or information that should be included. You are thenasked to evaluate the information in the BRD to determine the extent to which each of theapplicable criteria for validation and acceptance of toxicological test methods (ICCVAM
200313) have been appropriately addressed for the proposed use of the LLNA: DA. Adequate
validation14 is a prerequisite for a test method to be considered for use in regulatory decisionmaking by U.S. Federal agencies. The validation process characterizes the usefulness andlimitations of a test method for a specific intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA: DA (i.e., the proposed test method use, the proposed recommended standardized
protocol, the proposed test method performance standards, and any proposed additional
studies) and comment on whether the recommendations are supported by the information
provided in the draft LLNA: DA BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA: DA.
These include questions prepared by the ICCVAM Immunotoxicity Working Group (IWG)
to ensure that the assessment provides adequate information to facilitate U.S. Federal agency
decisions on the regulatory acceptability of this test method. The questions are also intended
to obtain guidance that will be helpful to federal agencies and other organizations that may
be involved in conducting or supporting further development, standardization, and/or
validation studies.
The overall question to consider is whether the validation status of the LLNA: DA has beenadequately characterized for its intended purpose, and is it sufficiently accurate and reliable
to be used for the identification of sensitizing substances and non-sensitizing substances inplace of the traditional LLNA procedure.
13

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
14
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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I.

Questions to the Panel: Review of the Draft LLNA: DA BRD for Errors andOmissions
1. In the draft LLNA: DA BRD, are there any errors in the BRD that should becorrected, or omissions of existing relevant data or information that should be
included?

II.

Questions to the Panel: Draft LLNA: DA BRD
1. Test Method Protocol
i. The traditional LLNA protocol (ICCVAM 1999; Dean et al. 2001)
recommends a minimum of five successfully treated animals per dose group.
Current validation of the LLNA: DA was performed using four animals perdose group. What impact might using fewer mice have on the accuracy
analysis of the LLNA: DA? Please explain your answer.
ii. The data generated for the substances analyzed in the LLNA: DA test methodcome from auricular lymph nodes that were pooled across mice in each dosegroup rather than being analyzed on an individual animal data. What impact
might the inclusion of pooled animal data have on the accuracy analysis of the
LLNA: DA? Please explain your answer.
iii. The LLNA: DA differs from the traditional LLNA in the treatment scheduleand by including a pre-treatment with 1% SLS prior to application of the test
substance. Do you consider these changes to be appropriate? Please explain
your answer.
2. Substances Used for the Validation Studies
i. Do you consider the LLNA: DA database representative of a sufficient rangeof chemical classes and physicochemical properties that it would be applicable
to any of the types of chemicals and products that are typically tested for skinsensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using
the LLNA: DA? What chemicals or products should be evaluated to fill thisdata gap? Please explain your answer.
3. Test Method Accuracy
i. The current accuracy analysis is based on overall concordance with thetraditional LLNA. Accuracy statistics compared to the guinea pig tests andhuman data/experience have also been provided. Are these comparisonsappropriate for assessing the accuracy of the LLNA: DA? Please explain youranswer.
ii. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: DA been adequately evaluatedand compared to the traditional LLNA (refer also to Table 6-1 of the draft
ICCVAM BRD)? If not, what other analyses should be performed? Please
explain your answer.
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex IIiii. There was one substance (2-mercaptobenzothiazole) that produced a “false
negative” response compared to the traditional LLNA when tested using the
LLNA: DA. The mean EC3 in the traditional LLNA for this substance is 2.5(n=2), it is positive in both the guinea pig and human, and has “high” peptide
reactivity as per Gerberick et al. (2007). Can you identify any characteristics
associated with this or similar substances, compared to the correctly identified
sensitizers, that might signal that this type of discordant response might occur,and therefore using the LLNA: DA to test such substances would not beappropriate or that negative results for such substances should indicate a needfor confirmatory testing? Please explain your answer.
iv. There was one substance (benzalkonium chloride) that produced a “false
positive” response compared to the traditional LLNA and guinea pig testwhen tested using the LLNA: DA. Can you identify any characteristics
associated with this or similar substances, compared to the correctly identified
non-sensitizers that might signal that this type of discordant response mightoccur, and therefore using the LLNA: DA to test such substances would not
be appropriate, or that positive results for substances with such properties may
warrant additional testing? Please explain your answer.
4. Test Method Reliability (Intra- and Inter-laboratory reproducibility)
i. Has the intralaboratory reproducibility of the LLNA: DA been adequately
evaluated and compared to the traditional LLNA (refer also to Table 7-1 ofthe draft LLNA: DA BRD)? If not, what other analyses should be performed?
Are any limitations apparent based on this intralaboratory reproducibility
assessment? Please explain your answer.
ii. Has the interlaboratory reproducibility of the LLNA: DA been adequately
evaluated and compared to the traditional LLNA (refer also to Tables 7-2 and7-3 of the draft LLNA: DA BRD)? If not, what other analyses should be
performed? Are any limitations apparent based on this interlaboratory
reproducibility assessment? Please explain your answer.
iii. The draft LLNA: DA BRD analyzes data from two interlaboratory validation
studies that used coded substances, as well as an intralaboratory validation
study with 31 substances that were not coded. Does the lack of coding of test
substances adversely impact or bias the current evaluation? In addition, it
appears that the lead laboratory established the dose levels tested in the twointerlaboratory validation studies and the participating laboratories did not
determine their own dose levels for testing. Does this adversely impact or bias
the current evaluation? Please explain your answer.
5. Data Quality
i. The studies evaluated in the draft BRD for the LLNA: DA were not conductedin accordance with Good Laboratory Practices (GLP) guidelines although
there were reportedly done in laboratories that conduct GLP studies and wereconducted “in the spirit” of GLP (K. Idehara, personal communication).

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Please discuss what impact this might have on the evaluation of the LLNA:
DA.
ii. The original records for these studies were requested but have not yet beenobtained. As a result, an independent audit could not be conducted to confirm
that the reported data is the same as the data recorded in laboratory notebooks.
Should any recommendations from ICCVAM be contingent upon thecompletion of such an audit and findings that there were no significant errors
in data transcription? Please explain your answer.
6. Consideration of all available data and relevant information
i. Based on the draft LLNA: DA BRD, have all the relevant data identified inpublished or unpublished studies that employ this test method been adequately
considered? Are there other comparative test method data that were notconsidered in the draft BRD, but are available for consideration? If yes, please
explain how to obtain such data.
IV.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA: DA
1. Test Method Usefulness and Limitations
i. Do you agree that the available data and test method performance (accuracy
and reliability) support the ICCVAM draft recommendations for the LLNA:
DA in terms of the proposed test method usefulness and limitations? Please
explain your answer.
ii. If restrictions on using radioactive materials are present, should the LLNA:
DA be routinely recommended for hazard identification of skin sensitizing
substances in lieu of having to possibly use guinea pig tests? Please explain
your answer.
iii. Even if limitations in using radioactive materials are not present, should the
LLNA: DA procedure or other valid and accepted non-radioactive method beroutinely recommended for hazard identification of skin sensitizing substances
instead of the traditional LLNA? Please explain your answer.
iv. From a public health perspective, is the recommended guidance for evaluating
negatives sufficient to address concerns associated with the false negative rateof 5% (1/19 substances) calculated for the LLNA: DA? Do you havesuggestions for additional guidance or limitations? Please explain youranswer.
v. From a testing strategy perspective, does the ICCVAM guidance addressconcerns associated with the false positive rate of 10% (1/10 substances)calculated for the LLNA: DA? Are there other suggestions for additional suchguidance or limitations? Please explain your answer.
2. Test method protocol
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: DA procedure in terms of the proposed test
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method standardized protocols? If not, what recommendations would you
make? Please explain your answer.
ii. Can the traditional LLNA limit dose procedure be applied to the LLNA: DA?
Please explain your answer.
3. Future Studies
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: DA in terms of the proposed future studies?
If not, then what recommendations would you make? Please explain youranswer.
4. Performance Standards
i. The LLNA: DA protocol differs from the ICCVAM-recommended protocol
for the traditional LLNA (ICCVAM 1999; Dean et al. 2001; EPA 2003) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes.
In addition, there are differences between the two protocols that relate to how
and when the test substance is applied and when the lymph nodes arecollected (Table 2-1 and Appendix A in the draft LLNA: DA BRD).
According to the proposed draft ICCVAM Performance Standards for the
traditional LLNA
(http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm), anychange to the LLNA protocol other than the method used to assess
lymphocyte proliferation is considered a major change. Do you agree thatthese should be considered major changes and therefore the usefulness andlimitations of the LLNA: DA should not be assessed using the draft ICCVAM
Performance Standards? Please explain your answer.
ii. Even if the draft ICCVAM Performance Standards do not apply to the LLNA:
DA, what impact should the accuracy analysis based on 13 of the 18 required
performance standards substances (only one false negative and no false
positives) have on the overall evaluation of test method accuracy? Please
explain your answer.
iii. Should separate performance standards be developed for the LLNA: DA?
Please explain your answer.

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Annex F4
Questions for the Peer Review Panel:
Non-Radioactive LLNA Protocol - LLNA: BrdU-FC Test Method

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Instructions for the Peer Review Panel: Non-Radioactive LLNA Protocol: LLNA: BrdUFC Test Method
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the LLNA: BrdU-FC (Local Lymph Node Assay: Bromodeoxyuridine detected byflow cytometry) for assessing the allergic contact dermatitis potential of chemicals and othersubstances. This test method, developed by MB Research Labs (Spinnerstown, PA), is a nonradiolabeled version of the traditional LLNA, and is based on measuring the incorporation of
bromodeoxyuridine (BrdU) into the DNA of dividing lymphocytes using flow cytometry as
in indicator of cell proliferation.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA: BrdU-FC. You are first asked to review the information in the draft ICCVAM
LLNA: BrdU-FC Background Review Document (BRD) for completeness, and to identify
any errors or omissions of existing relevant data or information that should be included. You
are then asked to evaluate the information in the BRD to determine the extent to which eachof the applicable criteria for validation and acceptance of toxicological test methods(ICCVAM 200315) have been appropriately addressed for the proposed use of the LLNA:
BrdU-FC. Adequate validation16 is a prerequisite for a test method to be considered for use inregulatory decision-making by U.S. Federal agencies. The validation process characterizes
the usefulness and limitations of a test method for a specific intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA: BrdU-FC (i.e., the proposed test method use, the proposed recommended
standardized protocol, the proposed test method performance standards, and any proposed
additional studies) and comment on whether the recommendations are supported by the
information provided in the draft LLNA: BrdU-FC BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA: BrdU-FC.
These include questions prepared by the ICCVAM Immunotoxicity Working Group (IWG)
to ensure that the assessment provides adequate information to facilitate U.S. Federal agency
decisions on the regulatory acceptability of this test method. The questions are also intended
to obtain guidance that will be helpful to federal agencies and other organizations that may
be involved in conducting or supporting further development, standardization, and/or
validation studies.
The overall question to consider is whether the validation status of the LLNA: BrdU-FC has
been adequately characterized for its intended purpose, and is it sufficiently accurate and
15

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
16
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

reliable to be used for the identification of sensitizing substances and non-sensitizing
substances in place of the traditional LLNA procedure.
I.

Questions to the Panel: Review of the Draft LLNA: BrdU-FC BRD for Errors
and Omissions
1. In the draft LLNA: BrdU-FC BRD, are there any errors in the BRD that should becorrected, or omissions of existing relevant data or information that should be
included?

II.

Questions to the Panel: Draft LLNA: BrdU-FC BRD
1. Test Method Protocol
i. The LLNA: BrdU-FC includes routine measurements of ear swelling as anindicator of excessive dermal irritation. Do you consider this procedure to bean appropriate approach? Do you think that this measurement should berecommended for routine inclusion into all LLNA protocols? Please explain
your answers.
ii. The LLNA: BrdU-FC also includes optional quantification of
immunophenotypic markers as an additional mechanism for distinguishingirritants from sensitizers. Do you consider this to be an appropriate approachto reduce false positives? Are the correct markers being considered or do you
recommend other/additional markers? Should these measurements berecommended for routine inclusion in the LLNA: BrdU-FC? Please explain
your answers.
iii. Please comment on the appropriateness of the "sequential strategy" used in theeLLNA: BrdU-FC (see Figure 2-1 of the draft BRD).
2. Substances Used for the Validation Studies
i. Do you consider the LLNA: BrdU-FC database representative of a sufficient
range of chemical classes and physicochemical properties that it would beapplicable to any of the types of chemicals and products that are typicallytested for skin-sensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using
the LLNA: BrdU-FC? What chemicals or products should be evaluated to fillthis data gap? Please explain your answers.
3. Test Method Accuracyi.

The current accuracy analysis is based on overall concordance with thetraditional LLNA. Accuracy statistics compared to the Guinea Pig tests andhuman data/experience have also been provided. Are these comparisonsappropriate for assessing the accuracy of the LLNA: BrdU-FC? Please explain
your answer.

ii.

Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: BrdU-FC been adequately
evaluated and compared to the traditional LLNA (refer also to Table 6-1 of
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

the draft ICCVAM BRD)? If not, what other analyses should be performed?
Please explain your answer.
iii.

Three substances (benzalkonium chloride, resorcinol, and Tween 80)
produced a “false positive” response compared to the traditional LLNA andguinea pig test when tested using the LLNA: BRDU-FC (Based on
immunophenotyping, benzalkonium chloride was subsequently classified as
an irritant rather than a sensitizer). Can you identify any characteristics
associated with these or similar substances, compared to the correctly
identified non-sensitizers that might signal that this type of discordant
response might occur, and therefore using the LLNA: BrdU-FC to test such
substances would not be appropriate, or that positive results for substanceswith such properties may warrant additional testing? Please explain youranswer.

4. Test Method Reliability (Intra- and Inter-laboratory reproducibility)i.

Has the intralaboratory reproducibility of the LLNA: BrdU-FC beenadequately evaluated and compared to the traditional LLNA (refer also toTable 7-1 of the draft LLNA: BrdU-FC BRD)? If not, what other analyses
should be performed? Are any limitations apparent based on this intralaboratory reproducibility assessment? Please explain your answer.

ii.

The draft LLNA: BrdU-FC BRD analyzes data from repeat testing of hexyl
cinnamic aldehyde (HCA) in six different vehicles and intralaboratory
reproducibility is assessed by coefficient of variation (CV). The calculated
CVs ranged from 30% to 53%. Based on these data, are there concerns with
the intralaboratory reproducibility of the LLNA: BrdU-FC? Please explain
your answer.

5. Data Qualityi.

The studies evaluated in the draft BRD for the LLNA: BrdU-FC were not all
conducted in accordance with Good Laboratory Practices (GLP) guidelines
although there were done in a laboratory that routinely conducts GLP studies(G. DeGeorge, personal communication). Please discuss what impact thismight have on the evaluation of the LLNA: BrdU-FC.

ii.

The original records for these studies were requested but have not yet beenobtained. As a result, an independent audit could not be conducted to confirm
that the reported data is the same as the data recorded in laboratory notebooks.
Do you agree that any recommendations from ICCVAM should be contingent
upon the completion of such an audit and findings that there were no
significant errors in data transcription? Please explain your answer.
Consideration of all available data and relevant informationiii.

Based on the draft LLNA: BrdU-FC BRD, have all the relevant data identified
in published or unpublished studies that employ this test method beenadequately considered? Are there other comparative test method data thatwere not considered in the draft BRD, but are available for consideration? If
yes, please explain how to obtain such data.
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III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-FC
1. Test Method Usefulness and Limitations
i. Do you agree that the available data and test method performance (accuracy
and reliability) support the ICCVAM draft recommendations for the LLNA:
BrdU-FC in terms of the proposed test method usefulness and limitations?
Please explain your answer.
ii. If restrictions on using radioactive materials are present, should the LLNA:
BrdU-FC be routinely recommended for hazard identification of skinsensitizing substances in lieu of having to possibly use guinea pig tests?
Please explain your answer.
iii. Even if limitations in using radioactive materials are not present, should the
LLNA: BrdU-FC procedure be routinely recommended for hazard
identification of skin sensitizing substances instead of the traditional LLNA?
If not, then why? Please explain your answer.
iv. Do the ICCVAM recommendations adequately address concerns associatedwith the false positive rate of 17% (3/18 substances) calculated for the LLNA:
BrdU-FC? Are there other suggestions for additional such guidance orlimitations that should be considered? Please explain your answer.
2. Test Method Protocol
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-FC procedure in terms of the proposed
test method standardized protocol? If not, then what recommendations would
you make? Please explain your answer.
ii. Can the traditional LLNA limit dose procedure be applied to the LLNA:
BrdU-FC? Please explain your answer.
3. Future Studies
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-FC in terms of the proposed futurestudies? What other recommendations would you make? Please explain youranswer.
4. Performance Standards
i. The LLNA: BrdU-FC protocol differs from the ICCVAM-recommended
protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes.
According to the proposed draft ICCVAM Performance Standards for the
traditional LLNA
(http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm), anychange to the LLNA protocol other than the method used to assess
lymphocyte proliferation is considered a major change. Do you agree that
protocol differences between the LLNA: BrdU-FC and the traditional LLNA
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex IIshould be considered only minor changes and therefore the validity of this test
method should be based only on the draft ICCVAM Performance Standards?
Please explain your answer.
ii. Even if the draft ICCVAM Performance Standards do not apply to the LLNA:
BrdU-FC, what impact should the accuracy analysis based on 13 of the 18
required performance standards substances have on the overall evaluation oftest method accuracy? Please explain your answer.
iii. Are there concerns that 3/6 sensitizers, for which EC3 data were available,
had EC3 values that were outside of the proposed 0.5x to 2.0x EC3
acceptability range developed based on the traditional LLNA? Please explain
your answer.

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Annex F5
Questions for the Peer Review Panel:
Non-Radioactive LLNA Protocol - LLNA: BrdU-ELISA Test Method

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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

Instructions for the Peer Review Panel: Non-Radioactive LLNA Protocol: LLNA:
BrdU-ELISA Test Method
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the LLNA: BrdU-ELISA (local lymph node assay with bromodeoxyuridine [BrdU]
detected by ELISA) for assessing the allergic contact dermatitis potential of chemicals andother substances. This test method, developed by Dr. Masahiro Takeyoshi (Tokyo, Japan), isa non-radiolabeled version of the traditional LLNA based on measuring levels ofincorporated BrdU in the auricular lymph nodes as an indicator of increased cellproliferation.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA: BrdU-ELISA. You are first asked to review the information in the draft
ICCVAM LLNA: BrdU-ELISA Background Review Document (BRD) for completeness,and to identify any errors or omissions of existing relevant data or information that should be
included. You are then asked to evaluate the information in the BRD to determine the extent
to which each of the applicable criteria for validation and acceptance of toxicological test
methods (ICCVAM 200317) have been appropriately addressed for the proposed use of the
LLNA: BrdU-ELISA. Adequate validation18 is a prerequisite for a test method to beconsidered for use in regulatory decision-making by U.S. Federal agencies. The validation
process characterizes the usefulness and limitations of a test method for a specific intended
use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA: BrdU-ELISA (i.e., the proposed test method use, the proposed recommended
standardized protocol, the proposed test method performance standards, and any proposed
additional studies) and comment on whether the recommendations are supported by the
information provided in the draft LLNA: BrdU-ELISA BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA: BrdU-ELISA.
These include questions prepared by the ICCVAM Immunotoxicity Working Group (IWG)
to ensure that the assessment provides adequate information to facilitate U.S. Federal agency
decisions on the regulatory acceptability of this test method. The questions are also intended
to obtain guidance that will be helpful to federal agencies and other organizations that may
be involved in conducting or supporting further development, standardization, and/or
validation studies.
The overall question to consider is whether the validation status of the LLNA: BrdU-ELISA
has been adequately characterized for its intended purpose, and is it sufficiently accurate and17

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
18
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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reliable to be used for the identification of sensitizing substances and non-sensitizing
substances in place of the traditional LLNA procedure.
I.

Questions to the Panel: Comments on the Draft LLNA: BRDU-ELISA BRD forErrors and Omissions
1. In the draft LLNA: BrdU-ELISA BRD, are there any errors in the BRD thatshould be corrected, or omissions of existing relevant data or information thatshould be included?

II.

Questions to the Panel: Draft LLNA: BrdU-ELISA BRD
1. Test Method Protocol
i. The data generated for the substances analyzed in the LLNA: BrdU-ELISA
test method come from auricular lymph nodes from four individual mice ineach dose group. The recommended ICCVAM LLNA protocol and OECD
Test Guideline 429 recommend a minimum of five animals per dose group forcollecting individual animal data. What impact might the use of four animals
per dose group have on the accuracy and reliability of the LLNA: BrdUELISA? Do you agree with the ICCVAM recommendation that future use ofthis test method protocol should include five animals per dose group? Please
explain your answer.
2. Substances Used for the Validation Studies
i. Do you consider the LLNA: BrdU-ELISA database representative of a
sufficient range of chemical classes and physicochemical properties that thetest method would be applicable to any of the types of chemicals and productsthat are typically tested for skin-sensitization potential? If not, what are therelevant chemical classes/properties (other than those that are identified aslimitations in the traditional LLNA) that should be tested with caution, or not
evaluated using the LLNA: BrdU-ELISA? What chemicals or products shouldbe evaluated to fill this data gap? Please explain your answers.
3. Test Method Accuracyi.

The current accuracy analysis using a stimulation index (SI) ≥3 or SI ≥1.3 toidentify sensitizers is based on overall concordance with the traditional
LLNA. Accuracy statistics compared to the Guinea Pig tests and humandata/experience have also been provided. Are these comparisons appropriate
for assessing the accuracy of the LLNA: BrdU-ELISA? Please explain youranswer.

ii.

Takeyoshi et al. (2007) performed an accuracy analysis using decision criteriaother than SI ≥3 to classify substances as sensitizers. Maximal accuracy for
the LLNA: BrdU-ELISA occurred when an SI ≥1.3 was used to distinguishbetween sensitizers and non-sensitizers. Using this decision criteria, they
achieved an accuracy of 91% (21/23), with a sensitivity of 100% (16/16) and a
specificity of 71% (5/7) (i.e., there were no false negatives and two false
positives). Does this analysis support a recommendation that the decision
criteria be based on an SI ≥1.3? Are there concerns with using a small
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

increase (i.e., 1.3-fold) above the vehicle control response as the basis for
identifying a positive response? Please explain your answers.
iii.

Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: BrdU-ELISA, using the SI ≥3
criterion, been adequately evaluated and compared to the traditional LLNA
(refer also to Table 6-1 of the draft ICCVAM BRD)? If not, what otheranalyses should be performed? Please explain your answer.

iv.

Using the SI ≥3 criterion, there were four substances (aniline, 4-chloroaniline,
2-mercaptothiazole, and hydroxycitronellal) when tested using the LLNA:
BrdU-ELISA that produced “false negative” responses compared to the
traditional LLNA. 4-Chloroanline and aniline are amines. 2Mercaptobenzothiazole is a heterocylic compound and hydroxycitronellal is ahydrocarbon. 2-Mercaptobenzothiazole is a liquid, but the other threesubstances are solids. Can you identify any characteristics associated withthese or similar substances, compared to the correctly identified sensitizers,that might signal that this type of discordant response might occur, andtherefore using the LLNA: BrdU-ELISA to test such substances would not beappropriate or that negative results for such substances should indicate a needfor confirmatory testing? Please explain your answer.

4. Test Method Reliability (Intra- and Inter-laboratory reproducibility)
i. Has the intralaboratory reproducibility of the LLNA: BrdU-ELISA beenadequately evaluated and compared to the traditional LLNA (refer also toTables 7-1 through 7-3 of the draft LLNA: BrdU-ELISA BRD)? If not, what
other analyses should be performed? Are any limitations apparent based on
this intra-laboratory reproducibility assessment? Please explain your answers.
ii. The substances evaluated for intralaboratory reproducibility of the LLNA:
BrdU-ELISA study were not coded. Does the lack of coding of test substances
adversely impact or bias the current evaluation? Please explain your answer.
iii. The Japanese Center for the Validation of Alternative Methods (JaCVAM)
has implemented a multi-laboratory validation study of the LLNA: BrdUELISA. Although the results from this study have yet to be reported, we arehoping to obtain information on the study design (i.e., with regard to number
and types of chemicals tested and the number of laboratories involved). If wedo, do you consider the design appropriate to adequately determine the extent
of interlaboratory reproducibility for the LLNA: BrdU-ELISA? If not, what
other analyses should be performed? Are any limitations apparent based on
this study design? Please explain your answer.
5. Data Quality
i. The studies evaluated in the draft BRD for the LLNA: BrdU-ELISA were notconducted in strict accordance with all provisions of the Good Laboratory
Practice (GLP) guidelines, although there were reportedly performed inlaboratories that conduct GLP studies (M. Takeyoshi, personal
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex IIcommunication). Please discuss what impact this might have on the evaluationof the LLNA: BrdU-ELISA.
ii. The original records for these studies were requested but were not available.
As a result, an independent audit could not be conducted to confirm that thereported data in peer reviewed publications and a poster presentation is thesame as the data recorded in laboratory notebooks. Should any
recommendations from ICCVAM be contingent upon the completion of suchan audit and findings that there were no significant errors in datatranscription? Please explain your answer.
6. Consideration of all available data and relevant information
i. Based on the draft LLNA: BrdU-ELISA BRD, have all the relevant data
identified in published or unpublished studies that employ this test method
been adequately considered? Are there other comparative test method datathat were not considered in the draft BRD, but are available for consideration?
If yes, please explain how to obtain such data.

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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-ELISA
1. Test Method Usefulness and Limitations
i. Do you agree that the available data and test method performance (accuracy
and reliability) support the ICCVAM draft recommendations for the LLNA:
BrdU-ELISA in terms of the proposed test method usefulness and limitations?
Please explain your answer.
ii. If restrictions on using radioactive materials are present, should the LLNA:
BrdU-ELISA be routinely recommended for hazard identification of skinsensitizing substances in lieu of using guinea pig tests due to the advantagesof fewer animals and the avoidance of pain and distress? Please explain youranswer.
iii. Even if limitations in using radioactive materials are not present, should the
LLNA: BrdU-ELISA procedure or other valid and accepted non-radioactive
method be routinely recommended for hazard identification of skin sensitizing
substances instead of the traditional LLNA? Please explain your answer.
iv. Does using a decision criterion of SI ≥1.3 instead of SI ≥3.0 resolve any
concerns with respect to potential false positives or false negatives that may
occur in this test method? Are there other suggestions for additional suchguidance or limitations that should be considered? Please explain youranswer.
2. Test Method Protocol
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA procedure in terms of the
proposed test method standardized protocols? If not, then what
recommendations would you make? Please explain your answer.
ii. Can the traditional LLNA limit dose procedure be applied to the LLNA:
BrdU-ELISA? Please explain your answer.
3. Future Studies
a. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA in terms of the proposed futurestudies? If not, then what recommendations would you make? Please explain
your answer.
i.

Performance Standards
i. The LLNA: BrdU-ELISA protocol differs from the ICCVAM-recommended
protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes.
According to the proposed draft ICCVAM Performance Standards for the
traditional LLNA
(http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm), anychange to the LLNA protocol other than the method used to assess
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

lymphocyte proliferation is considered a major change. Do you agree that
protocol differences between the LLNA: BrdU-ELISA and the traditional
LLNA should be considered only minor changes and therefore the validity ofthis test method should be based only on the draft ICCVAM PerformanceStandards? Please explain your answer.
ii. Even if the draft ICCVAM Performance Standards do not apply to the LLNA:
BrdU-ELISA, what impact should the accuracy analysis based on eight of the
18 required performance standards substances (only one false negative and nofalse positives) have on the overall evaluation of test method accuracy? Please
explain your answer.
iii. Are there concerns that 4/4 sensitizers, for which EC3 data were available,
had EC3 values that were outside of the recommended 0.5x to 2.0x EC3
acceptability range developed based on the traditional LLNA? Please explain
your answer.
iv. Should separate performance standards be developed for the LLNA: BrdUELISA? Please explain your answer.

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Annex F6
Questions for the Peer Review Panel:
Draft ICCVAM Performance Standards for the LLNA

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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

Instructions for the Peer Review Panel: Draft ICCVAM Performance Standards for the
LLNA
ICCVAM has developed draft LLNA performance standards consisting of essential test
method components, a minimum list of reference substances, and expected accuracy andreliability values. These are proposed for evaluating the acceptability of proposed test
methods that are mechanistically and functionally similar to the traditional LLNA test
method protocol previously recommended by ICCVAM.
The overall question for the Panel is whether these performance standards are consideredadequate for assessing the accuracy and reliability of test method protocols that are based on
similar scientific principles and that measure the same biological effect as the traditional
LLNA?
1) Purpose and Applicabilitya) ICCVAM proposes that these performance standards should only be applicable toversions of the LLNA that incorporate minor modifications to the traditional LLNA.
Currently, this is limited to the use of non-radioactive reagents to measurelymphocyte proliferation. It is considered essential that the modified LLNA should
otherwise adhere to all other aspects of the traditional LLNA protocol, as defined by
ICCVAM (1999) and Dean et al. (2001). This includes aspects such as: the sex andstrain of mouse used, the number of mice per dose group, the timing and site of test
article treatment, the duration between the last treatment and lymph node collection,
the inclusion of concurrent negative and positive control groups, the measured
endpoint (i.e., lymphocyte proliferation in the draining auricular lymph node), and thecollection of data at the level of the individual mouse. Do you agree that the use of
non-radioactive reagents for measuring cell proliferation in the lymph nodes, if that is
the only difference, constitutes a minor modification to the traditional LLNA
protocol? Is it necessary to keep the same decision criteria for distinguishing between
sensitizers and non-sensitizers (i.e., an SI of 3)? Please explain your answer.
b) Are there other procedural modifications that you consider minor and therefore could
be evaluated for equivalence to the traditional LLNA using the proposed performancestandards? If yes, please explain what they are and why.
c) Do you consider these performance standards to also be applicable to the LLNA limit
dose procedure? Please explain your answer.
2) Essential Test Method Componentsa) The essential test method components are based on the ICCVAM recommended
protocol (ICCVAM 1999; Dean et al. 2001), which is the basis for the current U.S.
Environmental Protection Agency (EPA 2003) test guideline (TG). There are somenotable differences between these protocols and the Organisation for Economic Cooperation and Development TG 429 for the LLNA (OECD 2002). When evaluationsof non-radioactive versions of the traditional LLNA are conducted using these
performance standards, is it necessary that the validation studies follow the ICCVAM
recommended protocol? Specifically, should the studies include: 1) a concurrent
positive control with each test substance; 2) using a minimum of five animals perdose group; and 3) measuring proliferation in lymph nodes from individual animals
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

rather than pooling lymph nodes across all animals in a dose group? Please explain
your answers.
b) Should the concurrent testing of the positive control and test substance be conductedin the same vehicle or can different vehicles be used? Please explain your answer.
3) Proposed Reference Substancesa) Do you agree with the selection and prioritization criteria used to select theperformance standards reference substances? Please explain your answer.
b) The rationale for the number of substances included on the "required" list of
substances (n=18) is provided in Appendix C of the draft ICCVAM PerformanceStandards. Do you consider this to be an adequate number upon which to evaluate theperformance of non-radioactive LLNA test methods, where the only protocol
modification is the method for assessing cell proliferation in the auricular lymph
nodes? If not, how many reference chemicals should be tested? Please explain youranswer.
c) Do you consider the types of substances included in the reference substance list, withregard to relative sensitization potency, physicochemical characteristics, and vehicles,
to be representative of the overall diversity of substances that are likely to be tested
for skin sensitization? Please explain your answer.
d) Are there other types of information relevant to skin sensitization that should beconsidered in order to demonstrate an adequately diverse reference list? If yes, please
explain what additional information should be included.
e) Are there other substances that you consider to be more appropriate for assessing thesensitivity (ability of the test method to correctly identify sensitizing substances) andspecificity (ability of the test method to correctly identify non-sensitizing substances)of non-radioactive LLNA test methods, and for which there is available LLNA,
guinea pig, and human data? If yes, please name the substances and explain why.
f) Four "discordant chemicals" (i.e., two LLNA false negatives and two false positives
compared to guinea pig tests or human data) are included as optional substances that
could be studied to evaluate if the proposed modifications might provide improved
performance relative to the traditional LLNA.
•

Please comment on the appropriateness of including these specific substances in
the reference list. Should different substances be included? Should more false
negative/positive substances be tested? If so, what are they? Please explain youranswers.

•

Do you consider their "optional" status appropriate, or should testing thesesubstances be required? Please explain your answer.

•

Would "correct" results with these four discordant chemicals be sufficient toconsider the alternative test method to be more predictive of skin sensitization
than the traditional LLNA? Please explain your answer.

4) Test Method Accuracy Standards
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex IIa) The draft ICCVAM Performance Standards state that the non-radioactive proposed
LLNA test method should exactly match the accuracy of the traditional LLNA whenevaluated with the minimum set of 18 reference substances. Do you agree that test
method accuracy should be based on a chemical-by-chemical match with regard to
identifying the chemicals as sensitizers or non-sensitizers? Please explain youranswer.
b) The draft ICCVAM LLNA Performance Standards recommend that, for eachsensitizer, the threshold concentration that induces a positive SI response should bewithin 0.5x to 2.0x of the concentration obtained for the EC3 in the traditional LLNA.
As described in Appendix D of the draft ICCVAM Performance Standards, statistical
approaches have been used in an attempt to identify an appropriate range, but thesecalculated ranges do not appear to be the most practical. In contrast, the NICEATM
LLNA database demonstrates that EC3 values from replicate tests for a sensitizing
chemical when tested using the same solvent are rarely outside of this proposed 0.5x
to 2.0x acceptability EC3 range. Please comment on the appropriateness of using thiscriterion to judge the equivalency of a non-radioactive version of the traditional
LLNA. If this approach is not acceptable, please explain why, and present analternative approach along with the basis for this approach.
c) For five of the 13 sensitizers on the draft ICCVAM reference substances list, thereference EC3 value is based on a single LLNA study (see Table C1 of the draft
ICCVAM Performance Standards). Please comment on the appropriateness of
including such chemicals in the list of recommended reference substances andwhether or not the 0.5x to 2.0x criteria should be applied to such substances. Please
explain your answer.
5) Test Method Reliability Standardsa) The draft ICCVAM Performance Standards state that acceptable intralaboratory
reproducibility will be indicated by a laboratory obtaining, in each of four
independent experiments conducted with at least one week between each experiment,
ECt values (the estimated concentration needed to produce an SI of a defined
threshold [e.g., EC3]) for hexyl cinnamic aldehyde (HCA) that are generally within
0.5x to 2.0x (i.e., 5% to 20%) of the historical mean EC3 concentration (10%) for thissubstance, based on existing available traditional LLNA data.
•

Do you consider the number of repeat experiments (n=4) to be adequate? Please
explain your answer.

•

Do you consider testing HCA adequate for demonstrating intralaboratory
reproducibility? If not, which substance(s) should be tested? Please explain youranswer.

•Is the required one-week interval between independent tests adequate and/or
appropriate? If not, please provide an alternative schedule and explain the basis
for your recommendation.

•

Do you consider the criteria for acceptability to be appropriate? If not, please
describe another criteria and explain the basis for your recommendation.
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex IIb) The draft ICCVAM Performance Standards state that acceptable interlaboratory
reproducibility will be indicated by each of three laboratories obtaining ECt valuesfor HCA and 2,4-dinitrochlorobenzene (DNCB) from a single experiment that are
generally within 0.5x to 2.0x (5% to 20% and 0.025 to 0.1%, respectively) of themean historical EC3 concentration (10% and 0.05%, respectively) obtained for these
two substances in the traditional LLNA.
•

Do you consider the single experiment per substance in each laboratory to beadequate? If not, please provide an alternative approach and explain why.

•

Do you consider testing HCA and DNCB to be adequate for demonstratinginterlaboratory reproducibility? If not, which substance(s) should be tested?
Please explain your answer.

•

Do you consider the criteria for acceptability to be appropriate? If not, please
describe another criteria and explain the basis for your recommendation.

6) Summary Questiona) If a radioactive or non-radioactive LLNA method were proposed with a “majorchange” (e.g., different mouse strain or use of male mice, change in the schedule fortest article administration, change in schedule for lymph node excision, etc.), whatcriteria should be used to evaluate the equivalence of this method to the traditional
LLNA?
•

Would a new set of performance standards be required for this method? Please
explain your answer.

•

How many reference substances might be considered adequate? Would the 18
minimum reference substances in the draft ICCVAM LLNA PerformanceStandards be sufficient? If more substances are considered necessary, how many
should there be tested and what should their characteristics be? Please explain
your answer.

•

Regardless of the number of reference substances, should the alternative LLNA
be required to obtain the same call (and potency for sensitizers) as the traditional
LLNA for the 18 minimum reference substances in the draft ICCVAM LLNA
Performance Standards? Please explain your answer.

•

Are there additional specific substances that should be used? If yes, what are
they? Please explain your answer.

•

What, if any, additional information would be considered necessary and why?

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Annex F7
Questions for the Peer Review Panel:
Use of the LLNA for Potency Determinations

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Instructions for the Peer Review Panel: Use of the LLNA for Potency Determinations
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the murine local lymph node assay (LLNA) for assessing the potential potency of
substances to cause allergic contact dermatitis.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA for potency determinations. You are first asked to review the information in thedraft ICCVAM LLNA for potency determinations Background Review Document (BRD) forcompleteness, and to identify any errors or omissions of existing relevant data or informationthat should be included. You are then asked to evaluate the information in this BRD todetermine the extent to which each of the applicable criteria for validation and acceptance of
toxicological test methods (ICCVAM 200319) have been appropriately addressed for the
proposed use of the LLNA for potency determinations. Adequate validation20 is aprerequisite for a test method to be considered for use in regulatory decision-making by U.S.
Federal agencies. This validation process characterizes the usefulness and limitations of a test
method for its intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA for potency determinations (i.e., the proposed test method use, the proposed
recommended standardized protocol, the proposed test method performance standards, andany proposed additional studies) and comment on whether the recommendations aresupported by the information provided in the draft BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA for potency determinations.
These include questions prepared by the ICCVAM IWG to ensure that the assessment
provides adequate information to facilitate U.S. Federal agency decisions on the regulatory
acceptability of this test method, and/or adequate guidance for organizations that may be
involved in conducting or supporting further development, standardization, and/or validation.
The overall question to consider is whether the validation status of the LLNA for potency
determinations has been adequately characterized, and is it sufficiently accurate and reliable
to be used for the identification of sensitizing substances according to their relative potencyclassification based on a comparison to either human or guinea pig responses.
I.

Questions to the Panel: Review for Errors and Omissions
1. Are there any errors or omissions of existing relevant data or information thatshould be included in the draft BRD?

19

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
20
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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ICCVAM Test Method Evaluation Report: Appendix F1, Annex II

II.

Questions to the Panel: LLNA for Potency Determinations Draft BRD
1. Do you consider the database of substances evaluated representative of a
sufficient range of chemical classes and physical chemical properties that it would
be applicable to any of the types of chemicals and products that are typicallytested for skin-sensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using thefor potency determinations? What chemicals or products should be evaluated tofill this data gap? Please explain your recommendation.
2. While coding of chemicals is recommended for prospective studies, thisevaluation is based on a retrospective evaluation of existing data, most of which
was not generated using coded chemicals to reduce bias. Does the lack of codingof test substances adversely impact or bias the current evaluation? Please provide
a rationale for your answer.
3. For some substances submitted using the LLNA, it was not possible to confirm
whether the data were generated using pooled animal data for each dose group (asallowed in Test Guideline [TG] 429 of the Organisation for Economic
Co-operation and Development [OECD]) rather than individual animal data (asrecommended in the ICCVAM 2001 protocol)? Cockshott et al. (2006) reported
that using individual animal data allowed for technical problems during anexperiment to be identified. Considering this, should the analysis of theperformance of the LLNA for potency determinations be limited to data fromstudies that can be confirmed as using individual animal data collection
procedures? What impact might the inclusion of pooled animal data have on theaccuracy analysis included in Section 6.0 of the draft ICCVAM BRD? Please
explain your answer.
4. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA for potency determinations beenadequately evaluated and compared to the human and guinea pig (refer also to
Section 6.0 of the draft ICCVAM BRD)? If not, what other analyses should be
performed?
5. The accuracy analysis (see Section 6.0 of the draft ICCVAM BRD) focuses on the
two-level categorization scheme proposed by the United Nations Globally
Harmonized System for Classification and Labelling for both human and guinea
data. Should other categorization schemes be considered?
6. Does the BRD adequately characterize the usefulness and limitations of the
LLNA for potency determinations based on the accuracy analyses? If not, what
additions or changes should be made to the current usefulness and limitations?
7. Has the reliability (e.g., intralaboratory repeatability, intra- and inter-laboratory
reproducibility) of the LLNA for potency determinations been adequately
evaluated (refer also to Section 7.0 of the draft ICCVAM BRD)? If not, what
other analyses should be performed?
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8. For some studies included in the draft BRD, it was not possible to determine
whether or not they had been conducted in accordance with Good Laboratory
Practice (GLP) guidelines. Neither was it possible to obtain the results of GLP
audits for all studies conducted in accordance with GLP guidelines. Please discuss
what impact this lack might have on the evaluation of the LLNA for potency
determinations and whether such studies should be excluded from any analysis.
9. As described in the draft BRD, original records for some of the non-GLP studies
included in this evaluation could not be obtained. As a result, an independent
audit could not be conducted to confirm that the reported data is the same as thedata recorded in laboratory notebooks. Considering this, should the results ofthese studies (all of which are currently included) be excluded from any of theperformance analyses? If yes, please explain.
10. Based on the draft BRD, have all the relevant data identified in published or
unpublished studies conducted using the LLNA for potency determinations beenadequately considered? If not, what other studies should to be considered?
III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA for Potency Determinations
1. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA for potency determinations in terms of the proposed test method
usefulness and limitations? Please explain your answer. If not, why
recommendations would you make?
•Should the LLNA be routinely recommended for the hazard classification of
the skin-sensitization potency of chemicals?

2. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed test method standardized protocol? Please
explain your answer. If not, then what recommendations would you make?
3. Should the relevant testing guidelines for the LLNA be updated to include thecalculation of an EC3 value?
4. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed future studies? Please explain youranswer. If not, then what recommendations would you make?

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ICCVAM Test Method Evaluation Report: Appendix F2Appendix F2
Summary Minutes of Independent Scientific Review Panel Meeting
March 4–6, 2008

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ICCVAM Test Method Evaluation Report: Appendix F2

Summary Minutes
Independent Scientific Peer Review Panel Meeting
Validation Status of New Versions and Applications of the Murine Local Lymph Node
Assay (LLNA): A Test Method for Assessing the Allergic Contact Dermatitis Potentialof Chemicals and Products
Consumer Product Safety Commission (CPSC), Headquarters
Bethesda, MD
March 4 – 6, 2008
8:30 a.m. – 5:30 p.m.
Peer Review Panel Members:
Michael Luster, Ph.D. (Peer Review
Panel Chair)
Nathalie Alépée, Ph.D.
Anne Marie Api, Ph.D.
Nancy Flournoy, M.S., Ph.D.
Thomas Gebel, Ph.D.
Kim Headrick, B. Admin., B.Sc.
Dagmar Jírová, M.D., Ph.D.

David Lovell, Ph.D
Howard Maibach, M.D.
James McDougal, Ph.D.

Senior Consultant to the NIOSH Health Effects
Laboratory, Morgantown, WV, U.S.
Associate Research Fellow, Pfizer PDRD MCTLaboratory, France
Vice President, Human Health Sciences, ResearchInstitute for Fragrance Materials, Woodcliff Lake,
NJ, U.S.
Professor and Chair, Dept. of Mathematics andStatistics, University of Missouri-Columbia,
Columbia, MO, U.S.
Regulatory Toxicologist, Federal Institute for
Occupational Safety and Health, Dortmund,
Germany
International Harmonization Senior Policy Advisor,
Health Canada, Ottawa, Ontario, Canada
Toxicologist, Research Manager, Head of ReferenceCenter for Cosmetics, Head of Reference Laboratory
for Experimental Immunotoxicology, National
Institute of Public Health, Czech Republic
Reader in Medical Statistics, Postgraduate Medical
School, University of Surrey, Guildford, Surrey,
U.K.
Professor, Dept. of Dermatology, University ofCalifornia-San Francisco, San Francisco, CA, U.S.
Professor and Director of Toxicology Research,
Dept. of Pharmacology and Toxicology, Boonshoft
School of Medicine, Wright State University,
Dayton, OH, U.S.
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ICCVAM Test Method Evaluation Report: Appendix F2Peer Review Panel Members:
Michael Olson, Ph.D.
Raymond Pieters, Ph.D.
Jean Regal, Ph.D.
Peter Theran, V.M.D.
Stephen Ullrich, Ph.D.

Michael Woolhiser, Ph.D.
Takahiko Yoshida, M.D., Ph.D.

Director of Occupational Toxicology, Corporate
Environment Health and Safety, GlaxoSmithKline,
RTP, NC, U.S.
Associate Professor, Immunotoxicology Group
Leader, Institute for Risk Assessment Sciences,
Utrecht University, Utrecht, The Netherlands
Professor, Dept. of Pharmacology, University of
Minnesota Medical School, Duluth, MN, U.S.
Massachusetts Society for the Prevention of Cruelty
to Animals, Novato, CA, U.S.
Dallas/Ft. Worth Living Legends Professor &Professor of Immunology, Post-graduate School of
Biomedical Science, University of Texas M.D.
Anderson Cancer Center, Houston, TX, U.S.
Technical Leader - Immunotoxicology, Toxicology
and Environmental Research and Consulting
Immunology, Dow Chemical, Midland, MI, U.S.
Professor, Dept. of Health Science, Asahikawa
Medical College, Hokkaido, JapanICCVAM and ICCVAM IWG Members:
Paul Brown, Ph.D.
FDA, Silver Spring, MD, U.S.
Ruth Barratt, Ph.D., D.V.M.
FDA, Rockville, MD, U.S.
Karen Hamernik, Ph.D.
EPA, Washington, DC, U.S.
Masih Hashim, Ph.D.
EPA, Washington, DC, U.S.
Abigail Jacobs, Ph.D. (IWG Co-Chair) FDA, Silver Spring, MD, U.S.
Kristina Hatlelid, Ph.D.
CPSC, Bethesda, MD, U.S.
Joanna Matheson, Ph.D. (IWG CoCPSC, Bethesda, MD, U.S.
Chair)Tim McMahon, Ph.D.
EPA, Washington, DC, U.S.
Amy Rispin, Ph.D.
EPA, Washington, DC, U.S.
William Stokes, D.V.M., D.A.C.L.A.M. NIEHS, RTP, NC, U.S.
Raymond Tice, Ph.D.
NIEHS, RTP, NC, U.S.
Ron Ward, Ph.D.
EPA, Washington, DC, U.S.
Marilyn Wind, Ph.D. (ICCVAM Chair) CPSC, Bethesda, MD, U.S.
Jiaqin Yao, Ph.D.
FDA, Silver Spring, MD, U.S.
ECVAM Observer:David Basketter, Ph.D.
Invited Experts:
George DeGeorge, Ph.D., D.A.B.T.
Kenji Idehara, Ph.D.
Masahiro Takeyoshi, Ph.D.

DABMEB Consultancy Ltd., Bedfordshire, U.K.
MB Research Laboratories, Spinnerstown, PA, U.S.
Daicel Chemical Industries, Hyogo, Japan
Chemicals Evaluation and Research Institute,
Saitama, Japan
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ICCVAM Test Method Evaluation Report: Appendix F2

Public Attendees:
Odette Alexander
Nancy Beck, Ph.D.
Ann Blacker, Ph.D.
Stuart Cagan, Ph.D.
Joan Chapdelaine, Ph.D.
Adriana Doi, Ph.D.
Carol Eisenmann, Ph.D.
Charles Hastings, Ph.D.
Kailash Gupta, D.V.M., Ph.D.
John Lyssikatos
Laurence Musset. Ph.D.
Carol O’Neil
Kui Lea Park, Ph.D.
Rafael Rivas
Terri Sebree
Libby Sommer
Merrill Tisdel
Jeffrey Toy, Ph.D.

Syngenta Crop Protection, Inc., Greensboro, NC,
U.S.
PCRM, Washington, DC, U.S.
Bayer CropScience, RTP, NC, U.S.
Shell Oil Company, Houston, TX, U.S.
Calvert Laboratories, Inc., Olyphant, PA, U.S.
BASF Corporation, RTP, NC, U.S.
Personal Care Products Council, Washington,
DC, U.S.
BASF Corporation, RTP, NC, U.S.
Retired CPSC, Bethesda, MD, U.S.
Hill Top Research, Miamiville, OH, U.S.
OECD, Paris, France
NuPathe, Conshohocken, PA, U.S.
National Institute of Toxicological Research,
KFDA, Seoul, Korea
AFRRI/USHUS, Bethesda, MD, U.S.
NuPathe, Conshohocken, PA, U.S.
EPA, Washington, DC, U.S.
Syngenta Crop Protection Inc., Greensboro, NC,
U.S.
FDA, Rockville, MD, U.S.

NICEATM:
William Stokes, D.V.M., D.A.C.L.A.M. Director
Raymond Tice, Ph.D.
Deputy Director
Debbie McCarley
Special Assistant to the Director
Support Contract Staff— Integrated Laboratory Systems, Inc. (ILS)David Allen, Ph.D.
Michael Paris
Thomas Burns, M.S.
Eleni Salicru, Ph.D.
Linda Litchfield
Judy Strickland, Ph.D., D.A.B.T.
Douglas Winters, M.S.
ILS, Inc.
Abbreviations: AFFRI = Armed Forces Radiobiology Research Institute; CPSC = U.S. Consumer Product Safety Commission; ECVAM =European Centre for the Validation of Alternative Methods; EPA = U.S. Environmental Protection Agency; FDA = U.S. Food and Drug
Administration; ICCVAM = Interagency Coordinating Committee on the Validation of Alternative Methods; ILS = Integrated Laboratory
Systems; IWG = Immunotoxicology Working Group; KFDA = Korea Food and Drug Administration; NICEATM = National Toxicology
Program Interagency Center for Evaluation of Alternative Toxicological Methods; NIEHS = National Institute of Environmental Health
Sciences; NIOSH = National Institute of Occupational Safety and Health; OECD = Organisation for Economic Co-operation andDevelopment; PCRM = Physicians Committee for Responsible Medicine; USDA = U.S. Department of Agriculture; USHUS = Uniformed
Services University of the Health Sciences.

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Tuesday, March 4, 2008
Call to Order and Introductions
Dr. Michael Luster (Peer Review Panel Chair) called the meeting to order at 8:30 a.m. andintroduced himself. He then asked all Peer Review Panel (hereafter Panel) members to
introduce themselves and to state their name and affiliation for the record. He then asked all the
National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM) staff, the Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) members, the ICCVAM Immunotoxicity
Working Group (IWG) members, the European Centre for the Validation of Alternative
Methods (ECVAM) observer, and members of the public to also introduce themselves. Dr.
Luster stated that there would be opportunity for public comments during each of the sevenlocal lymph node assay (LLNA)-related topics. He asked that all those interested in making acomment, register at the registration table and provide a written copy of their comments, if
available, to NICEATM staff. Dr. Luster emphasized that the comments would be limited toseven minutes per individual and that, while an individual would be welcome to makecomments during each commenting period, repeating the same comments at each comment
period would be inappropriate. He further stated that the meeting was being recorded and that
Panel members should speak directly their microphone. Finally, Dr. Luster noted that if the
Panel finished early with the assigned topics on the agenda for that day, they would proceed to
the next day’s topics if time permitted.
Welcome from the ICCVAM Chair
Dr. Marilyn Wind, U.S. Consumer Product Safety Commission (CPSC) and Chair of ICCVAM,
welcomed everyone to CPSC and to the Panel meeting. Dr. Wind stressed the importance of
this Panel’s efforts especially considering recent reports that allergies and asthma haveincreased markedly over the past number of years and that contact dermatitis is the mostcommon occupational illness in the United States. Dr. Wind thanked the Panel members forgiving their expertise, time, and effort and acknowledged their important role to the ICCVAM
test method evaluation process. Dr. Wind also emphasized the important role of the public andtheir comments in this process.
Welcome from the Director of NICEATM, and Conflict of Interest Statements
Dr. William Stokes, Director of NICEATM, stated the Panel meeting was being convened as a
National Institutes of Health (NIH) special emphasis panel and was being held in accordancewith the Federal Advisory Committee Act regulations. As such, Dr. Stokes indicated that hewould serve as the Designated Federal Official for this public meeting. He reminded the Panelthat they had signed a conflict-of-interest statement when they were selected for the Panel, in
which they identified any potential conflicts of interest. He then read this statement to provide
another opportunity for members of the Panel to identify any conflicts not previously declared.
Dr. Luster asked the Panel members to declare any direct or indirect conflicts based on Dr.
Stokes statements and to recuse themselves from discussion and voting on any aspect of the
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meeting where there might be a conflict. None of the Panel members declared a conflict ofinterest.
Overview of the ICCVAM Test Method Evaluation Process
Dr. Stokes provided an overview of the ICCVAM test method evaluation process. He statedthat the Panel was made up of 19 different scientists from eight different countries (Canada,
Czech Republic, France, Germany, Japan, The Netherlands, United Kingdom, and the UnitedStates). Dr. Stokes thanked the Panel members for the significant amount of time and effort thatthey had devoted to prepare for and attend the meeting. He explained that the purpose of the
Panel was to assist ICCVAM by carrying out an independent scientific peer review of theinformation provided on a series of proposed new versions of the LLNA and some expanded
applications of the assay. Dr. Stokes mentioned that the original LLNA peer review panel in1998 considered the LLNA a valid substitute for the guinea pig-based test in most testing
situations, but not all. He mentioned that three Panel members from the 1998 review are also on
the current Panel (i.e., Drs. Howard Maibach, Jean Regal, and Stephen Ullrich). Dr. Stokes also
reviewed the nomination that was received from CPSC in January 200721, which provides thebasis for the current evaluation.
Dr. Stokes then identified the 15 Federal agencies that comprise ICCVAM and summarized
ICCVAM’s mission. He noted that ICCVAM, as an interagency committee, does not carry out
research and development or validation studies. Instead, ICCVAM, in conjunction withNICEATM, carries out the critical scientific evaluation of proposed test methods with regard totheir usefulness and limitations for regulatory testing and then makes formal recommendations
to ICCVAM agencies.
Dr. Stokes provided a brief review of ICCVAM's history and summarized the ICCVAM
Authorization Act of 200022, detailing the purpose and duties of ICCVAM. He noted that one of
ICCVAM's duties is to review and evaluate new, revised, and alternative test methods
applicable to regulatory testing. He stated that all of the reports produced by NICEATM areavailable on the NICEATM-ICCVAM website or can be obtained upon request fromNICEATM. He also mentioned that ICCVAM provides guidance on test method development,
validation criteria, and processes, and helps to facilitate not only the acceptance of scientificallyvalid alternative methods, but also encourages international harmonization.
Dr. Stokes then described the ICCVAM test method evaluation process, which begins with atest method nomination or submission. NICEATM conducts a prescreen evaluation tosummarize the extent to which the proposed submission or nomination addresses the ICCVAM
prioritization criteria. A report of this evaluation is then provided to ICCVAM, which in turndevelops recommendations regarding the priority for evaluation. ICCVAM then seeks input on
their recommendations from the Scientific Advisory Committee on Alternative Toxicological
Methods (SACATM) and the public. Given sufficient regulatory applicability, sufficient data,
resources, and priority, a test method will move forward into a formal evaluation. A draft21
22

http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
http://iccvam.niehs.nih.gov/docs/about_docs/PL106545.pdf
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background review document (BRD), which provides a comprehensive review of all available
data and information, is prepared by NICEATM, in conjunction with an ICCVAM working
group designated for the relevant toxicity testing area (e.g., the IWG). In addition, ICCVAM
considers all of the available information and makes draft test method recommendations on the
proposed usefulness and limitations of the test methods, test method protocol, performancestandards, and future studies. The BRD and the draft ICCVAM test method recommendations
are made available to the Panel and the public for review and comment. The Panel peer reviews
the BRD and evaluates the extent to which it supports the draft ICCVAM test method
recommendations. A Panel report is published, which is then considered, along with public and
SACATM comments by ICCVAM in making final recommendations. These final
recommendations are forwarded to the ICCVAM member agencies for their consideration andpossible incorporation into relevant testing guidelines.
Dr. Stokes reviewed the ICCVAM criteria for adequate validation. He stated that validation isdefined by ICCVAM as the process by which the reliability and relevance of a procedure areestablished for a specific purpose, and that adequate validation is a prerequisite forconsideration of a test method by U.S. Federal regulatory agencies. Dr. Stokes listed the
ICCVAM acceptance criteria for test method validation and acceptance. He concluded bysummarizing the timeline of the review activities beginning with CPSC’s nomination in January
2007 and ending with the present Panel meeting.
ICCVAM Charge to the Panel
Dr. Stokes reviewed the charge to the Panel, which was to: (1) review the draft BRDs, the draft
Addendum to the traditional23 LLNA, and the draft performance standards for completeness andidentify any errors or omissions; (2) determine the extent to which each of the applicable
criteria for validation and regulatory acceptance had been addressed for the proposed revised or
modified versions of the LLNA; and (3) consider and provide comment on the extent to which
the ICCVAM draft test method recommendations including the proposed use, standardized
protocols, performance standards, and additional studies are supported by the information
provided in the draft BRDs and draft Addendum.
Dr. Stokes thanked the IWG and ICCVAM for their contributions to this project, andacknowledged the contributions from the participating liaisons from ECVAM and JaCVAM(Japanese Center for the Validation of Alternative Methods). He also acknowledged the
NICEATM staff for their support and assistance in organizing the Panel meeting and preparing
the materials being reviewed.

23

For the purposes of this document, the radioactive LLNA test method, which was first evaluated by ICCVAM
in 1999, and subsequently recommended to U.S. Federal agencies as a valid substitute for currently accepted
guinea pig test methods to assess the allergic contact dermatitis potential of many, but not all, types ofsubstances, is referred to as the traditional LLNA.
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Current Regulatory Testing Requirements and Hazard Classification Schemes forAllergic Contact Dermatitis and the Traditional LLNA Procedure
Dr. Joanna Matheson, Chair of the IWG, briefly reviewed the regulatory testing requirements of
U.S. Federal agencies for skin-sensitization hazard identification and provided a brief
description of the LLNA protocol.
Overview of the Agenda
Dr. Luster provided a brief synopsis of the agenda. He stated that there were six test methods
and applications, along with the draft LLNA performance standards for review and that thesame agenda would be followed for each: (1) introductory summary of the draft ICCVAM
recommendations from one of the NICEATM staff members; in addition, test method
developers would provide a brief description of the methodology for each of the three nonradioactive tests, (2) presentation of the Evaluation Group draft comments by the Evaluation
Group leader, (3) Panel discussion, (4) public comments, (5) recommendations and conclusions
by the Panel.
Overview of the Draft LLNA Limit Dose Procedure24 BRD and Draft ICCVAM Test
Method Recommendations
Dr. David Allen, Integrated Laboratory Systems, Inc., the NICEATM support contractor,
presented an overview of the draft ICCVAM BRD for the LLNA limit dose procedure. He
mentioned that the draft ICCVAM BRD provided a comprehensive review of the available dataand information regarding the usefulness and limitations of the LLNA limit dose procedure.
The method was reviewed for its accuracy in correctly identifying sensitizers and nonsensitizers, when compared to the traditional LLNA.
NICEATM published a series of Federal Register (FR) notices, including an FR notice(72FR27815, May 17, 2007) requesting original data from the LLNA. This FR notice was also
sent to over 100 potentially interested stakeholders for their input and comment. As a result,data on 255 substances tested in the LLNA were received. The resulting LLNA databaseconsisted of 471 studies of 466 unique substances, 211 of which were included in the original
ICCVAM 1999 evaluation. Dr. Allen briefly summarized the performance characteristics of the
LLNA limit dose procedure test method, which is detailed in the draft ICCVAM BRD25, andbriefly summarized the draft ICCVAM test method recommendations for the LLNA limit dose
procedure26
Panel Evaluation:
Dr. Michael Olson led the Panel discussion on the LLNA limit dose procedure and specificallythanked the members of his Evaluation Group (i.e., Drs. James McDougal, Raymond Pieters,
Jonathan Richmond [not present], and Takahiko Yoshida) for their collegial review of theinformation presented in the draft ICCVAM LLNA Limit Dose Procedure BRD. Dr. Olson also
24

Also know as the reduced LLNA (rLLNA).
http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-LD/LLNAldBRD07Jan08FD.pdf
26
http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-LD/IWGrecLLNA-LD07Jan08FD.pdf
25

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ICCVAM Test Method Evaluation Report: Appendix F2

thanked the NICEATM staff for their technical support during the BRD review process. He
then presented the draft responses to ICCVAM’s questions to the Panel for consideration by theentire Panel. The focus was on review of the BRD for errors and omissions, assessment of thevalidation status of the test method, and review of draft ICCVAM test method
recommendations. The Panel discussion and their recommended revisions to each section of thedraft ICCVAM BRD and recommendations are reflected in the Validation Status of New
Versions and Applications of the Murine Local Lymph Node Assay: A Test Method forAssessing the Allergic Contact Dermatitis Potential of Chemicals and Products, published in
May 2008 (hereafter, the Panel report27).
During the Panel’s evaluation, discussion arose regarding what might have resulted in the
inverted-U-shaped dose response that was seen with the false-negative substances in the LLNA
limit dose procedure. Dr. Olson responded that although it was difficult to understand what thecause might have been, he speculated that the top dose was either toxic at a systemic-effectlevel or that those substances were immunosuppressive at the highest dose level. He also statedthat there did not seem to be any structural features of the substances that could be attributed forthe false negative response in the LLNA limit dose procedure.
The Panel also discussed the use of concurrent versus intermittent positive controls in the
LLNA limit dose procedure. Dr. Olson indicated that the Evaluation Group had discussed thepossibility to allow intermittent positive controls for laboratories that exhibited repeatable andadequate performance with the LLNA but he indicated that it would be important to describe a
set of performance criteria that would determine when this practice would be acceptable.
Clearly, if the laboratory was not performing the assay routinely or if there were other reasons
to suspect variability in response with any substance, the positive control would be necessary.
Dr. Stokes indicated that this discussion was pertinent and indicated that the Panel’ssuggestions for what the performance criteria might be for intermittent positive control testing
would be of interest to the IWG. Dr. Stokes also wanted to clarify that the OECD TG, isconsistent with the EPA TG and the ICCVAM-recommended test method protocol for the
LLNA although the OECD TG allows additional latitude in how tests are run (i.e., four animals
per dose group, use of pooled data, and the option to not run a positive concurrent positive).
Public Comments:
Dr. Amy Rispin, EPA
Dr. Rispin stated that the ICCVAM LLNA report (199928) and standardized protocol (200129)
recommends the use of a concurrent positive control in addition to the concurrent negative
control required for each study. Subsequently, the OECD (Organisation for Economic Cooperation and Development) Test Guideline (TG) 429 (Skin Sensitisation: Local Lymph Node
Assay) was finalized (2002). She said that originally, OECD TG 429 was drafted without aconcurrent positive control but that language was added to include the recommended use of aconcurrent positive control until laboratories demonstrate competence. Subsequent to that, EPA
27

http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
29
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/LLNAProt.pdf
28

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put forth its LLNA guideline for sensitization30, which states that concurrent positive andnegative controls are to be included in each study. Dr. Rispin then added that U.S. Federal
regulatory agencies, most notably the EPA and FDA, received LLNA data from studies inwhich the positive control did not achieve the appropriate limits of performance (i.e., thecontrol values were not in the appropriate range) and therefore the studies were deemedunacceptable, underscoring the importance of a concurrent positive control for regulatory
acceptance in the United States.
In response to Dr. Rispin’s public comment, Drs. Ullrich and Theran asked how competence isdetermined and if laboratories have difficulties reaching a level of competence, respectively.
Dr. Abby Jacobs responded by stating that the FDA has seen large data variations inlaboratories that conduct the LLNA. It is often difficult to determine what the variations might
be due to (e.g., new technicians, tail vein injection, lymph node removal) and these variations
have been seen both in laboratories that are established and those that are not.
Dr. David Basketter, ECVAM Observer
Dr. Basketter said that the main point he wanted to address is that efforts should be made toharmonize the LLNA protocol with that described in OECD TG 429. He stated that although
there is referral to the “ICCVAM protocol” throughout the BRDs under consideration, OECD
TG 429 is more globally recognized for regulatory use of the LLNA and therefore should be thereferenced protocol. Dr. Basketter further stated that if the LLNA limit dose procedure followed
the ICCVAM protocol using five animals per group instead of following OECD TG 429, whichallows using four animals per group, there would only be a savings of one animal for substancesthat were negative. He stated that the goal of ECVAM was actually to halve the number ofanimals by omitting the mid- and low-dose groups and that this would achieve significant
animal savings since the likely prevalence of non-sensitizers is approximately two-thirds ofchemicals tested and non-sensitizers would not require further testing even if dose response
information for sensitizers was needed.
Dr. Basketter also mentioned that the retrospective evaluation of the LLNA being presented to
the Panel analyzed whether the top dose could identify a substance as a sensitizer and how thatcompares to the traditional LLNA’s performance. Since the traditional LLNA assay wasdetermined to be positive or negative based on a stimulation index (SI) of three, it isproblematic if the focus is on statistics when using the five-animal model as this would requirealso going back and re-evaluating all the preceding data using the statistical approach.
Dr. McDougal responded to Dr. Basketter’s comment by stating that one wouldn’t have to go
back and retrospectively re-evaluate previous data but that new data generated could beanalyzed statistically. This approach would include determining if the treatment group was
statistically different from the vehicle control group and then determining the biologicalrelevance. This might help to eliminate irritants.

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Panel Conclusions and Recommendations:Dr. Luster asked the Panel to review the conclusions and recommendations for the LLNA limit
dose procedure they had discussed earlier and to make any revisions, if necessary. One
particular question that was asked during the Panel’s conclusions and recommendations waswhether an OECD TG existed for the LLNA limit dose procedure. Dr. Stokes indicated that theOECD TG would need to be updated to allow for the provision of a limit dose procedure andthat’s why the Panel’s conclusions and recommendations are even more relevant. Dr. Stokes
indicated that ICCVAM has already submitted a proposal to update the OECD TG based on theoutcome of these deliberations and recommendations from the IWG.
The Panel agreed to use the term weight-of-evidence to refer to existing information that would
aid the LLNA limit dose procedure in identifying a substance as a sensitizer or a non-sensitizer.
The Panel also discussed the use of concurrent positive controls and recommended that alaboratory that is proficient at conducting the limit dose procedure can test a positive control atroutine intervals rather than concurrently (although the Panel did not identify what constituted
routine intervals). The Panel also discussed the use of individual versus pooled data and agreed
with the ICCVAM-recommended protocol that individual animal data should always becollected. The Panel concluded that individual animal response data are necessary in order toallow for statistical analyses of any differences between treated and control data. In addition,
having data from individual animals also allows for identification of technical problems andoutlier animals within a dose group. Dr. Luster asked the Panel if they agreed with the changes
and revisions made at this point and with the Panel conclusions and recommendations aspresented and revised. The Panel unanimously agreed. The Panel’s detailed recommendations
and conclusions on the LLNA limit dose procedure are included in their final Panel report31.
Overview of the Draft Addendum for the Applicability Domain of the LLNA and Draft
ICCVAM Test Method Recommendations
Dr. Eleni Salicru, Integrated Laboratory Systems, Inc. (the NICEATM support contractor),summarized the information provided in the draft ICCVAM Addendum to the ICCVAM LLNA
report (1999). This Addendum provided an updated assessment of the validity of the LLNA for
testing the sensitizing potential of mixtures, metals, and aqueous solutions. The database used
for this evaluation contained traditional LLNA data submitted as part of the original LLNA
evaluation (ICCVAM 1999), data extracted from peer-reviewed articles published after theoriginal evaluation, and data submitted to NICEATM in response to the FR notice (72 FR
27815, May 17, 2007) requesting such data. Dr. Salicru then summarized the performance
characteristics of the LLNA when used to test mixtures, metals, and aqueous solutions32, as wellas the draft ICCVAM test method recommendations for each of the three categories of test
substances33.

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Panel Evaluation:
Dr. McDougal, on behalf of his Evaluation Group, presented for consideration by the entire
Panel the draft responses to the questions asked of the Panel by ICCVAM. The Panel thendiscussed the completeness of the draft ICCVAM Addendum, identified any errors andomissions, and reviewed the draft ICCVAM test method recommendations with regard to theability of the LLNA to be used to test the sensitizing potential of mixtures, metals, and aqueous
solutions. The Panel discussion and their recommended revisions to each section of the draft
ICCVAM Addendum are reflected in the Panel report, published in May 200834. During thePanel’s evaluation of the LLNA’s applicability domain, the difficulty of testing metals in the
LLNA was discussed and Dr. Woolhiser asked if testing metals was also problematic in theguinea pig. Dr. Api indicated that with the metals, most of the data has come from the clinicalexperience because animal studies are not predicting accurately what is happening in the clinic.
Dr. Maibach indicated that metals have been tested in the guinea pig and that they are sensitizedeasily. Dr. Maibach further commented that metals in man need to be patch-tested for clinicalrelevance at a level close to the irritant dose and that a thoughtful series of algorithms isnecessary to determine this. He also pointed out that patch test results to some metals (e.g.,nickel, palladium) may indicate that a cell mediated reaction is occurring (i.e., contact allergy)
but it needs to be sorted out if this cell mediated reaction actually results in a disease (i.e.,allergic contact dermatitis) and this is where the LLNA could prove useful.
With regard to mixtures, Dr Api commented that based on her experience, when the mixture
tested in the LLNA contains a predominant material (loosely defined that as greater than 70
percent) then the LLNA for the mixture mirrors what occurs for that one material. Whenevidence indicates that the substance is a true mixture, some times the LLNA does what is
expected and other times the results are unexpected. In those cases, a weight-of-evidence
approach (e.g., structure-activity relationships, clinical evidence) is employed.
Public Comments:
Dr. Charles Hastings, BASF Corporation
Dr. Hastings, representing CropLife America (an industry association of companies in the crop
protection business), provided an overview of current activities in industry related to the use of
the LLNA to detect dermal sensitizers and the global issues that are of importance. Dr. Hastings
mentioned that CropLife America’s primary concern is the testing of pesticide mixtures andformulations. He stated that they support the use of the LLNA for testing the dermal
sensitization of mixtures and formulations as well as single ingredients.
Dr. Hastings mentioned that in the United States, EPA OPPTS (Office of Prevention, Pesticides
and Toxic Substances) Guideline 870.260035 allows for the use of the LLNA as the preferredalternative to the standard guinea pig test. Based on this recommendation, member companies
of CropLife America conducted a large number of LLNA studies for both active ingredients34

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ICCVAM Test Method Evaluation Report: Appendix F2and formulations in the European Union (E.U.) and were at the point of submitting data in the
United States, as well. Then, in early 2007, they were informed that EPA had concerns about
the validity of using the LLNA to test mixtures and formulations, and were advised to
discontinue using this test method for that purpose until it had been adequately validated. Dr.
Hastings stated that, in contrast to the EPA, E.U. regulators consider the LLNA acceptable fortesting pesticide formulations and actually prefer it to a guinea pig test.
Dr. Pieters asked if the E.U. has conducted any evaluations of the validity of the LLNA for
testing mixtures and formulations. Dr. Hastings replied that he was not certain if they had
performed an extensive evaluation or not but that the E.U. considered the LLNA a validated
method and therefore likely considered it appropriate to test not only the active ingredient but
also the formulation or mixture.
Dr. Hastings mentioned that one concern in terms of using the LLNA for testing mixtures orformulations, particularly in the E.U., is the testing of aqueous substances. Many of the industry
formulations are aqueous-based and may be incompatible with traditional LLNA vehicles. The
European Crop Protection Association sponsored a study that evaluated the use of an aqueous
vehicle known as Pluronic L92, which helps adhere the test material to the mouse ear. In the
study, they tested three aqueous pesticide formulations that contained known sensitizers, using
Pluronic L92 as the vehicle. As expected, the test results demonstrated sensitizing activity.
Regarding global considerations, Dr. Hastings mentioned that if the LLNA is not accepted for
mixture/formulation testing in the United States, industry will have no choice but to conductboth the LLNA, with 18 to 24 animals, and a guinea pig test, with 20 to 30 animals, for each
formulation they may develop for global distribution. This scenario counters the ICCVAM goalof “reducing, refining, and replacing” animal use in regulatory safety testing.
Dr. Hastings ended with the following conclusions:•

CropLife America believes the LLNA test can be used for pesticide formulations.

•

CropLife America supports the efforts of EPA and ICCVAM to confirm thevalidity of the LLNA for testing mixtures/formulations and encourages a quick
evaluation.

•

CropLife America is willing to help, as needed.

•

If and, when, it is determined that the LLNA is acceptable, CropLife America
requests that EPA notify them so they can then begin conducting the LLNA again
for the United States.

Dr. Api asked if CropLife America has data comparing pesticides that have been evaluated in
the LLNA and in guinea pigs and/or humans. Dr. Hastings replied that they do and thatgenerally there is not much discrepancy with guinea pig test results. Occasionally they mightsee a false positive compared to a guinea pig test, but he did not recall ever seeing a false
negative. In most cases, they would feel comfortable accepting an occasional false positive
because human health is still protected.

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Dr. David Basketter, ECVAM Observer
Dr. Basketter stated that he had personal reservations about testing complex mixtures andformulations in assays that were designed for testing substances (e.g., the LLNA) since nosingle test has ever been validated for testing mixtures. On another point, he stated that most of
the metals of importance have been tested in both the guinea pig and the LLNA and the “right”
answers have been generated. Thus, it does not seem worthwhile to produce new tests withrevised protocols for hazard and potency categorization for testing metals.
Panel Conclusions and Recommendations:Dr. Luster asked the Panel if they agreed with the comments and recommendations that weremade earlier during the Panel discussion. The Panel agreed with the draft ICCVAM
recommendation for continued collection of information from traditional LLNA evaluations of
mixtures, metals, and aqueous solutions with comparative data for guinea pig (i.e., guinea pig
maximization test [GPMT] or Buehler test [BT]) and human (i.e., human maximization test
[HMT] or human repeat insult patch test [HRIPT]) tests. However, the Panel suggested that,given resource limitations, it would be important to organize the recommendations based onrelative priority. Dr. Luster asked the Panel if they agreed with this suggestion aboutprioritization of activities; all members of the Panel agreed with one abstention. Dr. Howard
Maibach abstained from voting stating that he hoped this public meeting and the subsequent
Panel report would emphasize to industry the need for them to submit more data on mixtures,
metals, and aqueous substances in order to provide a clearer evidence of the validity of the
LLNA in testing these types of substances. The Panel’s detailed recommendations andconclusions on the applicability domain of the LLNA are included in their final Panel report36.
Method Description and Overview of the LLNA: Daicel Adenosine Triphosphate(LLNA: DA) Test Method
Dr. Kenji Idehara, Daicel Chemical Industries, Ltd. (private limited company), summarized thetechnical aspects of the LLNA: DA test method. He described the LLNA: DA as a nonradioisotopic version of the LLNA method in which lymph node adenosine triphosphate (ATP)content is used as a measure of cell proliferation instead of radiolabeled thymidine
incorporation. Dr. Idehara indicated that the LLNA: DA was developed six years ago at Daicel
Chemical Industries, Ltd., and that they use the test method regularly for in-house assessmentsof the skin-sensitization potential of chemical materials, intermediates, or products. He
summarized the protocol differences between the LLNA: DA and the traditional LLNA. In the
LLNA: DA, the application site is treated with 1% sodium lauryl sulfate (SLS) one hour before
each test substance (or vehicle control) application, and the test substance is applied to the testsite on day 7 as well as on days 1, 2, and 3. The auricular lymph nodes are excised fromindividual animals on day 8 rather than on day 6 and the amount of ATP in the lymph nodes ismeasured with a luciferin-luciferase assay. Dr. Idehara mentioned that these modifications (i.e.,
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proliferation in order to achieve an SI = 3 in the LLNA: DA, which allows for a more directcomparison to the traditional LLNA.
Dr. Idehara mentioned that after excision, ATP content gradually decreased with time.
Therefore, the overall assay time for measuring ATP content needs to be similar (i.e., withinapproximately 30 minutes) among all test animals. He noted that this was an important point forthis method and recommended that the LLNA: DA be conducted by at least two persons. Dr.
Idehara mentioned that ATP content assays are conducted using commercially available kits,
and his laboratory has experience with two different commercial sources in Japan, Kikkoman
and Lonzar.
Overview of the Draft LLNA: DA BRD and Draft ICCVAM Test Method
Recommendations
Dr. Allen then presented an overview of the draft ICCVAM BRD for the LLNA: DA test
method. He mentioned that the draft ICCVAM BRD provided a comprehensive review of theavailable data and information regarding the usefulness and limitations of the LLNA: DA todistinguish between sensitizers and non-sensitizers, compared to the traditional LLNA. The
objective of the BRD was to describe the current validation status of the LLNA: DA test
method, including its relevance and reliability, scope of substances tested, and the availabilityof a standardized protocol.
Dr. Allen mentioned that the data analyzed in the BRD included data provided by Daicel
Chemical Industries, Ltd., on 31 substances tested at their laboratories. In addition, data for 14
different coded substances were generated from a two-phased interlaboratory validation study
that included 17 total labs. Taken together, the total database represented in the LLNA: DA
BRD included 33 different substances. Dr. Allen briefly summarized the performance
characteristics of the LLNA: DA test method, which is detailed in the draft ICCVAM BRD37.
Dr. Allen concluded by briefly summarizing the draft ICCVAM test method recommendations
for the LLNA: DA test method38.
Panel Evaluation:
Dr. Michael Woolhiser thanked the Panel members of his Evaluation Group (i.e., Drs. Nathalie
Alépeé, Thomas Gebel, Sidney Green [not present], and Jean Regal) for their tireless efforts inreviewing their Evaluation Group's assigned documents. He also thanked the NICEATM staff
for their technical support during the review process. Dr. Woolhiser then presented the draft
responses to ICCVAM’s questions about this test method for consideration by the entire Panel.
This included their review of the draft BRD for errors and omissions, their overall assessmentof the validation status of the test method, and their comments on the draft ICCVAM test
method recommendations. The Panel discussion and their recommended revisions to eachsection of the draft ICCVAM BRD are reflected in the Panel report, published in May 200839.

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ICCVAM Test Method Evaluation Report: Appendix F2Adjournment
The meeting was adjourned for the day at 5:03 p.m., to reconvene at 8:30 a.m., Wednesday,
March 5, 2008.

Wednesday, March 5, 2008
Reconvening of the Panel Meeting
Dr. Luster reconvened the Panel Meeting at 8:30 a.m. He introduced himself and then asked
that all Panel members, followed by all others in attendance, introduce themselves as well.
Overview of the Draft LLNA: DA BRD and Draft ICCVAM Test Method
Recommendations
Panel Evaluation:
Dr. Woolhiser continued his presentation from the previous day of the draft responses to
ICCVAM’s questions to the Panel, for consideration by the entire Panel. The Panel discussion
and their recommended revisions to each section of the draft ICCVAM BRD are reflected in the
Panel report, published in May 200840. Dr. Woolhiser indicated that the Evaluation Group had
two main concerns with the LLNA: DA test method. The first concern related to pretreatment
with 1% SLS and understanding how this impacted the biology of the response. Second, thetime course of the study was different than the traditional LLNA because it extended the study
by one day and included an additional challenge. This brought forth a question about theimmunology of the response as it relates to the potential for elicitation and whether or not that isa significant change from the traditional LLNA, which is purely an induction model.
Public Comments:
Dr. George DeGeorge, MB Research Laboratories
In response to a question raised during the Panel discussion, Dr. DeGeorge commented thatusing lymph node weight as the readout to differentiate between sensitizers and non-sensitizers
in the LLNA is problematic because although there are more lymph node cells packed into a
node, each cell has less cytoplasm. The lymph nodes swell to a point, and then excrete water andbecome smaller lymphocytes that are countable. He cited examples from his laboratory withseveral different sensitizers, which demonstrate that lymphocytes in the node are smaller when alarge SI (e.g., SI = 25) is obtained relative to when a smaller SI (e.g., SI = 3) is obtained.
Dr. DeGeorge also commented that he agreed with a point made during the Panel discussionthat the LLNA: DA method and the LLNA: Bromodeoxyuridine Detected by ELISA (LLNA:
BrdU-ELISA) method should be considered separately, because they are so dissimilar.
In his final comment, Dr. DeGeorge stated that in the traditional LLNA, in the LLNA:
Bromodeoxyuridine Detected by Flow Cytometry (LLNA: BrdU-FC), and probably also in the
LLNA: DA, strong sensitizing substances do not need to be administered three times. For
instance, if one administers with a single, moderately high dose of dinitrochlorobenzene
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ICCVAM Test Method Evaluation Report: Appendix F2(DNCB) (i.e., one that would induce an SI of 20 to 40) and then measure lymph node cellproliferation on day 1, 2, 3, or 4, an increase in the number of cells in the node and the numberof cells that are positive for BrdU would likely be observed. Thus, administrations of additional
applications have the potential to cause cumulative irritation. Dr. DeGeorge stated that the
LLNA: DA method, which extends the assay to eight days instead of six days, should evaluate
what happens to lymph node cell number at earlier sample times. In addition, if the animals
receive just one application using a high dose, with or without the SLS, is there an increase in
the SI? If so, that would lead to the possibility that the extra applications are not necessary andmight lead to cumulative irritation.
Dr. David Basketter, ECVAM Observer
Dr. Basketter made a statement that from a clinical perspective, substances are typicallydescribed as significant sensitizers or not significant sensitizers, and within that latter groupsome of the substances may indeed be non-sensitizing. Thus, just because a substance has beenshown in an isolated case report to be a human sensitizer does not mean that there is sufficientevidence to consider it as positive for comparison with outcomes of predictive assays. It has to
be of sufficient importance (i.e., potency) to trigger a positive classification. Dr. Basketter
mentioned SLS, methyl salicylate, and isopropanol, as substances which will always be
positive in some human cases although they shouldn't be positive in a predictive assay.
Dr. Basketter also commented that caution should be given to making sensitization assumptions
based on chemical class references. As an example, eugenol and isoeugenol are structurally
similar and have similar physical properties, but they act by different chemical reaction
mechanisms and could fit into distinctly different chemical classes.
Dr. Basketter’s last comment acknowledged that much work has been done in terms ofvalidating the traditional LLNA. If one makes minor changes to the LLNA in terms of adifferent readout for proliferation, then they benefit from all the experience generated invalidating the traditional LLNA and less effort is needed to prove that the minor modification isvalid. In contrast, if more significant modifications are made, one cannot rely on that same
experience. Dr. Basketter cautioned that more importance should be placed on distinguishingwhether something has changed substantially enough such that you can no longer rely on the
traditional LLNA as a reference.
Dr. Masahiro Takeyoshi, Chemicals Evaluation and Research Institute
Dr. Takeyoshi made a short presentation about differences in LLNA sensitization
responsiveness among different strains of mice. He mentioned that this was an important issue
when evaluating the modified LLNA methods being developed in Japan. He showed
differences in responsiveness among three different mouse strains commonly used in Japan(i.e., BALB/cAnN, CBA/JN, and CD-1) tested with parabenzoquinone in his group’s nonradioactive LLNA (i.e., LLNA: BrdU-ELISA). The data indicated that the CBA/JN mousestrain exhibited a higher responsiveness, as indicated by an increased SI, to parabenzoquinone
than the other two mouse strains tested. Based on these results, CBA/JN mice were chosen for
testing substances in the LLNA: BrdU-ELISA test method. Dr. Takeyoshi also indicated thatbased on evaluating different SI cutoffs in the LLNA: BrdU-ELISA, 2F-178

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mercaptobenzothiazole, 3-(4-isopropylphenyl)isobutyraldehyde, and hydroxycitronellal, had
low responsiveness (i.e., SI values). He noted that 2-mercaptobenzothiazole is an OECD TG
429 recommended positive control for the LLNA however repeat tests could not detect thissubstance as positive when using an SI value of 1.7 or more. Dr. Takeyoshi suggested that asubstance-specific lower response might exist in the test system. Dr. Takeyoshi also
summarized LLNA data by Dr. Ullmann and coworkers with the contract lab RCC, Ltd. inwhich they investigated the responsiveness of six different mouse strains (CBA/CaOlaHsd,
CBA/Ca (CruBR), CBA/Jlbm (SPF), CBA/JNCrj, BALB/c and NMRI) to 25% 2mercaptobenzothiazole. The data indicated that CBA/JNCrj mice showed markedly lower
responsiveness compared to the other strains tested. These studies indicate that strain relateddifferences would not be negligible with regard to measuring different endpoints of cellular
proliferation in the LLNA because depending on the chemicals tested, responsiveness might
be potentially impacted. For instance, some of the discordance seen in the LLNA: DA test
method (e.g., 2-mercaptobenzothiazole) could be a strain specific effect.
Panel Conclusions and Recommendations:Dr. Luster asked the Panel to review their conclusions and recommendations and discuss any
revisions, if necessary. The Panel viewed the difference in treatment schedule between the
LLNA: DA and the traditional LLNA to potentially be significant if the treatment schedule forthe LLNA: DA corresponds to entering the elicitation phase of skin sensitization. The Panelwas concerned that the 1% SLS pretreatment step in the LLNA: DA might modify the inherent
sensitivity of the LLNA. They recommended that the test method developer (Daicel Chemical
Industries, Ltd.) justify the use of 1% SLS or consider an alternative decision criterion (i.e., an
SI threshold other than three) such that the 1% SLS pretreatment is no longer necessary. Dr.
Luster asked the Panel if they agreed with the recommendations and conclusions that the Panelmade along with the revisions; unanimously, the Panel agreed. The Panel’s detailed
recommendations and conclusions on the LLNA: DA test method are included in their final
Panel report.41
Method Description and Overview of the LLNA: BrdU-FC Test Method
Dr. George DeGeorge, MB Research Laboratories, presented an overview of the LLNA: BrdUFC test method. He stated that mice are dosed topically on the ears once daily for threeconsecutive days (i.e., days 1, 2, and 3), just like the traditional LLNA protocol. On day 6, themice receive an intraperitoneal injection with bromodeoxyuridine (BrdU), and five hours later,
the auricular lymph nodes are removed. The lymph nodes from individual animals areprocessed and, using flow cytometry, the number of BrdU-positive cells are counted fromtreated animals and compared to control animals as a measure of lymph node cell proliferation.
Dr. DeGeorge described in detail how the cells are processed and gated for flow cytometricanalysis. He mentioned that the cells are also permeabilized and treated with propidium iodide
which allows gates to be drawn around the G0, G1, S, and G2M phases of the cell cycle. Dr.
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DeGeorge projected specific examples of flow cytometry plots and histograms for DNCB,
hexyl cinnamic aldehyde (HCA), and positive and negative control data.
Dr. DeGeorge also described the tiered protocol for the assessment of sensitization potential
using the LLNA: BrdU-FC and how ear swelling measurements and additional
immunophenotypic endpoints (i.e., the enhanced LLNA: BrdU-FC) aid in distinguishing skinirritants from an irritating sensitizer.
Overview of the Draft LLNA: BrdU-FC BRD and Draft ICCVAM Test Method
Recommendations
Dr. Judy Strickland, Integrated Laboratory Systems, Inc. (the NICEATM support contractor),presented an overview of the draft ICCVAM BRD for the LLNA: BrdU-FC test method. She
stated that the draft ICCVAM BRD provided a comprehensive review of the available data andinformation regarding the usefulness and limitations of the LLNA: BrdU-FC test method.
Specifically, the test method was reviewed for its ability to distinguish between sensitizers andnon-sensitizers compared with the traditional LLNA. The objective of the BRD was to describe
the current validation status of the LLNA: BrdU-FC test method, including its relevance andreliability, scope of substances tested, and the availability of a standardized protocol.
Dr. Strickland indicated that MB Research Laboratories submitted data to NICEATM for the 48
substances analyzed in the BRD in response to an FR notice (72FR27815, May 17, 2007) thatrequested such data. Dr. Strickland briefly summarized the performance characteristics of the
LLNA: BrdU-FC test method, which is detailed in the draft ICCVAM BRD42, and the draft
ICCVAM test method recommendations for the LLNA: BrdU-FC test method43.
Panel Evaluation:
Dr. Raymond Pieters, on behalf of his Evaluation Group, presented the Evaluation Group's
review of the draft BRD and the draft test method recommendations for the LLNA: BrdU-FC
test method. Specifically, he presented the draft responses to ICCVAM’s questions to the Panelfor consideration by the entire Panel. This included their review of the draft BRD for errors andomissions, their overall assessment of the validation status of this test method, and theircomments on the draft ICCVAM test method recommendations. The Panel discussion and theirrecommended revisions to each section of the draft ICCVAM BRD are reflected in the Panelreport, published in May 200844. The applicability of the draft ICCVAM-recommended LLNA
performance standards to the LLNA: BrdU-FC test method was discussed, particularly withregard to the number of substances tested in the LLNA: BrdU-FC method and whether moredata would be necessary for review before the validation status of the assay could bedetermined. Dr. Stokes reminded the Panel that the proposed LLNA performance standards
didn't exist when the studies for the LLNA: BrdU-FC test method were performed. The
42
43
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questions should be whether the adequacy of the substances that have been tested is sufficientor if more studies need to be done to cover any gaps that might exist (e.g., range of potencies or
activity, chemical classes).
Public Comments
Dr. David Basketter, ECVAM Observer
Dr. Basketter commented on the statement that Dr. DeGeorge made during his overview of the
LLNA: BrdU-FC test method that HCA is irritating. He said that he is not convinced it is asignificant irritant. Based on previous data, they had to use 50% HCA in a 48 hour occlusive
application in the guinea pig in order to produce a mildly irritating response. Dr. Api added to
Dr. Basketter’s comment by stating that RIFM has also not found HCA to be an irritant whentested up to 20% in humans.
Dr. Basketter also commented that in the draft BRD for the LLNA: BrdU-FC, resorcinol wasnoted to be negative in the traditional LLNA and this is not correct. Dr. Basketter’s grouppublished results in 2007 in the journal Contact Dermatitis that resorcinol is clearly positive inthe traditional LLNA when tested at higher concentrations and therefore this should becorrected for the record.
Dr. George DeGeorge, MB Research Laboratories
Dr. DeGeorge wanted to clarify that the LLNA: BrdU-FC test method was compared to the
traditional LLNA to determine if the LLNA: BrdU-FC was more predictive of skinsensitization potential. He stated that in some cases it was better while in others it wasn't, but
overall, using human data as the gold standard reference, the LLNA: BrdU-FC exceeded the
traditional LLNA predictivity values and accuracy. He also noted that the additional endpoints
included in the LLNA: BrdU-FC allow for them to distinguish irritating substances thattypically are considered false positives in the LLNA.
Dr. DeGeorge also noted that since the LLNA: BrdU-FC is so similar to the traditional LLNA
the issue of refinement and reduction in animal use is not immediately apparent but if the assay
is done in as few as four mice per group with a periodic positive control (e.g., every six months)
this represents a significant decrease in animal numbers compared to guinea pig tests.
Furthermore, there is a refinement since mice are phylogenitically lower than guinea pigs, andundergo less pain and distress during the assay than guinea pigs undergo.
With regard to the discussion of coefficients of variation (CVs) and the 0.5x to 2.0x EC3 (i.e.,
the estimated concentration needed to produce a stimulation index of three) range, Dr.
DeGeorge suggested that a larger range might be more reasonable because the current range islikely too restrictive.
Dr. George also noted that ICCVAM requires interlaboratory validation if a test method is to betransferred to other laboratories. With regard to the LLNA: BrdU-FC, it is a “me-too” assay andonly has “minor” changes from the traditional LLNA and is currently only used in one
laboratory. Therefore, the current dataset should suffice for determining the validity of the
LLNA: BrdU-FC. In response to Dr. DeGeorge’s comment, Dr. Stokes stated that if a method isonly proposed to be used by one laboratory, having only intralaboratory data certainly would
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suffice but if it was proposed for broader use (e.g., adopted or endorsed by regulatory
authorities), then other laboratories would have to demonstrate interlaboratory reproducibility.
Dr. Luster asked if there was any mechanism available so that a company or small laboratory
could apply for funding to help support an inter-laboratory validation. Dr. Stokes indicated thatthey could nominate the test method for additional validation studies to ICCVAM. It would go
through a nomination review process and a prioritization would be given to that. The nomination
would then be considered by the member agencies as to whether funding would be provided.
Panel Conclusions and Recommendations:Dr. Luster asked the Panel to review their conclusions and recommendations and discuss any
revisions, if necessary. The Panel suggested that the utility of ear swelling or other methods todetect inflammation appeared warranted for inclusion in every variation of the LLNA
(including the traditional LLNA), but should be further investigated before routine inclusion in
the protocol is recommended. The Panel further agreed that the draft ICCVAM test method
recommendations for future studies highlighted the unanswered questions raised by theavailable data set. Specifically, conducting interlaboratory studies as a part of the validation
process is important.
The Panel considered the immunological markers suggested for the LLNA: BrdU-FC to beappropriate, but noted that other immunological markers for discrimination of irritant versus
sensitization phenomena were also available. In general, for any future work, efforts should bemade to decrease the variability and to thereby increase the power of the test in order to ensurethat more animals were not needed relative to the traditional LLNA or other modified LLNA
protocols.
Dr. Luster asked the Panel to indicate if they agreed with the recommendations and conclusions
that the Panel made along with the revisions; the Panel unanimously agreed. The Panel’s
detailed recommendations and conclusions on the LLNA: BrdU-FC test method are included intheir final Panel report.45
Method Description and Overview of the LLNA: BrdU-ELISA Test Method
Dr. Masahiro Takeyoshi, Chemicals Evaluation and Research Institute, presented an overview
of the LLNA: BrdU-ELISA test method. He stated that the LLNA: BrdU-ELISA test method isvery similar to the traditional LLNA test method. Unique to the LLNA: BrdU-ELISA test
method, after test substance applications on days 1, 2, and 3, BrdU is injected interperitoneallyon day 5. Approximately 24 hours after the BrdU injection, lymph nodes are collected, anddetection of the amount of BrdU incorporated into the DNA of lymph node cells is conductedwith an ELISA.
In the development process of this method, experiments were conducted to detect the mostefficient injection schedule of BrdU. Based on the various injection schedules tested, a single
injection protocol on day four was identified as the optimal injection schedule for BrdU
administration.
45

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Dr. Takeyoshi then showed a video of laboratory personnel preparing the lymph node cells for
BrdU detection by ELISA. He went on to describe data for the LLNA: BrdU-ELISA comparedto the traditional LLNA and how performance could be improved using alternative decision
criteria (i.e., an SI other than three as the threshold for a positive response).
Overview of the Draft LLNA: BrdU-ELISA BRD and Draft ICCVAM Test Method
Recommendations
Dr. Salicru presented an overview of the draft ICCVAM BRD for the LLNA: BrdU-ELISA test
method. She noted that the draft ICCVAM BRD provided a comprehensive review of theavailable data and information regarding the usefulness and limitations of the LLNA: BrdUELISA test method. Specifically, the test method was reviewed for its ability to distinguishbetween sensitizers and non-sensitizers compared with the traditional LLNA and guinea pig test
methods. The objective of the BRD was to describe the current validation status of the LLNA:
BrdU-ELISA test method, including its relevance and reliability, scope of substances tested,
and the availability of a standardized protocol.
Dr. Salicru stated that data from a total of 29 substances were considered in the accuracy
analysis for the LLNA: BrdU-ELISA, and they were all tested in one laboratory. Dr. Salicru
briefly summarized the performance characteristics of the LLNA: BrdU-ELISA test method,which are detailed in the draft ICCVAM BRD46, and the draft ICCVAM test method
recommendations for the LLNA: BrdU-ELISA test method47.
Panel Evaluation:
Ms. Kim Headrick, presented her Evaluation Group’s (Drs. Anne Marie Api, Howard Maibach,Peter Theran, and Stephen Ullrich), review of the draft BRD and draft ICCVAM test method
recommendations for the LLNA: BrdU-ELISA test method. Specifically, she presented the draft
responses to ICCVAM’s questions to the Panel for consideration by the entire Panel. This
included their review of the draft BRD for errors and omissions, their overall assessment of thevalidation status of the test method, and their comments on the draft ICCVAM test method
recommendations. The Panel discussion and their recommended revisions to each section of thedraft ICCVAM BRD are reflected in the Panel report, published in May 2008.48
Public Comments:
Dr. David Basketter, ECVAM Observer
Dr. Basketter noted that when the traditional LLNA was first suggested as an alternative to theguinea pig tests, it went through a comprehensive validation process, and one of the concerns
was that it should perform reliably and distinctly better than the guinea pig assays. He
emphasized that this point should be kept in mind when thinking about the modified LLNA
protocols with alternative end points that are currently being reviewed. He stated that thecurrent rigor of examination for the modified LLNA protocols being reviewed for validation is46

http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/BrdUELISAbrd07Jan08.pdf
http://iccvam.niehs.nih.gov/methods/immunotox/llna-ELISA/BrdUELISARecs07Jan08FD.pdf
48
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47

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ICCVAM Test Method Evaluation Report: Appendix F2higher than that for the traditional LLNA. He speculated that in the not-too-distant future, in
vitro alternatives are likely to be going through a similar review process and it is going tobecome ever more difficult to put these alternatives in place, not because there is ill-will against
the selections but because of the high standard of being good scientists. Thus, it is importantthat pragmatic decisions are made using the tools that are available.
Dr. George DeGeorge, MB Research Laboratories
Dr. DeGeorge commented that he agreed with Dr. Basketter’s statements. He said that based on
his experience in this peer review process, it is unlikely that he would bring any of the three invitro test methods that MB Research Laboratories is developing for consideration by ICCVAM,
given the many high hurdles that have to be negotiated.
In response to the comments by Drs. Basketter and DeGeorge, Dr. McDougal commented that it
does not seem unreasonable to raise the bar for what is expected of new or modified tests. Dr.
Luster added that understandably, the focus on animal refinement and reduction is paramount,but that as scientists we have to ensure that the bar is maintained sufficiently high so that as theyears go by scientific quality is not compromised.
Panel Conclusions and Recommendations:Dr. Luster asked the Panel to review their conclusions and recommendations and discuss any
revisions, if necessary. The Panel concluded that the available data and test method
performance for the LLNA: BrdU-ELISA support the draft ICCVAM test method
recommendations that it may be useful for identifying substances as potential skin sensitizers
and non-sensitizers, but that more information and existing data must be made available before
the LLNA: BrdU-ELISA can be recommended for use. The Panel also stated that a detailed
protocol was needed, in addition to sufficient quantitative data for broader analysis on a larger
set of balanced reference substances that take into account physicochemical properties andsensitization potency, as well as an appropriate evaluation of interlaboratory reproducibility.
The Panel’s main concern with this test method was that the accuracy of the LLNA: BrdUELISA at SI ≥ 3 was inadequate and not equivalent to the traditional LLNA. Furthermore,
although using a decision criterion of SI ≥ 1.3 improved the test’s performance in identifying
sensitizers from non-sensitizers, it did not resolve concerns about the test method, particularly
considering that power calculations suggest a much larger number of animals per group would
be required to identify a positive response. Thus, the Panel also concluded that it might be more
appropriate to use a statistically based decision criterion rather than a stimulation index toclassify substances as sensitizers, and that this should be further investigated. Dr. Luster asked
the Panel to indicate if they agreed with the recommendations and conclusions that the Panelmade along with the revisions; unanimously, the Panel agreed. The Panel’s detailed
recommendations and conclusions on the LLNA: BrdU-ELISA test method are included in their
final Panel report49.

49

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Overview of the Draft ICCVAM Performance Standards for the LLNA
Dr. Allen presented an overview of the draft ICCVAM Performance Standards for the LLNA.
He briefly summarized the overall purpose of performance standards (i.e., to provide a basis forevaluating the performance of a proposed test method that is mechanistically and functionallysimilar to the validated test method) and the three elements encompassed within such
performance standards (i.e., essential test method components, a minimum list of reference
substances, and accuracy/reliability values). He noted that the proposed applicability of thesedraft ICCVAM LLNA performance standards is for the evaluation of LLNA protocols thatdeviate from the ICCVAM-recommended LLNA protocol only with respect to the method forassessing lymphocyte proliferation (e.g., using non-radioactive instead of radioactive reagents).
Dr. Allen then provided an overview of the essential test method components, the minimum listof reference substances, and the accuracy/reliability values as detailed in the draft ICCVAM
LLNA Performance Standards50.
Panel Evaluation:
Dr. Woolhiser, on behalf of his Evaluation Group, presented the Evaluation Group’s responses
to the ICCVAM questions asked about the draft ICCVAM LLNA Performance Standards forthe entire Panel to consider. The overall question for the Panel was whether these performancestandards were considered adequate for assessing the accuracy and reliability of test method
protocols that were based on similar scientific principles and that measured the same biologicaleffect as the traditional LLNA. The Panel discussion and their recommended revisions to thedraft ICCVAM LLNA Performance Standards are reflected in the Panel report published in
May 200851.
Adjournment
The meeting was adjourned at 5:42 p.m., to reconvene at 8:30 a.m., Thursday, March 6, 2008.

Thursday, March 6, 2008
Reconvening of the Panel Meeting
Dr. Luster reconvened the Panel Meeting at 8:30 a.m. He introduced himself and then asked
that all Panel members and all others in attendance introduce themselves as well.
Overview of the Draft ICCVAM LLNA Performance Standards
Panel Evaluation:
Dr. Woolhiser reviewed some of the important points highlighted during the previous day's
discussion on this topic, and then continued to summarize the remaining comments of his
Evaluation Group on the questions asked by ICCVAM on the draft ICCVAM LLNA
Performance Standards for consideration by the entire Panel. As mentioned above, the Panel

50
51

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discussion and their recommended revisions to the draft ICCVAM LLNA PerformanceStandards are reflected in the Panel report published in May 200852.
Dr. Woolhiser noted was that there were general comments on the topic order for the Panel’s
review. He asked if Dr. Stokes would comment on the rationale for the topic order. Dr. Stokes
indicated that as the IWG deliberated the order of topics for this review, consideration wasgiven to the fact that the three non-radioactive methods had undergone validation studies prior
to the creation of LLNA performance standards. Thus, the non-radioactive test methods werereviewed before the performance standards, so as to not bias the Panel’s assessment of each test
method’s performance. The performance standards could then be considered for theirapplication to future test methods.
Public Comments:
Dr. Amy Rispin, EPA
Dr. Rispin stated that her intent was to provide some additional regulatory perspective on someof the points that have been discussed. When Federal agencies evaluate the validation status ofa test method under ICCVAM, they conduct a comprehensive analysis of overall performance(i.e., accuracy and reliability) in the context of making regulatory decisions with data from thetest method. Thus, in a regulatory situation, equal or greater accuracy compared to the reference
test method is the expectation. If the number of animals can be decreased only at the expense of
accuracy, the acceptability of such a test method for the particular regulatory purpose would
need to be carefully considered. Certain methods, instead of being complete replacements,
might have to be relegated to the role of screens, where positives would be accepted, but
negatives would require further testing - a less than ideal situation.
Dr. Rispin commented that performance standards are the regulating agencies basis for theacceptability of variations of accepted test methods. If an agency receives data from a modified
LLNA method that has not been reviewed and validated in the ICCVAM process, there isunlikely to be a comprehensive peer review of it within the agency, given resource limitations.
Therefore, the question of major versus minor departures from the functional criteria is
important to ICCVAM and its member agencies. One cannot anticipate that there will beanything other than these performance standards to adequately evaluate the usefulness andlimitations of a new method.
Dr. David Basketter, ECVAM Observer
Dr. Basketter first commented on a point that Dr. Thomas Gebel alluded to during the Panel’s
discussion of the draft ICCVAM LLNA Performance Standards ,which was that if a newlaboratory performed the traditional LLNA to assess 18 or 22 chemicals, they probably
wouldn’t get a complete match. Dr. Basketter disagreed with Dr. Gebel’s statement and viewedthat a competent laboratory performing the LLNA would get it 100% correct.
Dr. Basketter then provided some comments that he stated were "from the ECVAM perspective.”
He stated that the ECVAM performance standards tried to address adhering to a standard52

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protocol and that any change to the protocol other than the method for evaluating lymph node
proliferation (e.g., strain, species, number of applications, time) was considered not to be minor,
and therefore such a protocol would not be applied to these performance standards. By restricting
the performance standards to minor changes, ECVAM was trying to minimize the number ofchemicals required to evaluate sensitivity. Furthermore, the EC3 value could be used to see if thetest method could classify substances in the appropriate range of sensitization potency.
ECVAM initially chose their reference substances in order to determine whether a modified
method (differing only in the method for measuring cell proliferation) would give the sameanswer as the traditional LLNA. Thus, there was no intent to compare to the guinea pig or
human data.
Dr. Basketter speculated that it is doubtful that data from multiple LLNA studies on the same
substance are available and therefore it is unlikely that much larger sample sizes from which to
calculate mean EC3 values and associated ranges will be obtained.
Dr. Basketter concluded by stating that ECVAM will not include more false positives and false
negatives in its list. It has included one false positive and false negative in order to harmonize
with ICCVAM but they don’t see an added statistical value of just having one more false
positive and false negative.
Karen Hamernik, EPA
Dr. Hamernik concurred with the comments that Dr. Rispin made previously, that performancestandards, if developed such that they are too generalized with respect to minor versus majorchanges, would be problematic for regulatory agencies when they are reviewing submissions
that include data from a modified LLNA protocol. Dr. Hamernik also asked for clarificationfrom the Panel on a statement made during their discussions that a test for concordance formeasuring the accuracy of classification (i.e., yes/no answer) should be done and that achemical-for-chemical match is not necessary. Dr. Flournoy responded that concordance is not
absolute but a continuum. Dr. Luster further clarified that the Panel discussion was based on thefact that the traditional LLNA is not a perfect match when compared to the guinea pig tests.
Because there are false negatives and false positives compared to the guinea pig, there should
be some flexibility so that an absolute chemical-by-chemical match is not required. In addition,a scientifically valid explanation can be provided for any discordance. Dr. Stokes emphasizedthat this was an important point and that additional clarity on the differences between achemical-by-chemical match and overall accuracy need to be carefully considered before thefinal test method accuracy requirements are defined.
Panel Conclusions and Recommendations:Dr. Luster asked the Panel to review the conclusions and recommendations for the ICCVAM
LLNA performance standards they had discussed earlier and to make any revisions, if
necessary. The Panel indicated that modified LLNA protocols that are undergoing validation
should contain essential test method components that follow the ICCVAM-recommended

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protocol53, unless adequate scientific rationale for deviating from this protocol was provided.
The Panel also identified aspects of the LLNA that should be required as part of the test method
validation process, if more extensive changes to the protocol are being considered: (1)application of the test substance to the skin with sampling of the lymph nodes draining that site,(2) measurement of cell proliferation in the draining lymph node, (3) absence of a skin reactionthat could be indicative of the onset of the elicitation phase of skin sensitization, (4) datacollected at the level of the individual animal to allow for an estimate of the variance within
control and treatment groups,54 and (5) if dose response information is needed, there are anadequate number of dose groups (n ≥ 3) with which to accurately characterize the dose response
for a given test substance.
The Panel also recommended that statistical tests to analyze the data might allow for a moreaccurate interpretation. They recommended that a suitable variance-stabilizing transformation(e.g., log transformation, square root transformation) be applied in all statistical analyses and inreporting summary standard deviations. The Panel also recommended that a more rigorous
evaluation be conducted of what would be considered an appropriate range of ECt values (i.e.,estimated concentration needed to produce a stimulation index that is indicative of a positive
response) to include as a requirement. This would be a statistical evaluation that considers thevariability of ECt values generated among the sensitizers included on the performancestandards reference substances list and the statistical multiple comparisons problem.
Dr. Luster asked the Panel if they agreed with the changes and revisions made at this point andwith the Panel conclusions and recommendations as presented and revised. The members of the
Panel agreed with one abstention; Dr. McDougal abstained from voting stating that he still had
a concern about what constitutes a “major/minor” change. The Panel’s detailed
recommendations and conclusions on the ICCVAM LLNA performance standards are includedin their final Panel report.55
Overview of the Draft LLNA Potency Determinations BRD and Draft ICCVAM Test
Method Recommendations
Dr. Strickland presented an overview of the draft ICCVAM BRD for the use of the LLNA to
determine skin-sensitization potency. She mentioned that the draft ICCVAM BRD provided acomprehensive review of the available data and information regarding the usefulness andlimitations of the LLNA as a stand-alone assay for hazard categorization of skin-sensitization
potency. In the BRD, the LLNA was evaluated for its ability to categorize substances for skinsensitization potency using EC3 values.
Dr. Strickland noted that the analyses conducted in the BRD were based on LLNA studies
obtained from ICCVAM (1999), the published literature, and data received in response to an FR
notice (72 FR 27815, May 17, 2007) requesting original data from the LLNA. As a result, theanalyzed data included 170 substances with LLNA, human, and/or guinea pig data. Dr.
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Individual animal data will allow the application of a formal statistical test, if deemed necessary, and will also
allow power calculations associated with the modified LLNA test.
55
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Strickland noted that three sets of data were analyzed and briefly summarized the results which
are detailed in the draft ICCVAM BRD.56 Dr. Strickland also briefly summarized the draft
ICCVAM test method recommendations for potency determinations.57
Panel Evaluation:
Ms. Headrick presented her Evaluation Group’s draft responses to ICCVAM’s questions to the
Panel for consideration by the entire Panel. This included their review of the draft BRD forerrors and omissions, their overall assessment of the validation status of the test method, andtheir comments on the draft ICCVAM test method recommendations. The Panel discussion andtheir recommended revisions to each section of the draft ICCVAM BRD and recommendations
are reflected in the Panel report published in May 2008.58
During the course of the discussion on the potency applicability of the LLNA, Dr. Woolhiser
asked what the basis for the human threshold concentration cutoff values of 250 and 500µg/cm2 were. Dr. Wind replied that a number of experts and clinicians from throughout theworld went back and looked at what, in their countries, they demarcated as strong sensitizers.
The proposed Globally Harmonized System of Classification and Labeling of Chemicals (GHS)
subcategory guidance values for the LLNA, guinea pig tests (GPMT, BT) and human data
(HMT and HRIPT) were made on the basis of an impact analysis of 175 chemicals. In addition,
the two proposed cut-offs were evaluated by the GHS Expert Group on Sensitization based
upon chemicals already regulated as strong sensitizers to ensure their inclusion within the GHS
categorization scheme. Clinical members of the Expert Group also confirmed relevance of the
cut-off values such that clinically important skin sensitizers fell into the appropriate
subcategory. The proposed guidance values were also in line with the European Commission’s
Expert Working Group recommendations.
Public Comments:
Dr. David Basketter, ECVAM Observer
Dr. Basketter commented that reviewing the potency data by splitting it into pooled andunpooled groups could be interesting but might be difficult since the majority of available data
likely comes from pooled groups. Furthermore, much of the deliberation concluding that
individual animal data must be used, was derived from analyses based only or largely on pooleddata from four animals.
Dr. Basketter further stated that he viewed the analyses, which make the assumption that thehuman threshold data is the gold standard, as fundamentally flawed. Human data comes fromstudies conducted at different times, with different protocols, according to varying quality
standards, and by different people. Therefore, there is no definitive knowledge of the
reproducibility of the data. However, he considers the analyses adequate for recommending the
LLNA as a part of a weight-of-evidence decision on human sensitization potency
categorizations.
56

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58
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Dr. Amy Rispin, EPA
Dr. Rispin noted that there has been much discussion about various ways of handling thepotency data. The OECD expert task force on skin sensitization needs to see an analyticalcomparison of what is considered to be the most appropriate approach for evaluating the data.
The question for categorization purposes is, What is the ideal testing modality for separatingstrong versus weak sensitizers for potency categorization? A regulator who must assign acategorization is going to be confronted with all available test data and must know which data
should be given the greatest weight in their evaluation.
Dr. Rispin noted that the OECD task force also reviewed the draft BRD on potency
determinations and sent a list of several questions to the Panel, some of which have beenanswered, many of which have not been. One of the questions is, can the LLNA protocols be
refined (e.g., by selection of solvents or choice of other test parameters) to improve correlation?
She concluded by noting that she hopes that the additional analyses that the Panel has suggested
will bring some clarity to the matter.
Panel Conclusions and Recommendations:Dr. Luster asked the Panel to review the conclusions and recommendations for the LLNA
potency determinations they had discussed earlier and to make any revisions, if necessary. The
Panel agreed with the draft ICCVAM recommendation that the LLNA should not be used as astand-alone assay for categorizing skin sensitizers as strong versus weak, but that it could be
used as part of a weight-of-evidence evaluation (e.g., along with quantitative structure-activity
relationships, peptide reactivity, human evidence, historical data from other experimental
animal studies) for this purpose. The Panel also agreed with ICCVAM’s recommendation thatany LLNA studies conducted for the purpose of evaluating skin-sensitization potency should
use the ICCVAM-recommended LLNA protocol. In addition, the Panel stated that the relevant
testing guidelines for the traditional LLNA should be revised to include the procedure forcalculating an EC3 value. Dr. Luster asked the Panel if they agreed with the changes andrevisions made at this point and with the Panel conclusions and recommendations as presented
and revised; the Panel unanimously agreed. The Panel’s detailed recommendations andconclusions on the LLNA potency determinations are included in their final Panel report.59
Concluding Remarks
Dr. Luster, on behalf of the Panel, thanked the NICEATM-ICCVAM staff for their continued
assistance during the review process and the Panel meeting. He also thanked Drs. Joanna
Matheson and Abby Jacobs, the IWG co-chairs, and Dr. Marilyn Wind, ICCVAM Chair and
IWG member, for the hard work they put into the project. Dr. Luster also thanked the Panel and
the Panel Chairs for their involvement in the huge task of reviewing seven topics. He
commented that, for future reference for ICCVAM, the Panel in their individual groups wereable to do a good job in reviewing the materials, but because they were so focused on their
particular topics due to serious time constraints, there may not have been the full benefit of theirexpertise for other topics in all cases.
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Drs. Wind and Stokes thanked the Panel again for their hard work, thoughtful and objective
deliberations, and advice. Dr. Stokes further thanked the invited test method developers fortheir excellent summaries of their method for the benefit of the Panel, and CPSC for hosting thePanel meeting. He mentioned that there has been discussion about obtaining additional existingdata (i.e., on mixtures, on one or more of the non-radiolabeled test methods), and that should
these data become available in a timely manner and if NICEATM is able to assimilate andanalyze the data, the Panel might be reconvened by teleconference to review the data. Dr.
Stokes concluded by saying he looked forward to further working with the Panel members tocomplete their Panel report.
Adjournment
The meeting was adjourned and concluded at 3:20 p.m.

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Appendix G
Federal Register Notices and Public Comments
G1 Federal Register Notices..........................................................................................G-3
G2 Public Comments Received in Response to Federal Register Notices .................G-17
G3 Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) Comments: SACATM Meeting on June 18-19, 2008 ......................G-51

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G-2

ICCVAM Test Method Evaluation Report: Appendix G1Appendix G1Federal Register Notices
G1-1 72 FR 27815 (May 17, 2007)—The Murine Local Lymph Node Assay:
Request for Comments, Nominations of Scientific Experts, andSubmission of Data ............................................................................................. G-5
G1-2 72 FR 52130 (September 12, 2007)—Draft Performance Standards for the
Murine Local Lymph Node Assay: Request for Comments ............................. G-9
G1-3 73 FR 1360 (January 8, 2008)—Announcement of an Independent
Scientific Peer Review Panel Meeting on the Murine Local Lymph Node
Assay; Availability of Draft Background Review Documents; Request forComments ......................................................................................................... G-11
G1-4 73 FR 29136 (May 20, 2008)—Peer Review Panel Report on the Validation
Status of New Versions and Applications of the Murine Local Lymph
Node Assay (LLNA): A Test Method for Assessing the Allergic Contact
Dermatitis Potential of Chemicals and Products: Notice of Availability andRequest for Public Comments.......................................................................... G-15

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G-4

ICCVAM Test Method Evaluation Report: Appendix G1Federal Register / Vol. 72, No. 95 / Thursday, May 17, 2007 / Notices

27815(NIEHS), National Institutes of Health(NIH).
ACTION: Request for comments,
submission of relevant data, andnominations of scientific experts.
SUMMARY: The Interagency Coordinating

National Toxicology Program (NTP)
Interagency Center for the Evaluationof Alternative Toxicological Methods(NICEATM); the Murine Local Lymph
Node Assay: Request for Comments,
Nominations of Scientific Experts, andSubmission of Data

Committee on the Validation of
Alternative Methods (ICCVAM) received
a nomination from the U.S. Consumer
Product Safety Commission (CPSC) toevaluate the validation status of: (1) The
murine local lymph node assay (LLNA)
as a stand-alone assay for determining
potency (including severity) for the
purpose of hazard classification; (2) the‘‘cut-down’’ or ‘‘limit dose’’ LLNA
approach; (3) non-radiolabeled LLNA
methods; (4) the use of the LLNA for
testing mixtures, aqueous solutions, andmetals; and (5) the current applicability
domain (i.e., the types of chemicals andsubstances for which the LLNA has
been validated). ICCVAM reviewed thenomination, assigned it a high priority,and proposed that NICEATM andICCVAM carry out the following
activities in its evaluation: (1) Initiate areview of the current literature andavailable data, including the preparationof a comprehensive background review
document, and (2) convene a peer
review panel to review the various
proposed LLNA uses and procedures forwhich sufficient data and information
are available to adequately assess theirvalidation status. ICCVAM also
recommends development of
performance standards for the LLNA. At
this time, NICEATM requests: (1) Publiccomments on the appropriateness andrelative priority of these activities, (2)nominations of expert scientists toconsider as members of a possible peer
review panel, and (3) submission of data
for the LLNA and/or modified versionsof the LLNA.
DATES: Submit comments, data, andnominations by June 15, 2007. Relevant
data will also be accepted after this dateand considered when feasible.
ADDRESSES: Dr. William S. Stokes,
NICEATM Director, NIEHS, P.O. Box12233, MD EC–17, Research Triangle
Park, NC 27709, (fax) 919–541–0947,(e-mail) niceatm@niehs.nih.gov. Courier
address: NICEATM, 79 T.W. Alexander
Drive, Building 4401, Room 3128,
Research Triangle Park, NC 27709.
Responses can be submitted
electronically at the ICCVAM–NICEATM Web site: http://
iccvam.niehs.nih.gov/contact/
FR_pubcomment.htm or by e-mail, mail,or fax.

AGENCY: National Institute of

FOR FURTHER INFORMATION CONTACT:

Environmental Health Sciences

Other correspondence should be

DEPARTMENT OF HEALTH AND
HUMAN SERVICES

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Federal Register / Vol. 72, No. 95 / Thursday, May 17, 2007 / Notices

directed to Dr. William S. Stokes (919–541–2384 or niceatm@niehs.nih.gov).
SUPPLEMENTARY INFORMATION:
Background
ICCVAM previously evaluated thevalidation status of the LLNA as a standalone alternative method to the Guinea
Pig Maximization Test (GPMT) and theBuehler Assay (NIH publication No. 99–
4494; available at http://
iccvam.niehs.nih.gov/methods/
immunotox/llna.htm). Based on thisevaluation, ICCVAM recommended the
LLNA as a valid substitute for theguinea pig methods for most testing
situations. The Environmental
Protection Agency, Food and Drug
Administration, and the CPSC
subsequently accepted the method as avalid substitute. The OECD also adopted
the LLNA as OECD Test Guideline 429.
In January 2007, the CPSC submitted
a nomination to NICEATM (http://
iccvam.niehs.nih.gov/SuppDocs/
submission.htm) requesting thatICCVAM assess the validation status of:• The LLNA as a stand-alone test forpotency determinations (including
severity) for the purpose of hazard
classification.
• LLNA protocols that do not require
the use of radioactive materials.
• The LLNA ‘‘cut-down’’ or ‘‘limit
dose’’ procedure.
• The ability of the LLNA to test
mixtures, aqueous solutions, andmetals.
• The current applicability domain(i.e., the types of chemicals andsubstances for which the LLNA has
been determined to be useful).
Since 2003, ICCVAM has routinely
developed performance standards fortest methods; however, they were notdeveloped for the LLNA, which wasreviewed in 1999. Accordingly,
ICCVAM proposes to now develop
performance standards for the LLNA.
Performance standards communicate
the basis by which new proprietary andnonproprietary test methods have been
determined to have sufficient relevanceand reliability for specific testing
purposes. Performance standards basedon test methods accepted by regulatory
agencies can be used to evaluate thereliability and relevance of other test
methods that are based on similarscientific principles and measure or
predict the same biological or toxic
effect. On January 24, 2007, ICCVAM
unanimously endorsed with a highpriority: (1) Developing performancestandards for the LLNA and (2)initiating a review of the available dataand information associated with the
CPSC nominated activities. A
determination of which (if any) of thenominated activities will move forward
will be made subsequent to this review
and after consideration of comments by
the public and the Scientific Advisory
Committee on Alternative Toxicological
Methods (SACATM). If a decision ismade to proceed with evaluation ofthese test methods, ICCVAM and
NICEATM propose convening a peer
review panel to review the usefulness
and limitations of each of the LLNA
methods listed above. The panel would
also formulate conclusions on theadequacy of draft ICCVAM performancestandards, any proposed future
validation studies, and draft ICCVAMproposed standardized test method
protocols.
Request for Public Comments andNominations of Scientific Experts
NICEATM requests public commentson the appropriateness and relative
priority of the nominated activities.
NICEATM also requests thenominations of scientists with relevant
knowledge and experience to serve on
the panel if a panel meeting occurs.
Areas of relevant expertise include, but
are not limited to: physiology,
pharmacology, immunology, skinsensitization testing in animals,
development and use of in vitro
methodologies, biostatistics, knowledge
about the use of chemical datasets forvalidation of toxicity studies, andhazard classification of chemicals andproducts. Each nomination should
include the person’s name, affiliation,
contact information (i.e., mailingaddress, e-mail address, telephone andfax numbers), curriculum vitae, and abrief summary of relevant experienceand qualifications.
Request for Data
NICEATM invites the submission ofdata from standard LLNA testing (i.e.,OECD TG 429) with mixtures, aqueous
solutions, and/or metals, as well as
corresponding data from human andother animal studies. In addition,
NICEATM invites the submission ofdata supporting the use of (1) the LLNA
as a stand-alone test for determining
potency (including severity) for the
purpose of hazard classification, (2) the
LLNA ‘‘cut-down’’ or ‘‘limit dose’’
procedure, and (3) LLNA protocols that
do not require the use of radioactivity.
Although data can be accepted at any
time, data submitted by June 15, 2007,
will be considered during the ICCVAM
evaluation process. Submitted data will
be used to further evaluate theusefulness and limitations of the LLNA
and may be incorporated into future
NICEATM and ICCVAM reports andpublications as appropriate. The data

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will also be included in a database tosupport the investigation of other test
methods for assessing skin sensitization.
When submitting chemical andprotocol information/test data, pleasereference this Federal Register noticeand provide appropriate contact
information (name, affiliation, mailingaddress, phone, fax, e-mail, andsponsoring organization, as applicable).
NICEATM prefers data to besubmitted as copies of pages from study
notebooks and/or study reports, if
available. Raw data and analyses
available in electronic format may also
be submitted. Each submission for achemical should preferably include thefollowing information, as appropriate:• Common and trade name.
• Chemical Abstracts Service Registry
Number (CASRN).
• Chemical class.
• Product class.
• Commercial source.
• LLNA protocol used.
• Individual animal responses.
• The extent to which the study
complied with national or international
Good Laboratory Practice (GLP)
guidelines.
• Date and testing organization.
• Sensitization data from other test
methods.
Consideration by SACATM
On June 12, 2007, SACATM will meetat the Marriott Bethesda North Hoteland Conference Center in Bethesda,
Maryland. The agenda includes
consideration of the nominated LLNA
activities, priorities, and proposed
activities http://ntp.niehs.nih.gov/go/7441) and an opportunity for oral publiccomments. The SACATM meeting wasannounced in a separate Federal
Register notice (Federal Register Vol.
72, No. 83, pp. 23831–32, May 1, 2007).
Background Information on ICCVAM
and NICEATMICCVAM is an interagency committee
composed of representatives from 15
federal regulatory and research agencies
that use or generate toxicologicalinformation. ICCVAM conducts
technical evaluations of new, revised,and alternative methods with regulatory
applicability and promotes the scientificvalidation and regulatory acceptance of
toxicological test methods that moreaccurately assess the safety and hazardsof chemicals and products and thatrefine, reduce, or replace animal use.
The ICCVAM Authorization Act of 2000
(42 U.S.C. 285l–3, available at http://
iccvam.niehs.nih.gov/about/
PL106545.htm) establishes ICCVAM as apermanent interagency committee of theICCVAM Test Method Evaluation Report: Appendix G1Federal Register / Vol. 72, No. 95 / Thursday, May 17, 2007 / Notices
NIEHS under NICEATM. NICEATM
administers ICCVAM and provides
scientific and operational support for
ICCVAM-related activities. NICEATM
and ICCVAM work collaboratively toevaluate new and improved test
methods applicable to the needs offederal agencies. Additional information
about ICCVAM and NICEATM isavailable on the following Web site:
http://iccvam.niehs.nih.gov.
Dated: May 8, 2007.
David A. Schwartz,
Director, National Institute of Environmental
Health Sciences and National Toxicology
Program.
[FR Doc. E7–9544 Filed 5–16–07; 8:45 am]
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Federal Register / Vol. 72, No. 176 / Wednesday, September 12, 2007 / Notices
DEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Toxicology Program (NTP)
Interagency Center for the Evaluationof Alternative Toxicological Methods(NICEATM); Draft PerformanceStandards for the Murine Local Lymph
Node Assay: Request for Comments
AGENCY: National Institute ofEnvironmental Health Sciences(NIEHS), National Institutes of Health(NIH).
ACTION: Request for comments.
SUMMARY: The Murine Local Lymph

Node Assay (LLNA) is the firstalternative test method evaluated andrecommended by the Interagency
Coordinating Committee on the
Validation of Alternative Methods(ICCVAM). It was subsequently
accepted by regulatory authorities todetermine the allergic contact dermatitis
potential of chemicals and products. In
January 2007, the U.S. Consumer
Product Safety Commission (CSPC)
submitted a nomination requesting thatNICEATM and ICCVAM assess thevalidation status of (1) The LLNA as astand-alone assay for potency
determination for hazard classification
purposes; (2) modified LLNA protocols;(3) the LLNA limit test; (4) the use of
LLNA to test mixtures, aqueous
solutions, and metals; and (5) theapplicability domain for LLNA. In orderto facilitate the review of the modified
LLNA protocols, ICCVAM proposeddeveloping performance standards for
the LLNA. In May 2007, a Federal
Register notice was published (Vol. 72,
No. 95, pages 27815–27817, May 17,2007) requesting comments and data
relevant to these nominated activities.
In June 2007, the Scientific Advisory
Committee on Alternative Toxicological
Methods (SACATM) endorsed thenominated activities as high priorities
for ICCVAM. In response to SACATM
comments, along with those provided
by the public in response to theprevious Federal Register notice,
ICCVAM also endorsed these activities
as high priorities. ICCVAM
subsequently prepared draft
performance standards for the LLNA
and now requests public comments on
this draft document, which is available
on the NICEATM/ICCVAM Web site at:(http://iccvam.niehs.nih.gov/methods/
immunotox/immunotox.htm) or bycontacting NICEATM (see FOR FURTHERINFORMATION CONTACT below).
DATES: Submit comments on or before
October 29, 2007.
ADDRESSES: Dr. William S. Stokes,
NICEATM Director, NIEHS, P.O. Box

G-912233, MD EC–17, Research Triangle
Park, NC 27709, (fax) 919–541–0947, (email)niceatm@niehs.nih.gov. Courier address:
NICEATM, 79 T.W. Alexander Drive,
Building 4401, Room 3128, Research
Triangle Park, NC 27709. Responses canbe submitted electronically at the
ICCVAM–NICEATM Web site: http://
iccvam.niehs.nih.gov/contact/
FR_pubcomment.htm or by e-mail, mail,or fax.
FOR FURTHER INFORMATION CONTACT:Other correspondence should bedirected to Dr. William S. Stokes (919–541–2384 or niceatm@niehs.nih.gov).
SUPPLEMENTARY INFORMATION:
Background
The LLNA is an alternative test
method used for skin sensitization
testing that reduces the number ofanimals needed, reduces the timerequired for testing, and cansubstantially reduce or avoid pain anddistress associated with traditionalguinea pig testing methods. The LLNA
was the first alternative test method
evaluated and recommended by
ICCVAM and based on therecommendations of ICCVAM and an
independent scientific peer review
panel, the LLNA has been accepted by
U.S. and international regulatory
authorities as an alternative to theguinea pig maximization test andBuehler test for assessing allergic
contact dermatitis (EPA 2003; ISO 2002;
OECD 2002). Since 2003, ICCVAM has
routinely developed performancestandards for test methods; however,
because the concept of performancestandards was not developed by
ICCVAM until 2003, they were notdeveloped during the ICCVAM
evaluation of the LLNA in 1998 (NIH
Publication No. 99–4494, available:(http://iccvam.niehs.nih.gov/docs/
immunotox_docs/llna/llnarep.pdf).
In January 2007, CSPC submitted anomination requesting that NICEATM
and ICCVAM assess the validation
status of (1) The LLNA as a stand-alone
assay for potency determination forclassification purposes; (2) modified
LLNA protocols; (3) the LLNA limit test;(4) the use of LLNA to test mixtures,aqueous solutions, and metals; and (5)the applicability domain for LLNA.
ICCVAM endorsed the nomination andalso decided to develop performancestandards to facilitate evaluation ofmodified LLNA protocols to the
traditional LLNA. In May 2007, aFederal Register notice was publishedrequesting comments and data relevantto these activities (Vol. 72, No. 95, pages
27815–27817, May 17, 2007; available,

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Federal Register / Vol. 73, No. 5 / Tuesday, January 8, 2008 / Notices

DEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Institutes of HealthNational Toxicology Program (NTP);NTP Interagency Center for the
Evaluation of Alternative Toxicological
Methods (NICEATM); Announcementof an Independent Scientific Peer
Review Panel Meeting on the Murine
Local Lymph Node Assay; Availabilityof Draft Background Review
Documents; Request for Comments
AGENCY: National Institute ofEnvironmental Health Sciences(NIEHS), National Institutes of Health(NIH).
ACTION: Meeting announcement andrequest for comments.

pwalker on PROD1PC71 with NOTICESSUMMARY: NICEATM in collaboration

with the Interagency Coordinating
Committee on the Validation of
Alternative Methods (ICCVAM)
announces an independent scientificpeer review panel meeting to evaluate
modifications and new applications for
the Murine Local Lymph Node Assay(LLNA). The LLNA is an alternative test
method that can be used to determine
the allergic contact dermatitis potentialof chemicals and products. The panel
will review the following:• The validation status of threemodified LLNA test method protocols
that use non-radioactive probe
chemicals.
• The validation status of a LLNA
limit dose procedure.
• The use of the LLNA to test
mixtures, aqueous solutions, and metals(applicability domain for the LLNA).
• The use of the LLNA to determine
potency (potential for causing allergic
contact dermatitis).
• Revised draft recommended
performance standards for the LLNA.
At this meeting, the panel will peer
review the draft background review
documents and revised draft LLNA
performance standards for each topic
and evaluate the extent that establishedvalidation and acceptance criteria havebeen appropriately addressed. The
panel will also comment on the extent

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pwalker on PROD1PC71 with NOTICES

that the review documents support draft
ICCVAM recommendations on proposed
test method protocols, proposed uses of
the LLNA, and the revised draft LLNA
performance standards.
NICEATM invites public commentson the draft background review
documents, draft ICCVAM test
recommendations, draft test method
protocols, and revised draft LLNA
performance standards. All documents
will be available on the NICEATM–ICCVAM Web site at http://
iccvam.niehs.nih.gov/methods/
immunotox/immunotox.htm by January
8, 2008.
DATES: The meeting is scheduled forMarch 4–6, 2008, from 8:30 a.m. to 5
p.m. each day. The meeting is open to
the public free of charge, withattendance limited only by the space
available. In order to facilitate planningfor this meeting, persons wishing toattend are asked to register by February
20, 2008, via the NICEATM–ICCVAM
Web site (http://iccvam.niehs.nih.gov/
contact/reg_LLNAPanel.htm). The
deadline for written comments is
February 22, 2008.
ADDRESSES: The meeting will be held at
the U.S. Consumer Product Safety
Commission (CPSC) Headquarters,
Bethesda Towers Bldg., 4330 East West
Highway, Bethesda, MD.
FOR FURTHER INFORMATION CONTACT:
Comments may also be submitted via
the NICEATM–ICCVAM Web site at
http://iccvam.niehs.nih.gov/contact/FR_
pubcomment.htm. Comments or othercorrespondence can be sent to Dr.
William S. Stokes, NICEATM Director,
NIEHS, P.O. Box 12233, MD EC–17,
Research Triangle Park, NC, 27709,(phone) 919–541–2384, (fax) 919–541–
0947, (e-mail) niceatm@niehs.nih.gov.
Courier address: NICEATM, 79 T.W.
Alexander Drive, Building 4401, Room
3128, Research Triangle Park, NC 27709.
SUPPLEMENTARY INFORMATION:
Background
The LLNA is a reduction andrefinement alternative test method forskin sensitization testing because it
reduces the number of animals needed
and can substantially reduce or avoid
pain and distress compared to
traditional guinea pig testing methods
for sensitization. The LLNA was the firstalternative test method evaluated andrecommended by ICCVAM (NIH
Publication No. 99–4494, available at:
http://iccvam.niehs.nih.gov/docs/
immunotox_docs/llna/llnarep.pdf).
Based on the recommendations of
ICCVAM and an independent scientificpeer review panel, U.S. andinternational regulatory authorities haveaccepted the LLNA as an alternative tothe guinea pig maximization test andBuehler test for assessing allergic
contact dermatitis (ISO 2002; OECD
2002; EPA 2003). This review will
evaluate the potential for broader use of
the LLNA for regulatory testing ofchemicals and products for allergic
contact dermatitis potential, enabling
further reduction and refinement (lesspain and suffering) of animal use forthis purpose. In January 2007, the CPSC
submitted a nomination requesting thatNICEATM and ICCVAM assess thevalidation status of (1) the LLNA as astand-alone assay for potency
determination for hazard classification
purposes; (2) modified LLNA protocols;(3) the LLNA limit test; (4) the use of the
LLNA to test mixtures, aqueous
solutions, and metals; and (5) theapplicability domain for the LLNA. In
June 2007, the Scientific Advisory
Committee on Alternative Toxicological
Methods (SACATM) endorsed these
activities as high priorities for ICCVAM.
NICEATM on behalf of ICCVAM alsosought input from the public on these
activities (Federal Register: Vol. 72, No.
95, pages 27815–27817, May 17, 2007).
After considering these inputs, ICCVAM
endorsed these activities as highpriorities. ICCVAM is also developing
performance standards to facilitate
evaluation of modified LLNA protocols
compared to the traditional LLNA.
Although ICCVAM has routinely
developed performance standards fortest methods since 2003, they were notdeveloped as part of the ICCVAM
evaluation of the LLNA in 1998. These
draft performance standards for the
LLNA were made public and comments
were requested via the Federal Register(Vol. 72, No. 176, pages 52130–52131,
Sept. 12, 2007). The May 2007 Federal
Register notice requested data fromstudies using the LLNA or modified
versions of the LLNA.
Drawing on the submitted data andliterature sources, ICCVAM and
NICEATM drafted background review
documents for each of the modifications
and new applications of the LLNA.
ICCVAM has also developed draft test
method recommendations regarding the
proposed usefulness, limitations, andvalidation status of these test methods.
ICCVAM will convene an independentscientific panel to peer review the draft
background review documents for thetest methods and determine whether thedata and analyses in the draft
documents support the draft ICCVAM
test method recommendations. The
panel will also be asked to comment on
the adequacy of the revised draft
performance standards, proposed futurestudies, draft standardized test method
protocols, and recommended reference
substances. NICEATM will ask thepanel to consider all available
information, including the scientificstudies cited in the draft review
documents, public comments, and any
new information identified during thepeer review, for developing theirconclusions and recommendations.
Peer Review Panel Meeting
The purpose of this meeting is toconduct a scientific peer review of therevised draft performance standards andan evaluation of modifications and new
applications for the LLNA. The LLNA isan alternative test method that can beused to determine the allergic contact
dermatitis potential of chemicals andproducts. The panel will review thefollowing:• The LLNA as a stand-alone assay
for potency determination for hazard
classification purposes• Modified LLNA protocols• The LLNA limit test
• The use of the LLNA to test
mixtures, aqueous solutions, and metals(applicability domain for the LLNA)• The use of the LLNA to determine
potency (potential for causing allergic
contact dermatitis).
The panel will consider the draft
background review documents for eachof these methods and evaluate theextent that established validation andacceptance criteria are appropriately
addressed for each test method (asdescribed in the ICCVAM document,
Validation and Regulatory Acceptanceof Toxicological Test Methods: A Reportof the ad hoc Interagency Coordinating
Committee on the Validation of
Alternative Methods, NIH Publication
No. 97–981, available at http://
iccvam.niehs.nih.gov/docs/about_docs/
validate.pdf). The panel will thencomment on the extent to which thedraft ICCVAM recommendations aresupported by the information provided
in the background review document foreach topic. It is anticipated that thepanel will address the topics in thefollowing order:
1. The LLNA limit test.
2. The applicability domain of the
LLNA including its suitability for
mixtures, aqueous solutions, andmetals.
3. The LLNA as a stand-alone assay
for potency determination for hazard
classification.
4. The revised draft performancestandards for the LLNA.
5. The modified LLNA test method
protocols using non-radioactive
materials.

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Federal Register / Vol. 73, No. 5 / Tuesday, January 8, 2008 / Notices

Additional information about themeeting, including a roster of the panel
members and the draft agenda, will bemade available two weeks prior to themeeting on the NICEATM-ICCVAM Web
site (http://iccvam.niehs.nih.gov). This
information will also be available afterthat date by contacting NICEATM (seeFOR FURTHER INFORMATION CONTACT

above).
Attendance and Registration
This public meeting will take place
March 4–6, 2008, at the CPSC
Headquarters, Bethesda Towers Bldg.,
4330 East West Highway, Bethesda, MD(an area map, driving directions, and
CPSC contact information are available
at http://www.cpsc.gov/about/contact.html). The meeting will begin at
8:30 a.m. and is scheduled to conclude
at approximately 5 p.m. each day,although adjournment on March 6 may
occur earlier or later depending upon
the time needed for the expert panel tocomplete its work. It is also possiblethat the panel may conclude its
deliberations on March 5 and not needto meet on March 6. Persons needing
special assistance in order to attend,such as sign language interpretation orother reasonable accommodation,
should contact 919–541–2475 (voice),
919–541–4644 TTY (text telephone,through the Federal TTY Relay System
at 800–877–8339), or e-mailniehsoeeo@niehs.nih.gov. Requests
should be made at least seven days inadvance of the event.

pwalker on PROD1PC71 with NOTICES

Availability of the Draft Background
Review Documents and Draft ICCVAM
Recommendations
NICEATM prepared draft backgroundreview documents on each of thesemodifications or applications of the
LLNA that describe the current
validation status of the modified test
methods and applications and containall of the data and analyses supporting
this proposed validation status. The
draft background review documents,
draft ICCVAM test method
recommendations, draft test method
protocols, and revised draft test method
performance standards are available
from the NICEATM–ICCVAM Web site(http://iccvam.niehs.nih.gov/methods/
immunotox/immunotox.htm) or bycontacting NICEATM (see FOR FURTHERINFORMATION CONTACT above).
Request for Public Comments
NICEATM invites the submission of
written comments on the draft
background review documents, draft
ICCVAM test method recommendations,draft test method protocols, and revised
draft test method performancestandards. Written comments should besubmitted preferably electronically via
the NICEATM–ICCVAM Web site or bye-mail (niceatm@niehs.nih.gov); thedeadline for submission of writtencomments is February 22, 2008. Whensubmitting written comments, pleaserefer to this Federal Register notice andinclude appropriate contact information(name, affiliation, mailing address,
phone, fax, e-mail, and sponsoringorganization, if applicable). Writtencomments may also be sent by mail, fax,or e-mail to Dr. William Stokes (see FOR
FURTHER INFORMATION CONTACT above).
All comments received will be placedon the NICEATM–ICCVAM Web site(http://iccvam.niehs.nih.gov) andidentified by the individual’s name andaffiliation or sponsoring organization (ifapplicable). Comments will also be sent
to the panel and ICCVAM agency
representatives and made available atthe meeting.
This meeting is open to the public,
and time will be provided for thepresentation of oral comments by the
public at designated times during thepeer review. Members of the public whowish to present oral statements at themeeting should contact NICEATM (seeFOR FURTHER INFORMATION CONTACT

above) no later than February 20, 2008,
and provide contact information (name,affiliation, mailing address, phone, fax,
e-mail, and sponsoring organization, if
applicable). Up to seven minutes will beallotted per speaker, one speaker perorganization. Persons registering to
make comments are asked to provide
NICEATM a written copy of theirstatement by February 27, 2008, so that
copies can be distributed to the panel
prior to the meeting. If this is notpossible, please bring 40 copies of yourcomments to the meeting for
distribution and to supplement therecord. Written statements can
supplement and expand the oralpresentation.
Summary minutes and the panel’s
final report will be available following
the meeting on the NICEATM–ICCVAM
Web site (http://iccvam.niehs.nih.gov).
ICCVAM will consider the panel’s
conclusions and recommendations andany public comments received when
finalizing their test method
recommendations and performancestandards for these methods.

and alternative methods with regulatory
applicability, and promotes thescientific validation and regulatory
acceptance of toxicological test methods
that more accurately assess the safety
and hazards of chemicals and products
and that refine, reduce, or replace
animal use. The ICCVAM Authorization
Act of 2000 (42 U.S.C. 285l–3, available
at http://iccvam.niehs.nih.gov/docs/
about_docs/PL106545.pdf) establishes
ICCVAM as a permanent interagency
committee of the NIEHS under
NICEATM. NICEATM administers
ICCVAM and provides scientific andoperational support for ICCVAM-related
activities. NICEATM and ICCVAM work
collaboratively to evaluate new andimproved test methods applicable to theneeds of Federal agencies. Additional
information about ICCVAM and
NICEATM is available on the
NICEATM–ICCVAM Web site at http://
iccvam.niehs.nih.gov.
References
EPA. 2003. EPA OPPTS 870.2600 Test
Guideline—Skin Sensitization. Available:
http://www.epa.gov/opptsfrs/publications/
OPPTS_Harmonized/870_Health_Effects_
Test_Guidelines/Drafts/870–2600.pdf.
ISO. 2002. ISO 10993–10 Biologicalevaluation of medical devices—Part 10:
Tests for irritation and delayed-type
hypersensitivity. Geneva: International
Organization for Standardization.
OECD. 2002. OECD Guideline for the Testingof Chemicals—Test Guideline 429: Skin
Sensitization: Local Lymph Node Assay(adopted 24 April 2002). Paris:
Organisation for Economic Co-operation
and Development.
Dated: December 19, 2007.
Samuel H. Wilson,
Acting Director, National Institute ofEnvironmental Health Sciences and National
Toxicology Program.
[FR Doc. E7–25553 Filed 1–7–08; 2:42 pm]
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Background Information on ICCVAM
and NICEATMICCVAM is an interagency committee
composed of representatives from 15
Federal regulatory and research agencies
that use or generate toxicologicalinformation. ICCVAM conducts
technical evaluations of new, revised,

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ICCVAM Test Method Evaluation Report: Appendix G1

29136

Federal Register / Vol. 73, No. 98 / Tuesday, May 20, 2008 / Notices
DEPARTMENT OF HEALTH AND
HUMAN SERVICES
National Toxicology Program (NTP);NTP Interagency Center for the
Evaluation of Alternative Toxicological
Methods (NICEATM); Peer Review
Panel Report on the Validation Statusof New Versions and Applications of
the Murine Local Lymph Node Assay(LLNA): A Test Method for Assessing
the Allergic Contact Dermatitis
Potential of Chemicals and Products:
Notice of Availability and Request for
Public Comments
AGENCY: National Institute ofEnvironmental Health Sciences(NIEHS), National Institutes of Health(NIH).
ACTION: Request for comments.

jlentini on PROD1PC65 with NOTICESSUMMARY: NICEATM, in collaboration

with the Interagency Coordinating
Committee on the Validation of
Alternative Methods (ICCVAM),
convened an independent international
scientific peer review panel on March
4–6, 2008 to evaluate new versions andapplications of the LLNA for assessing
the allergic contact dermatitis potentialof chemicals and products. The peer
review panel (‘‘the Panel’’) report fromthis meeting is now available. The
report contains (1) the Panel’s
evaluation of the validation status of themethods and (2) the Panel’s comments
and conclusions on draft ICCVAM test
method recommendations. NICEATM
invites public comment on the Panel’sreport. The report is available on the
NICEATM–ICCVAM Web site at http://
iccvam.niehs.nih.gov/methods/
immunotox/llna_PeerPanel.htm or bycontacting NICEATM at the address
given below.
DATES: Written comments on the Panelreport should be received by July 7,
2008.
ADDRESSES: Comments should besubmitted preferably electronically via
the NICEATM–ICCVAM Web site at
http://iccvam.niehs.nih.gov/contact/
FR_pubcomment.htm. Comments canalso be submitted by e-mail toniceatm@niehs.nih.gov. Writtencomments can be sent by mail or fax toDr. William S. Stokes, Director,
NICEATM, NIH/NIEHS, P.O. Box 12233,
MD EC–17, Research Triangle Park, NC
27709, (phone) 919–541–2384, (fax)919–541–0947. Courier address:
NICEATM, 79 T.W. Alexander Drive,
Building 4401, Room 3128, Research
Triangle Park, NC 27709.
FOR FURTHER INFORMATION CONTACT: Dr.
William S. Stokes, Director, NICEATM(919–541–2384 or
niceatm@niehs.nih.gov).

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SUPPLEMENTARY INFORMATION:

Background
In January 2007, the Consumer
Product Safety Commission submitted anomination to NICEATM and ICCVAM
to assess the validation status of (1) The
use of the LLNA to determine potency
for hazard classification purposes; (2)
LLNA protocols using non-radioactive
procedures; (3) the LLNA limit dose
procedure; and (4) the use of the LLNA
to test mixtures, aqueous solutions, andmetals (i.e., an updated assessment of
the applicability domain of the LLNA).
In June 2007, the Scientific Advisory
Committee on Alternative Toxicological
Methods (SACATM) endorsed these
activities as high priorities for ICCVAM.
NICEATM, on behalf of ICCVAM, alsosought input from the public on these
activities and requested data fromstudies using the LLNA or modified
versions of the LLNA (Federal RegisterVol. 72, No. 95, pages 27815–27817,
May 17, 2007). After considering allcomments received, ICCVAM endorsed
carrying out these activities as highpriorities. ICCVAM also developed draft
LLNA performance standards tofacilitate evaluation of modified LLNA
protocols that are functionally andmechanistically similar to the
traditional LLNA. These draft LLNA
performance standards were madepublic and comments were requested
via the Federal Register (Vol. 72, No.
176, pages 52130–52131, Sept. 12,2007).
ICCVAM and NICEATM prepareddraft background review documents(BRDs) that provided comprehensive
reviews of available data and relevant
information for each of themodifications and new applications of
the LLNA. ICCVAM also developed
draft test method recommendations
regarding the proposed usefulness andlimitations, standardized protocols, andfuture studies. Both the draft BRDs anddraft recommendations were madeavailable for public comment, and apublic peer review meeting wasannounced in the Federal Register (Vol.
73, No. 5, pages 1360–1362, Jan. 8,
2008).
The Panel met in public session on
March 4–6, 2008. The Panel reviewed
the draft ICCVAM BRDs forcompleteness, errors, and omissions ofany existing relevant data or
information. The Panel evaluated theinformation in the BRDs to determine
the extent to which each of theapplicable criteria for validation andacceptance of toxicological test methods(ICCVAM, 2003) had been appropriately
addressed. The Panel then considered
the ICCVAM draft test method

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20MYN1ICCVAM Test Method Evaluation Report: Appendix G1Federal Register / Vol. 73, No. 98 / Tuesday, May 20, 2008 / Notices
recommendations (i.e., proposed test
method uses, proposed recommended
standardized protocol, proposed test
method performance standards, andproposed additional studies) andcommented on whether therecommendations were supported by
the information provided in the draft
BRDs.
The Panel’s conclusions andrecommendations are detailed in thePeer Review Panel Final Report:
Validation Status of New Versions andApplications of the Murine Local Lymph
Node Assay (LLNA): A Test Method forAssessing the Allergic Contact
Dermatitis Potential of Chemicals andProducts (available at http://
iccvam.niehs.nih.gov/methods/
immunotox/llna_PeerPanel.htm). The
draft BRDs, draft ICCVAM test method
recommendations, and the draft LLNA
Performance Standards are available at
http://iccvam.niehs.nih.gov/methods/
immunotox/immunotox.htm.

jlentini on PROD1PC65 with NOTICES

Request for Comments
NICEATM invites the submission of
written comments on the Panel’s report.
When submitting written comments,
please refer to this Federal Register
notice and include appropriate contact
information (name, affiliation, mailingaddress, phone, fax, e-mail, andsponsoring organization, if applicable).
All comments received will be madepublicly available on the NICEATM–ICCVAM Web site at http://ntpapps.niehs.nih.gov/iccvampb/
searchPubCom.cfm. In addition, there
will be an opportunity for oral publiccomments on the Panel’s report during
an upcoming meeting of SACATM
scheduled for June 18–19, 2008.
Information concerning the SACATM
meeting will be published in a separate
Federal Register notice and available on
the SACATM Web site at http://
ntp.niehs.nih.gov/go/7441.
ICCVAM will consider the Panelreport along with SACATM and publiccomments when finalizing test method
recommendations. An ICCVAM test
method evaluation report, which will
include the final ICCVAM
recommendations, will be forwarded to
relevant Federal agencies for theirconsideration. The evaluation reportwill also be available to the public on
the NICEATM–ICCVAM Web site andby request from NICEATM (seeADDRESSES above).

toxicological information. ICCVAM
conducts technical evaluations of new,revised, and alternative methods withregulatory applicability and promotes
scientific validation, regulatory
acceptance, and national andinternational harmonization of
toxicological test methods that moreaccurately assess safety and hazards ofchemicals and products and that refine,
reduce, and replace animal use. The
ICCVAM Authorization Act of 2000 (42U.S.C. 285l-3, available at http://
iccvam.niehs.nih.gov/docs/about_docs/
PL106545.pdf) established ICCVAM as apermanent interagency committee of the
NIEHS under NICEATM. NICEATM
administers ICCVAM and provides
scientific and operational support for
ICCVAM-related activities. NICEATM
and ICCVAM work collaboratively toevaluate new and improved test
methods applicable to the needs ofFederal agencies. Additional
information about ICCVAM and
NICEATM can be found at the
NICEATM–ICCVAM Web site (http://
iccvam.niehs.nih.gov).
Additional information about
SACATM, including the charter, roster,and records of past meetings, can befound at http://ntp.niehs.nih.gov/go/
167.
References
ICCVAM, 2003, ICCVAM Guidelines for
the Nomination and Submission of New,
Revised, and Alternative Test Methods. NIH
Publication No. 03–4508. Research Triangle
Park, NC: NIEHS. Available at: http://
iccvam.niehs.nih.gov.
Dated: May 8, 2008.
Samuel H. Wilson,
Acting Director, National Institute ofEnvironmental Health Sciences and National
Toxicology Program.
[FR Doc. E8–11195 Filed 5–19–08; 8:45 am]
BILLING CODE 4140–01–P

Background Information on ICCVAM,NICEATM, and SACATMICCVAM is an interagency committee
composed of representatives from 15
Federal regulatory and research agencies
that use, generate, or disseminate

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20MYN129137

ICCVAM Test Method Evaluation Report: Appendix G2Appendix G2
Public Comments Received in Response to Federal Register Notices
Responses to 72 FR 27815 (May 17, 2007)—The Murine Local Lymph Node Assay:
Request for Comments, Nominations of Scientific Experts, and Submission of Data
1. Dr. Michael Olson (GlaxoSmithKline)............................................................... G-19
2. Robert Guest (SafePharm Laboratories, Ltd., Derby, United Kingdom) ............. G-21
3. Dr. Catherine Willett (People for the Ethical Treatment of Animals), Sara
Amundson (Humane Society Legislative Fund), Dr. Martin Stephens (Humane
Society of the United States), Kristie Stoick (Physicians Committee for
Responsible Medicine), Sue Leary (Alternatives Research & Development
Foundation), Tracie Letterman (American Anti-Vivisection Society)................. G-23
4. Data Submissions .............................................................................................. G-29
Responses to 72 FR 52130 (September 12, 2007)—Draft Performance Standards
for the Murine Local Lymph Node Assay: Request for Comments
1. Dr. Catherine Willett (People for the Ethical Treatment of Animals), Sara
Amundson (Humane Society Legislative Fund), Dr. Martin Stephens (Humane
Society of the United States), Kristie Stoick (Physicians Committee for
Responsible Medicine), Sue Leary (Alternatives Research & Development
Foundation), Tracie Letterman (American Anti-Vivisection Society)................. G-31
Responses to 73 FR 1360 (January 8, 2008)—Announcement of an Independent
Scientific Peer Review Panel Meeting on the Murine Local Lymph Node Assay;
Availability of Draft Background Review Documents; Request for Comments
1. Dr. G. Frank Gerberick (Procter & Gamble) ...................................................... G-35
2. Dr. Catherine Willett (People for the Ethical Treatment of Animals) and Kristie
Stoick (Physicians Committee for Responsible Medicine) ................................. G-45
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ICCVAM Test Method Evaluation Report: Appendix G2

Responses to 73 FR 29136 (May 20, 2008)—Peer Review Panel Report on the Validation
Status of New Versions and Applications of the Murine Local Lymph Node Assay(LLNA): A Test Method for Assessing the Allergic Contact Dermatitis Potential ofChemicals and Products: Notice of Availability and Request for Public Comments
No comments received

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ICCVAM Test Method Evaluation Report: Appendix G2Page 1 of 2

Date: Thu, 7 Jun 2007 09:00:44 -0400
Subject: NTP NICEATM Nomination of experts and response to call for data LLNA
Ref.: Federal Register vol. 72 no. 95, p. 27815, 17 May 2007
Dr. Stokes Responding to the request for comment on the US CPSC proposal to ICCVAMNICEATM for evaluation of the validation status of the murine local lymph node
assay, I am pleased to submit the following information for consideration. (Theviews expressed in item 1.) below are solely my own and do not necessarilyreflect the corporate position of GSK.)
1.) Appropriateness and relative priority of items comprising the proposed reviewof the status of the LLNA: It seems entirely justified that the proposed review
should be undertaken based on the large volume of high quality peer-reviewed
information published on performance, data evaluation and proposed protocol
modifications of the LLNA in the period since the original ICCVAM-sponsored
LLNA validation exercise. As proposed by US CPSC, ICCVAM-NICEATM
preparation of a comprehensive background review should precede activation ofa study panel. Regarding the priority of items for the background review aspresented in the Federal Register notice, I suggest that the priority sequence
should be slightly rearranged to highlight items 1, 5, 4, 2 and 3 (as identified in
the Fed. Reg. notice) in priority sequence. Thus, from most to least pressing: 1.
development of data to allow the LLNA to be used as a stand-alone tool indetermining potency / severity of sensitising potential of chemicals; 2. evaluationand extension of the domain of applicability of the LLNA; 3. use of the LLNA for
testing mixtures, aqueous solutions, and metals; 4. development of an animalsparing cut-down approach to the LLNA focused on use of untreated vs. single
high-concentration test group; and 5. assessment of the status of LLNA methods
using non-radiolabeled tracer for end-point analysis.
2.) Nomination of expert scientists to serve on a possible LLNA review panel: I
am pleased to offer the name of my GSK colleague Frederick J. Guerriero as apossible panel member. Mr Guerriero is a key member of the GSK Occupational
Toxicology working group and in this capacity has had the responsibility of
protocol development, study contracting and evaluation of a large number of LLN
assays over the past 7-8 years. In addition, Mr Guerriero has previously servedon the NICEATM study panel which evaluated in vitro alternatives for evaluationof ocular irritant/corrosion effects of chemicals. As a secondary potential
candidate for the study panel, I would also be pleased to volunteer my service
which is based in similar experience to that of Mr. Guerriero.
3.) Submission of LLNA data: Over the past 5 years GSK has transitioned to soleuse of the LLNA as a means for evaluating the sensitising potential of a wide
variety of chemical materials used in the synthesis of pharmaceuticals. The
spectrum of substances which have been evaluated includes commodity
chemicals used as starting materials, proprietary synthetic intermediates ofvarying structural complexity, and active pharmaceutical entities. All of these

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ICCVAM Test Method Evaluation Report: Appendix G2Page 2 of 2

assessments have been conducted by the "traditional" control + 3 concentration
protocol using 3H-thymidine label. A small proportion of materials also havecompanion data evolved with the M&K or Beuhler dermal sensitisation protocol.
Although the composite data are not presently in a readily transmitted form, I
believe that we could be in position to share results of assessment of ca.190
chemicals if materials from the pharmaceutical sector would be of interest in theassessment which NICEATM is planning.
I will send this letter in print form with mailing today. I look forward to your reply indue course.
Sincerely yours Michael J. Olson, Ph.D.
Director, Occupational Toxicology
Corporate Environment, Health and Safety
GlaxoSmithKline

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ICCVAM Test Method Evaluation Report: Appendix G2Page 1 of 2

Subject: FR Notice Comments - 72FR27815 - LLNA
Date: Friday, June 15, 2007 1:43 PM
Dear Dr Stokes,
Safepharm Laboratories Ltd., UK (SPL) has conducted Local lymph node
assays on behalf of sponsoring companies since 1997. The assays have been
conducted on a wide variety of chemicals and chemical preparations. Since
August 2002 the use of other animal models for evaluation of skin sensitisation
potential for regulatory purposes (e.g. methods that require the use of guineapigs) has been permissible in the UK only if a valid scientific reason can beprovided as to why a LLNA cannot be conducted. In effect, the LLNA is the only
method that can be used in the UK for assessment of skin sensitisation
potential forregulatory purposes. We therefore support the proposed activities
of ICCVAM-NICEATM as detailed in the Federal Register vol. 72, No. 95,
p.27815-27817, 17 May 2007 in response to the U.S. CPSC nomination ofJanuary 10, 2007.
We have witnessed concerns in some areas of the chemical industry, withregard to the applicability of the LLNA for testing of preparations, mixtures andirritant substances, and also with regard to the fact that the LLNA has notalways provided results consistent with existing knowledge of the test
substance or related test substances. We do not know if all of these concerns
are justified, but they can only serve to reduce confidence in the predictive
capability of the assay. This is not desirable when the assay offers significant
scientific and animal welfare advantages over guinea pig models for many
product types, and in a country where the assay is effectively the only available
method for evaluation of skin sensitisation potential for regulatory purposes. An
assessment of the applicability domain of the assay in its current form and theuse of the assay for testing mixtures, preparations, aqueous solutions, irritant
substances and metals is therefore very much welcomed. It seems very
appropriate to initiate a review of the current peer-reviewed literature andavailable data, in order to prepare a comprehensive background review
document, conduct a review of the validation status of the LLNA for its various
uses and to develop relevant performance standards.
It is noted that at its 26th meeting held on 26-27th April 2007 at the European
Centre for the Validation of Alternative Methods (ECVAM), the non-commission
members of ECVAM Scientific and Advisory Committee (ESAC) considered the
reduced version of the LLNA (rLLNA) to be scientifically validated, but only whenused as a screening test to distinguish between sensitisers and nonsensitisers and with due regard to the conditions set forth in the official ESAC
statement of 27th April 2007. This statement was based on the outcome of areview of LLNA data for 211 chemicals1. The review of existing and newlyprovided LLNA data proposed by ICCVAM-NICEATM therefore presents an ideal
opportunity to assess further the validity of the rLLNA for screening purposes.
As a contract research organisation, SPL is unable to provide data for review by
ICCVAM-NICEATM without the permission of its Sponsors, although we

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ICCVAM Test Method Evaluation Report: Appendix G2Page 2 of 2

consider it may be possible to provide a summary of study outcomes, coupled
to general product type, should this be of interest to ICCVAMNICEATM.
In conclusion, Safepharm Laboratories Ltd. welcomes the proposed activities
of ICCVAM-NICEATM in response to the U.S. CPSC nomination of January 10,
2007, and will be pleased to explore ways in which our experience may be ofuse in the process.
Yours sincerely,
Robert L. Guest
Head of Alternative and Acute Toxicology
Safepharm Laboratories Ltd.
1

I Kimber, RJ Dearman, CJ Betts, GF Gerberick, CA Ryan, PS Kern, GY
Patlewicz, DA Basketter (2006). The local lymph node assay and skinsensitization: a cut-down screen to reduce animal requirements? Contact
Dermatitis 2006: 54:181-185

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ICCVAM Test Method Evaluation Report: Appendix G2June 15, 2007
Dr William S Stokes
Director, NICEATM
National Institute of Environmental Health Sciences
PO Box 12233, MD EC-17
Research Triangle Park, NC 27709
Re: 72 FR 27815; May 17, 2007; National Toxicology Program (NTP) Interagency Centerfor the Evaluation of Alternative Toxicological Methods (NICEATM); the Murine
Local Lymph Node Assay (LLNA): Request for Comments, Nominations of
Scientific Experts, and Submission of Data
Dear Dr. Stokes:
These comments are submitted on behalf of the Alternatives Research and Development
Foundation, the American Anti-Vivisection Society, Humane Society Legislative Fund, The
Humane Society of the United States, People for the Ethical Treatment of Animals and the
Physicians Committee for Responsible Medicine. The parties to this submission are national
animal protection, health, and scientific advocacy organizations with a combined constituency
of more than 10 million Americans who share the common goal of promoting reliable andrelevant regulatory testing methods and strategies that protect human health and theenvironment while reducing, and ultimately eliminating, the use of animals.
In January, 2007, (ICCVAM) received a nomination from the U.S. Consumer Product Safety
Commission (CPSC) to evaluate the validation status of: (1) The murine local lymph node assay(LLNA) as a stand-alone assay for determining potency (including severity) for the purpose ofhazard classification; (2) the ‘‘cut-down’’ or ‘‘limit dose’’ LLNA approach; (3) nonradiolabeled LLNA methods; (4) the use of the LLNA for testing mixtures, aqueous solutions,and metals; and (5) the current applicability domain (i.e., the types of chemicals and substances
for which the LLNA has been validated).
ICCVAM reviewed the nomination, assigned it a high priority, and proposed that NICEATM
and ICCVAM carry out the following activities in its evaluation: (1) Initiate a review of thecurrent literature and available data, including the preparation of a comprehensive backgroundreview document, and (2) convene a peer review panel to review the various proposed LLNA
uses and procedures for which sufficient data and information are available to adequately assess
their validation status. ICCVAM also recommends development of performance standards for
the LLNA. At this time, NICEATM requests: (1) Public comments on the appropriateness andrelative priority of these activities, (2) nominations of expert scientists to consider as membersof a possible peer review panel, and (3) submission of data for the LLNA and/or modified
versions of the LLNA.
At the meeting of the Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) on June 12, 2007, several comments were made that suggested ICCVAM wasassuming a relatively rapid review of these issues. However, this is not borne out by the CSPC

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ICCVAM Test Method Evaluation Report: Appendix G2

nomination which does not mention an expedited process. In addition, ICCVAM has
recommended the creation of a background review document (BRD) and review by an expert
peer review panel, with no mention of an expedited process. The cost/benefit of this LLNA
review has not been evaluated, and SACATM was asked to vote to accept or reject
NICEATM/ICCVAM’s decision to proceed without offering any alternatives. Doubts about
the cost/benefit of this project caused one SACATM member to vote against proceeding.
Despite the fact that ICCVAM documents, including the Guidelines for the Nomination andSubmission of New, Revised, and Alternative Test Methods,1 mention the possibility of an
expedited review process, it would appear that this process has only been used in one case.
Despite repeated critiques of ICCVAM for failing to act expeditiously, we are still unable tolocate a description of the expedited review process in ICCVAM literature and the parameters
for applying it.
In light of the fact that the LLNA has been used by regulatory agencies for classifying skinsensitizers for years and both research data and regulatory use of the LLNA have been
extensively reviewed in the literature, yet another review of this widely accepted method isunwarranted. The only circumstance under which this proposal is acceptable is if ICCVAM
quickly reviews the existing literature and makes an expedited evaluation regarding therelevance of this information to Agency regulatory needs. ICCVAM’s limited resources shouldbe spent validating and promoting for regulatory acceptance any of the number of non-animal
methods for skin sensitization that are currently in development.
In March 1999, ICCVAM published a final peer review report concluding that the LLNA is avalid alternative to currently accepted guinea pig test methods.2 The U.S. EPA, FDA, andOSHA announced their acceptance of the LLNA as an alternative to the guinea pig
maximization test for assessing allergic contact dermatitis in October 1999. That same year,
ESAC, the Scientific Advisory Committee of the European Centre for the Validation ofalternative Methods (ECVAM), also endorsed the LLNA for regulatory use.
In September 2000, the European Centre for Ecotoxicology and Toxicity of Chemicals(ECETOC) published a comprehensive review of sensitization test methods with respect tohazard identification and labeling, (and?) to determine whether the various methods areappropriate for determining relative potency and risk assessment.3 The conclusions from thisreview included: (1) the LLNA is a viable and complete alternative to traditional guinea pig test
methods for the purposes of skin sensitization hazard identification, and (2) the LLNA issuitable for the determination of relative skin sensitizing potency and the adaptation of thismethod for derivation of comparative criteria such as EC3 values provides an effective andquantitative basis for such measurements. This report further recommends that “the LLNA isthe recommended method for new assessments of relative potency and/or for the investigationof the influence of vehicle or formulation on skin sensitizing potency.”1

http://iccvam.niehs.nih.gov/SuppDocs/SubGuidelines/SD_subg034508.pdf
http://iccvam.niehs.nih.gov/methods/immunotox/immunotox.htm
3
ECETOC. 2000. Skin Sensitization Testing for the Purpose of Hazard Identification and Risk Assessment.
2

2

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ICCVAM Test Method Evaluation Report: Appendix G2More recent work has further verified the use of the LLNA as a stand-alone method for
estimating potency for regulatory purposes, including a 2005 study that concludes that there isa “clear linear relationship between LLNA-derived EC3 values and historical human skin patch
data.”4 A 2007 review concludes that “The LLNA, when conducted according to publishedguidelines, provides a robust method for skin sensitization testing that not only provides
reliable hazard identification in formation but also data necessary for effective risk assessment
and risk management.” In addition, a retrospective analysis of the regulatory use of the LLNA
in the EU was published in 2006 and concluded that “the LLNA is satisfactory for routine
regulatory use.” 5 We acknowledge that the LLNA must be validated for determining
sensitization potency for regulatory use; however, we urge ICCVAM to take an abbreviated test
validation approach, as was recommended by the recent International Programme on Chemical
Safety Workshop on Skin Sensitization in Chemical Risk Assessment:6 “An abbreviated test
validation approach may be appropriate to assess the validity of potency assessment based on
the LLNA and its appropriateness for predicting sensitizing induction potency in humans.”
The ‘‘cut-down’’ or ‘‘limit dose’’ LLNA approach (reduced, or rLLNA) has recently beenreviewed by an ECVAM peer review panel. In April, 2007, ESAC issued a statement
supporting the use of the rLLNA “within tiered-testing strategies to reliably distinguish between
chemicals that are skin sensitizers and non-sensitizers “thereby reducing animal use by as muchas 50%.”7 The statement also notes the following limitations: that “the test results provided by
the rLLNA do not allow the determination of the potency of a sensitising chemical,” and that“negative test results associated with testing using concentrations of less than 10% should
undergo further evaluation”
The applicability and limitations of this modification of the LLNA have been clearly
established. Therefore, in lieu of a lengthy review of this method, ICCVAM should
expeditiously review and endorse the ESAC peer review and circulate harmonized testing
recommendations regarding this assay to US agencies before year’s-end and NICEATM shouldcollaborate with ECVAM to address the question of concentration threshold.
Other recent work has included the development of several applications of non-radioactive
detection methods for the LLNA, including BrdU incorporation, methods measuring the releaseof various cytokines, and methods using fluorescent markers and quantification by flow
cytometry. In many cases, these methods have been shown to be as sensitive as protocols
involving radio-labeling.8 In addition, in NIH-sponsored and contract work, MB Research has
shown that “for a large range of chemicals, the FC-LLNA EC3 values were consistent with
4

Basketter et al. Predictive identification of human skin sensitization thresholds. Contact Dermatitis. 2005; 53 (5):260-267.
5
Cockshott et al., The local lymph node assay in practice: a current regulatory perspective. Hum Exp Toxicol
2006; 25 (7): 387-394.
6
http://www.who.int/ipcs/methods/harmonization/areas/sensitization_summary.pdf
7
http://ecvam.jrc.it/publication/ESAC26_statement_rLLNA_20070525-1.pdf
8
Takeyoshi et al. Advantage of using CBA/N strain mice in a non-radioisotopic modification of the local lymph
node assay. J Appl Toxicol. 2006. 26:5-9. Takeyoshi et al. Novel approach for classifying chemicals according toskin sensitizing potency by non-radioisotopic modification of the local lymph node assay. J Appl Toxicol. 2005.
25:120-134. Suda et al. Local lymph node assay with non-radioisotope alternative endpoints. J Toxicol Sci. 2002.
27:205-218.

3

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ICCVAM Test Method Evaluation Report: Appendix G2

those reported in ICCVAM LLNA validation studies.”9 Both ECVAM and Japanese Center for
the Validation of Alternative Methods (JaCVAM) are currently reviewing these methods and,rather than initiate a full independent review, ICCVAM must collaborate with these ongoing
efforts.
With regard to the assessment of the LLNA for aqueous mixtures and metals, the informationthat is currently available should allow ICCVAM to make a rapid determination of theapplicability and limitations of the LLNA for these classes of chemicals and, if it cannot, we do
not endorse further validation efforts in this regard, but recommend the pursuit of in vitro
methods for this purpose.
Several non-animal methods for estimating sensitivity are under development, including
quantitative structure activity relationship (QSAR) modeling that shows a high concordancewith both guinea pig and LLNA data,10 quantification of peptide reactivity, which also shows ahigh concordance with LLNA data,11 and human cell cultures.12 We urge ICCVAM to secure
an interagency grant from the CPSC to fund the validation of one or more of these non-animal
methods. Clearly, ICCVAM and the CPSC both benefit from the sharing of resources, as the
CPSC nominated the method and ICCVAM will be tasked with the final work product.
ICCVAM should consider taking an approach similar to the European Sens-it-iv project,13
which involves the coordinated efforts of more than two dozen groups from industry, academia
and other organizations, all working toward the common goal of developing in vitro methods toassess immunotoxicity. ICCVAM should consider facilitating the creation of such a goaloriented task force.
To summarize, given the fact that the LLNA has been used by regulatory agencies forclassifying skin sensitizers for years and both research data and regulatory use of the LLNA
have been extensively reviewed in the literature and by other countries, yet another lengthy
review of this widely accepted method is clearly unwarranted. Instead, we urge ICCVAM toperform an expedited review of the existing information regarding the LLNA’s performance andlimitations and to issue recommendations to US agencies with all due speed. In the interest of
eventual replacement of animals in sensitization testing, ICCVAM must spend its time andresources promoting the development and regulatory use of non-animal methods, which it can
do by engaging in integrated approaches to in vitro immunotoxicity.
Sincerely,9

http://www.mbresearch.com/TOXNOTE/TOXNOTE-LLNA.pdf
Fedorowicz et al., Structure-activity models for contact sensitization. Chem Res Toxicol. 2005; 18(6): 954-969.
11
Gerberick et al. Quantification of chemical peptide reactivity for screening contact allergens: a classification treemodel approach. 2007; 97(2): 417-427.
12
Schoeters et al. Microarray analyses in dendritic cells reveal potential biomarkers for chemical-induced shinsensitization. 2007; 44(12): 3222-3233.
13
http://www.sens-it-iv.eu/
10

4

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ICCVAM Test Method Evaluation Report: Appendix G2/s/Catherine Willett, PhD
Science Policy Advisor
Regulatory Testing Division
People for the Ethical Treatment of Animals/s/Sara Amundson
Executive Director
Humane Society Legislative Fund/s/

Dr. Martin Stephens
Vice President for Animal Research Issues
Humane Society of the United States/s/
Kristie Stoick, MPH
Research Analyst
Physicians Committee for Responsible Medicine/s/
Sue A. Leary
President
Alternatives Research & Development Foundation/s/
Tracie Letterman, Esq.
Executive Director
American Anti-Vivisection Society

5

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ICCVAM Test Method Evaluation Report: Appendix G2

Data Submissions
In response to the FR notices, the following public commenters provided data for evaluation of
the rLLNA. Data are available in Appendix D (see Annex III of the Final Background Review
Document).
•Dr. Dori Germolec, National Institute for Environmental Health Sciences•

Dr. Hans Werner Vohr, Bayer HealthCare (Wuppertal-Elberfeld, Germany)•

Dr. Melissa Kirk and Daniel Cervins, MB Research Labs•

Dr. Michael Olson, GlaxoSmithKline•

Dr. Kirill Skirda, European Committee of Organic Surfactants and their Intermediates•

Dr. Phil Botham, Syngenta (Cheshire, United Kingdom)•

Dr. Peter Ungeheuer, The European Federation for Cosmetic Ingredients (Frankfurt,
Germany)•

Dr. Eric Debruyne, Bayer CropScience (France)

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ICCVAM Test Method Evaluation Report: Appendix G2Page 1 of 3

Subject: FR Notice Comments - 72FR52130: LLNA Performance Standards
Date: Monday, October 29, 2007 4:31 PM
Dr William S Stokes
Director, NICEATM
National Institute of Environmental Health Sciences
PO Box 12233, MD EC-17
Research Triangle Park, NC 27709
Re: 72 FR 52130; September 12, 2007; National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods(NICEATM); Draft Performance Standards for the Murine Local Lymph Node
Assay: Request for Comments.
Dear Dr. Stokes:
These comments are submitted on behalf of the Alternatives Research andDevelopment Foundation, the American Anti- Vivisection Society, Humane
Society Legislative Fund, The Humane Society of the United States, People for
the Ethical Treatment of Animals, and the Physicians Committee for Responsible
Medicine. The parties to this submission are national animal protection, health,and scientific advocacy organizations with a combined constituency of more than
10 million Americans who share the common goal of promoting reliable andrelevant regulatory testing methods and strategies that protect human health andthe environment while reducing, and ultimately eliminating, the use of animals.
In January, 2007, (ICCVAM) received a nomination from the U.S. Consumer
Product Safety Commission (CPSC) to evaluate the validation status of: (1) The
murine local lymph node assay (LLNA) as a stand-alone assay for determining
potency (including severity) for the purpose of hazard classification; (2) the "cutdown"˙ or "limit dose" LLNA approach; (3) non-radiolabeled LLNA methods; (4)the use of the LLNA for testing mixtures, aqueous solutions, and metals; and (5)the current applicability domain (i.e., the types of chemicals and substances forwhich the LLNA has been validated). The development of these performancestandards is an initial response to this nomination, and ICCVAM is requesting
comment on these performance standards.
Although we fully support the development of performance standards that
expedite the validation of new protocols that are similar to previously validated
methods, we reiterate our disappointment that ICCVAM/ NICEATM has chosen
to apply its limited resources to the lengthy process of developing performancestandards for such a narrow scope of applicability. These performance standards
apply only to modifications of the "standard LLNA" that involve incorporation of
non-radioactive methods of detecting lymphocyte proliferation.
A major aspect of the ICCVAM Authorization Act of 2000 (Public Law 106-545,
42 U.S.C. 285l-3) is the charge to "reduce, refine, and/or replace the use of

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ICCVAM Test Method Evaluation Report: Appendix G2Page 2 of 3

animals in testing where feasible." The performance standards described in thisFR notice apply to modifications of the standard LLNA that do not affect the
number of animals used in this method. The only conceivable reduction could
occur if the availability of accepted non-radioactive methods of detection would
allow more laboratories to perform the LLNA, and if they then choose the LLNA
over the Guinea Pig Maximization test or the Buehler Test. The issue of how thisexercise (development of performance standards with this limited applicability)
addresses ICCVAM's mandate of reducing, refining or replacing the use ofanimals is not currently mentioned in the draft document and needs to beadequately explained.
In addition, the draft performance standards require the use of a minimum of 20
reference compounds. The criteria by which the compounds were chosen andthe characteristics of the compounds are described; however, there is no
justification for the requirement of such a large number of compounds for thisparticular method modification. The methods to which these performancestandards apply will differ from the "standard LLNA" only in the method of
detection of lymphocyte proliferation; therefore the element of concern issensitivity of the detection method. All other aspects of the methods to beevaluated will be identical to the standard LLNA, including delivery and biologicalresponse. It is therefore not necessary to test representatives for every chemicalclass or every solvent that has been tested in the standard LLNA. The importantcharacteristic of the reference compound is the magnitude of proliferation
response that is generated, and the list of reference compounds chosen should
be limited to those that represent the range of response seen with the standard
LLNA.
Finally, it is the belief of the parties to this submission that the limited resources
available to ICCVAM/NICEATM would be better spent on activities that would
have greater impact on the reduction, refinement or replacement of animal use,such as evaluating the use of human cell lines or one of the available in vitro skinmodels as a replacement for the LLNA.
Sincerely,
Catherine Willett, PhD
Science Policy Advisor
Regulatory Testing Division
People for the Ethical Treatment of Animals
Sara Amundson
Executive Director
Humane Society Legislative Fund
Dr. Martin Stephens
Vice President for Animal Research Issues
Humane Society of the United States

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ICCVAM Test Method Evaluation Report: Appendix G2Page 3 of 3

Kristie Stoick, MPH
Research Analyst
Physicians Committee for Responsible Medicine
Sue A. Leary
President
Alternatives Research & Development Foundation
Tracie Letterman, Esq.
Executive Director
American Anti-Vivisection Society

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ICCVAM Test Method Evaluation Report: Appendix G2

The Procter & Gamble Company
Central Product Safety
Miami Valley Laboratories
P.O. Box 538707
Cincinnati, Ohio 45253-8707
www.pg.com
February 22, 2008
William S. Stokes, D.V.M., DACLAM
RADM, U.S. Public Health Service
Director, National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods
Executive Director, Interagency Coordinating Committee on
the Validation of Alternative Toxicological Methods
National Institute of Environmental Health Sciences, NIH, DHHS
P.O. Box 12233
Research Triangle Park, NC 27709
Dear Dr. Stokes
Thank you for the opportunity to review and comment on the documents prepared by ICCVAM and
NICEATM related to a number of the modifications/proposed uses for the traditional LLNA that will beconsidered by an independent international expert panel in early March.
The teams have done a great job summarizing the available data on the LLNA and for the most part weare in agreement with the conclusions and recommendations outlined in the documents. What makes the
LLNA such a valuable tool for skin sensitization hazard identification and risk assessment is that thestrengths and limitations of the assay are recognized so well. I am not sure there is another toxicologicaltest that is more understood and evaluated than the LLNA. I am certain that most experts in the field ofskin allergy would agree that the older guinea pig skin sensitization test methods are considerably lessunderstood, specifically related to their lack of evaluation through a formal validation process. Our hopeis that this peer review of the LLNA will lead to a better appreciation of the LLNA and more important
help researchers develop non-animal test methods for evaluating potential skin sensitizing chemicals byusing the robust and quantitative natureof the LLNA as a foundation to compare new alternative methods.
For your review and consideration our LLNA experts (Cindy Ryan, Pierre Aeby, Petra Kern and myself)have prepared comments on the LLNA documents posted on the website. I hope you will find them usefuland please let us know if you need any additional information.
Sincerely,
G. Frank Gerberick, Ph.D.
Research Fellow Victor Mills Society

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ICCVAM Test Method Evaluation Report: Appendix G2DRAFT ICCVAM Recommendations: LLNA Potency
Comparison of LLNAEC3 values to human data:
An evaluation of the ability of the LLNA to predict the relative sensitization potency ofchemicals in humans necessitates the use of human sensitization data for comparative purposes.
In order for such a comparison to provide meaningful information, one must be aware of andunderstand the limitations in each of the datasets. The human data used in the comparison are
derived from either HRIPT or HMT studies in which single test concentrations, expressed asµg/cm2, were used for the induction phase of the test protocol. Therefore, a test concentration
could be defined as the NOEL, when in reality it may just be the highest concentration tested todate which did not induce sensitization and there is a probability that higher levels would also
fail to induce. This certainly could be the case if a LOEL for the particular chemical has notbeen identified. Indeed, it is difficult to compare LLNA EC3 concentrations against a human
NOEL or an arbitrary value of the LOEL/10 (which is intended to represent an estimation of aprobable induction threshold value). On one side, the LLNA data were generated using a test
protocol designed to produce quantitative values with dose response information which permit
the calculation of the LLNA EC3 and on the other side, the human data were generated by avariety of different human repeated insult patch test and human maximization test protocols
which, by design are more qualitative in nature, and unless a series of studies were conducted,
provide limited if any information on an induction dose response.
It is concerning that in the evaluation of the LLNA to predict skin sensitization potency inhumans key values for the comparison are “pragmatically determined”, as is indicated in lines
335-337 of the background review document “Next, the optimal EC3 value that maximized
obtaining the correct skin sensitization calls for strong and weak sensitizers (using one or theother proposed decision criterion) was pragmatically determined.” Similar wording is used inlines 801-804. The method or rationale for this “pragmatic determination” are not clearly
evident in the document. A sound statistical approach should have been used instead and would
have provided a more scientifically robust comparison.
Comparison of LLNA EC3 values to guinea pig data:
To assess the ability of the LLNA to predict skin sensitization potency in Guinea Pigs is not
relevant to the purpose of this review. Guinea pig tests such as the Buehler (BT) and Guinea Pig
Maximization tests (GPMT) were designed for the purpose of hazard identification and arepoorly suited for potency estimations. While the ECETOC Technical Report No. 87, Contact
Sensitisation: Classification According to Potency proposes methods to categorize allergenic
potency based on BT and GPMT data, it demands that the study was conducted in full accord
with OECD TG 406 and advises judicious interpretation of the data as does a similar European
Union commission expert review. While the BT and GPMT have served the toxicology
community well for many years as predictive skin sensitization hazard methods, it is important torecognize that, unlike the LLNA, neither of these tests has been formally validated by arecognized organization nor has the inter-laboratory variability been adequately investigated.

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ICCVAM Test Method Evaluation Report: Appendix G2

In several sections of the background review document, for examples Lines 321-324 and lines
714-717, it is indicated that for each substance with comparative LLNA and guinea pig data,
potency was evaluated by comparing the LLNA EC3 concentration against the percentage of
responding guinea pigs in the BT or GPMT and the associated induction concentration.
Comparing LLNA EC3 concentration against the percentage of responding guinea pigs is not
appropriate in our opinion and resulting data are of very different natures; the LLNA measures
events associated with the induction of skin sensitization and provides objective, quantitative
dose response information whereas data derived from the guinea pig tests are based on a
subjective evaluation of skin responses occurring at the elicitation phase of sensitization andprovides no dose response information on the induction phase.
It appears that the authors understand the difficulty of comparing LLNA EC3 values withpotency classifications based on guinea pig data. In line 395 of the background review document
it states that “…for substances that had more than one EC3 or guinea pig response, the geometricmean EC3 value and the weight of evidence GP classification category was used. Although thedata generated by the GPMT and the BT is categorical, using the weight of evidence
categorization provided some measure of a mean response across multiple studies.” Considering
the admitted difficulties encountered in dealing with multiple sets of guinea pig-derived data, theauthors should be consistent and not make any conclusion based on such comparison.
Proposed classification categories for sensitization:
While cut-off values for potency classification are proposed based on either Buehler test and
GPMT responses (Table 1-1) we would caution the use of such data in the absence of any othersupporting data due to the nature of the test design. In addition, the proposed scheme uses theintradermal induction dose of the GPMT along with the % responders as the basis forclassification. We believe that the topical induction concentration should be considered as it is
the more relevant route of exposure and the concentration used for intradermal injection is often
limited by the addition of Freund’s Complete Adjuvant.
The proposed classification (as well as the one proposed by ECETOC TR No. 87) considers only
data from guinea pig tests which are defined as ‘positive’ by the accepted TG 406 definition of asensitizing chemical (i.e. induces 30% or 15% positive responses in the GPMT or BT
respectively). It is possible that a weakly sensitizing chemical tested in a guinea pig test could
elicit positive responses in 20% or 25% of the test animals in a GPMT or 10% in the BT, andwould be considered as a non-sensitizer and thus would not be classified according to the
proposed scheme while a chemical with any LLNA EC3 value would be assigned to one of the 2
proposed categories. Data obtained through the LLNA allows for a continuous spectrum of EC3
values and thus provides a rank ordering of relative potencies which offer more opportunities for
categorization beyond two categories. And on the other side, Human and GP tests which aredesigned to provide yes/no answers have various threshold values creatively proposed in order toforce results in the same two categories.
In the proposed two level classification scheme for sensitization potency (Table 1-1), the criteria
for classification for category 1 are given as “A high frequency of occurrence….” OR “Aprobability of occurrence of a high sensitization rate in humans…” and for category 2 are given
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ICCVAM Test Method Evaluation Report: Appendix G2

as “A low or moderate frequency ….” OR “A probability of occurrence of a low to moderatesensitization rate in humans…”. The frequency of sensitization or the sensitization rate within
an exposed population concerns the prevalence of allergic contact sensitization to a particularchemical, which is entirely different from the inherent potency of the chemical. Therefore theuse of such criteria to classify potency is not appropriate. The likelihood of a chemical inducing
skin sensitization within an exposed population (i.e. the probable sensitization rate) depends on
two key elements: the intrinsic allergenic potency of the chemical AND the conditions and extent
of the allergen exposure (e.g. frequency, duration, exposure conditions, etc.). Clinically, thenature, extent and duration of exposure are commonly the predominant determinants ofprevalence. The relative potency of a chemical concerns the amount of chemical required toinduce sensitization. In general, the more potent the allergen, the lower the dose per unit area
required to induce sensitization. Prevalence data are derived from diagnostic patch testing ofpatients with suspect allergic contact dermatitis, often presenting with clinical disease, indermatology clinics. The diagnostic patch test itself is designed to detect the weakest degrees ofallergy by using occluded exposure conditions for 48 hours and highest allergen concentrations
possible to elicit a reaction. For example, the standard patch test concentration for nickel sulfate
is 2.5%. Applied in a diagnostic patch test using an 8 mm Finn chamber delivers a dose per unit
area of 750 µg/cm2, well above the identified human induction threshold of 154 µg/cm2 (seeTable 2 of Appendix A of the LLNA potency background review documents). Many times thenature of the exposure conditions leading to the induction of allergy for these patients is notclearly defined. At best the published results of thousands of such diagnostic patch tests can beused to evaluate trends in patch test reactions.
One example often used to illustrate the difference between potency and prevalence is nickel. It
is a very common contact allergen with a relatively high sensitization rate in the US and Europe.
However, experimental evidence indicates that nickel is a relatively weak contact allergen, with
LLNA EC3 of 140 µg/cm2 and a human induction threshold of 154 µg/cm2 for nickel sulfate.
The high prevalence is due to the wide distribution, frequent exposure and the nature of
exposure, often through ‘compromised’ skin such as body piercing.
Conversely, the preservative methylchloroisothiazolinone/methylisothiazolinone (MCI/MI) is awell known contact allergen considered to be of strong to extreme potency with LLNA EC3 of
2.25 µg/cm2 and a human induction NOEL of 1.25 µg/cm2. In Europe, the prevalence rate ofallergy to MCI/MI is stable at 1-3% of patch-tested patients. Considering the number of
MCI/MI-containing cosmetics and toiletries that are on the market, the opportunities for
exposure and the allergenic potency of the preservative one would expect a much higher
incidence rate. The prevalence rate for this potent allergen is kept low because of regulatory
guidelines/limits on the level of MCI/MI permissible in certain products, thus limiting the dose
per unit area of the exposure. Thus, the clinical prevalence of the strong allergen MCI/MI is low
whereas for nickel, a known weak allergen, the prevalence is considerably higher which is
opposite of what would be expected if only looking at potency and not considering exposure.
The proposed two level classification scheme for sensitization potency (Table 1-1) does notaccurately reflect the range of allergenic potencies that have been demonstrated by both animaland human data. LLNA EC3 values and human induction thresholds clearly span several ordersof magnitude as shown by the data in Table 2 of Appendix A of the LLNA potency background
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ICCVAM Test Method Evaluation Report: Appendix G2

review documents. Human threshold values range from 1.25 µg/cm2 for MCI/MI, to 250 µg/cm2
for isoeugenol, to 2755 µg/cm2 for farnesol, to 20,690 µg/cm2 for benzyl benzoate. Clinicalexperience with allergic contact dermatitis would also indicate that discrete classes of sensitizing
potency exist (Contact Derm, 2000, 42:344-348).

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ICCVAM Test Method Evaluation Report: Appendix G2DRAFT ICCVAM Recommendations: LLNA Applicability Domain
Draft Recommendations – Use of the LLNA to Test Mixtures:
A dataset of 18 mixtures was evaluated, 15 of which had guinea pig data and none had humandata. As a result, the LLNA data were compared to the guinea pig data. Since the database is
severely limited due to the lack of human data, there is no proof that the guinea pig data would
be representative of the human response. Thus, using the guinea pig data as the standard to which
the LLNA data should be compared is not appropriate.
In addition, the usefulness of these data is limited further by the fact that information on theingredients is known for only one of the 15 mixtures and 11 were tested in the LLNA in an
aqueous vehicle, the performance of which is also being assessed in this same report.
High quality LLNA mixture data is published in Lalko et al. (2006), cited in section 7.6 of
Addendum No. 1 to the ICCVAM report. This publication concerns the evaluation of essential
oils and includes analytical data on the composition of the oils as well as LLNA data on theidentified major constituents. These data should have been included in the evaluation and notjust mentioned as other available scientific reports.
Since the database is severely limited due to the lack of human data, we agree with therecommendation that an assessment of the suitability of the LLNA for testing mixtures should
not be conducted until a sufficient quantity of quality data become available. A similar logic ofcourse also applies to guinea pig test methods.
Draft Recommendations – Use of the LLNA to Test Metal Compounds:
The reference dataset contains human data for 17 metal compounds representing 13 different
metals. Since the allergenic potential in humans of most all of the known metals has been
established, one questions the importance of or need for an assessment of the LLNA’s ability todetect metal allergens. However, we agree with the recommendation that the LLNA is useful forthe testing of metal compounds. Whether or not the LLNA is useful for testing nickel
compounds is of limited importance as nickel is a well known human contact allergen.
In addition, since only 1 of the 14 metal compounds with LLNA and human data was tested inboth in an aqueous vehicle, the comparison does not add much value to the assessment,
especially in light of the fact that the performance of the LLNA using aqueous vehicles is being
assessed in this same report.
Draft Recommendations – Use of the LLNA to Test Substances in Aqueous Solutions:
A dataset of 21 substances tested in aqueous solutions was evaluated, 4 of which had had humandata. Since the database is severely limited due to the lack of human data, we agree with therecommendation that an assessment of the suitability of the LLNA for testing substance inPage 6
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ICCVAM Test Method Evaluation Report: Appendix G2

aqueous solutions should not be conducted until a sufficient quantity of quality data become
available.

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ICCVAM Test Method Evaluation Report: Appendix G2DRAFT ICCVAM Recommendations: LLNA Limit Dose Procedure
Draft Recommendations – Limit Dose Procedure:
We agree with the recommendation that the LLNA limit dose procedure is appropriate for hazard
identification purposes.
We must point out that a 10% concentration threshold for defining non-sensitizing chemicals is
not, as suggested in line 44 of the recommendation, proposed by Kimber et al. (2006) as theabsolute cut-off. In the discussion section of that same paper, Kimber et al. indicate that for the
purposes of that article the 10% threshold was used and that that figure “should not be regarded
as inviolable.” They go on to say that a case could be made for using, for instance, either 15% or20%. In the 2005 Gerberick et al. paper (Compilation of historical local lymph node data forevaluation of skin sensitization alternative methods. Dermatitis, 16(4):157-202), compounds that
did not induce a positive response at any concentration tested, with the highest concentration
being at least 20% or greater, were categorized as non-sensitizing.
In addition, the 10% threshold concentration at which all which all negative results would beconsidered valid did not originate in the cited Kimber et al 2006 publication. The originalreference is Cockshott et al., 2006, Human and Experimental Toxicology, 25:387-394 in which
the performance of the LLNA was evaluated in a regulatory context. In that paper, a negative
result obtained with the highest concentration tested at 10% would be considered a valid result if
the positive control, a mild to moderate sensitizer, gave a positive response. In other words, achemical which is negative at a top concentration of 10% does not represent a significant humansensitization hazard. This is similar to the definition of a non-sensitizing chemical in the Guinea
Pig Maximization Test (GPMT) or Buehler test as one which induces less than 30% or 15%
positive responses respectively. Therefore, if a chemical elicits positive responses in 20% or
25% of the test animals in a GPMT, it would be considered as a non-sensitizer from a regulatory
perspective.
Comments on DRAFT ICCVAM Recommendations: LLNA Non-Radioactive Methods
DRAFT ICCVAM Recommendations: LLNA BrdU ELISA Procedure
We agree with the recommendation that more information and data are needed on this method inorder to conduct a meaningful assessment of the BrdU ELISA procedure’s performance relative
to the traditional LLNA. It is especially important to have information regarding the interlaboratory performance of this assay.
We do have one suggestion for consideration. Table 6-2 of the Background Review Documentsshows a comparison of standard LLNA EC3 values and 0.5x-2x range for the performancestandard chemicals and EC3 values calculated from the BrdU ELISA LLNA. Since analternative SI cutoff for the BrdU ELISA LLNA was identified that provides greater accuracy

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ICCVAM Test Method Evaluation Report: Appendix G2

than an SI = 3 cutoff i.e., SI = 1.3, a comparison of BrdU ELISA EC1.3 values to standard
LLNA EC3 values would be helpful.
DRAFT ICCVAM Recommendations: LLNA BrdU FC Procedure
We agree with the recommendation that more information and data are needed on this method inorder to conduct a meaningful assessment of the BrdU-FC procedure’s performance relative tothe traditional LLNA. While the total number of chemicals tested (45) is sufficient, it isespecially important to have information regarding the inter-laboratory performance of thisassay. The background review document speculates that the transferability of the LLNA: BrdUFC and the eLLNA: BrdU-FC would be similar to the traditional LLNA. However, we do notthink that will be the case. Flow cytometry is not a trivial technique. It is certainly more error
prone than scintillation counting and often the quality of the results is very dependant on trained
personnel and precise procedures.
Only 13 of the 18 minimum performance standard reference chemicals have been tested in the
LLNA BrdU-FC procedure. This may not be sufficient to assess the test performance according
to the ICCVAM Performance Standards for the LLNA. In addition, rather than focusing on thenumber of chemicals for which the BrdU-FC procedure produced equivocal results or did not
obtain 100% concordance with the ICCVAN LLNA performance standard reference chemicals,
we believe that it would be of greater value to investigate potential causes for those results. Suchinformation would provide some understanding of the limitations of the methods.
Since the purpose of this evaluation of the LLNA BrdU-FC procedure is to assess its ability to bea non-radioactive alternative to the traditional LLNA, is a comparison with Guinea Pig data
justified?
The provided test protocol indicates that at least 6 mice be employed for an irritation prescreen
and a possible 12 more be used for the optional quantitative irritation test. Therefore, thismethod has the potential to use more mice than the traditional LLNA. This requirement for
greater animal usage must be taken into consideration when evaluating the BrdU-FC Procedureand it must be determined that the quality or quantity of information provided by this method
exceeds that which would be obtained with the traditional LLNA. In other words, are theadditional mice required by the BrdU-FC worth any possible additional information that would
be gained compared to conducting a traditional LLNA?
DRAFT ICCVAM Recommendations: LLNA DA Procedure
Beyond the method to assess lymph node cell proliferation, the test protocol for the LLNA DA
contains several key deviations from the OECD Test Guideline 429 recommended protocol andthe Essential Test Method Components as described in the Draft ICCVAM PerformanceStandards for the LLNA . As indicated in the recommendation document (lines 77-79), the
LLNA DA has made major modification to the traditional LLNA in both the test substancetreatment and sampling schedule. Therefore, this method is outside of the requirements of thedraft ICCVAM Performance Standards for the LLNA and should not be consider for validation
as an LLNA alternative at this time.
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ICCVAM Test Method Evaluation Report: Appendix G2

February 22, 2008
Dr William S Stokes
Director, NICEATM
National Institute of Environmental Health Sciences
PO Box 12233, MD EC-17
Research Triangle Park, NC 27709
Re: 73 FR 25553; January 8, 2008; National Toxicology Program (NTP); NTP Interagency
Center for the Evaluation of Alternative Toxicological Methods (NICEATM);
Announcement of an Independent Scientific Peer Review Panel Meeting on the Murine
Local Lymph Node Assay; Availability of Draft Background Review Documents;
Request for Comments
Dear Dr. Stokes:
These comments are submitted on behalf of People for the Ethical Treatment of Animals andthe Physicians Committee for Responsible Medicine. The parties to this submission are
national animal protection, health, and scientific advocacy organizations with a combined
constituency of more than two million Americans who share the common goal of promoting
reliable and relevant regulatory testing methods and strategies that protect human health andthe environment while reducing, and ultimately eliminating, the use of animals.
Please take note of the following thoughts and transmit them to the Peer Review Panel (PRP)
accordingly.
In January, 2007, (ICCVAM) received a nomination from the U.S. Consumer Product Safety
Commission (CPSC) to evaluate the validation status of: (1) The murine local lymph node assay(LLNA) as a stand-alone assay for determining potency (including severity) for the purpose ofhazard classification; (2) the ‘‘cut-down’’ or ‘‘limit dose’’ LLNA approach; (3) nonradiolabeled LLNA methods; (4) the use of the LLNA for testing mixtures, aqueous solutions,and metals; and (5) the current applicability domain (i.e., the types of chemicals and substances
for which the LLNA has been validated).
Now more than a year later, ICCVAM is preparing for a peer review meeting to evaluate its
recommendations and findings on these four items. It is unclear when final recommendations
will be transmitted to federal agencies, but if ICCVAM’s review of in vitro pyrogenicity
methods is any indication, it may be at least another year.
Since this review of the LLNA and the proposed recommendations contained therein will leadto little reduction or refinement of animal use in sensitization, the resources that ICCVAM
devote to this exercise should be kept to a minimum, and any forthcoming recommendations
should be transmitted to agencies immediately following the Peer Review.
We have divided our comments into sections following the FR Notice:

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ICCVAM Test Method Evaluation Report: Appendix G2

LLNA limit dose procedures (the reduced or rLLNA) —draft Background Review
Document (BRD) and other related documents
In April, 2007, ESAC issued a statement supporting the use of the rLLNA “within tiered-testing
strategies to reliably distinguish between chemicals that are skin sensitizers and non-sensitizers“thereby reducing animal use by as much as 50%.”1
In spite of the ESAC recommendations, ICCVAM has conducted its own data call in and data
review. The reviewed database is comprehensive and contains a broad cross-section of thechemical universe. The performance characteristics were all above 95% (false negative andpositive rates are very low or zero). Even though this additional review was largely
unnecessary, we are pleased that ICCVAM’s draft recommendations concluded favorably forthe rLLNA procedure and urge the Peer Review Panel to concur. ICCVAM should forward
recommendations regarding the use of the rLLNA to federal agencies immediately following thePeer Review.
Mixtures, metals, and aqueous solutions—draft Updated Assessment of the Validity of the
LLNA for Mixtures, Metals, and Aqueous Solutions and related documents
ICCVAM has evaluated available data with respect to the use of LLNA in predicting the skinsensitization potential of mixtures, metals, and aqueous solutions. In all cases, the limited
availability of data prevented a conclusive recommendation for the use of the LLNA; for metals,
the LLNA is recommended only as part of a weight-of-evidence approach, which does not
significantly promote a reduction in the use of animals.
Clearly this approach to expanding the applicability domain of the LLNA has not proved
terribly fruitful, and we do not endorse further validation efforts in this regard, but recommend
all resources are directed towards the pursuit of in vitro methods for this purpose.
Potency—draft BRD and related documents
Once again, ICCVAM has reviewed all availed data and come to a conclusion that is in
opposition to that of other experts in the field. For more than 10 years data has been
accumulating indicating the potential for the LLNA to make a determination of thesensitization potency of a chemical.2 Several publications by Basketter and others (many ofwhich are referenced in the BRD) as well as the eloquent argument by Basketter et al.
presented in Appendix A, conclude that LLNA is appropriate for determining potency. In
September 2000, the European Centre for Ecotoxicology and Toxicity of Chemicals(ECETOC) published a comprehensive review of sensitization test methods with respect tohazard identification and labeling, to determine whether the various methods are appropriate
for determining relative potency and risk assessment.3 The conclusions from this review
included: (1) the LLNA is a viable and complete alternative to traditional guinea pig test1

http://ecvam.jrc.it/publication/ESAC26_statement_rLLNA_20070525-1.pdf
Kimber I, Basketter D A. Contact sensitization: A new approach to risk assessment.
Human and Ecological Risk Assessment 1997: 3: 385 - 395.
3
ECETOC. 2000. Skin Sensitization Testing for the Purpose of Hazard Identification and Risk Assessment.
2

2

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ICCVAM Test Method Evaluation Report: Appendix G2

methods for the purposes of skin sensitization hazard identification, and (2) the LLNA issuitable for the determination of relative skin sensitizing potency and the adaptation of thismethod for derivation of comparative criteria such as EC3 values provides an effective andquantitative basis for such measurements. This report further recommends that “the LLNA isthe recommended method for new assessments of relative potency and/or for the investigationof the influence of vehicle or formulation on skin sensitizing potency.”
More recent work has further verified the use of the LLNA as a stand-alone method for
estimating potency for regulatory purposes, including a 2005 study that concludes that there isa “clear linear relationship between LLNA-derived EC3 values and historical human skin patch
data.”4 A 2007 review concludes that “The LLNA, when conducted according to publishedguidelines, provides a robust method for skin sensitization testing that not only provides
reliable hazard identification in formation but also data necessary for effective risk assessment
and risk management.” In addition, a retrospective analysis of the regulatory use of the LLNA
in the EU was published in 2006 and concluded that “the LLNA is satisfactory for routine
regulatory use.” 5
Despite all of this, ICCVAM’s review of the LLNA for potency determination does not support
such a finding, even though, according to the BRD, the LLNA was better overall at predicting
sensitization potency than guinea pig data. It is clear from the BRD that different data
treatments result in different R2 values, and the BRD should more clearly discuss the reasons
those analysis decisions were made. Further, the BRD should explain in detail why conclusions
were drawn that are opposite to that of the evidence they reference.
We urge the PRP to take into account the submission in Appendix A of the draft LLNApotency BRD, which details why the LLNA is a scientifically appropriate method of potency
determination, and the subsequent submitted comment by Dr. David Basketter, a recognizedexpert in the field of skin sensitization, when making its final report to ICCVAM.
Non-radioactive methods—draft BRDs and related documents
Three new methods of measuring lymphocyte proliferation have been proposed. Unlike thetraditional LLNA, these new methods do not use a radioactive indicator, which could increase
the use of the LLNA in facilities that cannot use radioactive material. The new methods include
two variants of a bromodioxyuridine system [BrdU: ELISA and BrdU: Flow Cytometry (FC)]and the LLNA: DA.
When compared to human data, the LLNA: BrdU-FC had a higher accuracy rate, higher
sensitivity, the same specificity, the same false positive rate, and a lower false negative rate
than the traditional LLNA. Despite this performance, the assay does not achieve complete
concordance with the proposed LLNA Performance Standards the PRP will be evaluating. Thisis also the case with for the LLNA-DA method, which compares identically to human data, yet
4Basketter et al. Predictive identification of human skin sensitization thresholds. Contact Dermatitis. 2005; 53 (5):260-267.
5
Cockshott et al., The local lymph node assay in practice: a current regulatory perspective. Hum Exp Toxicol
2006; 25 (7): 387-394.

3

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ICCVAM Test Method Evaluation Report: Appendix G2

falls short when compared to the traditional LLNA. While reasons for this are not clear, it isworth an examination of whether we should compare new methods to the methods they are
replacing or to the endpoint of actual interest.
The BrdU: ELISA has been recommended for use by ICCVAM pending receipt of additional
information and using alternative decision criteria. We support this finding. Because of theincomplete concordance between these methods and the traditional LLNA, ICCVAM qualified
their acceptance and recommends a “weight-of-evidence” approach. While it is usually goodscientific practice to evaluate any test method results in weight-of-evidence manner,qualifications such as these undercut the recommendations and introduce undue confusion to
the reader. In our view, this gives a company a clear incentive to conduct more testing, when in
reality the methods evaluated have acceptable performance and should simply berecommended.
Performance Characteristics
Although we fully support the development of performance standards that expedite thevalidation of new protocols that are similar to previously validated methods, we reiterate ourdisappointment that ICCVAM/ NICETAM has chosen to apply its limited resources to thelengthy process of developing performance standards for such a narrow scope of applicability.
These performance standards apply only to modifications of the “standard LLNA” that involve
incorporation of non-radioactive methods of detecting lymphocyte proliferation.
In addition, the draft performance standards require the use of a minimum of 22 referencecompounds. The criteria by which the compounds were chosen and the characteristics of thecompounds are described; however, there is no justification for the requirement of such a large
number of compounds for this particular method modification. The methods to which these
performance standards apply will differ from the “standard LLNA” only in the method of
detection of lymphocyte proliferation; therefore the element of concern is sensitivity of thedetection method. All other aspects of the methods to be evaluated will be identical to thestandard LLNA, including delivery and biological response. It is therefore not necessary to test
representatives for every chemical class or every solvent that has been tested in the standard
LLNA. The important characteristic of the reference compound is the magnitude ofproliferation response that is generated, and the list of reference compounds chosen should be
limited to those that represent the range of response seen with the standard LLNA.
In addition, a major criterion for the selection of the above compounds is that there are Guinea
pig data available; more appropriately, chemicals should be chosen on the basis of available
human data.
Conclusions and Future directions
This exercise is a good example of actions undertaken by ICCVAM which result in frustrationin the animal protection community. In the future we hope that ICCVAM will take a more
holistic approach to determine the ways in which it spends its limited time and resources so as toensure maximum benefit for animals in laboratories.

4

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ICCVAM Test Method Evaluation Report: Appendix G2Several non-animal methods for estimating sensitivity are under development, including
quantitative structure activity relationship (QSAR) modeling that shows a high concordancewith guinea pig and LLNA data,6 quantification of peptide reactivity, which also shows a highconcordance with LLNA data,7,8 and human cell cultures.9,10 We urge ICCVAM to secure aninteragency grant from the CPSC to fund the validation of one or more of these non-animal
methods. Clearly, ICCVAM and the CPSC both benefit from the sharing of resources, as the
CPSC nominated the method and ICCVAM will be tasked with the final work product.
ICCVAM should consider taking a more pro-active approach similar to the European Sens-it-iv
project,11 which involves the coordinated efforts of more than two dozen groups from industry,
academia and other organizations, all working toward the common goal of developing in vitro
methods to assess immunotoxicity.
Sincerely,/s/Catherine Willett, PhD
Science Policy Advisor
Regulatory Testing Division
People for the Ethical Treatment of Animals/s/
Kristie Stoick, MPH
Scientific and Policy Advisor
Physicians Committee for Responsible Medicine

6

Fedorowicz et al. Structure-activity models for contact sensitization. Chem Res Toxicol. 2005; 18(6): 954-969.
Gerberick et al. Quantification of chemical peptide reactivity for screening contact allergens: A classification treemodel approach. Toxicol. Sci. 2007; 97(2): 417-427.
8
Natsch and Emter. Skin sensitizers induce antioxidant response element dependent genes: Application to the invitro testing of the sensitization potential of chemicals. Tox Sci. 2008; 102(1): 110-119.
9
Sakaguchi, et al., Development of an in vitro skin sensitization test using human cell lines; huna Cell Line
Activation Test (h-CLAT) II. An inter-laboratory study of the h-CLAT. Toxicol. In vitro. 2005; 20 (5): 774-784.
10
Schoeters et al. Microarray analyses in dendritic cells reveal potential biomarkers for chemical-induced shinsensitization. Mol. Immunol. 2007; 44(12): 3222-3233.
11
http://www.sens-it-iv.eu/
7

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ICCVAM Test Method Evaluation Report: Appendix G3Appendix G3
Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) Comments
SACATM Meeting on June 18–19, 2008
The following is excerpted from the final minutes and speaker presentations of the SACATM
meeting convened on July 18-19, 2008. The full meeting minutes are available online at
http://ntp-server.niehs.nih.gov/?objectid=AF6CC417-F1F6-975E-75B5F3FF7DF1CDDC.

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ICCVAM Test Method Evaluation Report: Appendix G3

Validation Status of New Versions and Applications of the
Murine Local Lymph Node Assay
Dr. Marilyn Wind presented the “Report on the Independent Scientific Peer Review Meeting:
Validation Status of New Versions and Applications of the Murine Local Lymph Node Assay(LLNA), a Test Method for Assessing the Contact Dermatitis Potential of Chemicals andProducts – Introduction and Overview,” authored by Dr. Joanna Matheson.
In 2007, the timeline for the ICCVAM evaluations included the nomination from the CPSC,
endorsement by ICCVAM, and SACTAM’s endorsement of the recommendation. In 2008, the
LLNA Peer Review Panel met and a report was made available. The new/updated LLNA
applications and protocols reviewed by the Peer Review Panel included:•

LLNA limit dose procedure•

LLNA for testing mixtures, metals, and aqueous solutions•

Non-radioactive LLNA: DA method•

Non-radioactive LLNA: BrdU-FC method•

Non-radioactive LLNA: BrdU-ELISA method•

Draft ICCVAM LLNA performance standards•

LLNA for potency determinations.

The documents prepared by the NICEATM and ICCVAM Immunotoxicity Working Group(IWG) for each new/updated LLNA applications included the draft background review
document (BRD), the draft ICCVAM test method recommendations (usefulness and limitations,recommended protocol, future studies), and questions for the Peer Review Panel.
Dr. Wind presented an overview of the LLNA test method protocol.
•

The LLNA protocol was initially described by Kimber et al. (1986).

•

The purpose of the LLNA is to identify chemical sensitizers through quantificationof lymphocyte proliferation.

•

The LLNA uses a minimum of three dose levels. The highest dose level should be
the maximum soluble concentration that does not cause systemic toxicity orexcessive local irritation.
–

A stimulation index (SI) is calculated as the ratio of radioactivity incorporated
into the cells of auricular lymph nodes of the treated animals to that of thevehicle control animals. The threshold for classifying a substance as a skinsensitizer is an SI ≥ 3.

–

In order for an LLNA study to be considered acceptable, the concurrent positive
control must yield an SI ≥ 3.

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ICCVAM Test Method Evaluation Report: Appendix G3

A condensed version of the LLNA test method protocol was provided.
•

Test substance is applied to mouse ears on Days 1, 2, and 3.

•

On Day 6, mice are injected with radiolabeled thymidine (or an analogue ofthymidine).

•

Radiolabeled thymidine is incorporated into the DNA of proliferating cells and theauricular lymph nodes are removed.

•

The amount of radiolabeled thymidine in the lymph nodes is determined as ameasure of lymphocyte proliferation. The ratio of incorporated radioactivity in theauricular lymph nodes of treated vs. control mice (i.e., SI) is calculated, which leads
to classification of a compound as negative or as a sensitizer.

The sole difference between the LLNA limit dose (rLLNA) test method protocol and that of the
traditional LLNA protocol is that only a single dose, the highest dose that does not induce
systemic toxicity or excessive local irritation, is used.
The LLNA Limit Dose Test Method Database contains information that is included in the BRD,
which is based on a retrospective review of traditional LLNA data that were either submitted aspart of the original LLNA evaluation (ICCVAM 1999), extracted from peer-reviewed
publications, or submitted to NICEATM in response to an FR notice requesting available dataand information. Data from 471 studies representing 466 unique substances are available (211
substances were included in the 1998 ICCVAM evaluation of the traditional LLNA). Resultswith the LLNA limit dose test procedure almost always agree with results from the traditional
LLNA. Kimber et al. (2006) showed a 98.6% accuracy for 211 substances, and ICCVAM(2008) showed a 98.9% accuracy for 466 substances.
Dr. Wind provided the draft ICCVAM recommendations for the LLNA Limit Dose Test
Method.
•The LLNA limit dose procedure should be used for the hazard identification of skinsensitizing substances if dose response information is not needed. Use all other
LLNA protocol specifications recommended by ICCVAM (ICCVAM 1999, Dean etal. 2001).

•

Users should be aware that the limit dose is the highest soluble concentration that
does not induce overt systemic toxicity and/or excessive local irritation. A small
possibility of a false negative result exists (1.6% [5/313]) when compared to the
traditional LLNA.

Overview of the Peer Review Panel Report
Dr. Michael Luster presented the “Overview of the LLNA Independent Scientific Peer Review
Panel Report,” starting with the charge to the Panel to review the draft BRDs and to evaluate
the extent to which applicable validation and acceptance criteria of toxicological test methods
have been appropriately addressed. Further the Panel was to consider the ICCVAM draft test
method recommendations for proposed method uses and limitations, recommended
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ICCVAM Test Method Evaluation Report: Appendix G3

standardized protocols, test method performance standards, and proposed future studies. The
Panel was to then comment on the extent to which these items are supported by the information
provided in the BRD. The Panel evaluated the LLNA modifications and applications as listed
above.
Dr. Luster provided highlights of the final Independent Scientific Peer Review Panel report andrecommended that the report should be consulted for a detailed description of the Panel’s
conclusions and recommendations. He provided evaluation highlights for all LLNA
modifications and applications. The following points were made about the LLNA Limit Dose
Procedure (rLLNA).
•

The procedure follows the traditional ICCVAM LLNA protocol except for thenumber of doses tested. This procedure uses only the high dose group (requires 40%
fewer animals).

•

In general, the Panel concurred with the draft ICCVAM recommendation that the
LLNA limit dose procedure should be routinely recommended for the hazard
identification of skin sensitizing chemicals when dose response information is not
required. The Panel also recommended that the limit dose procedure can be used as
an initial test when dose-response information is required.

•

The Panel also recommended that if dose-response information is required, as a way
to further reduce animal use, the LLNA limit dose procedure should be routinely
recommended as the initial test to identify sensitizers before conducting the
traditional LLNA since negative results would not require further testing.

•

Additionally, the Panel suggested that the test be referred to as the “reduced LLNA”(rLLNA) to be consistent with ECVAM terminology.

SACATM Discussion
There were no public comments specific to the rLLNA.
Dr. Grantley Charles concurred with the recommendation that the rLLNA protocol should
include a discussion of how to determine the maximum dose if only a single dose is to be used
in a screen process. An investigator must be able to define excessive irritation; otherwise, thetesting may produce a bell-shaped response curve. The updated ICCVAM LLNA protocol has
been revised to add specific guidance on determining the maximum concentration to be tested
to avoid overt systemic toxicity and excessive local irritation.
Dr. Marion Ehrich suggested that the rLLNA appears favorable because
153/153 nonsensitizing agents and 308/318 sensitizing agents were predicted.
Dr. Luster said that the Panel made a very strong suggestion at the panel meeting that there besome histology associated with the rLLNA procedure. Histology was part of the plan, and it isembedded in the text.
Dr. Donald Fox stated that use of the reduced LLNA procedure could encourage the alternativeuse of animals.
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ICCVAM Test Method Evaluation Report: Appendix H

Appendix H
Relevant Skin Sensitization Regulations and Testing Guidelines
H1

Relevant Skin Sensitization Test Regulations............................................. H-3

H2

OECD Guideline 429: Skin Sensitisation – Local Lymph Node Assay...... H-5

H3OECD Guideline 406: Skin Sensitisation .................................................. H-15

H4

EPA Health Effects Test Guidelines OPPTS 870.2600:
Skin Sensitization....................................................................................... H-27

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H-2

ICCVAM Test Method Evaluation Report: Appendix H1Appendix H1
Relevant Skin Sensitization Test Regulations
United States
Agency/Center or Office

Regulated Products

Legislation
Federal Food, Drug, and Cosmetic
Act (U.S.C. Title 21, Chapter 9)

FDA/CDER

Pharmaceuticals
Public Health Service Act (U.S.C.
Title 42, Chapter 6A)
Toxic Substances Control Act(U.S.C. Title 15, Chapter 53)

Statutory
Requirements
21 CFR 312
21 CFR 314

40 CFR 158.50
40 CFR 158.100
40 CFR 158.340

EPA/OPPTS

Pesticides

CPSC

Consumer ProductsFederal Hazardous Substances Act(U.S.C. Title 15, Chapters 12611278)

16 CFR 1500.3

OSHA

Chemicals

Occupational Safety and HealthAct of 1970 (U.S.C. Title 29,
Chapter 15)

29 CFR 1910.1200

Legislation

Statutory
Requirements

Federal Insecticide, Fungicide, andRodenticide Act (U.S.C. Title 7,
Chapter 6)

EuropeAgency/Center or Office

Regulated Products

EU

Dangerous
Preparations(Chemicals andchemical mixtures)

EU

Pesticides

Directive 1999/45/EC of theEuropean Parliament and of theCouncilof 31 May 1999
Annex V to Directive 67/548/EECof 27 June 1967
Directive 91/414/EEC of theEuropean Parliament and of theCouncil of 15 July 1991

International
Agency/Center or Office

Regulated Products

GHS

Chemicals

Legislation

H-3

Statutory
Requirements
GHS Part 3, Chapter
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H-4

ICCVAM Test Method Evaluation Report: Appendix H2Appendix H2OECD Test Guideline 429: Skin Sensitisation – Local Lymph Node Assay

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H-6

ICCVAM Test Method Evaluation Report: Appendix H2

OECD/OCDE

429
Adopted:24th April 2002

OECD GUIDELINE FOR THE TESTING OF CHEMICALSSkin Sensitisation: Local Lymph Node Assay

INTRODUCTION
1.
The OECD Test Guideline Programme periodically reviews progress in test method development
and refinement, both in terms of scientific advances and animal welfare, to determine whether existing Test
Guidelines should be updated and whether new Guidelines should be developed. Toward that end, a new
assay for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA) has
been sufficiently validated and accepted to justify its adoption as a new Test Guideline (1)(2)(3). This is
the second Guideline to be promulgated for assessing skin sensitisation potential of chemicals in animals.
The other Guideline (406) utilises guinea pig tests, notably the guinea pig maximisation test and theBuehler test (4)..
2.
The LLNA provides certain advantages with regard to both scientific progress and animal
welfare. It studies the induction phase of skin sensitisation and provides quantitative data suitable for dose
response assessment. The details of the validation of the LLNA and a review of the associated work havebeen published (5)(6)(7)(8). In addition, it should be noted that the mild/moderate sensitisers, which arerecommended as suitable positive control substances for guinea pig test methods, are also appropriate foruse with the LLNA (6)(8)(9).

INITIAL CONSIDERATIONS
3.
The LLNA provides an alternative method for identifying skin sensitising chemicals and for
confirming that chemicals lack a significant potential to cause skin sensitisation. This does not necessarily
imply that in all instances the LLNA should be used in place of guinea pig tests, but rather that the assay isof equal merit and may be employed as an alternative in which positive and negative results generally nolonger require further confirmation.
4.
The LLNA is an in vivo method and, as a consequence, will not eliminate the use of animals in
the assessment of contact sensitising activity. It has, however, the potential to reduce the number ofanimals required for this purpose. Moreover, the LLNA offers a substantial refinement of the way inwhich animals are used for contact sensitisation testing. The LLNA is based upon consideration of
immunological events stimulated by chemicals during the induction phase of sensitisation. Unlike guinea
pig tests the LLNA does not require that challenged-induced dermal hypersensitivity reactions be elicited.
Furthermore, the LLNA does not require the use of an adjuvant, as is the case for the guinea pig
maximisation test. Thus, the LLNA reduces animal distress. Despite the advantages of the LLNA overtraditional guinea pig tests, it should be recognised that there are certain limitations that may necessitate
the use of traditional guinea pigs tests (e.g., false negative findings in the LLNA with certain metals, false
positive findings with certain skin irritants)(10).

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OECD/OCDE

PRINCIPLE OF THE TEST5.
The basic principle underlying the LLNA is that sensitisers induce a primary proliferation oflymphocytes in the lymph node draining the site of chemical application. This proliferation is proportional
to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an
objective, quantitative measurement of sensitisation. The LLNA assesses this proliferation as a doseresponse in which the proliferation in test groups is compared to that in vehicle treated controls. The ratioof the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, isdetermined, and must be at least three before a test substance can be further evaluated as a potential skinsensitiser. The methods described here are based on the use of radioactive labelling to measure cellproliferation. However, other endpoints for assessment of proliferation may be employed provided there isjustification and appropriate scientific support, including full citations and description of the methodology.

DESCRIPTION OF THE ASSAY
Selection of animal species
6.
The mouse is the species of choice for this test. Young adult female mice of CBA/Ca or CBA/J
strain, which are nulliparous and non-pregnant, are used. At the start of the study, animals should be
between 8-12 weeks old, and the weight variation of the animals should be minimal and not exceed 20% of
the mean weight. Other strains and males may be used when sufficient data are generated to demonstratethat significant strain and/or gender-specific differences in the LLNA response do not exist.

HOUSING AND FEEDING CONDITIONS
7.
Animals should be individually housed. The temperature of the experimental animal room should
be 22ºC (+ 3ºC). Although the relative humidity should be at least 30% and preferably not exceed 70%
other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, the sequence
being 12 hours light, 12 hours dark. For feeding, conventional laboratory diets may be used with anunlimited supply of drinking water.

PREPARATION OF ANIMALS
8.
The animals are randomly selected, marked to permit individual identification (but not by any
form of ear marking), and kept in their cages for at least 5 days prior to the start of dosing to allow for
acclimatisation to the laboratory conditions. Prior to the start of treatment all animals are examined toensure that they have no observable skin lesions.
Reliability check
9.
Positive controls are used to demonstrate appropriate performance of the assay and competencyof the laboratory to successfully conduct the assay. The positive control should produce a positive LLNA
response at an exposure level expected to give an increase in the stimulation index (SI) >3 over thenegative control group. The positive control dose should be chosen such that the induction is clear but notexcessive. Preferred substances are hexyl cinnamic aldehyde (CAS No 101-86-0) andmercaptobenzothiazole (CAS No 149-30-4). There may be circumstances in which, given adequate
justification, other control substances, meeting the above criteria, may be used. While ordinarily a positive
control group may be required in each assay, there may be situations in which test laboratories will haveavailable historic positive control data to show consistency of a satisfactory response over a six-month or
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429

more extended period. In those situations, less frequent testing with positive controls may be appropriate
at intervals of no greater than 6 months. Although the positive control substance should be tested in thevehicle that is known to elicit a consistent response (e.g., acetone:olive oil), there may be certain regulatory
situations in which testing in a non-standard vehicle (clinically/chemically relevant formulation) will also
be necessary. In such situations the possible interaction of a positive control with this unconventional
vehicle should be tested.

TEST PROCEDURE
Number of animals and dose levels
10.
A minimum of four animals is used per dose group, with a minimum of three concentrations of
the test substance, plus a negative control group treated only with the vehicle for the test substance, and apositive control, as appropriate. In those cases in which individual animal data are to be collected, aminimum of five animals per dose group are used. Dose and vehicle selection should be based on therecommendations given in reference (2). Doses are selected from the concentration series 100%, 50%,
25%, 10%, 5%, 2.5%, 1%, 0.5% etc. Existing acute toxicity and dermal irritation data should beconsidered, where available, in selecting the three consecutive concentrations so that the highest
concentration maximises exposure whilst avoiding systemic toxicity and excessive local skin irritation(2)(11). Except for absence of treatment with the test substance, animals in the control groups should be
handled and treated in a manner identical to that of animals in the treatment groups.
11.
The vehicle should be selected on the basis of maximising the test concentrations and solubility
whilst producing a solution/suspension suitable for application of the test substance. In order ofpreference, recommended vehicles are acetone/olive oil (4:1 v/v), dimethylformamide, methyl ethyl
ketone, propylene glycol and dimethyl sulphoxide (2)(10), but others may be used if sufficient scientificrationale is provided. In certain situations it may be necessary to use a clinically relevant solvent or thecommercial formulation in which the test substance is marketed as an additional control. Particular careshould be taken to ensure that hydrophilic materials are incorporated into a vehicle system, which wets theskin and does not immediately run off. Thus, wholly aqueous vehicles are to be avoided.
Experimental schedule
12.

The experimental schedule of the assay is as follows:• Day 1:
Individually identify and record the weight of each animal. Open application of 25µL of theappropriate dilution of the test substance, the vehicle alone, or the positive control (asappropriate), to the dorsum of each ear.
• Days 2 and 3:
Repeat the application procedure carried out on day 1.
• Days 4 and 5 :No treatment.
• Day 6 :Record the weight of each animal. Inject 250µL of phosphate-buffered saline (PBS)
containing 20 µCi (7.4e+5 Bq) of 3H-methyl thymidine into all test and control mice via thetail vein. Alternatively inject 250 µL PBS containing 2 µCi (7.4e + 4 Bq) of 125I­
iododeoxyuridine and 10-5M fluorodeoxyuridine into all mice via the tail vein. Five hours(5 h) later, the animals are killed. The draining auricular lymph nodes from each ear are
excised and pooled in PBS for each experimental group (pooled treatment group approach);
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OECD/OCDE
alternatively pairs of lymph nodes from individual animals may be excised and pooled in
PBS for each animal (individual animal approach). Details and diagrams of the node
identification and dissection can be found in Annex I of the ICCVAM Immunotoxicology
Working Group LLNA Protocol (10).

Preparation of cell suspensions
13.
A single cell suspension of lymph node cells (LNC) either from pooled treatment groups orbilaterally from individual animals is prepared by gentle mechanical disaggregation through 200 µm-meshstainless steel gauze. Lymph node cells are washed twice with an excess of PBS and precipitated with 5%
trichloroacetic acid (TCA) at 4oC for 18h(2). Pellets are either re-suspended in 1 mL TCA and transferredto scintillation vials containing 1.0 mL of scintillation fluid for 3H-counting, or transferred directly to
gamma counting tubes for 125I-counting.
Determination of cellular proliferation (incorporated radioactivity)
14.
Incorporation of 3H-methyl thymidine is measured by β-scintillation counting as disintegrations
per minute (DPM). Incorporation of 125I-iododeoxyuridine is measured by 125I-counting and also isexpressed as DPM. Depending on the approach used, the incorporation will be expressed as
DPM/treatment group (pooled approach) or DPM/animal (individual approach).

OBSERVATIONSClinical observations
15.
Animals should be carefully observed once daily for any clinical signs, either of local irritation at
the application site or of systemic toxicity. All observations are systematically recorded with individual
records being maintained for each animal.

Body weights
16.
As stated in paragraph 12, individual animal body weights should be measured at the start of thetest and at the scheduled kill of the animals.

CALCULATION OF RESULTS
17.
Results are expressed as the Stimulation Index (SI). When using the pooled approach, the SI is
obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of
the pooled vehicle control group; this yields a mean SI. When using the individual approach, the SI isderived by dividing the mean DPM /mouse within each test substance group and the positive control groupby the mean DPM/mouse for the solvent/vehicle control group. The average SI for vehicle treated controls
is then 1.
18.
Use of the individual approach to calculate the SI will enable the performance of a statisticalanalysis of the data. In choosing an appropriate method of statistical analysis, the investigator should
maintain an awareness of possible inequalities of variances and other related problems that may necessitate
a data transformation or a non-parametric statistical analysis. An adequate approach for interpreting the
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429

data is to evaluate all individual data of treated and vehicle controls, and derive from these the best fitting
dose response curve, taking confidence limits into account (10)(12)(13). However, the investigator shouldbe alert to possible “outlier” responses for individual animals within a group that may necessitate the use ofan alternative measure of response (e.g. median rather than mean) or elimination of the outlier.
19.
The decision process with regard to a positive response includes a stimulation index ≥ 3, together
with consideration of dose-response and, where appropriate, statistical significance (3)(6)(10)(13)(14).
20.
If it is necessary to clarify the results obtained, consideration should be given to various
properties of the test substance, including whether it has a structural relationship to known skin sensitisers,
whether it causes excessive skin irritation, and the nature of the dose response seen. These and otherconsiderations are discussed in detail elsewhere (7).

DATA AND REPORTING
Data
21.
Data should be summarised in tabular form showing the mean and individual DPM values andstimulation indexes for each dose (including vehicle control) group.
Test report
22.

The test report should contain the following information:
Test substance:-

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

Vehicle:-identification data (purity; concentration, where appropriate; volume used);justification for choice of vehicle.

Test animals:-strain of mice used;microbiological status of the animals, when known;
number, age and sex of animals;source of animals, housing conditions, diet, etc.

Test conditions:-details of test substance preparation and application;
justification for dose selection (including results from range finding study, if conducted);­vehicle and test substance concentrations used, and total amount of substance applied;details of food and water quality (including diet type/source, water source).
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OECD/OCDE

Reliability check:-a summary of results of latest reliability check, including information on substance,
concentration and vehicle used;concurrent and/or historical positive and negative control data for testing laboratory.

Results:--

individual weights of animals at start of dosing and at scheduled kill;a table of mean/median (pooled approach) and individual (individual approach) DPM
values, as well as the range of values for both approaches, and stimulation indices foreach dose (including vehicle control) group;
statistical analysis, where appropriate;
time course of onset and signs of toxicity, including dermal irritation at site ofadministration, if any, for each animal.

Discussion of results:-

A brief commentary on the results, the dose-response analysis, and statistical analyses,
where appropriate, with a conclusion as to whether the test substance should beconsidered a skin sensitiser.

LITERATURE(1)Kimber, I. and Basketter, D.A. (1992). The murine local lymph node assay; collaborative studies
and new directions: A commentary. Food and Chemical Toxicology 30, 165-169.

(2)

Kimber, I. Dearman, R.J. Scholes E.W, and Basketter, D.A (1994). The local lymph node assay:
developments and applications. Toxicology, 93, 13-31.

(3)Kimber, I. Hilton, J. Dearman, R.J. Gerberick, G.F. Ryan, C.A. Basketter, D.A. Lea, L. House,
R.V. Ladies, G.S. Loveless, S.E. Hastings, K.L. (1998). Assessment of the skin sensitisation
potential of topical medicaments using the local lymph node assay: An interlaboratory exercise.
Journal of Toxicology and Environmental Health, 53 563-79 (1998).

(4)OECD (1992). Guideline 406 Skin Sensitisation.

(5)

Chamberlain, M. and Basketter, D.A. (1996). The local lymph node assay: status of validation.
Food and Chemical Toxicology, 34, 999-1002.

(6)

Basketter, D.A. Gerberick, G.F. Kimber, I. and Loveless, S.E. (1996). The local lymph node assay
A viable alternative to currently accepted skin sensitisation tests. Food and Chemical Toxicology,
34, 985-997.

(7)

Basketter, D.A. Gerberick, G.F. and Kimber, I. (1998). Strategies for identifying false positive
responses in predictive sensitisation tests. Food and Chemical Toxicology. (in press) 36 327-33.

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429

(8)

Van Och, F.M.M, Slob, W., De Jong, W.H., Vandebriel, R.J., Van Loveren, H. (2000). A
quantitative method for assessing the sensitising potency of low molecular weight chemicals using
a local lymph node assay: employment of a regression method that includes determination of
uncertainty margins. Toxicology 146 49-59.

(9)Dearman, R.J. Hilton, J. Evans, P. Harvey, P. Basketter, D.A. and Kimber, I. (1998). Temporalstability of local lymph node assay responses to hexyl cinnamic aldehyde. Journal of Applied
Toxicology 18 281-4.

(10)

National Institute of Environmental Health Sciences (1999). The murine local lymph node Assay:
A test method for assessing the allergic contact dermatitis potential of chemicals/compounds: The
results of an independent peer review evaluation coordinated by the Interagency Coordinating
Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology
Program Center for the Evaluation of Alternative Toxicological Methods (NICETAM). NIH
Publication No: 99-4494, Research Triangle Park, N.C. (http://iccvam.niehs.nih.gov).

(11)

OECD (2002). Guideline 404 Acute Dermal Irritation/Corrosion.

(12)

Basketter, D.A. Selbie, E. Scholes, E.W. Lees, D. Kimber, I. and Botham, P.A. (1993). Resultswith OECD recommended positive control sensitisers in the maximisation. Buehler and local
lymph node assays. Food and Chemical Toxicology, 31, 63-67.

(13)

Basketter DA, Lea LJ, Dickens A, Briggs D, Pate I, Dearman RJ, Kimber I (1999). A comparisonof statistical approaches to the derivation of EC3 values from local lymph node assay dose
responses. J. Appl. Toxicology, 19, 261-266.

(14)

Basketter DA, Blaikie L, Derman RJ, Kimber I, Ryan CA, Gerberick GF, Harvey P, Evans P,
White IR, and Rycroft RTG (2000). Use of local lymph node assay for the estimation of relative
contact allergenic potency. Contact Dermatitis, 42, 344-48.

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406
Adopted:17.07.92

OECD GU ID ELIN E FOR TES TIN G OF CHEMICALS
Adopted by the Council on 17th July 1992
Skin Sensitisation

IN TROD U CTION

1.
OECD Guidelines for Testing of Chemicals are periodically reviewed in light of scientificprogress. In such reviews, special attention is given to possible improvements in relation to animal
welfare. This updated version of the original guideline 406, adopted in 1981, is the outcome of ameeting of OECD experts held in Paris in May 1991.
2.
Currently, quantitative structure-activity relationships and in vitro models are not yet
sufficiently developed to play a significant role in the assessment of the skin-sensitisation potential of
substances which therefore must continue to be based on in vivo models.
3.
The guinea pig has been the animal of choice for predictive sensitisation tests for several
decades. Two types of tests have been developed: adjuvant tests in which sensitisation is potentiatedby the injection of Freunds Complete Adjuvant (FCA), and non-adjuvant tests. In the original
guideline 406, four adjuvant tests and three non-adjuvant tests were considered to be acceptable. In
this updated version, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman whichuses adjuvant (1)(2)(3)(4) and the non-adjuvant Buehler Test (5)(6) are given preference over othermethods and the procedures are presented in detail. It is recognised, however, that there may becircumstances where other methods may be used to provide the necessary information on sensitisation
potential.
4.
The immune system of the mouse has been investigated more extensively than that of theguinea pig. Recently, mouse models for assessing sensitisation potential have been developed thatoffer the advantages of an endpoint which is measured objectively, short duration and minimal animaltreatment. The mouse ear swelling test (MEST) and the local lymph node assay (LLNA) appear tobe promising. Both assays have undergone validation in several laboratories (7)(8)(9)(10)(11) and it
has been shown that they are able to detect reliably moderate to strong sensitisers. The LLNA or the
MEST can be used as a first stage in the assessment of skin sensitisation potential. If a positive result
is seen in either assay, a test substance may be designated as a potential sensitiser, and it may not benecessary to conduct a further guinea pig test. However, if a negative result is seen in the LLNA or
MEST, a guinea pig test (preferably a GPMT or Buehler Test) must be conducted using the proceduredescribed in this guideline.
5.

Definitions used are set out in the Annex.

GEN ERAL PRIN CIPLE OF S ENSITISATION TES TS IN GU IN EA PIGS

6.
The test animals are initially exposed to the test substance by intradermal injection and/or
epidermal application (induction exposure). Following a rest period of 10 to 14 days (induction
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period), during which an immune response may develop, the animals are exposed to a challenge dose.
The extent and degree of skin reaction to the challenge exposure in the test animals is compared withthat demonstrated by control animals which undergo sham treatment during induction and receive thechallenge exposure.

ELEMEN TS COMMON TO S ENSITISATION TES TS IN GU IN EA PIGS
Sex of animals

7.
Male and/or female healthy young adult animals can be used. If females are used they should
be nulliparous and non-pregnant.
Housing and feeding conditions

8.
The temperature of the experimental animal room should be 20oC (+ 3oC) and the relative
humidity 30-70 per cent. Where the lighting is artificial, the sequence should be 12 hours light, 12hours dark. For feeding, conventional laboratory diets may be used with an unlimited supply of
drinking water. It is essential that guinea pigs receive an adequate amount of ascorbic acid.
Preparation of the animals

9.
Animals are acclimatised to the laboratory conditions for at least 5 days prior to the test.
Before the test, animals are randomised and assigned to the treatment groups. Removal of hair is byclipping, shaving or possibly by chemical depilation, depending on the test method used. Care should
be taken to avoid abrading the skin. The animals are weighed before the test commences and at theend of the test.
Reliability check10.
The sensitivity and reliability of the experimental technique used should be assessed every six
months by use of substances which are known to have mild-to-moderate skin sensitisation properties.
11.
In a properly conducted test, a response of at least 30% in an adjuvant test and at least 15%
in a non-adjuvant test should be expected for mild/moderate sensitisers. Preferred substances are hexyl
cinnamic aldehyde (CAS No. 101-86-0), mercaptobenzothiazole (CAS No. 149-30-4) and benzocaine(CAS No. 94-09-7). There may be circumstances where, given adequate justification, other control
substances meeting the above criteria may be used.
Remo val of the te st substanc e

12.
If removal of the test substance is considered necessary, this should be achieved using wateror an appropriate solvent without altering the existing response or the integrity of the epidermis.

D ES CRIPTION OF THE GU IN EA- PIG MAXIMISATION TES T METHOD
N umber of animals

13.
A minimum of 10 animals is used in the treatment group and at least 5 animals in the control
group. When fewer than 20 test and 10 control guinea pigs have been used, and it is not possible toconclude that the test substance is a sensitiser, testing in additional animals to give a total of at least
20 test and 10 control animals is strongly recommended.

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Dose levels

14.
The concentration of test substance used for each induction exposure should be well-tolerated
systemically and should be the highest to cause mild-to-moderate skin irritation. The concentration
used for the challenge exposure should be the highest non-irritant dose. The appropriate
concentrations can be determined from a pilot study using two or three animals. Consideration should
be given to the use of FCA-treated animals for this purpose.
Induction: Intradermal Injections
D ay 0 - tre ated group

15.
Three pairs of intradermal injections of 0.1 ml volume are given in the shoulder region which
is cleared of hair so that one of each pair lies on each side of the midline.
Injection 1:

a 1:1 mixture (v/v) FCA/water or physiological saline

Injection 2:

the test substance in an appropriate vehicle at the selected concentration

Injection 3:

the test substance at the selected concentration formulated in a 1:1 mixture(v/v) FCA/water or physiological saline.

16.
In injection 3, water soluble substances are dissolved in the aqueous phase prior to mixing
with FCA. Liposoluble or insoluble substances are suspended in FCA prior to combining with theaqueous phase. The concentration of test substance shall be equal to that used in injection 2.
17.
Injections 1 and 2 are given close to each other and nearest the head, while 3 is given towards
the caudal part of the test area.
D ay 0 - control group18.
Three pairs of intradermal injections of 0.1 ml volume are given in the same sites as in thetreated animals.
Injection 1:

a 1:1 mixture (v/v) FCA/water or physiological saline

Injection 2:

the undiluted vehicle

Injection 3:

a 50% w/v formulation of the vehicle in a 1:1 mixture (v/v) FCA/water orphysiological saline.

Induction: Topical Application
D ay 5-7 - tre ated and control groups

19.
Approximately twenty-four hours before the topical induction application, if the substance is
not a skin irritant, the test area, after close-clipping and/or shaving is painted with 0.5 ml of 10%
sodium lauryl sulphate in vaseline, in order to create a local irritation.
D ay 6-8 - tre ated group

20.
The test area is again cleared of hair. A filter paper (2 x 4 cm) is fully-loaded with test
substance in a suitable vehicle and applied to the test area and held in contact by an occlusive dressing
for 48 hours. The choice of the vehicle should be justified. Solids are finely pulverised and
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incorporated in a suitable vehicle. Liquids can be applied undiluted, if appropriate.
D ay 6-8 - control group

21.
The test area is again cleared of hair. The vehicle only is applied in a similar manner to thetest area and held in contact by an occlusive dressing for 48 hours.
Challenge: Topical Application
D ay 20-22 - tre ated and control groups

22.
The flanks of treated and control animals are cleared of hair. A patch or chamber loaded withthe test substance is applied to one flank of the animals and, when relevant, a patch or chamber loadedwith the vehicle only may also be applied to the other flank. The patches are held in contact by an
occlusive dressing for 24 hours.
Observations - tre ated and control groups

23.

-

approximately 21 hours after removing the patch the challenge area is cleaned andclosely-clipped and/or shaved or depilated if necessary;-

approximately 3 hours later (approximately 48 hours from the start of the challenge
application) the skin reaction is observed and recorded according to the grades shown
below;-

approximately 24 hours after this observation a second observation (72 hours) is madeand once again recorded.

Blind reading of test and control animals is encouraged.

TAB LE: MAGNUSSON AN D KLIGMAN GRAD IN G S CALE FOR THE EVALU ATION
OF CHALLEN GE PATCH TES T R EACTION S

0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Rechalleng e

24.
If it is necessary to clarify the results obtained in the first challenge, a second challenge (i.e.
a rechallenge), where appropriate with a new control group, should be considered approximately one
week after the first one. A rechallenge may also be performed on the original control group.
Clinical observations

25.
All skin reactions and any unusual findings, including systemic reactions, resulting frominduction and challenge procedures should be observed and recorded. Other procedures, e.g.
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doubtful reactions.

D ES CRIPTION OF THE B U EHLER TES T METHOD
N umber of animals

26.
A minimum of 20 animals is used in the treatment group and at least 10 animals in the control
group.
Dose levels

27.
The concentration of test substance used for each induction exposure should be the highest tocause mild irritation. The concentration used for the challenge exposure should be the highest
non-irritating dose. The appropriate concentration can be determined from a pilot study using two or
three animals.
28.
For water soluble test materials, it is appropriate to use water or a dilute non-irritating solutionof surfactant as the vehicle. For other test materials 80% ethanol/water is preferred for induction andacetone for challenge.
Induction: Topical application
D ay 0 - tre ated group29.
One flank is cleared of hair (closely-clipped). The test patch system should be fully loadedwith test substance in a suitable vehicle (the choice of the vehicle should be justified; liquid test
substances can be applied undiluted, if appropriate). The test patch system is applied to the test areaand held in contact with the skin by an occlusive patch or chamber and a suitable dressing for 6 hours.
30.
The test patch system must be occlusive. A cotton pad is appropriate and can be circular or
square, but should approximate 4-6 cm2. Restraint using an appropriate restrainer is preferred to assure
occlusion. If wrapping is used, additional exposures may be required.
D ay 0 - control group31.
One flank is cleared of hair (closely-clipped). The vehicle only is applied in a similar mannerto that used for the treated group. The test patch system is held in contact with the skin by an
occlusive patch or chamber and a suitable dressing for 6 hours. If it can be demonstrated that a shamcontrol group is not necessary, a naive control group may be used.
D ays 6-8 and 13-15 - tre ated and control groups

32.
The same application as on day 0 is carried out on the same test area (cleared of hair ifnecessary) of the same flank on day 6-8, and again on day 13-15.
Challeng e
D ay 27-29 - tre ated and control groups

33.
The untreated flank of treated and control animals is cleared of hair (closely-clipped). An
occlusive patch or chamber containing the appropriate amount of test substance is applied, at the
maximum non-irritant concentration, to the posterior untreated flank of treated and control animals.
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When relevant, an occlusive patch or chamber with vehicle only is also applied to the anterior
untreated flank of both treated and control animals. The patches or chambers are held in contact bya suitable dressing for 6 hours.
Observations - tre ated and control groups

34.

-

approximately 21 hours after removing the patch the challenge area is cleared of hair;-approximately three hours later (approximately 30 hours after application of thechallenge patch) the skin reactions are observed and recorded according to the grades
shown in the Guinea-Pig Maximisation Test (see paragraph 23);-

approximately 24 hours after the 30 hour observation (approximately 54 hours after
application of the challenge patch) skin reactions are again observed and recorded.

Blind reading of test and control animals is encouraged.
Rechalleng e

35.
If it is necessary to clarify the results obtained in the first challenge, a second challenge (i.e.
a rechallenge), where appropriate with a new control group, should be considered approximately one
week after the first one. The rechallenge may also be performed on the original control group.
Clinical observations

36.
All skin reactions and any unusual findings, including systemic reactions, resulting frominduction and challenge procedures should be observed and recorded. Other procedures, e.g.
histopathological examination, measurement of skin fold thickness, may be carried out to clarify
doubtful reactions.

D ATA AN D R EPOR TIN G (GPMT and B uehler Te st)
D ata

37.
Data should be summarised in tabular form, showing for each animal the skin reactions at eachobservation.
Te st report

38.

The test report must include the following information:
Test substance:-

physical nature and, where relevant, physicochemical properties;
identification data.

Vehicle:-justification of choice of vehicle.

Test animals:-strain of guinea-pig used;
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number, age and sex of animals;source, housing conditions, diet, etc.;
individual weights of animals at the start and at the conclusion of the test.

Test conditions:-

technique of patch site preparation;
details of patch materials used and patching technique;result of pilot study with conclusion on induction and challenge
concentrations to be used in the test;
details of test substance preparation, application and removal;
vehicle and test substance concentrations used for induction andchallenge exposures and the total amount of substance applied for
induction and challenge.

Reliability check:-a summary of the results of the latest reliability check including information on
substance, concentration and vehicle used.

-on each animal including grading system;
narrative description of the nature and degree of effects
observed;
any histopathological findings.

Results:-

Discussion of the results.
If a screening assay is performed before the guinea pig test the description or reference of thetest, including details of the procedure, must be given together with results obtained with the test andreference substances.

LITERATURE(1)

Magnusson B. and Kligman A.M. (1969). The identification of contact allergens by animal
assay. The guinea pig maximisation test. J. Invest. Dermatol., 52, 268.

(2)

Magnusson B. and Kligman A.M. (1970). Allergic Contact Dermatitis in the Guinea Pig.
Charles G. Thomas; Springfield, Illinois.

(3)

Magnusson B. (1980). Identification of contact sensitizers by animal assay. Cont. Derm., 6,
46.

(4)

Magnusson B., Fregert S. and Wahlberg J. (1979). Determination of skin sensitization
potential of chemicals. Predictive testing in guinea pigs. Arbete och Hälsa, 26(E).

(5)

Buehler E.V. (1965). Delayed contact hypersensitivity in the guinea pig. Arch. Dermatol.,
91, 171.

(6)

Ritz H.L. and Buehler E.V. (1980). Procedure for conducting the guinea pig assay. Current
Concepts in Dermatology, Drill V.A. and Lazar P. (eds), Academic Press, New York, N.Y.,
25-40.
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(7)

Kimber I., Hilton J. and Weisenberger C. (1989). The murine local lymph mode assay for
identification of contact allergens: a preliminary evaluation of in situ measurement oflymphocyte proliferation Contact Dermatitis, 21, 215-220.

(8)

Kimber I., Hilton J. and Botham P.A. (1990). Identification of contact allergens using themurine local lymph node assay: comparisons with the Buehler OccludedPatch Test in guinea
pigs. Journal of Applied Toxicology, 10(3), 173-180.

(9)

Kimber I., Hilton J., Botham P.A., Basketter D.A., Scholes E.W., Miller K., Robbins M.C.,
Harrison P.T.C., Gray T.J.B. and Waite S.J. (1991). The murine local lymph node assay:
results of an inter-laboratory trial. Toxicology Letters, 55, 203-213.

(10)

Basketter D.A., Scholes E.W., Kimber I., Botham P.A., Hilton J., Miller K., Robbins M.C.,
Harrison P.T.C. and Waite S.J. (1991). Interlaboratory evaluation of the local lymph node
assay with 25 chemicals and comparison with guinea pig test data. Toxicology Methods, 1,30-43.

(11)

Gad S.C., Dunn B.J., Dobbs D.W., Reilly C. and Walsh R.D. (1986). Development andvalidation of an alternative dermal sensitisation test: the mouse ear swelling test (MEST).
Toxicol. Appl. Pharmacol., 84, 93-114.

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AN N EXD EFIN ITION S

Skin sensitisation (allergic contact dermatitis) is an immunologically mediated cutaneous reaction toa substance. In the human, the responses may be characterised by pruritis, erythema, oedema, papules,
vesicles, bullae or a combination of these. In other species the reactions may differ and only erythemaand oedema may be seen.
Induction exposure: an experimental exposure of a subject to a test substance with the intention of
inducing a hypersensitive state.
Induction period: a period of at least one week following an induction exposure during which a
hypersensitive state may develop.
Challenge exposure: an experimental exposure of a previously treated subject to a test substancefollowing an induction period, to determine if the subject reacts in a hypersensitive manner.

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ICCVAM Test Method Evaluation Report: Appendix H4Appendix H4
EPA Health Effects Test Guidelines
OPPTS 870.2600: Skin Sensitization

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ICCVAM Test Method Evaluation Report: Appendix H4United States
Environmental Protection
AgencyPrevention, Pesticides
and Toxic Substances(7101)EPA 712–C–03–197
March 2003

Health Effects Test
Guidelines
OPPTS 870.2600
Skin Sensitization

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ICCVAM Test Method Evaluation Report: Appendix H4

INTRODUCTION
This guideline is one of a series of test guidelines that have beendeveloped by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing ofpesticides and toxic substances, and the development of test data that mustbe submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization thatblended the testing guidance and requirements that existed in the Officeof Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), theOffice of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub­lished by the Organization for Economic Cooperation and Development(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing proceduresthat must be performed to meet the data requirements of the U. S. Environ­mental Protection Agency under the Toxic Substances Control Act (15U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act(7 U.S.C. 136, et seq.).
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on disks or paper
copies: call (202) 512–0132. This guideline is also available electronicallyin PDF (portable document format) from EPA’s Internet Web site at http://www.epa.gov/opptsfrs/home/guidelin.htm.

i

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ICCVAM Test Method Evaluation Report: Appendix H4

OPPTS 870.2600

Skin sensitization.

(a) Scope—(1) Applicability. This guideline is intended to meet test­
ing requirements of both the Federal lnsecticide, Fungicide, andRodenticide Act (FIFRA) (7 U.S.C. l36, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source materials used in developing this har­monized OPPTS test guideline are OPPTS Harmonized Test Guidelines
Series 870, Guideline 870.2600 Skin Sensitization, dated August 1998; 40
CFR 798.4100 Dermal Sensitization; OECD 406 Skin Sensitization (adopt­ed July 1992); and OECD 429 Skin Sensitization: Local Lymph Node
Assay (adopted April 2002).
(b) Purpose. The purpose of the selected test is to identify substanceswith skin sensitization potential. Determination of the potential to cause
or elicit skin sensitization reactions (allergic contact dermatitis) is an im­
portant element in evaluating a substance’s toxicity. Information derived
from skin sensitization tests serves to identify possible hazards to a popu­
lation exposed repeatedly to a test substance. Testing is not required if
the test material is a known skin sensitizer. If it is suspected that the test
material is a strong dermal irritant, see OPPTS 870.1000, paragraph(d)(2)(iii).
(c) Definitions. The following definitions apply to this test guideline.
The definitions in Section 3 of TSCA and in 40 CFR Part 792—Good
Laboratory Practice Standards (GLP) also apply to this test guideline.
Challenge exposure is an exposure of a previously treated subject toa test substance following an induction period to elicit a contact hypersensitivity response.
Induction exposure is the administration of a test substance to thetest subject with the intention of inducing contact sensitization.
Induction period is a period of at least 1 week following an induction
exposure during which sensitization may develop.
Skin sensitization (allergic contact dermatitis) is an immunologicallymediated cutaneous reaction to a substance. In the human, the responses
may be characterized by pruritis, erythema, edema, papules, vesicles,
bullae, or a combination of these. In other mammalian species, the reac­
tions may differ and only erythema and edema may be seen.
Stimulation index (SI) is the ratio of

3H-methyl

thymidine or

125I-iododeoxyuridine (125IU) incorporation into test group lymph nodes

relative to that recorded for solvent/vehicle control group lymph nodes.
(d) Test procedures—(1) Methods. Any of the following test meth­ods is considered to be acceptable:

1

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ICCVAM Test Method Evaluation Report: Appendix H4(i) Local Lymph Node Assay (LLNA) test, or(ii) Guinea-Pig Maximization Test (GPMT), or(iii) Buehler test.
(2) Choice of assays. See OPPTS 870.1000 for a general discussionof factors to be considered prior to performing the test. In addition, thefollowing considerations apply:(i) The LLNA (see references in paragraphs (g)(1) through (g)(6) of
this guideline) is a preferred alternative method, where applicable, to the
traditional guinea pig test because it demonstrates an equivalent predictionof human allergic contact dermatitis as compared to the other sensitization
tests, provides quantitative data and an assessment of dose-response, gives
consideration to animal welfare concerns, and is suitable for testing col­ored substances. It should be recognized that there are certain testing situa­
tions that may necessitate the use of traditional guinea pig tests. The tester
should note that the LLNA may not be appropriate for all types of test
materials, such as certain metallic compounds, high molecular weight pro­teins, strong dermal irritants and materials that do not sufficiently adhere
to the ear for an acceptable period of time during treatment. When using
the LLNA, particular care should be taken to ensure that hydrophilic materials are incorporated into a vehicle system that wets the skin and does
not immediately run off. Thus, wholly aqueous vehicles or test materials
and runny liquids are to be avoided. In all instances, the tester must docu­
ment that appropriate techniques were used to facilitate adherence to themouse ear for an adequate exposure duration. It may be possible to use
the LLNA to test some of these materials if appropriate techniques areused to facilitate adherence.
(ii) In situations for test materials where the LLNA is not applicableor may provide unreliable or problematic results, the GPMT or Buehler
tests are recommended (see references in paragraphs (g)(7) through (g)(14)of this guideline).
(iii) Although the LLNA, GPMT, or Buehler tests are considered tobe acceptable tests, it is recognized that other tests may give useful results.
If other tests are used, the investigator must provide justification/reasoning
for use of other procedures and methods and protocols must be provided.
A positive and negative control group must be included in each test.
(e) Test methods—(1) LLNA method—(i) Principle of the method.
The basic principle underlying the LLNA is that skin sensitizers induce
proliferation of lymphocytes in the lymph nodes draining the site of chem­
ical application. Generally, under appropriate test conditions, this prolifera­tion is proportional to the dose applied, and provides a means of obtaining
an objective, quantitative measurement of sensitization. The test measures
cellular proliferation as a function of in vivo radioisotope incorporation

2

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ICCVAM Test Method Evaluation Report: Appendix H4

into the DNA of dividing lymphocytes. The LLNA assesses this prolifera­tion in the draining auricular lymph nodes located in the cervical region
at the bifurcation of the jugular vein. Lymphocyte proliferation in test
groups is compared to that in concurrent solvent/vehicle-treated controls.
A positive control is added to each assay to provide an indication of appro­
priate assay performance.
(ii) Animal selection—(A) Sex and strain of animals. Young adultfemale mice (nulliparous and non-pregnant) of the CBA/Ca or CBA/J
strain should be used at age 8–12 weeks. All animals are to be agematched (preferably within a one-week time frame). Females are used because the existing database is predominantly based on this gender. Males
and other strains of mice should not be used until it is sufficiently dem­
onstrated that significant strain-specific and/or gender-specific differencesin the LLNA response do not exist.
(B) Housing and feeding. The temperature of the experimental ani­
mal room should be 21 ± 3 oC and the relative humidity 30–70%. When
artificial lighting is used, the light cycle should be 12 hours light: 12 hours
dark. For feeding, standard laboratory mouse diets are to be used withan unlimited supply of drinking water. The mice must be acclimatized
for at least 5 days prior to the start of the test. Animals must be housed
individually. Healthy animals are randomly assigned to control and treat­ment groups having statistically homogeneous body weights. The animals
are uniquely identified prior to being placed on study. Although a variety
of techniques exist to uniquely mark mice, any method that involves iden­tification via ear marking (e.g., ear tags) must not be used.
(iii) Test conditions—(A) Preparation of doses. Solid test substances are to be dissolved in appropriate solvents or vehicles and diluted,
if appropriate, prior to dosing of the animals. Stable suspensions mightalso be acceptable. Liquid test substances may be dosed directly or diluted
prior to dosing. Fresh preparations of the test substance are to be prepareddaily unless stability data demonstrate the acceptability of storage.
(B) Solvent/vehicle. The solvent/vehicle is to be selected on the basisof maximizing the test concentration while producing a solution/suspension
suitable for application of the test substance. In order of preference, rec­ommended solvents/vehicles are acetone/olive oil (4:1 v/v), N,N­
dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethyl
sulfoxide, but others may be used if appropriately justified. The selectedsolvent/vehicle must not interfere with or bias the test result and should
be selected to achieve the maximum concentration/skin exposure of thetest substance. Ensure that hydrophilic materials are incorporated into avehicle system that wets the skin and does not immediately run off. Thus,
wholly aqueous vehicles are to be avoided.

3

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ICCVAM Test Method Evaluation Report: Appendix H4(C) Controls. (1) Concurrent negative (solvent/vehicle) and positive
controls are to be included in each test. In some circumstances, it may
be useful to include a naive control. Except for treatment with the test
substance, animals in the control groups are to be handled in an identicalmanner to animals of the treatment groups.
(2) Positive controls are used to ensure the appropriate performanceof the assay. The positive control must produce a positive LLNA responseat an exposure level expected to give an increase in the stimulation index(SI) of three or greater (SI ≥ 3) over the solvent or vehicle control group.
The positive control dose is to be chosen such that the induction is clear
but not excessive. Preferred positive control substances are hexyl cinnamic
aldehyde (HCA) and mercaptobenzothiazole. There may be circumstanceswhere, given adequate justification, other positive control substances may
be used. However, benzocaine should not be used as a positive control
in the LLNA.
(3) The positive control substance is tested in the vehicle that is
known to elicit a consistent response (i.e., acetone/olive oil). If a nonstandard vehicle (chemically relevant formulation) is used with a positive
control, the non-standard vehicle (chemically relevant formulation) must
be tested for a local lymph node response prior to the initiation of thestudy and the results reported.
(iv) LLNA test procedure—(A) A minimum of five animals areused per dose group. At least three consecutive doses of the test substance are to be used. A solvent/vehicle control group and a positive con­trol group are also required. Doses are normally selected from within theconcentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, 0.1%.
In general, dose selection is based on factors such as toxicity, solubility,
irritancy and any other available information such as the results of othertesting and structure-activity relationships. To avoid false negatives, test
as high a concentration as possible. Generally, the maximum concentration
tested is the highest achievable level that avoids overt systemic toxicity
and excessive local irritation. To identify the appropriate maximum test
substance dose, an initial toxicity test, conducted under identical experi­mental conditions except for an assessment of lymph node proliferative
activity, may be necessary. To support an ability to identify a dose-re­sponse relationship, data must be collected on at least three test substancetreatment doses, in addition to the concurrent solvent/vehicle control
group. Where the LLNA study results are negative, the concurrent positive
control must induce a SI ≥ 3 relative to its solvent/vehicle-treated control.
(B) LLNA experimental procedure. The LLNA experimental proce­dure is to be performed by appropriately trained staff as follows:(1) Day 1. Record the body weight of each mouse prior to dermal
applications. Apply 25 µL/ear of the appropriate dilution of the test sub-

4

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ICCVAM Test Method Evaluation Report: Appendix H4stance, or the positive control, or the solvent/vehicle control alone to thedorsum of both ears. A positive displacement pipettor may facilitate appli­
cation of the test material.
(2) Days 2 and 3. Repeat the application procedure as carried out
on day 1.
(3) Days 4 and 5. No treatment.
(4) Day 6. Record the body weight of each mouse. Inject 250 µLof sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl
thymidine or 250 µL PBS containing 2 µCi 125IU and 10-5 M
fluorodeoxyuridine into each experimental mouse via the tail vein. Five
hours later, the draining (auricular) lymph node of each ear is excisedand pooled in PBS for each animal. A single cell suspension of lymph
node cells (LNC) is prepared for each mouse. The single cell suspension
is prepared in PBS by either gentle mechanical separation through 200mesh stainless steel gauze or another acceptable technique for generating
a single cell suspension. The LNC are washed twice with an excess of
PBS and the DNA precipitated with 5% trichloroacetic acid (TCA) at 4oC for approximately 18h.
(5) For the 3H-methyl thymidine method, pellets are resuspended in
1 mL TCA and transferred to 10 mL of scintillation fluid. Incorporationof 3H-methyl thymidine is measured by B-scintillation counting as disinte­
grations per minute (dpm) for each mouse and expressed as dpm/mouse.
For the 125IU method, the 1 mL TCA pellet is transferred directly into
gamma counting tubes. Incorporation of 125IU is determined by gamma
counting and also expressed as dpm/mouse.
(C) Observations. At a minimum, observe mice once daily for any
clinical signs, either of local irritation at the application site or of systemic
toxicity. Weighing mice prior to treatment and at the time of necropsy
will aid in assessing systemic toxicity. All observations are systematicallyrecorded, with records being maintained for each individual mouse.
(D) Measurements and calculation of results. (1) The proliferative
response of lymph node cells from the pooled lymph nodes of each indi­vidual animal is expressed as the number of radioactive disintegrations
per minute (dpm) per animal, subtracting out any background dpm. Thenthe group mean dpm, along with an appropriate measure of inter-animal
variability (i.e., mean ± standard deviation), is calculated for each test
group (i.e., positive, solvent/vehicle, and any other control groups) andthe solvent/vehicle group. Final results are expressed as the SI which is
calculated as a ratio (i.e., SI = mean dpm of test group divided by mean
dpm of solvent/vehicle control group).
(2) In addition to an assessment of the magnitude of the ratio esti­mate, SI, conduct statistical analyses which include both an overall assess-

5

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ICCVAM Test Method Evaluation Report: Appendix H4ment (e.g. ANOVA) of the dose-response relationships and pairwise com­
parisons of the SIs of the test groups, positive control group and any othercontrol group versus that of the solvent/vehicle control group. In choosing
an appropriate method of statistical analysis, the investigator should beaware of possible inequality of variances and other related problems that
may necessitate a data transformation or a nonparametric statistical anal­ysis.
(v) Data interpretation and reporting for LLNA—(A) Data Inter­
pretation. (1) A substance is regarded as a skin sensitizer in the LLNA
if at least one concentration of the test material results in a 3-fold or great­er increase in 3H-methyl thymidine or 125IU incorporation in the lymph
node cells of test group lymph nodes relative to that recorded for solvent/vehicle control lymph nodes, as indicated by the SI. However, the mag­nitude of the SI should not be the sole factor used in determining thebiological significance of a skin sensitization response. A quantitative as­
sessment must be performed by statistical analysis of individual animal
data in order to provide a more complete evaluation of the test substance(see paragraph (e)(1)(iv)(D)(2) of this guideline). Factors to be consideredin evaluating the biological significance of a response or outcome of thetest include the results of the SI determinations, statistical analyses, thestrength of the dose-response relationship, chemical toxicity, solubility,
and the consistency of the solvent/vehicle and positive control responses.
(2) Strong irritants may yield false positive results in the LLNA due
to the initiation of a significant lymphocyte proliferation. However, thedose-response information from the assay may help to uncover a strong
irritant response since, for instance, it has been shown that the proliferationinduced by irritation usually results in a shallow dose-response relationship. Concurrent evaluation of ear swelling may also provide helpful infor­
mation on differentiating weak sensitizers from strong irritants.
(B) Test report. The test report for LLNA must contain the following
specific information:(1) Test substance. (i) Identification data and CAS number, if known,
and EPA registration number, if applicable;(ii) Physical nature and purity;(iii) Physicochemical properties relevant to the conduct of the study;(iv) Stability of the test substance, if known; and(v) Lot number of the test substance.
(2) Solvent/vehicle. (i) Solvent/vehicle used and its purity;(ii) Justification for choice of solvent/vehicle, if appropriate; and

6

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ICCVAM Test Method Evaluation Report: Appendix H4(iii) Solubility and stability of the test substance in the solvent/vehi­
cle.
(3) Test animals. (i) Strain of mice used;(ii) Acclimation information;(iii) Number, age, and sex of mice;(iv) Source, housing conditions, diet, etc.;(v) Individual body weight of the animals at the start and end of thetest, including body weight range, mean, and associated error term for eachgroup;(vi) Health and microbiological/pathogen status of the mouse; and(vii) Details of animal food and water quality;(4) Test conditions. (i) Details of test substance preparation;(ii) Details of the administration of the test substance;(iii) Detailed description of treatment and sampling schedules; and(iv) Methods for measurement of toxicity.
(5) Results. (i) Positive and negative (solvent/vehicle) control data
in tabular form;(ii) Data from range-finding study, if conducted;(iii) Doses used;(iv) Rationale for dose level selection;(v) Signs of toxicity;(vi) Dpm/mouse values for each mouse within each treatment groupand control group;(vii) Group mean dpm/mouse and associated error term for each treat­ment group and control group;(viii) The SI calculated, compared to the concurrent solvent/vehicle
control group, for each test substance treatment dose group, the concurrent
positive control group, and any other concurrent control group;(ix) Individual mouse dpm data must be presented in tabular form,
along with the group mean dpm, its associated error term and the SI for
each dose group;(x) Criteria for considering studies as positive or negative (including
information on any qualitative or quantitative measure of ear swelling);

7

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ICCVAM Test Method Evaluation Report: Appendix H4(xi) Dose-response relationship;(xii) Statistical analyses and method applied;(xiii) Concurrent and negative control data as established in the test­
er’s laboratory; and(xiv) Concurrent positive control data.
(6) Discussion of the results.
(7) Conclusions.
(8) The reporting requirements specified under 40 CFR Part l58 (forpesticides) and 40 CFR Part 792, Subpart J (for toxic substances) should
be followed.
(2) GPMT and Buehler Methods—(i) Principle of the test meth­ods. Following initial exposure to a test substance, the animals are sub­jected, after a period of not less than 1 week, to a challenge exposure
with the test substance to establish whether a hypersensitive state has been
induced. Sensitization is determined by examining the reaction to the chal­lenge exposure and comparing this reaction with that of the initial induc­tion exposure. The test animals are initially exposed to the test substanceby intradermal and/or epidermal application (induction exposure). Fol­lowing a rest period of 10 to 14 days (the induction period), during which
an immune response may develop, the animals are exposed to a challengedose. The extent and degree of skin reaction to the challenge exposure
is compared with that demonstrated by control animals that undergo shamtreatment during induction and then receive the challenge exposure.
(ii) Animal selection—(A) Species and strain. The young adultguinea pig is preferred. Young adult commonly used laboratory strains
must be employed.
(B) Housing and feeding. The temperature of the experimental ani­
mal room should be 20 ± 3 oC with the relative humidity 30–70 percent.
Where the lighting is artificial, the sequence should be 12 h light/12 hdark. Conventional laboratory diets may be used with an unlimited supplyof drinking water. It is essential that guinea pigs receive an adequate
amount of ascorbic acid.
(C) Number and sex. The number and sex will depend on the method
chosen. Either sex may be used in the Buehler test and the GPMT. If
females are used, they must be nulliparous and not pregnant. The Buehler
test recommends using a minimum of 20 animals in the treatment andat least 10 as controls. At least 10 animals in the treatment group and
5 in the control group must be used with the GPMT, with the stipulationthat if it is not possible to conclude that the test substance is a sensitizer
after using fewer than 20 test and 10 control guinea pigs, the testing of

8

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ICCVAM Test Method Evaluation Report: Appendix H4

additional animals to give a total of at least 20 test and 10 control animals
is strongly recommended(D) Control animals. (2) Every 6 months, assess the sensitivity andreliability of the experimental technique in naive animals by the use of
positive control substances known to have mild-to-moderate skin-sensi­
tizing properties. In a properly conducted test, a response of at least 30
percent in an adjuvant test and at least 15 percent in a nonadjuvant test
is expected for mild-to-moderate sensitizers. Preferred substances arehexylcinnamic aldehyde (CAS No.101–86–0), mercaptobenzothiazole(CAS No. 149–30–4), benzocaine (CAS No. 94–09–7), dinitro-chloro-ben­zene (CAS No. 97–00–7), or DER 331 epoxy resin (CAS No. 25068–
38–6). There may be circumstances where, given adequate justification,other control substances meeting the above criteria may be used.
(2) To ensure that the response to the challenge reaction in treatedanimals is truly of allergic origin and not due to skin irritancy, a shamtreated vehicle-only control is included in the test strategy. This shamtreated control group is treated in exactly the same manner as the test
animals, except that during the induction phase the test article is omitted.
The selected vehicle must not interfere or alter the test results.
(E) Dose levels. The dose level will depend on the test method se­lected. In the Buehler test, select the concentration of the induction dosesuch that it is high enough to cause mild irritation, and the challenge dosesuch that it is the highest non-irritating concentration. In the GPMT, theconcentration of the induction dose must be well tolerated systemically,
and must be high enough to cause mild-to-moderate skin irritation; the
GPMT challenge dose must use the highest non-irritating concentration.
(F) Observation of animals. (1) Skin reactions are to be graded andrecorded after the challenge exposures at the time specified by the method­
ology selected. This is usually at 24 and 48 hours. Additional notations
are to be made as necessary to fully describe unusual responses.
(2) Regardless of the test method selected, initial and terminal bodyweights must be taken and recorded.
(G) Procedures. The procedures to be used are those described by
the test method chosen. Brief summaries are given here, but the testeris referred to the original literature for more complete guidance on con­ducting the Buehler test (see references in paragraphs (g)(7) through(g)(10) of this guideline) or the GPMT (see references in paragraphs(g)(11) through (g)(14) of this guideline).
(1) The Buehler test uses topical administration via a closed patchon days 0, 6–8, and 13–15 for induction, with topical challenge of theuntreated flank for 6 hours on day 27–28. Readings are made approxi­
mately 24 hours alter removing the challenge patch, and again 24 hours

9

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ICCVAM Test Method Evaluation Report: Appendix H4after that. If the results are equivocal, the animals may be rechallenged
one week later, using either the original control group or a new control
group for comparison.
(2) The GPMT uses intradermal injection with and without Freund’s
complete adjuvant (FCA) for induction, followed on days 5–8 by topicalirritation/induction, followed by topical challenge for 24 hours on day 20–
22. Readings are made approximately 24 hours after removal of the chal­lenge dose, and again after another 24 hours. As with the Buehler test,
if the results are equivocal, the animals may be rechallenged 1 week later.
If only 10 animals were used initially and gave equivocal results, the useof an additional 10 experimental and 5 control animals is strongly rec­ommended.
(3) Blind reading of both test and control animals is recommended.
(4) Removal of the test material is accomplished with water or an
appropriate solvent, without altering the existing response or the integrityof the epidermis.
(5) Hair is removed from the site of application by clipping, shaving,or possibly by depilation, depending on the test selected.
(iii) Data and reporting for GPMT and Buehler Methods. Data
must be summarized in tabular form, showing for each individual animal
the skin reaction, results of the induction exposure, and the challenge exposure at times indicated by the method chosen. As a minimum, the erythemaand edema must be graded and any unusual finding must be recorded.
(A) Evaluation of the results. The evaluation of results will provide
information on the proportion of each group that became sensitized andthe extent (slight, moderate, severe) of the sensitization reaction in eachindividual animal.
(B) The following specific information is to be reported for the
GPMT and Buehler Methods.
(1) A description of the method used and the commonly accepted
name.
(2) Information on the positive control study, including the positive
control substance used, the method used, and the time conducted.
(3) The number, species, strain, age, source, and sex of the test ani­mals.
(4) Individual body weights of the animals at the start of the test
and at the conclusion of the test.
(5) A brief description of the grading system.

10

H-40

ICCVAM Test Method Evaluation Report: Appendix H4(6) Each reading made on each individual animal.
(7) The chemical identification and relevant physicochemical prop­
erties of the test substance.
(8) Manufacturer, source, purity, and lot number of test substance.
(9) Physical nature, and, where appropriate, concentration and pH
value for the test substance.
(10) The vehicles used for induction and challenge and justification
for their use, if other than water or physiological saline. Any material thatmight reasonably be expected to react with or enhance or retard absorptionof the test substance must be reported.
(11) The total amount of test substance applied for induction and chal­lenge, and the technique of application in each case.
(12) Description of any pre-test conditioning, including diet, quar­antine and treatment of disease.
(13) Description of caging conditions including number (and anychange in number) of animals per cage, bedding material, ambient tem­
perature and humidity, photoperiod, and identification of diet of test ani­mals.
(14) Histopathological findings, if any.
(15) Discussion of results.
(16) A list of references cited in the body of the report, i.e., references
to any published literature used in developing the test protocol, performing
the testing, making and interpreting observations, and compiling and evalu­
ating the results.
(17) The reporting requirements as specified under 40 CFR Part l58(for pesticides) and 40 CFR Part 792, Subpart J (for toxic substances)
should be followed(f) Screening tests. The mouse ear swelling test (MEST) (see ref­erences in paragraphs (g)(15) through (g)(18) of this guideline) may be
used as a screening test to detect moderate to strong sensitizers. If a posi­tive result is seen in this assay, the test substance may be designated apotential sensitizer, and it may not be necessary to conduct a further testin guinea pigs. If the MEST does not indicate sensitization, the test substance should not be designated a nonsensitizer without confirmation inan accepted test using guinea pigs or LLNA if appropriate.
(g) References. The following references should be consulted for ad­
ditional background information on this test guideline.

11

H-41

ICCVAM Test Method Evaluation Report: Appendix H4(1) The Murine Local Lymph Node Assay: A Test Method for Assess­ing the Allergic Contact Dermatitis Potential of Chemicals/Compounds.
Interagency Coordinating Committee on the Validation of Alternative
Methods (ICCVAM), National Institutes of Environmental Health
Sciences, NIH Publication No. 99–4494.3 (1999). (Document available at
http://iccvam.niehs.nih.gov/methods/llnadocs/llnarep.pdf.) Description andpicture of auricular lymph node dissection available at at http://
iccvam.niehs.nih.gov/methods/llnadocs/LLNAProt.pdf.
(2) Kimber, I. et al. The murine local lymph node assay for identifica­tion of contact allergens: a preliminary evaluation of in situ measurementof lymphocyte proliferation. Contact Dermatitis 21:215–220 (1989)(3) Kimber, I. et al. Identification of contact allergens using the mu­rine local lymph node assay: comparisons with the Buehler Occluded Patch
Test in guinea pigs. Journal of Applied Toxicology 10:173–180 (1990).
(4) Kimber, I. et al. The murine local lymph node assay: results ofan interlaboratory trial. Toxicology Letters 55:203–213 (1991).
(5) Basketter, D.A. et al. Interlaboratory evaluation of the local lymph
node assay with 25 chemicals and comparison with guinea pig test data.
Toxicology Methods 1:30–43 (1991).
(6) Basketter, D.A. et al. Identification of metal allergens in the local
lymph node assay. American Journal of Contact Dermatitis 10 (4):207–
212 (1999).
(7) Buehler, E.V. Delayed contact hypersensitivity in the guinea pig,
Archives of Dermatology 91:171–177 (1965).
(8) Ritz, H.L. and Buehler, E.V. Planning, conduct and interpretationof guinea pig sensitization patch test in Current Concepts in Cutaneous
Toxicity, ed. V. Drill and P. Lazar. Academic, New York, NY. pp. 25–42 (l980).
(9) Buehler, L.V. Occ1usive patch method for skin sensitization inguinea pigs: the Buehler method. Food and Chemical Toxicology 32:97–
101 (1994).
(10) Buehler, E.V. Prospective testing for delayed contact hypersensitivity in guinea pigs: the Buehler method; in Methods in
Immunotoxico1ogy, Vol. 2, ed. C. Burleson, A. Munson, and J. Dean.
Wiley, NY, pp. 343–356(1995).
(11) Magnusson, B. and Kligman, A.M. The identification of contact
allergens by animal assay. The guinea pig maximization test. Journal of
Investigative Dermatology 52: 268–276 (1969).

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ICCVAM Test Method Evaluation Report: Appendix H4(12) Magnusson, B. and Kligman, A.M. Allergic contact dermatitis
in the guinea pig. Charles C. Thomas, Springfield, IL (1970).
(13) Magnusson, B. Identification of contact sensitizers by animal
assay. Contact Dermatology 6:46 (1980).
(14) Magnusson, B. et al. Determination of skin sensitization potentialof chemicals. Predictive testing in guinea pigs. Arbete och Halsa: 26(E)(1979).
(15) Gad, S.C. et al. Development and validation of an alternative
dermal sensitization test: the mouse ear swelling test (MEST). Toxicology
and Applied Pharmacology 8 4:93–114 (1986).
(16) Maisey, J. and Miller, K., Assessment of the ability of mice fed
on Vitamin-A supplemented diet to respond to a variety of potential contact sensitizers. Contact Dermatitis 15:17–23 (l986).
(17) Thorne, P.S. et al., The noninvasive mouse ear swelling assay,
I. Refinements for detecting weak contact sensitizers. Fundamental andApplied Toxicology 7:790–806 (1991).
(18) Thorne, P.S. et al. The noninvasive mouse ear swelling assay.
II. Testing the contact sensitizing potency of fragrances. Fundamental andApplied Toxicology7:807–820 (1991).

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ICCVAM Test Method Evaluation Report: Appendix H4[This Page Intentionally Left Blank]

H-44

ICCVAM TEST METHOD EVALUATION REPORTThe Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and Products

NIH Publication Number 09-6439

ICCVAM TEST METHOD EVALUATION REPORTThe Reduced Murine Local Lymph Node Assay:
An Alternative Test Method Using Fewer Animals to Assess theAllergic Contact Dermatitis Potential of Chemicals and Products

U.S. Department of Health and Human Services

NIH Publication Number 09-6439

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

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

NICEATMICCVAM

National Toxicology Program
Interagency Center for the Evaluationof Alternative Toxicological Methods

Interagency Coordinating
Committee on the Validationof Alternative Methods

INDEPENDENT SCIENTIFIC
PEER REVIEW PANEL REPORT
Validation Status of New Versions
and Applications of the
Murine Local Lymph Node Assay:
A Test Method for Assessing theAllergic Contact Dermatitis Potentialof Chemicals and Products

May 2008

Independent Scientific Peer Review Panel Report:
Validation Status of New Versions and Applications of the
Murine Local Lymph Node Assay: A Test Method for Assessing
the Allergic Contact Dermatitis Potential of Chemicals andProducts
May 2008

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

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

Independent Peer Review Panel Report

May 2008

This document is available electronically at:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/LLNAPRPRept2008.pdf

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

Independent Peer Review Panel Report

May 2008

Table of Contents
List of Tables ..................................................................................................................... iii
List of Abbreviations and Acronyms.................................................................................iv
Members of the Independent Scientific Peer Review Panel .............................................vi
Preface ................................................................................................................................ix
Executive Summary ........................................................................................................ xiii
1.0

Murine Local Lymph Node Assay (LLNA) Limit Dose Procedure ................... 1-1
1.1 Comments on the Draft Background Review Document (BRD) for
Completeness, Errors, and Omissions ........................................................... 1-1
1.2 Comments on the Validation Status of the LLNA Limit Dose
Procedure ..................................................................................................... 1-4
1.3 Comments on the Draft ICCVAM Test Method Recommendations
on the LLNA Limit Dose Procedure ............................................................. 1-8

2.0

LLNA for Testing Aqueous Solutions, Metals, and Mixtures............................ 2-1
2.1 Comments on the Draft Addendum for Completeness, Errors, andOmissions..................................................................................................... 2-1
2.2 Comments on the Validation Status of the Traditional LLNA for
Testing Aqueous Solutions, Metals and Mixtures ......................................... 2-1
2.3 Comments on the Draft ICCVAM Test Method Recommendations
on the Traditional LLNA for Testing Aqueous Solutions, Metals,
and Mixtures ................................................................................................ 2-3

3.0

Non-Radioactive LLNA Protocol - The LLNA: Daicel Adenosine Triphosphate(LLNA: DA) Test Method ................................................................................... 3-1
3.1 Comments on the Draft BRD for Completeness, Errors, andOmissions..................................................................................................... 3-1
3.2 Comments on the Validation Status of the LLNA: DA ................................. 3-2
3.3 Comments on the Draft ICCVAM Test Method Recommendations
on the LLNA: DA ........................................................................................ 3-6iIndependent Peer Review Panel Report

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4.0

Non-Radioactive LLNA Protocol - The LLNA: Bromodeoxyuridine Detected by
Flow Cytometry (BrdU-FC) Test Method ...........................................................4-1
4.1 Comments on the Draft BRD for Completeness, Errors, andOmissions .....................................................................................................4-1
4.2 Comments on the Validation Status of the LLNA: BrdU-FC .........................4-3
4.3 Comments on the Draft ICCVAM Test Method Recommendations
on the LLNA: BrdU-FC ................................................................................4-6

5.0

Non-Radioactive LLNA Protocol - The LLNA: Bromodeoxyuridine Detected by
ELISA (BrdU-ELISA) Test Method ....................................................................5-1
5.1
5.2
5.3

Comments on the Draft BRD for Completeness, Errors, andOmissions .....................................................................................................5-1
Comments on the Validation Status of the LLNA: BrdU-ELISA ...................5-3
Comments on the Draft ICCVAM Test Method Recommendations
on the LLNA: BrdU-ELISA ..........................................................................5-7

6.0

Draft ICCVAM LLNA Performance Standards.................................................6-1
6.1 Comments on the Proposed Purpose and Applicability ..................................6-1
6.2 Comments on the Essential Test Method Components...................................6-2
6.3 Comments on the Proposed Reference Substances.........................................6-3
6.4 Comments on the Test Method Accuracy Standards ......................................6-5
6.5 Comments on the Test Method Reliability Standards.....................................6-7
6.6 Summary.......................................................................................................6-8
6.7 Additional Statistical Comments ...................................................................6-9

7.0

Use of the LLNA for Potency Determinations.....................................................7-1
7.1 Comments on the Draft BRD for Completeness, Errors, andOmissions .....................................................................................................7-1
7.2 Comments on the Validation Status of the Traditional LLNA to
Determine Skin Sensitization Potency ...........................................................7-1
7.3 Comments on the Draft ICCVAM Test Method Recommendations
for the Use of the LLNA for Potency Determination .....................................7-5

8.0

References.............................................................................................................8-1

Appendix A: Peer Review Panel Member Biosketches .................................................A-1
Appendix B: Questions for the Peer Review Panel........................................................ B-1

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List of Tables
Table 1-1

Power Calculations for the Traditional LLNA ....................................... 1-5

Table 3-1

Power Calculations for the LLNA: DA ................................................... 3-3

Table 4-1

Power Calculations for the LLNA: BrdU-FC ......................................... 4-8

Table 5-1

Power Calculations for the LLNA: BrdU-ELISA................................... 5-2

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List of Abbreviations and Acronyms
ACD
ANOVA
AOO
BRD
BrdU
BT
CD4
CPSC
CRO
CV
DMF
DMSO
DNCB
EC3
ECt
ECVAM
ELISA
eLLNA: BrdU-FC
EPA
FC
FR
GLP
GPMT
GSK
HCA
HMT
HRIPT
HTdR
ICCVAMISO
IWG
JaCVAM
LLNA
LLNA: BrdU-ELISA
LLNA: BrdU-FC
LLNA: DA
LNC
LOEL
MEK
NICEATM

Allergic contact dermatitis
Analysis of varianceAcetone: olive oil (4:1)
Background Review Document
Bromodeoxyuridine
Buehler Test
Cluster of differentiation 4
U.S. Consumer Product Safety Commission
Clinical research organization
Coefficient of variation
Dimethylformamide
Dimethylsulfoxide
Dinitrochlorobenzene
Estimated concentration needed to produce a stimulation index
of three
Estimated concentration needed to produce a stimulation index
that is indicative of a positive response
European Centre for the Validation of Alternative Methods
Enzyme Linked Immunosorbent Assay
Enhanced LLNA with BrdU detected by flow cytometry
U.S. Environmental Protection Agency
Flow cytometry
Federal Register
Good Laboratory PracticeGuinea Pig Maximization Test
GlaxoSmithKline
Hexyl cinnamic aldehyde
Human Maximization Test
Human Repeat Insult Patch Test
3
H-Methyl Thymidine
Interagency Coordinating Committee on the Validation of
Alternative Methods
International Organization for Standardization
Immunotoxicity Working Group
Japanese Center for Validation of Alternative Methods
Local Lymph Node Assay
LLNA with BrdU detected by ELISALLNA with BrdU detected by FC
LLNA: Daicel Adenosine Triphosphate
Lymph node cells
Lowest observed effect level
Methyl ethyl ketone
NTP Interagency Center for the Evaluation of Alternative
Toxicological MethodsivIndependent Peer Review Panel Report

NIEHS
NIH
NOEL
NTP
OD
OECD
QSAR
REACH
rLLNA
SAR
SD
SI
SDS
SLS
TG
Th
vs.

May 2008

National Institute of Environmental Health Sciences
National Institutes of Health
No observed effect level
National Toxicology Program
Optical density
Organisation for Economic Co-operation and Development
Quantitative structure-activity relationship
Registration, Evaluation, and Authorisation of Chemicals
Reduced LLNA
Structure-activity relationshipStandard deviation
Stimulation index
Sodium dodecyl sulfate
Sodium lauryl sulfate
Test Guideline
T-helperVersusv

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Members of the Independent Scientific Peer Review Panel
Michael Luster, Ph.D. (Panel Chair), Senior Consultant to the National Institute ofOccupational Safety and Health (NIOSH) Health Effects Laboratory, Morgantown, WV
Nathalie Alépée, Ph.D., Associate Research Fellow, Pfizer PDRD MCT Laboratory, France
Anne Marie Api, Ph.D., Vice President, Human Health Sciences, Research Institute for
Fragrance Materials, Woodcliff Lake, NJ
Nancy Flournoy, M.S., Ph.D. (Biostatistician), Professor and Chair, Dept. of Mathematics& Statistics, University of Missouri-Columbia, Columbia, MO
Thomas Gebel, Ph.D., Regulatory Toxicologist, Federal Institute for Occupational Safety &Health, Dortmund, Germany
Sidney Green, Ph.D.,1 Graduate Professor, Howard University, Washington, DC
Kim Headrick, B. Admin., B.Sc., International Harmonization Senior Policy Advisor,
Health Canada, Ottawa, Ontario, Canada
Dagmar Jírová, M.D., Ph.D., Toxicologist, Research Manager, Head of Reference Centerfor Cosmetics, Head of Reference Laboratory for Experimental Immunotoxicology, National
Institute of Public Health, Czech RepublicDavid Lovell, Ph.D. (Biostatistician), Reader in Medical Statistics, Postgraduate Medical
School, University of Surrey, Guildford, Surrey, U.K.
Howard Maibach, M.D., Professor, Department of Dermatology, University of CaliforniaSan Francisco, San Francisco, CA
James McDougal, Ph.D., Professor and Director of Toxicology Research, Department ofPharmacology and Toxicology, Boonshoft School of Medicine, Wright State University,
Dayton, OH
Michael Olson, Ph.D., Director of Occupational Toxicology, Corporate Environment Healthand Safety, GlaxoSmithKline, Research Triangle Park, NC
Raymond Pieters, Ph.D., Associate Professor, Immunotoxicology Group Leader, Institutefor Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands
Jean Regal, Ph.D., Professor, Department of Pharmacology, University of Minnesota
Medical School, Duluth, MN
Jonathan Richmond, MB ChB, FRCSEd,1 Head of Division, Animals ScientificProcedures Division, Home Office, London, U.K.
Peter Theran, V.M.D., Massachusetts Society for the Prevention of Cruelty to Animals,
Novato, CA

1Drs. Green and Richmond were unable to attend the public meeting on March 4-6, 2008. However, they wereinvolved in the review of the background review documents and concur with the conclusions andrecommendations included in this report.
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Stephen Ullrich, Ph.D., Dallas/Ft. Worth Living Legends Professor & Professor of
Immunology, Post-graduate School of Biomedical Science, University of Texas M.D.
Anderson Cancer Center, Houston, TX
Michael Woolhiser, Ph.D., Technical Leader - Immunotoxicology, Toxicology &Environmental Research & Consulting Immunology, Dow Chemical, Midland, MI
Takahiko Yoshida, M.D., Ph.D., Professor, Department of Health Science, Asahikawa
Medical College, Hokkaido, Japan

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Preface
In 1999, the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) recommended the murine local lymph node assay (LLNA) to U.S. Federal
agencies as a valid substitute for currently accepted guinea pig test methods to assess theallergic contact dermatitis potential of many, but not all, types of substances. The
recommendation was based on a comprehensive evaluation of the validation status of the
LLNA that included an assessment by an international independent scientific peer review
panel (hereafter, Panel). The Panel report and the ICCVAM LLNA test method
recommendations (ICCVAM 1999) are available at the National Toxicology Program
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)ICCVAM website.1 The LLNA was subsequently incorporated into national and international
test guidelines for the assessment of skin sensitization (OECD 2002; ISO 2002; EPA 2003).
For this Panel report, this LLNA will be referred to as the “traditional” LLNA.
On January 10, 2007, the U.S. Consumer Product Safety Commission (CPSC) formallyrequested through NICEATM that ICCVAM assess the validation status of:2•

The traditional LLNA as a stand-alone assay for potency determinations(including severity) for the purpose of hazard classification•

Three modifications of the traditional LLNA not requiring the use ofradioactive materials•

The LLNA limit dose procedure (also referred to as the "reduced" LLNA)•

The ability of the traditional LLNA to test mixtures, metals, and aqueous
solutions (i.e., to re-evaluate the applicability domain for the traditional
LLNA)

NICEATM, in coordination with ICCVAM and the ICCVAM Immunotoxicity Working
Group, prepared a comprehensive draft background review document (BRD) for each
modified version of the traditional LLNA test method being evaluated, as well as a draft
applicability domain addendum to the final BRD published previously on the traditional
LLNA. Each draft BRD and the draft addendum detailed the available data and informationfrom the published literature and submissions received in response to a 2007 Federal
Register (FR) notice that had requested data related to CPSC’s nomination (FR notice Vol.
72, No. 95, p. 27815-27817, May 17, 2007). In addition, ICCVAM developed draft LLNA
Performance Standards intended for use in validating alternative test methods that arefunctionally and mechanistically similar to the traditional LLNA. Finally, ICCVAM, basedon the information contained in each of the draft BRDs and the draft addendum, developed
draft test method recommendations.
The various supporting documents and the draft ICCVAM recommendations were provided
to a new international Panel for an independent scientific review. In addition, NICEATM
announced the availability of these documents on the NICEATM-ICCVAM website
1

The 1999 ICCVAM Panel report and recommendations can be obtained at:
http://iccvam.niehs.nih.gov/docs/immunotox_docs/llna/llnarep.pdf
2
The CPSC nomination can be obtained at:
http://iccvam.niehs.nih.gov/methods/immunotox/llnadocs/CPSC_LLNA_nom.pdf
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May 2008(http://iccvam.niehs.gov) for public comment in a FR notice (Vol. 73, No. 5, p. 1360-1362,
January 8, 2008) and via the ICCVAM listserv. The FR notice also announced the public
Panel meeting, to be convened at the CPSC Headquarters in Bethesda, MD on March 4–6,
2008.
The Panel was charged with:•

Reviewing each ICCVAM draft BRD and the draft addendum forcompleteness and identifying any errors or omissions of existing relevant data
or information•

Evaluating the information in each draft BRD and the draft addendum todetermine the extent to which each of the applicable criteria for validation andacceptance of toxicological test methods (ICCVAM 2003) had been
appropriately addressed for the recommended use of the new versions andapplications of the traditional LLNA•

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

Evaluating the draft ICCVAM LLNA Performance Standards and considering
whether they were adequate for assessing the accuracy and reliability ofalternative test methods that are functionally and mechanistically similar to the
traditional LLNA

During our public meeting in March 2008, the Panel discussed each charge, listened to publiccomments, and developed conclusions and recommendations for ICCVAM on each of thenominated activities. The Panel wished to emphasize that they were to consider two overall
questions. They were to consider: (1) whether the validation status of the each of the above
proposed modifications or alternative uses of the LLNA had been adequately characterized
for its intended purpose according to established ICCVAM validation criteria (available on
the NICEATM-ICCVAM website, http://iccvam.niehs.gov), and (2) whether proposed
modifications or alternative uses of the LLNA are sufficiently accurate and reliable to be
used for the identification of sensitizing substances and non-sensitizing substances in place ofthe traditional LLNA procedure.
This report details the Panel's independent conclusions and recommendations. ICCVAM willconsider this report, along with all relevant public comments, as it develops final test method
recommendations. The final ICCVAM test method recommendations will be forwarded to
U.S. Federal agencies for their consideration in accordance with the ICCVAM Authorization
Act of 2000 (Public Law 106-545).
The Panel gratefully acknowledges the efforts of NICEATM staff in coordinating thelogistics of the peer review Panel meeting and in preparing materials for their review. The

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Panel also thanks each of the test method developers, Drs. George DeGeorge (LLNA: BrdUFC), Kenji Idehara (LLNA: DA), and Masahiro Takeyoshi, (LLNA: BrdU-ELISA) for
providing summaries and additional clarifications of the non-radioactive test methods underreview. Finally, as Panel Chair, I want to thank each Panel member for her or his thoughtful
and objective review of these LLNA-related activities.
Michael Luster, Ph.D.
Chair, LLNA Peer Review PanelMay 2008

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Executive Summary
This report describes the conclusions and recommendations of an international independentscientific peer review panel (hereafter, Panel). This Panel was charged by the Interagency
Coordinating Committee on the Validation of Alternative Methods (ICCVAM) withevaluating the validation status of new versions and applications of the murine local lymph
node assay (LLNA) for assessing the allergic contact dermatitis (ACD) potential ofchemicals and products. The LLNA, which was first evaluated in 1999 by ICCVAM, ishereafter referred to as the “traditional LLNA” to distinguish it from other versions
considered by the Panel. The new versions and applications considered include:•

The LLNA limit dose procedure (also referred to as the "reduced" LLNA1)•

The ability of the traditional LLNA to test mixtures, metals, and aqueous
solutions (i.e., a re-evaluation of the applicability domain for the traditional
LLNA)

•

Three modifications of the traditional LLNA not requiring the use ofradioactive materials:– LLNA: DA (Local Lymph Node Assay: Daicel Adenosine Triphosphate)– LLNA: BrdU-FC (Local Lymph Node Assay: Bromodeoxyuridine
detected by flow cytometry)– LLNA: BrdU-ELISA (Local Lymph Node Assay: Bromodeoxyuridine
detected by ELISA)•

The traditional LLNA as a stand-alone assay for potency determinations(including severity) for the purpose of hazard classification

The Panel also evaluated the draft ICCVAM LLNA Performance Standards and considered
whether they were adequate for assessing the accuracy and reliability of alternative test
methods that are functionally and mechanistically similar to the traditional LLNA.
LLNA Limit Dose Procedure
The Panel agreed that the LLNA limit dose procedure, which normally allows for testing at
one dose level, should be routinely recommended for hazard identification when used fortesting purposes which do not require dose response information, because it would offer
time, cost, throughput and logistical benefits as well as using fewer animals. In instances
when a necessity to measure relative skin sensitization potency for the purpose of risk
assessment was present, then the traditional LLNA should be used in order to generate dose
response information. Still, the Panel recommended use of the LLNA limit dose procedure as1

As described in this report, the Panel agreed that consideration should be given to applying the same term to
the LLNA limit dose procedure since in various places throughout the draft BRD it was referred to differently
as either the “cut-down”, the “limit dose”, or the “reduced LLNA” (i.e., “rLLNA”). Since the European Centre
for the Validation of Alternative Methods (ECVAM) has already established a naming convention of “rLLNA”,
the Panel recommended adopting the ECVAM terminology to harmonize the terminology used among theinternational validation agencies. However, because the ICCVAM documents that were reviewed use "LLNA
limit dose procedure" that term is retained in this report.
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the initial testing procedure to identify sensitizers and non-sensitizers before conducting the
traditional LLNA even when dose response information is required since if the test substancewere negative in the limit dose procedure, it would not be necessary to conduct a multipledose LLNA test.
The draft background review document (BRD) for the LLNA limit dose procedure provides acomprehensive review of available data and information for assessing the usefulness andlimitations of this modified version of the LLNA for the purpose of skin sensitization hazard
classification. The Panel evaluated the draft BRD for completeness, errors, and omissions
and recommended that it be updated to reflect their suggestions/corrections relating to
general, statistical, and specific editorial issues. In particular, the Panel noted that thedifferences in terminology used for this procedure caused confusion and recommended thatan internationally harmonized term be adopted. They suggested referring to the procedure as
the “reduced LLNA” (i.e. “rLLNA”) since that is being used by the European Centre for the
Validation of Alternative Methods (ECVAM).
The Panel concluded that the stimulation index (SI) based on the ratio of 3.0 as the cutoff
value was indicative of a response that was sufficiently greater than the control and would beconsidered an immunologically relevant response, but recommended that statistical analyses
be used to definitively establish that a response induced by a test substance is significantly
different from the vehicle control. The Panel agreed that the LLNA protocol recommended
by ICCVAM (ICCVAM 1999; Dean et al. 2001) should be the standard protocol for all
future LLNA limit dose studies using the traditional LLNA protocol. Specifically,
prospective LLNA limit dose procedure studies should require that lymph nodes be collectedfrom individual animals instead of pooling them with other animals in a treatment group,
which is also currently permitted by the Organisation for Economic Co-operation andDevelopment (OECD) Test Guideline 429 (OECD 2002). Individual animal response data
are necessary in order to statistically analyze for differences between treated and control data.
In addition, having data from individual animals also allows for identification of technicalproblems and outlier animals within a dose group. Based on power calculations provided as
supplemental information, the Panel agreed that five animals per dose group is an appropriate
number to recommend for LLNA limit dose studies following the traditional LLNA protocol.
It should be noted that the Panel’s analysis of the LLNA limit dose dataset was not restricted
to studies with confirmed individual animal data, and that the Panel considered data known
to have been generated using pooled group data. The Panel stated that, internationally, bothindividual and pooled animal data have likely been used both for regulatory decisions and forin-house decisions relating to product development and risk management. In addition, thefact that the retrospective data analysis set out in the draft LLNA limit dose procedure BRD
did not distinguish between individual or pooled animal data suggested that both met thequality standards for inclusion in the draft BRD.
Although they did not reach consensus, the Panel suggested that for laboratories in which the
LLNA is “routinely” performed and have demonstrated the ability to consistently obtain
positive results, hexyl cinnamic aldehyde (HCA) or another positive control (e.g., a substancethat matches the chemical class of the test substances) could be run at intervals for quality
control purposes rather than concurrent with each experiment. The Panel cited Kimber et al.
(2006), which describes “routine” use of the “rLLNA” utilizing only a vehicle and a highdose group, as a rationale for this suggestion. However, the Panel does not recommend

xivIndependent Peer Review Panel Report

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omitting the concurrent positive control in laboratories that perform the LLNA only
“occasionally”.
Based on the analyses presented in the draft BRD, the Panel considered the accuracy of the
LLNA limit dose procedure to have been adequately evaluated and compared to the
traditional LLNA, mindful of the limitations associated with a retrospective evaluation. For
instance, it cannot be assumed that the compounds tested in the retrospective studies werealways tested at the highest possible dose unless such information was explicitly indicated. In
this regard, the Panel recommended that a more detailed description of what is considered“avoidance of excessive irritation” and “evidence of systemic toxicity” be included in any
LLNA protocol in order to aid in choosing the most appropriate high (i.e., limit) dose,
although specific indicators of “systemic toxicity or excessive irritation” were not formally
discussed.
The Panel agreed that it was appropriate to assume that the intra- and inter-laboratory
reproducibility of the LLNA limit dose procedure and the traditional LLNA would be
similar, because reproducibility is more dependent on the method than on the number of dosegroups. However, reducing the number of test substances dose groups from three to one
might reduce the sensitivity of the assay. The traditional LLNA may have a greater chance ofcorrectly identifying a sensitizer even in the presence of one or more technical errors sincedata from three dose groups are being considered and an SI ≥3.0 at any dose group would
result in the substance being classified as a sensitizer. However, for the purpose of adopting
an assay that uses fewer animals and provides increased throughput for testing purposes,
these hypothetical considerations are not a sufficient reason to argue against use of the limit
dose LLNA procedure.
LLNA for Testing Aqueous Solutions, Metals, and Mixtures
The draft ICCVAM recommendations state that, although more data are needed to assess theuse of the LLNA for testing for mixtures and aqueous solutions before a recommendation canbe made, the traditional LLNA appears to be useful for the testing of metal compounds, withthe exception of nickel. The Panel agreed with these draft ICCVAM recommendations.
Regarding the use of the LLNA for testing mixtures, the Panel acknowledged that the abilityof ICCVAM to develop draft test method recommendations was limited not only by theamount of data available, but the relatively poor concordance of traditional LLNA outcomesin comparison to those obtained in guinea pig tests, and recommended that this be noted in
the final ICCVAM recommendations. The term “mixtures” can represent an infinite numberof materials and it would be more beneficial to specify types or formulations of mixtures thatare being examined.
Regarding metals, the Panel concluded that the accuracy statistics for the traditional LLNA
when compared to results obtained from evaluation in humans supported use of the
traditional LLNA as a hazard identification tool for metals, with the exception of nickel,
which produces variable responses. One minority opinion stated that the results for nickel
compounds were not entirely questionable and that the traditional LLNA might also be
suitable for testing nickel compounds. Thus, the Panel recommended further evaluation of
the variable results obtained for nickel in the context of the available literature on allergic
contact dermatitis to nickel in humans.

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Regarding substances tested in aqueous solutions, the Panel suggested expanding the brief
section of the draft test method recommendations discussing the test method protocol for the
traditional LLNA to specifically point out how the conclusions of the applicability domain
evaluation may affect the standard traditional LLNA protocol. For instance, it could besuggested that aqueous test solutions be avoided due to problems associated with skinapplication. It would be preferable for a hierarchy of organic solvents to be considered as
dosing vehicles, with emphasis on using a vehicle to which humans may actually be exposedin circumstances linked to occupational sensitization.
The Panel agreed with the draft ICCVAM recommendation for continued accrual of
information from traditional LLNA evaluations of mixtures, metals, and aqueous solutions
with comparative data for guinea pig (i.e., guinea pig maximization test [GPMT] or Buehler
test [BT]) and human (i.e., human maximization test [HMT] or human repeat insult patch test
[HRIPT]) tests. However, the Panel suggested that, given resource limitations, it would be
important to organize the recommendations based on relative priority.
The draft Addendum to the original validation report for the traditional LLNA (ICCVAM
1999) provided a comprehensive review of currently available data and information forevaluating the usefulness and limitations of the traditional LLNA for assessing the skinsensitization potential of mixtures, metal compounds, and substances tested in aqueous
solutions. The Panel evaluated the draft Addendum for completeness, errors, and omissions
and concluded that there were no apparent errors or omissions, although they did state thatthe term “mixtures” was used too broadly (i.e., can represent an infinite number of materials)
and it would be more beneficial to specify types or formulations of mixtures that are being
examined.
The Panel did not identify any classes of chemicals missing from the dataset used to review
the utility of the traditional LLNA for testing aqueous solutions. However, while they did notpropose an alternative, the Panel expressed concern over the most appropriate definition for
an aqueous solution (defined in the draft Addendum as any solution containing ≥20% water).
For the mixtures included in the analysis, the Panel noted that quantitative compositions had
not been provided and therefore they could not comment on whether these mixtures wererepresentative of the types of mixtures typically tested in the traditional LLNA. With respect
to metals (none of which are mixtures), there was a paucity of important representatives ofcommercially useful metals such as platinum, palladium, iron, zinc, manganese and silver inthe data set. The Panel suggested that to enlarge the group of metal non-sensitizers,
substances used as cosmetic ingredients (e.g., titanium dioxide) and aluminum compounds
currently used in antiperspirants might be considered.
The Panel agreed that, although it was important to identify data obtained according to GLP
guidelines, data obtained from non-GLP studies should not be excluded automatically fromthis retrospective analysis. The Panel concluded that other factors could be used to identify
high quality data. Examples include data published in peer-reviewed journals or obtained
from a study conducted in a laboratory that has GLP capabilities.
The Panel concluded that, considering the limited comparative data that were available,
particularly for mixtures and aqueous solutions, the accuracy assessment of the traditional
LLNA for testing mixtures, metals, and aqueous solutions when compared to available
human and/or guinea pig test results was as comprehensive as was possible. The limited

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amount of comparative data made it unfeasible to draw definitive conclusions for mixtures
and aqueous solutions from the available accuracy statistics.
Non-Radioactive LLNA Protocol - The LLNA: DA Test Method
The Panel concluded that the available data and test method performance support the
ICCVAM draft recommendations for the LLNA: Daicel Adenosine Triphosphate test method(LLNA: DA), and that the test method may be useful for identifying substances as potential
skin sensitizers and non-sensitizers, but that this recommendation is contingent upon receipt,
review, and analyses of additional existing data and information from the test method
developer. Therefore, this non-radioactive version of the traditional LLNA cannot currently
be recommended for the hazard identification of skin sensitizing substances, regardless ofwhether or not there are restrictions on the use of radioactive materials, until such time as thisexisting data has been received and confirmed.
The draft LLNA: DA BRD was compiled to provide a comprehensive review of available
data and information evaluating the usefulness and limitations of the LLNA: DA test method
to assess the allergic contact dermatitis potential of chemicals and other substances. The
Panel evaluated the draft BRD for completeness, errors, and omissions and recommended
that their suggestions/corrections relating to general, statistical, and specific editorial issues
be incorporated into future revisions.
The Panel agreed that five animals per dose group should be recommended for validation ofmodified LLNA test methods. The Panel, however, noted that supplemental power
calculations for the LLNA: DA test method indicated that the power for detecting a threefold increase in the treatment group was estimated to be 95% for a sample size of three mice
per dose group. Thus, the Panel identified the use of three animals per dose group as apotential opportunity to reduce animal number when using modified assays in the future,assuming all essential validation requirements can be successfully met. A minority opinion
expressed by five Panel members was that if laboratories were operating under OECD
guidance (OECD 2002) and a reliable validation dataset had been generated, then pooled datafrom a least four animals per dose group could be considered.
Generally, the Panel viewed the difference in treatment schedule between the LLNA: DA andthe traditional LLNA to be potentially significant if the LLNA: DA induced the elicitation
phase of skin sensitization. The Panel was concerned that the 1% sodium lauryl sulfate (SLS)
pretreatment step in the LLNA: DA might modify the inherent sensitivity of the LLNA. They
recommended that the test method developer (Daicel Chemical Industries, Ltd.) justify theuse of 1% SLS or consider an alternative decision criterion (i.e., an SI threshold other than
3.0) such that the 1% SLS pretreatment is no longer necessary.
The Panel considered the database of substances tested in the LLNA: DA to be representativeof a sufficient range of chemicals expected to be tested for skin sensitization potential, andconcluded that the accuracy analysis had made appropriate comparisons to the traditional
LLNA, guinea pig tests, and human data/experience. The Panel could not identify specificcharacteristics associated with the one false negative (i.e., 2-mercaptobenzothiazole) or the
one false positive (i.e., benzalkonium chloride), but reemphasized that the potential impact ofpretreatment with 1% SLS in this context needed to be considered.

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With regard to test method reliability, the Panel concluded the intralaboratory reproducibilityof the LLNA: DA had not been adequately evaluated. They noted that the two sensitizers
tested had similar chemical structures (i.e., eugenol and isoeugenol) and that it was unclear if
the tests were truly independent. The Panel also noted that the interlaboratory reproducibilityof the assay could not be adequately evaluated given the lack of original laboratory data andlimitations in the study design. In particular, they cited the use of pooled lymph nodes fromthe mice in each treatment group and the testing of each substance at predetermined doselevels established by the lead laboratory as study design limitations. Still, a Panel minority
considered pooled data acceptable and the setting of dose levels for all laboratories based on
results from the lead laboratory to be reasonable.
The Panel also commented that ideally, test substances should be coded during the validationof a new assay, although they did not feel that a lack of coding constituted a reason forrejecting the current LLNA: DA dataset. The Panel also commented that although GLP
compliance is highly recommended for validation studies, the current studies should not berejected solely on the basis of a lack of GLP compliance. However, the Panel considered it
important to obtain the original records for all validation studies (which have been requested
by NICEATM) in order to confirm that the reported data were the same as the data recordedin the laboratory notebooks.
With regard to the 5% (1/19) false negative and 10% (1/10) false positive rates obtained with
the LLNA: DA, the Panel commented that it was important to identify reasons why thesubstances gave “false” results, taking into consideration factors such as intended use of thesubstances and the target population. They agreed that it might be useful to follow asuspected inaccuracy with an investigation of the mechanistic basis for the discordance since
it may help to establish a biologically-based rationale for the discordance.
The Panel noted that the available LLNA: DA data did not support all of the ICCVAM draft
recommendations in the proposed test method standardized LLNA: DA protocol. First,
although the Panel agreed with the ICCVAM protocol that recommends five animals perdose group, they noted that supplemental statistical information provided for the LLNA: DA
test method implied that using less than five animals per dose group was acceptable (e.g., a3.0-fold increase in the SI value would likely be detected with 99% confidence when using
four animals per dose group). In addition, the Panel considered it important to adequately
characterize the effect of the 1% SLS pretreatment step in the LLNA: DA, and it should bedemonstrated that the day 8 applications do not induce a skin reaction that could beindicative of the onset of the elicitation phase of skin sensitization. Keeping these points in
mind, the Panel agreed that if the limit dose procedure was applicable to the traditional
LLNA, then it would also be applicable to the LLNA: DA in order to further reduce thenumber of animals used.
The Panel also stated that the available data supported the ICCVAM draft recommendations
for the LLNA: DA in terms of future studies, which included performing a morecomprehensive evaluation using more non-sensitizers within and across laboratories. A
minority opinion stated by one Panel member was that although testing more sensitizers
might be warranted for interlaboratory validation studies, a sufficient number of nonsensitizers (n=11) had already been tested within the same laboratory.

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The Panel also commented that the protocol differences between the LLNA: DA and the
traditional LLNA could not clearly be constituted as “major” or “minor” changes. However,they considered this issue largely irrelevant if a test method was able to correctly predict thedermal sensitization potential of a test substance. Consequently, the Panel concluded that thecurrent draft ICCVAM Performance Standards could be applicable to the LLNA: DA as amechanistically and functionally similar test method.
Non-Radioactive LLNA Protocol - The LLNA: BrdU-FC Test Method
Overall, the Panel concluded that the available data and test method performance of the
LLNA with bromodeoxyuridine (BrdU) detected by flow cytometry (LLNA: BrdU-FC)
supported the draft ICCVAM recommendations that it may be useful for identifying
substances as potential skin sensitizers and non-sensitizers, but that more information andexisting data must be made available before the LLNA: BrdU-FC can be recommended forroutine use. The Panel concluded that the test method usefulness and limitations identified in
the draft ICCVAM recommendations accurately summarized the limits of the information
supplied and the additional information that would need to be generated or provided forfurther consideration of the test method. As a result, the Panel concluded that the LLNA:
BrdU-FC could not currently be considered as a scientifically valid replacement alternative tothe traditional LLNA. Still, the Panel suggested that the test method recommendation should
clearly state that the test method was not “invalid”, but simply that there was currently not
sufficient evidence and information to state that it had been adequately validated.
The draft LLNA: BrdU-FC BRD was compiled to provide a comprehensive review ofavailable data and information evaluating the usefulness and limitations of the LLNA: BrdUFC test method to assess the ACD-inducing potential of substances. The Panel evaluated thedraft BRD for completeness, errors, and omissions and recommended that theirrecommendations/corrections relating to general, statistical, and specific editorial issues be
incorporated into future revisions.
The LLNA: BrdU-FC included routine measurements of ear swelling as an indicator ofexcessive skin irritation. The Panel viewed that this, or any other quantitative measurementof skin irritation, should be carefully considered for inclusion in all LLNA protocols. The
Panel considered inclusion of optional quantification of immunophenotypic markers as anadditional mechanism for distinguishing irritants from sensitizers to be useful, as it might
reduce the frequency of false positives (i.e., substances which are actually skin irritants) and
improve comparisons with human data. However, they considered application of
immunological markers too detailed and costly for routine LLNA use (i.e., for hazard
classification purposes) and more suited for research purposes.
The Panel noted that the substances tested in the LLNA: BrdU-FC seemed representative of a
sufficient range of chemical classes and physical chemical properties, and thus that the test
method appeared applicable to many of the types of chemicals and products that are typicallytested for skin sensitization potential. However, the Panel considered the total databaseavailable for evaluation of the validation status of the LLNA: BrdU-FC to be relatively small
compared to the large number of substances assessed in the traditional LLNA. Therefore, thePanel recommended caution when making conclusions related to its concordance with thexixIndependent Peer Review Panel Report

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traditional LLNA. Still, the accuracy of the LLNA: BrdU-FC was considered adequately
evaluated and comparable to the traditional LLNA.
The Panel concluded that intralaboratory reproducibility was not adequately assessed and it
should be better evaluated in order to support the validation of this test method. The Panelsuggested that although the studies evaluated in the draft BRD were not GLP-compliant, thisshould not affect acceptance of the data for an evaluation of the validation status of this test
method. However, some sources of variability in the intralaboratory data, such as failure toappreciate differences in composition of dosing solutions between experiments caused by test
article instability or other phenomena, might be obscured if not in complete compliance with
GLP guidelines. Thus, the Panel suggested that any additional studies undertaken to validate
the test method should ideally be GLP-compliant.
The Panel agreed that the available data supported the ICCVAM draft recommendations for
the LLNA: BrdU-FC procedure in terms of the proposed test method standardized protocol.
They suggested that the utility of ear swelling or other methods to detect inflammation
appeared warranted in every variation of the LLNA (including the traditional LLNA), butshould be further investigated before routine inclusion in the protocol is recommended. The
Panel also concluded that the traditional LLNA limit dose procedure could be applied to the
LLNA: BrdU-FC, keeping in mind the limitations associated with a “limit dose” procedure.
The Panel further agreed that the ICCVAM draft recommendations for future studies
highlighted the unanswered questions raised by the available data set. Specifically,
conducting interlaboratory studies as a part of the validation process is important. The Panelconsidered the immunological markers suggested for the LLNA: BrdU-FC to be acceptable,
but that additional immunological markers for discrimination of irritant versus sensitization
phenomena were also possible. In general, for any future work, efforts should be made to
decrease the variability and to thereby increase the power of the test in order to ensure that
more animals were not needed relative to the traditional LLNA or other alternative LLNA
protocols.
The Panel considered the protocol differences between the LLNA: BrdU-FC and the
traditional LLNA to be “minor” changes, and therefore concluded that assessment of thevalidity of this test method could be based on the draft ICCVAM LLNA PerformanceStandards. The Panel also cautioned, however, that a clear definition of what constituted a“major” versus a “minor” change, or a different protocol altogether could be better addressed
once the recommendations for the current draft ICCVAM LLNA Performance Standards
were finalized.
Non-Radioactive LLNA Protocol - The LLNA: BrdU-ELISA Test Method
The Panel concluded that the available data and test method performance for the LLNA with
BrdU detected by enzyme-linked immunosorbent assay (LLNA: BrdU-ELISA) support the
ICCVAM draft recommendations that it may be useful for identifying substances as potential
skin sensitizers and non-sensitizers, but that more information and existing data must be
made available before the LLNA: BrdU-ELISA can be recommended for use. The Panel also
stated that a detailed protocol was needed, in addition to sufficient quantitative data forbroader analysis on a larger set of balanced reference substances that take into accountxxIndependent Peer Review Panel Report

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physicochemical properties and sensitization potency, as well as an appropriate evaluation ofinterlaboratory reproducibility.
The draft LLNA: BrdU-ELISA BRD was compiled to provide a comprehensive review ofavailable data and information evaluating the usefulness and limitations of the LLNA: BrdUELISA test method to assess the ACD-inducing potential of chemicals and other substances.
The Panel evaluated the draft BRD for completeness, errors, and omissions andrecommended that their suggestions/corrections relating to general, statistical and specificeditorial issues be incorporated into the final document.
The Panel’s main concern with the test method was that the accuracy of the LLNA: BrdUELISA at SI ≥3.0 was inadequate and not equivalent to the traditional LLNA. Furthermore,
although using a decision criterion of SI ≥1.3 improved the test’s performance in identifying
sensitizers from non-sensitizers, it did not resolve concerns about the test method. Based on a
power analysis for the LLNA: BrdU-ELISA, which was provided to the Panel as
supplemental information, the Panel concluded that it was difficult to justify using a SI ≥1.3
as the cutoff value, given the much larger number of animals that would be required to detect
a 1.3-fold increase above vehicle controls with similar power to the traditional LLNA when
five animals per dose group are used. For a three-fold increase, the supplemental statisticalanalyses indicated that a sample size of four was sufficient. Still, the Panel agreed with the
ICCVAM recommendation to use five animals per dose group and to collect individual
animal data. They concluded that this would allow for more robust calculations in the eventthat an outlier prevented some of the data from being included in the analysis. A minority
opinion by five Panel members was stated that if laboratories were operating under OECD
guidance (OECD 2002) and a reliable validation dataset had been generated, then pooled datafrom a least four animals could be considered.
The Panel noted that in organizations where the use or disposal of radioactive materials was
restricted, the potential to use the LLNA: BrdU-ELISA could reduce the number of animals
needed per test compared to the traditional LLNA and would result in less pain and suffering
compared to using traditional guinea pig test methods. However, if the SI ≥1.3 was chosen as
the decision criterion because of its improved accuracy compared to SI ≥3.0, the Panel statedthat the number of mice needed to perform the LLNA: BrdU-ELISA test should be comparedto the number of guinea pigs that would be needed for skin sensitization tests in order toassess if the LLNA: BrdU-ELISA actually reduced overall animal use for skin sensitization
testing.
In general, the Panel considered the number of substances tested in the LLNA: BrdU-ELISA
too few, and that data from more substances tested using the traditional LLNA, guinea pig
tests, and human tests should have been included. The Panel also did not consider theavailable data from the LLNA: BrdU-ELISA to be representative of a sufficient range ofchemical classes and physical chemical properties. The limited dataset prevents an evaluationof whether the test method would be considered applicable to any of the types of chemicals
and products typically tested for skin sensitization potential.
However, the Panel concluded that the appropriate comparisons between the traditional
LLNA, guinea pig test and human data had been made. The Panel agreed that the false
negative rate for hazard identification using the SI ≥3.0 in the LLNA: BrdU-ELISA wasexcessive (i.e., using this SI threshold value, the LLNA: BrdU-ELISA misclassified 29% andxxi

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39% of the substances classified as sensitizers in the traditional LLNA or in humans,
respectively).
The Panel also considered that the intralaboratory reproducibility of the LLNA: BrdU-ELISA
was not adequately evaluated and compared to the traditional LLNA. The Panel indicatedthat the number of substances was too few, and in some cases there was a wide variation in
repeat tests of the same substance. The Panel recommended a more comprehensive
evaluation of the intralaboratory reproducibility of the test method, using different SI values,
and that the analysis of the variability of the estimated concentration needed to produce a
positive SI value (ECt values) be conducted on a log scale.
The Panel also noted that interlaboratory reproducibility for the LLNA: BrdU-ELISA could
not be evaluated because neither the design of the study sponsored by the Japanese Center for
Validation of Alternative Methods nor any of the resulting data had been provided in advanceof their evaluation. The Panel agreed that a multi-laboratory validation study using abalanced set of chemicals would adequately characterize the interlaboratory reproducibilityof the LLNA: BrdU-ELISA.
In general, the Panel agreed that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA procedure in terms of the proposed test
method standardized protocols. However, as noted above, a minority opinion by five Panelmembers was that there could be circumstances in which pooled data from at least fouranimals could also be acceptable. The Panel also stated that if the LLNA: BrdU-ELISA wasfound to be equivalent to the traditional LLNA in the future that it would be appropriate toapply the LLNA limit dose procedure to the test. The Panel also agreed with ICCVAM’s test
method recommendations for future studies and emphasized that more data were needed inorder to determine the appropriate threshold value for the decision criterion. The Panelconcluded that it might be more appropriate to use a statistically-based decision criterion
rather than a stimulation index to classify substances as sensitizers, and that this should befurther investigated.
The Panel agreed that the LLNA: BrdU-ELISA protocol differed from the traditional LLNA
only in the method used to assess lymphocyte proliferation and as such concluded that thisrepresented a “minor” change (as defined in the current draft ICCVAM LLNA PerformanceStandards) and separate performance standards for the LLNA: BrdU-ELISA were notneeded.
Draft ICCVAM LLNA Performance Standards
The draft ICCVAM LLNA Performance Standards are intended to evaluate the acceptabilityof proposed test methods that are mechanistically and functionally similar to the traditional
LLNA. ICCVAM proposed that the applicability of the draft ICCVAM LLNA PerformanceStandards be restricted to protocols that incorporate “minor” modifications to the traditional
LLNA procedure, defined as changes only to the method for measuring lymphocyte
proliferation. The Panel agreed that different methods of measuring lymphocyte proliferation
represent “minor” modifications, but recommended that, instead of trying to define “minor”
modifications, a better strategy might be to define criteria that would need to be satisfied inorder to ensure that the alternative test method was mechanistically and functionally similar
to the traditional LLNA (e.g., only measure cell proliferation associated with the inductionxxii

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phase of a skin sensitization reaction). The Panel considered that the draft performancestandards were also appropriate for evaluating other modifications. Examples of acceptable
modifications included test animal sex, strain, the use of rats rather than mice, the number ofanimals per group, and timing of test article treatment. One minority opinion considered thepotential impact of changes to protocol components other than the method of measuring
lymphocyte proliferation to be significant and therefore would require more extensive
validation, which was not defined.
The Panel indicated that alternative LLNA protocols that are undergoing validation should
contain essential test method components that follow the ICCVAM-recommended protocol(ICCVAM 1999; Dean et al. 2001), unless adequate scientific rationale for deviating fromthis protocol was provided.
The Panel also identified aspects of the LLNA that should be required as part of the test
method validation process: (1) application of the test substance to the skin with sampling of
the lymph nodes draining that site, (2) measurement of cell proliferation in the draining
lymph node, (3) absence of a skin reaction that could be indicative of the onset of theelicitation phase of skin sensitization, (4) data collected at the level of the individual animal
to allow for an estimate of the variance within control and treatment groups (using thisvariance, a power analysis needs to be conducted to demonstrate that the modified method is
utilizing a sufficient number of animals per treatment group to permit hazard identification
with at least 95% power), and (5) if dose response information is needed, there are anadequate number of dose groups (n ≥3) with which to accurately characterize the dose
response for a given test substance.
The Panel noted that the list of substances included in the draft ICCVAM LLNA
Performance Standards was sufficiently representative of the types of materials that are likely
to be tested for skin sensitization. However, among the 13 sensitizers in the list of "required"substances, only five were considered to have robust data (i.e., traditional LLNA data basedon at least three independent studies).
To evaluate performance for use in hazard identification, the Panel concluded that all 22
substances in the draft ICCVAM-recommended list should be tested and accuracy statistics
calculated (Note: this list of substances includes "required" substances as well as "optional"
false negative and false positive substances, of which only 8/22 have "robust" datasets [n ≥ 3
as defined by the Panel]). To the extent possible, a rationale for any discordant results should
be provided. However, the most potent sensitizers (e.g., dinitrochlorobenzene [DNCB])
should always be identifiable. Also, considerable weight should be given to the balance
between animal welfare and human safety when considering the adequacy of test method
accuracy. Based on the limited data available for the sensitizers on the list and the lack ofstandardization of test methods from which the results were obtained, the current database
does not support inclusion of ECt values as a component of the accuracy evaluation.
The Panel agreed with the draft ICCVAM recommendations for evaluating test method
reliability. These recommendations included obtaining ECt values that are generally within
0.5x to 2.0x of the mean historical EC3 (i.e., estimated concentrations needed to produce an
SI of 3) values for hexyl cinnamic aldehyde (HCA) (intralaboratory, n=4 experiments in one
laboratory), or HCA and DNCB (interlaboratory, n=1 experiment in three laboratories).
However, the Panel recommended that the criteria for independent tests should be specified

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May 2008(e.g., different animal shipment, different reagents, different operator). The Panel concluded
that the proposed criteria for acceptability appeared to be appropriate in this case, because
only one or two substances were being evaluated (i.e., a statistical multiple comparisons2
problem does not exist). The Panel also suggested that historical control data using HCA and
DNCB in the same vehicle could be used to demonstrate adequate intra- and/or interlaboratory reproducibility.
The Panel also recommended that statistical tests to analyze the data might allow for a moreaccurate interpretation. They recommended that a suitable variance-stabilizing
transformation (e.g., log transformation, square root transformation) be applied in all
statistical analyses and in reporting summary standard deviations. The Panel also
recommended that a more rigorous evaluation be conducted of what would be considered an
appropriate range of ECt values to include as a requirement. This would be a statisticalevaluation that considers the variability of ECt values generated among the sensitizers
included on the performance standards reference substances list and the statistical multiple
comparisons problem.
Use of the LLNA for Potency Determinations
The Panel agreed with the draft ICCVAM recommendation that the LLNA should not be
used as a stand-alone assay for categorizing skin sensitizers as strong vs. weak, but that it
could be used as part of a weight-of-evidence evaluation (e.g., along with quantitative
structure-activity relationships, peptide reactivity, human evidence, historical data from otherexperimental animal studies) for this purpose. The Panel also agreed with the draft ICCVAM
recommendation that any LLNA studies conducted for the purpose of evaluating skinsensitization potency should use the ICCVAM-recommended LLNA protocol. In addition,
the Panel viewed that the relevant testing guidelines for the traditional LLNA should be
revised to include the procedure for calculating an EC3 value.
A draft BRD was compiled by ICCVAM that provided a comprehensive review of available
data and information and an evaluation of the usefulness and limitations of the traditional
LLNA for the categorization of substances with regard to skin sensitization potency. The
Panel evaluated the draft BRD for completeness, errors, and omissions and noted alternative
analyses that would allow for a more complete evaluation of the use of the traditional LLNA
for skin sensitization potency categorizations (see below).
The Panel agreed that the database of substances evaluated for potency determinations was
sufficient and represented a range of chemical classes and physicochemical properties
applicable to products typically tested for skin sensitization potential. The Panel also
concluded that since the database was compiled from existing data, the lack of substancecoding likely had no impact on the retrospective evaluation presented in the draft BRD. Still,
the Panel recommended the coding of test substances in any future validation studies. The

2

When multiple experiments are conducted and multiple observations, comparisons, or hypothesis tests are
conducted, the chance of observing rare events increases. Suppose, for example, that an interval is establishedsuch that 5% of observations from a particular population of data are outside that interval. Then if k independent
experiments generate data from this population (e.g., a standard normal distribution), the chances that all 20
results will lie inside the interval is (1.0 - 0.05)k (N. Flournoy, personal communication).

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Panel generally agreed that potency determinations based on traditional LLNA results should
ideally be limited to data from studies that evaluated lymph node proliferation in individual
animals so that outliers and technical errors could be identified. However, they also agreed
that pooled animal data should not be excluded automatically from a retrospective analysis.
The Panel indicated that the relevance of the LLNA for potency determinations had beenadequately compared and evaluated to human (i.e., HMT or HRIPT) and guinea pig (i.e.,
GPMT or BT) data. A minority opinion stated by one Panel member was that the relevanceof the traditional LLNA to human clinical observations had not been sufficiently determined.
In general, the Panel agreed that the proposed two-level categorization scheme (weak vs.
strong sensitizers) for both human and guinea pig data was appropriate. However, a minority
opinion stated by two Panel members was that a moderate category should be included sincecertain compounds might be on the border between weak and strong sensitizers. Thus, theysuggested that the five-category scheme proposed by Kimber et al. (2003), which includes
non-sensitizers, might be recommended.
The Panel concluded that the decision criteria providing the best overall performance was theuse of <250 µg/cm2 to distinguish between strong and weak sensitizers in humans and the useof an LLNA EC3 ≤9.4% to distinguish between strong and weak sensitizers in the LLNA.
The Panel stated that more data would be needed to determine if values different from these
two would be more appropriate. The Panel also recommended that safety factors other than
10 for the lowest observed effect level (LOEL) be evaluated to determine if improved results
could be obtained. The Panel also suggested an analysis that directly compares the LOEL
values without using a safety factor (i.e., using LOEL data only) and an analysis that onlyuses no observed effect level data. The Panel further stated that traditional LLNA tests basedon pooled or individual lymph nodes for a dose group should be evaluated independently toassess the impact of using pooled data on the accuracy analysis for skin sensitization
potency. Finally, the Panel stated that the effect of different vehicles should be recognized asa limitation in the current data analysis and a likely contributor to the variability observed
within and across laboratories.
The Panel stated that data from studies that could not be confirmed as being GLP-compliant,but that were from peer-reviewed literature or sources with high-quality laboratory
management practices, were still appropriate to include in the accuracy analysis. However,the Panel stated that, ideally, GLP compliance should be the standard, as it is clearly the only
objective way to judge the credibility of the data.
The Panel recommended that more data should be collected to determine the optimal
threshold in humans for distinguishing between strong and weak sensitizers. In addition, thePanel discouraged conducting additional animal studies unless such studies would be
expected to lead to an overall reduction in animal use. The Panel recommended that the
LOELs from Akkan et al. (2003) be used instead of the DSA05 (i.e., the dose per skin arealeading to a sensitization incidence of 5%) values from Schneider and Akkan (2004) in all of
the potency analyses. A minority opinion by one Panel member stated that it was acceptable
to use the DSA05 values from Akkan et al. (2003) as LOEL values in the evaluation. Thispanelist mentioned that the DSA05 value is a LOEL value adjusted to 5% incidence of
induction in order to correct for human studies leading to different inductions. Furthermore,
the panelist stated that because the DSA05 is corrected for an induction rate of 5%, it wouldxxv

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be better to compare with the traditional LLNA EC3 than to use the default uncorrected
LOEL.

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1.0

Murine Local Lymph Node Assay (LLNA) Limit Dose Procedure1

1.1

Comments on the Draft Background Review Document (BRD) for
Completeness, Errors and Omissions

1.1.1

General Comments

The international independent scientific peer review panel (hereafter, Panel) was asked if
there were errors in the draft LLNA limit dose procedure BRD that should be corrected, if
omissions of existing relevant data had been identified, or if there was additional informationthat should be included. The Panel agreed that consideration should be given to applying thesame term to the LLNA limit dose procedure since in various places throughout the draft
BRD it was referred to differently as either the “cut-down”, the “limit dose”, or the “reduced
LLNA” (rLLNA). Since the European Centre for the Validation of Alternative Methods(ECVAM) has already established a naming convention of “rLLNA”, the Panelrecommended adopting the ECVAM terminology to harmonize the terminology used among
the international validation agencies.
The Panel recommended that since the validation of the LLNA limit dose procedureencompassed data that was analyzed retrospectively, a discussion of the limitations of a
retrospective evaluation of previously published LLNA results should be included in the final
BRD. In particular, the assumption that the highest dose in the retrospective dose-response
study would be equivalent to the highest possible dose tested in the limit dose procedure
should be addressed. Discussing such a limitation would be important since it bears directlyon the validation of the limit dose procedure.
Further, since determination of the appropriate “limit dose” is critical to the LLNA limit dose
procedure, the Panel suggested that a discussion of how to arrive at the maximal
concentration for test substance dosing should be included in the final BRD. The final BRD
should also specifically define what is meant by the terms “avoidance of excessive irritation"and “systemic toxicity” to aid in choosing the most appropriate maximum dose. In thisregard, the Panel suggested that a systematic and quantitative measurement of ear thickness
and systemic toxicity be considered or evaluated for routine inclusion in the LLNA protocol.
The Panel discussed modifying the Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) LLNA protocol requirement for testing concurrent positive
controls (ICCVAM 1999; Dean et al. 2001) as a means of further streamlining the LLNA
limit dose procedure (i.e., reducing animal number, cost, etc.). Although the Panel did notreach consensus, a suggestion was made that for laboratories in which the LLNA is“routinely” performed and which had demonstrated the ability to consistently obtain positive
results, hexyl cinnamic aldehyde (HCA) or another positive control (e.g., a substance thatmatches the chemical class of the test substances) could be run at intervals for quality control
purposes rather than concurrent with each experiment. The Panel noted that Kimber et al.
(2006) have described the “routine” use of the “rLLNA” utilizing only a vehicle and a highdose group. The Panel also recommended that for laboratories that perform the LLNA only
“occasionally”, a concurrent positive control should be used. However, in their discussions,1

Also referred to as the "reduced" LLNA.
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the Panel was not able to conclude what would constitute “routine” or “occasional” LLNA
use or what would be an appropriate interval between positive control testing when aconcurrent positive control is not used.
The Panel also noted that including the following additional information in the final BRD
might prove informative if included:

1.1.2

•

An indication of any procedural problems reported for the tests•

An indication of the range of historical values obtained with the negative andpositive controls (the positive control historical range might give insight into
the need for a concurrent positive control)•

Any discussion of global harmonization should expand on why the draft BRD
did not place more reliance upon Organisation for Economic Co-operation andDevelopment (OECD) Test Guideline (TG) 429 (OECD 2002) as a normativereference•

For two of the substances tested multiple times (HCA and linalool alcohol),different doses were used and discordant results were obtained. It should be
noted for which (if either) of these tests, the highest dose tested was consistent
with the dose selection principles set out in the draft BRD

General Statistical Comments

The Panel also had some statistical comments related to the LLNA limit dose procedure.
First, the Panel viewed that a reference to the supplemental statistical information in which
Dr. Joseph Haseman performed power calculations on the traditional LLNA would be useful
in determining if the sample size used in the LLNA limit dose procedure was adequate for
evaluating skin sensitization potential. Also, the Panel concluded that although a stimulationindex (SI) based on the ratio of 3.0 as the decision criterion for a sensitizer is informative,
statistical analysis determining if the test substance is significantly different from the control
substance should be recommended.
1.1.3

Comments with Specific References to the Text

The Panel also identified the following minor formatting and grammatical errors, andinformation gaps in the draft BRD:•

The manner of notating numerical data in the draft BRD tables was notconsistent (e.g., in some places the value “one” was shown as 1, elsewhere as
1.0; in a few places the SI was shown over the percent concentration used).

•Lines 291-294: The statement was made that “chemical class information is
included to provide an indication of the variety of structural elements present
in the substances that were evaluated in this analysis, but it is not intended tosuggest an impact of structure on sensitization potential”. The latter concept is
not entirely correct; the portion of the sentence stating “but it is not intended
to suggest an impact of structure on sensitization potential” should be omitted.
Consideration should be given to using the large database of chemicals to
selectively modify structure-activity relationship (SAR) software for improved
predictivity. This could likely be accomplished by communication with

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software developers to point out availability of the newly expanded ICCVAM
dataset developed for evaluation of the LLNA limit dose procedure.
•Lines 299-300: The sentence is incomplete; “non-sensitizers” should be
inserted at the end of the sentence.

•Line 358: The citation to Sailstad et al. (2001) was not listed in Section 12.0
(References) and should be included.

•Lines 365-384 (Section 1.1.2): Consideration should be given to expanding
the background on the mechanism and natural history of allergic contact
dermatitis (ACD). Some additional detail regarding the biochemistry and cellbiology of immune induction and elicitation would be useful as an orientation
to how the LLNA functions as an integrated method of detection for ACD.

•

Lines 366–368: The introductory sentence on the prevalence of ACD as an
occupational health issue would benefit from amplification to also indicatethat ACD is of variable severity with some potentially severe ramifications,and that ACD is recurrent upon rechallenge possibly requiring workplaceaccommodation or change of employment.

•Lines 366-368: There is no reference provided for the statistic from the U.S.
Department of Labor Bureau of Labor Statistics cited in Section 1.1.2 of thedraft BRD.

•

Line 433: The reason for further evaluation of negative results withconcentrations less than 10% should be clarified (Kimber et al. 2006).

•

Lines 435-436: This bullet point re-plays conclusions made in the summary of
the “rLLNA” issued by the scientific advisory committee of the European
Centre for the Validation of Alternative Methods (ECVAM). However, in thedraft BRD the statement has been altered and should instead read "..., asappropriate, per OECD TG 429 (OECD 2002)." rather than citing ICCVAM(1999) and Dean et al. (2001).

•

Lines 452-453: The intent of the sentence would be clarified by modifying toread "...to identify potential human skin sensitizers through quantification oflymphocyte proliferation in the test method."•Lines 496-500: Reading of the Kimber et al. (2006) citation does not indicatea recommendation for a concurrent positive control group. Thus, the sentencein the draft BRD that reports use of vehicle and positive control groups in thelimit dose procedure based on the Kimber et al. (2006) paper is incorrect.

•

Lines 509-510: This sentence should also mention that the LLNA limit dose
procedure, as published by Kimber et al. (2006), is rationalized not only as ameans of bioresource economy but as a valid means of streamlining the LLNA
for regulatory screening assessment purposes under regulation such as
Registration, Evaluation and Authorisation of Chemicals (REACH).

•

Lines 520-523: A footnote might be needed to explain why OECD TG 429(OECD 2002) is not referenced here.

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Lines 629-636: Data donated by GlaxoSmithKline (GSK) to the National
Toxicology Program Interagency Center for the Validation of Alternative
Toxicological Methods (NICEATM)-ICCVAM LLNA review were generated
under GLP conditions at a clinical research organization (CRO) repeatedly
audited for GLP compliance by GSK. This information should be added to the
text.

•

Section 6.1: The presentation of data and associated discussion regarding
limitations in accepting a 10% dose concentration cut-off should be
repositioned for emphasis. This information is important in developing
suggestions for a standard LLNA limit dose procedure. Data from Appendix D
could be reduced to a small table or figure and be integrated into the body ofthe final BRD.

•

Lines 722-723: This appears to be an incomplete sentence.

•Lines 815-822: Data donated by GSK to the NICEATM-ICCVAM LLNA
review were generated under GLP conditions at a CRO repeatedly audited for
GLP compliance by GSK. This information should be added to the text.

•

Table following line 1126: There is a typographical error in the 2 x 2 table. It
appears that the cell for Negative (New Test) x Total (Reference Test) should
read "c + d" rather than "a + d".

1.2

Comments on the Validation Status of the LLNA Limit Dose Procedure

1.2.1

Test Method Protocol

For the proposed LLNA limit dose procedure, ICCVAM recommended that the number ofanimals used in each group should be the same as that recommended by ICCVAM for the
traditional LLNA based on its 1998 evaluation (i.e., at least five animals per group), and that
individual animal data should be collected and reported (ICCVAM 1999; Dean et al. 2001).
The Panel was asked whether they agreed that these are appropriate protocol requirements
for the limit dose procedure. The Panel agreed that, based on the supplemental power
calculations for the traditional LLNA performed by Dr. Joseph Haseman (see Table 1-1), a
minimum of five animals per treatment group should be recommended for all future studies
employing the limit dose procedure. In addition, the collection of individual animal data, as
recommended by ICCVAM for the traditional LLNA (ICCVAM 1999; Dean et al. 2001),
should also apply to all future studies following the LLNA limit dose procedure. Similarly,
application of the LLNA limit dose procedure to a modified LLNA protocol would require
adherence to a validated protocol with the exception of omitting the middle and low dosegroups. Respective power calculations would indicate if application of the LLNA limit dose
procedure to a validated modified LLNA protocol would allow fewer animals per dosegroup.

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Table 1-1

May 2008

Power Calculations for the Traditional LLNA
3.0-foldincrease

2.5-foldincrease

2.0-foldincrease

1.5-foldincrease

1.3-foldincrease

Mean Rx response

1034.4

862.0

689.6

517.2

448.24Log (Mean Rx
response)

6.942

6.759

6.536

6.248

6.105

Difference (log scale)

1.099

0.916

0.693

0.405

0.262

Difference/SD

2.40

2.00

1.51

0.88

0.57

Power for N=595%

80-90%

50-80%<50%<50%

Power for N=4

90%

80%

50%

<50%

<50%

Power for N=350-80%

50-80%

<50%

<50%

<50%

Other Power––

95% (N=11)95% (N=29)

95% (N=68)

Other Power––

90% (N=9)90% (N=23)

90% (N=54)

The power calculations above are based on a one-sided p<0.05 Student’s t test applied to log-transformed datafrom vehicle control LLNA tests.
Abbreviations: Fold-increase=Required increase above the vehicle control for a positive response (i.e., thestimulation index); N=number of animals; Rx=Treatment; SD=standard deviation.

The primary rationale for both provisions is to underpin robust statistical analysis of LLNA
results. Furthermore, the use of individual animal data would allow for the evaluation of
dosing errors or other anomalies that might be masked by the use of pooled animal data.
1.2.2

Substances Used for the Validation Studies

The Panel was asked whether they considered the traditional LLNA database representativeof a sufficient range of chemical classes and physical chemical properties such that it would
be applicable to any of the types of chemicals and products that are typically tested for skinsensitization potential. If not, the Panel was asked which relevant chemical classes/properties(other than those identified as limitations in the traditional LLNA) should be tested withcaution, or not evaluated using the limit dose procedure, and which chemicals or products
should be evaluated to fill this data gap. The Panel agreed that, in general, the traditional
LLNA database included in the LLNA limit dose procedure evaluation was representative ofa sufficient range of chemical classes and physical chemical properties and that it should beapplicable to any of the types of chemicals and products that are typically tested in the
traditional LLNA for skin sensitization potential. It was notable that the substances includedin this evaluation provided a diverse chemical database. Since much is known about themechanism of sensitization, the LLNA should theoretically identify any chemical that works
by migration of haptens to the lymph node. However, the Panel noted that substance classes
that are sometimes problematic in the LLNA (i.e., metals) would also likely be problematicin the LLNA limit dose procedure. There were also some substance classes that had limited
or no representation in the draft BRD (i.e., mixtures/formulations, higher molecular weightbiopharmaceuticals, and medical device materials). Thus, in general, the LLNA (and the
LLNA limit dose procedure) is best used as part of a weight-of-evidence appraisal in which
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attributes such as physical chemical parameters, SAR evaluation, and indications of otherbiological activity involving potential chemical-to-biological macromolecule interactions, arecarefully considered along with LLNA results to evaluate dermal sensitizing potential.
Because the LLNA limit dose procedure was based on a retrospective evaluation of existing
data, most of which was not generated using coded chemicals, the Panel was asked whether alack of coding of test substances adversely impacted or biased the current evaluation. The
Panel considered that although coding of chemicals should be recommended for prospective
validation studies, this evaluation was likely not adversely impacted or biased because of a
retrospective evaluation of existing data. This is supported by the fact that many of thechemical structures included in the analyses do not appear to contain known structural motifs
associated with ACD or chemical hypersensitivity and therefore there was no a priori
expectation that the chemical tested would be a sensitizer. The Panel viewed it important toconsider the issue of coding or bias in prospective validation studies.
For some substances submitted using the traditional LLNA test method, it was not possible toconfirm whether the data were generated using pooled animal data for each dose group (asallowed in OECD TG 429 [OECD 2002]). ICCVAM (1999), Dean et al. (2001), and EPA(2003) recommend the use of statistical analyses to help interpret LLNA study results, which
necessitates data collected at the level of the individual animal. Furthermore, Cockshott et al.
(2006) reported that using individual animal data allowed for technical problems or other
outliers during an experiment to be identified. The Panel was asked what impact theinclusion of pooled animal data might have on the accuracy analysis of the LLNA limit dose
procedure. The Panel concluded that, although it would be important to note whether
individual or pooled animal data were reported, the retrospective analysis of the LLNA limit
dose procedure versus the traditional LLNA should not be limited to studies with confirmed
individual animal data. The Panel stated that internationally, both individual and pooled
animal data have likely been used both for regulatory decisions and for in-house decisions
relating to product development and risk management. Also, the fact that the retrospective
data analysis presented in the draft LLNA limit dose procedure BRD did not distinguishbetween individual or pooled animal data suggested that both met the quality standards forinclusion in the draft BRD.
1.2.3

Test Method Accuracy

The Panel was asked whether the relevance (e.g., accuracy/concordance, sensitivity,
specificity, false positive and false negative rates) of the LLNA limit dose procedure had
been adequately evaluated and compared to the traditional LLNA (refer also to Table 6-1 ofthe draft ICCVAM BRD). The Panel concluded that the relevance of the LLNA limit dose
procedure had been adequately evaluated and compared to the traditional LLNA.
Comparisons resulting in an accuracy of 98.9% (461/466), a sensitivity of 98.4% (308/313)
and a specificity of 100% (153/153) for the LLNA limit dose procedure when compared tothe traditional LLNA were sufficient to consider it adequately validated for use in theevaluation of skin sensitization, mindful of its known limitations that are described
elsewhere. Still, the Panel noted that it was important to keep in mind that a prospective
analysis may not have the same accuracy as this retrospective analysis.
Furthermore, there were five substances for which the highest concentration tested produced
an SI <3.0, while lower concentrations of these substances produced an SI >3.0 (see Table 6-1-6

Independent Peer Review Panel Report

May 2008

2 of the draft ICCVAM BRD). These substances were classified as false negatives compared
to what was obtained in the traditional LLNA. The Panel was asked to identify any
characteristics associated with these or other substances that might signal that this type ofabnormal dose response might occur, and therefore, that using the LLNA limit dose
procedure would not be appropriate. The Panel could not identify any common
characteristics associated with the five false negative substances that would explain the nonlinear dose response obtained. It was not known if any procedural problems were reportedwith these studies or what values were returned by the negative/positive control groups (inrelation to other historical positive control values).
Thus, the Panel suggested that it might be worthwhile to examine whether LLNA results withthese five false negative substances should be repeated. If the difference turned out to berepeatable, there could be hypothetical reasons to explain why the higher doses did not pass
the SI threshold of 3.0. For example, under certain experimental conditions, the target
lymphocytes may be selectively induced to a highly sensitive state by some chemicals at
higher doses and may undergo either induction of apoptosis or inhibition of cell proliferation.
Still, there was no evidence that these substances were immunomodulators that might havedifferentially stimulated or depressed the immune response depending on the dose andexposure. In any case, understanding false negatives is encouraged to ensure adequate
protection of public health.
The Panel was asked whether the draft BRD adequately characterized the usefulness andlimitations of the LLNA limit dose procedure based on the accuracy analyses. Overall, thePanel agreed that the draft BRD adequately characterized the usefulness and limitations of
the LLNA limit dose procedure based on the accuracy analyses. Since the LLNA limit dose
procedure and the traditional LLNA have close concordance, there was no need for detailed
discussion in the draft BRD. However, it was not explicitly stated in the draft BRD thatcompared to a fully conducted traditional LLNA, a false positive result in the LLNA limit
dose procedure is not possible (i.e., if the single dose used in the proposed limit dose
procedure gives an SI ≥3.0, then so would the top dose in the traditional LLNA).
Furthermore, prospective testing with the LLNA limit dose procedure to predict thesensitization potential of an unknown chemical was not discussed.
1.2.4

Test Method Reliability

The Panel was asked if it was appropriate to assume that the intra- and inter-laboratory
reproducibility of the LLNA limit dose procedure and the traditional LLNA would be
similar, based on the fact that they use identical protocols with the exception of the numberof doses used (i.e., would reducing the number of test substance dose groups from three to
one reduce the reliability of the assay?). The Panel agreed that it was appropriate to assume
that the intra- and inter-laboratory reproducibility of the LLNA limit dose procedure and the
traditional LLNA would be similar, because reproducibility is more dependent on the methodthan on the number of dose groups. However, reducing the number of test substances dosegroups from three to one could reduce the sensitivity of the assay (i.e., the ability to correctly
identify sensitizers). The traditional LLNA may have a greater chance of correctly
identifying a sensitizer even in the presence of one or more technical errors since there aredata from three dose groups for consideration and an SI ≥3.0 at any dose group would resultin the substance being classified as a sensitizer. However, for the purpose of adopting anassay that uses fewer animals and provides increased throughput for screening purposes,
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these hypothetical considerations are not a sufficient reason to argue against use of the limit
dose LLNA procedure.
1.2.5

Data Quality

For some studies included in the draft BRD, it was not possible to determine whether or notthey had been conducted in accordance with GLP guidelines. Furthermore, original records
for some of the non-GLP studies included in this evaluation could not be obtained. As aresult, an independent audit could not be conducted to confirm that the reported data was thesame as the data recorded in laboratory notebooks. Neither was it possible to obtain the
results of GLP audits for all studies conducted in accordance with GLP guidelines. The Panelwas asked whether the results of such studies (all of which are currently included) should be
excluded from the performance analyses. The Panel concluded that it was important to note if
the data were obtained from studies conducted according to GLP guidelines, as ideally thisshould be the case. However, the Panel concluded that the data resulting from theretrospective studies that could not be confirmed as GLP-compliant should not be excluded
from the performance analysis. Since there was not an indication that the reliability of thedata presented for consideration may have been compromised, omitting any data would
likely lessen the impact of the analysis. Furthermore, data obtained from peer-reviewed
literature or final reports were likely of sufficient quality.
1.2.6

Consideration of All Available Data and Relevant Information

The Panel was asked if all the relevant data identified in published or unpublished studies
conducted using the traditional LLNA had been adequately considered in the draft BRD. If
not, the Panel was asked what other traditional LLNA data needed to be considered and how
such data could be obtained. The Panel considered that the draft BRD had taken into accounta large majority of the relevant data identified in published and unpublished traditional
LLNA studies. The data received as a result of the Federal Register (FR) notices and the key
literature citations seemed to be inclusive of the relevant data for this analysis. Althoughadditional data that could have been included might exist, it was deemed unlikely that thecurrent outcome (which is based on 466 substances) would be altered given the very smallchange in accuracy statistics relative to Kimber et al. (2006), which was based on 211
substances.
1.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA Limit Dose Procedure

1.3.1

Test Method Usefulness and Limitations

The Panel was asked to comment on whether the available data supported the ICCVAM draft
recommendations for the LLNA limit dose procedure in terms of the proposed test method
usefulness and limitations (i.e., that the LLNA limit dose procedure should be routinely
recommended for hazard identification when dose response information is not required). The
Panel considered that, based on the available information, the draft recommendations
appeared valid, but made the following suggestions:•

Further emphasis should be given to using the LLNA limit dose procedure asa part of a comprehensive weight-of-evidence evaluation of dermal sensitizing
potential (e.g., including physical chemical evaluation, SAR information,

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May 2008

including likelihood of dermal penetration, ability of materials to adductbiomacromolecules).
•Such information in addition to LLNA results might also be useful in
confirming or questioning LLNA outcomes terms of in human hazard
identification, since it should be emphasized that a major application of themethod is to prospectively detect harmful chemicals.

•

Solubility or thermodynamic activity data, beyond visual assessment (e.g., useof chemically-specific methods to document solubility), should be used toconfirm the appropriateness of the maximum dose tested.

•

Vehicle selection for the LLNA can affect the results and may not allow
accurate comparisons between chemicals applied in different vehicles. In
choosing the best vehicle, consider measured solubility information for the
potential vehicle. Then, it would be important to take into account how thevehicle affects the amount of the chemical that can be applied to the ear. More
importantly, the impact that vehicle selection has on the amount of appliedchemical that actually gets into the mouse to induce the sensitization response
should be evaluated. Some of the recommended LLNA vehicles (e.g., 4:1
acetone:olive oil (AOO), dimethylsulfoxide (DMSO), methyl ethyl ketone)
could be expected to disrupt the barrier properties of the skin. Additionally,
although propylene glycol might allow an increased amount of chemical to be
applied, it might also inhibit the penetration of a chemical by enhancing
partitioning in the vehicle relative to the skin.

The Panel was asked whether the LLNA limit dose procedure should be routinely
recommended for the hazard identification of skin sensitizing chemicals when dose response
information is not required. With the points noted above in mind, the Panel agreed that itshould be routinely recommended since the LLNA limit dose procedure offers time, cost,throughput, and logistical benefits as well as using fewer animals. Still, the investigatorshould keep in mind what is known of the chemical regarding general toxicity and note
scenarios where abnormal dose-response relationships in the traditional LLNA might resultin false negatives in the limit dose procedure (see Table 6-2 of the ICCVAM BRD).
The Panel was then asked whether the LLNA limit dose procedure should be routinely
recommended as the initial test to identify sensitizers before conducting the traditional
LLNA, as a way to further reduce animal use, even if dose response information is required,since negative results would not require further testing. The Panel agreed that use of the
LLNA limit dose procedure, as the initial testing procedure to identify sensitizers and nonsensitizers before conducting the traditional LLNA, is justifiable even when dose response
information is required. This is applicable in the occupational and public health setting whereobtaining hazard information is of critical importance. There is a benefit since dose-response
information generated in subsequent testing in the traditional LLNA for substances that werepositive in the limit dose procedure then gives further assurance of detecting hazardous
substances and allowing a potency estimate. The benefits of screening out the negatives(which do not require dose response information) is clear; however the animal welfare gains
will depend on the proportion of test substances in any class that turn out to be non-

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sensitizers and there might be possible consequences of the delays resulting from a further
round of testing for those materials that are identified as sensitizers.
Based on the existing database, there is a false negative rate of 1.6% (5/313 positive
compounds) for the LLNA limit dose approach compared to the results obtained in the
traditional LLNA. The Panel was asked whether they considered that this is adequately
addressed by the proposed cautionary language and weight-of-evidence consideration for
negative substances. The Panel agreed that the small rate of false negatives was adequately
addressed in the draft test method recommendations by giving cautionary and weight-ofevidence consideration to the negative substances (and any possible false positive results).
Furthermore, given that the dose responses for these five materials were rather unusual, it
was not known whether these studies were repeatable, whether any procedural problems
were reported with these studies, or what values were returned by the negative/positive
control groups (in relation to other historical positive control values). In general, the Panelviewed that the false negative rate of 1.6% would likely be unimportant when the larger
differences between the animal model and humans are considered.
1.3.2

Test Method Protocol

The Panel was asked whether they agreed that the available data support the ICCVAM draft
recommendations for the LLNA limit dose procedure in terms of the proposed standardized
test method protocol. The Panel agreed and recommended adherence to the ICCVAM (1999)
LLNA protocol for future studies of the LLNA limit dose procedure with the exception ofomitting the middle and low dose groups. Similarly, application of the LLNA limit dose
procedure to a modified LLNA protocol would require adherence to the modified LLNA
protocol with the exception of omitting the middle and low dose groups. Adhering to the useof individual animals for future studies was specifically stressed because it would allow foran estimate of inter-animal variability.
The recommended ICCVAM protocol (ICCVAM 1999; Dean et al. 2001; EPA 2003), as
well as OECD TG 429 (OECD 2002), specifies that the highest dose tested should be thehighest soluble concentration that does not induce systemic toxicity and/or excessive skinirritation. However, Kimber et al. (2006) concluded that negative results obtained fromstudies where the highest concentration tested was below 10% should be considered invalid,
and adopted a 10% application concentration as a threshold of confidence for categorizationof a chemical as being negative while noting that the figure should not be considered as
inviolable. The Panel was asked whether the data presented in the draft BRD (i.e., 51/313
positive substances in the NICEATM database were negative at concentrations equal to or
above 10%, but were positive at even higher concentrations) were adequate to conclude that
this threshold concentration is not appropriate. The Panel viewed that this point should beclarified. ICCVAM recommended that no threshold should be used to determine the validityof conduct of the LLNA limit dose procedure. Instead, formal attempts to maximize dosedelivery including documentation of solubility of the test substance in the vehicle usedshould be undertaken.
The Panel was asked whether additional testing should be required if a negative result was
obtained for a test substance in a study where the highest concentration that could be tested(based on systemic toxicity or excessive local irritation, as described in ICCVAM [1999],
Dean et al. [2001], and EPA [2003]) was <10%. The Panel considered that, if a negative

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result was obtained for a test substance under these conditions, additional testing should not
be required, because at that point it would likely be a toxic effect and not sensitization. In
contrast, the imperative should be to minimize the number of false negatives. For thispurpose, rigorous examination of maximum solubility or other parameters to ensure testing at
maximum concentration should be employed. In addition, weight-of-evidence considerations
such as SAR and physicochemical characteristics should be documented. More animal
testing to verify negative results should only be undertaken if the weight-of-evidence
suggests that it would be appropriate.
The Panel was asked if the current approach for selecting the “limit dose” was appropriate orwhether there is a threshold concentration for the LLNA at which a negative result could
always be considered as an acceptable result. The Panel agreed that the current
recommendation to select a maximum applied dose in the LLNA limit dose procedure isappropriate. However, the data presented in the draft BRD implied that at present it is notpossible to establish a uniform concentration threshold for the “limit dose”. Thus, it seemedjustifiable that preliminary experimentation (as would be typically performed during a dose
range finding study) should be conducted on vehicle selection, test substance solubility, andstability in the vehicle.
1.3.3

Future Studies

The Panel was asked if they agreed that the available data support the ICCVAM draft
recommendations for the LLNA limit dose procedure in terms of the proposed future studies.
Although limited in scope, the Panel considered that the available data supported the
ICCVAM draft recommendations for additional studies. The Panel agreed that attempts bemade to investigate if maximum solubility was achieved (e.g., use of chemically-specificmethods to document solubility). For hazard assessment, it was considered troublesome that
there were so many vehicle choices, because the vehicle could have a significant effect onwhether (and how much) a test substance penetrated the skin barrier. Observed vehicle
effects may relate to dermal penetration as well as to immunomodulation. The Panelconsidered it desirable to follow the hierarchy of vehicles recommended in the ICCVAM(ICCVAM 1999; Dean et al. 2001) protocol. In addition, dedicated attempts must be made toinvestigate solubility in AOO mixtures before using other vehicles. Regardless of the vehicle
used, it is important to ensure that a vehicle does not promote lymph node cell proliferation.
The Panel also suggested that it might be informative to test both known mild and severe
sensitizers concurrently in all recommended vehicles to evaluate if a specific vehicle
choice(s) might influence the results.
Although the false negative rate in the current analysis was small, a need exists to better
understand factors that could lead to false negative results with future use of the LLNA limit
dose procedure. Thus, consideration should also be given to formal statistical assessments toverify group size and use of individual animal data in routine performance of the LLNA limit
dose procedure. Criteria should be established to verify proficiency with the LLNA limit
dose procedure. Such criteria could be used to answer questions about the necessity toperform concurrent positive controls.

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1.3.4

May 2008

Comments with Specific References to the Text

The Panel also identified the following comments and/or corrections to the draft ICCVAM
test method recommendations document on the LLNA limit dose procedure that should beconsidered by ICCVAM:•

Lines 26-28: Conclusions given here regarding the relative potency ratings ofthe five materials classified as false negative in the analysis in Section 6.2 ofthe draft BRD were newly introduced. This assessment should also have been
considered for inclusion in Section 6.2 of the draft BRD.

•

Line 28: The citation of Gerberick et al. (2004) was not accompanied by areference.

•Lines 62-70: The listing of substances not amenable to test in the LLNA could
have been expanded to also include agents with anticipated pharmacodynamic
action as immune suppressants.

•

Line 69: The citation of Gaspari et al. (2007) was not accompanied by areference.

•Lines 75-79: Dependent upon other considerations, this portion of the text
could have been modified to (1) clarify recommendations regarding routine
use of concurrent positive control (i.e., possible exception for laboratories
conducting a high volume of LLNA work in which periodic positive control
for quality control purposes might suffice), and (2) expand on the details
regarding indications of excessive irritation and/or systemic toxicity to aid inchoice of maximal test dose.

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2.0

LLNA for Testing Aqueous Solutions, Metals, and Mixtures

2.1

Comments on the Draft Addendum for Completeness, Errors, and Omissions

In regard to the draft Addendum to the traditional LLNA BRD, the Panel was asked tocomment on any errors that should be corrected or omissions of relevant data/informationthat should have been included. The Panel concluded that there were no apparent errors oromissions to the draft Addendum.
2.2

Comments on the Validation Status of the Traditional LLNA for Testing
Aqueous Solutions, Metals, and Mixtures

2.2.1

Substances Used for the Validation Studies

The Panel was asked whether the database of substances evaluated was representative of a
sufficient range of mixtures, metal compounds, and substances in aqueous solutions that are
typically tested for skin sensitization potential. While there were limited data available on theeffects of mixtures, metals, and aqueous solutions on skin sensitization potential, the Panelconsidered the database to be generally representative. The Panel indicated that there did notseem to be obvious classes of chemicals missing from the data set used to evaluate the utilityof the traditional LLNA for testing aqueous solutions. However, quantitative compositions
for the mixtures included in the analysis had not been provided. Thus, it was difficult todetermine if those mixtures were representative of the types of mixtures typically tested in
the traditional LLNA. With respect to metals, there was a paucity of commercially useful
metals such as platinum, palladium, iron, zinc, manganese, and silver compounds. To enlarge
the group of metal non-sensitizers, substances used as cosmetic ingredients (e.g., titanium
dioxide) and aluminum compounds currently used in antiperspirants might be considered.
However, the Panel considered that the inclusion of an array of other metals and at least one
zinc and manganese salt likely weighted the data set appropriately and it appeared
sufficiently broad to support conclusions about the utility of the traditional LLNA for testing
the skin sensitization potential of metals.
Substances or mixtures that were tested in an aqueous or an organic:aqueous vehicle werelabeled as aqueous solutions. For the purpose of this evaluation, a substance or mixture
containing at least 20% water was defined as an aqueous solution. The Panel was asked
whether this criterion was appropriate for defining an aqueous solution. The Panel was
uncertain about the appropriateness of this definition of an aqueous solution, but did not offer
an alternative definition. However, the Panel indicated that an organic:aqueous solution thatis not miscible would likely produce varying results because of partitioning of the chemical
into either phase.
The Panel was asked whether the lack of coding of test substances might adversely impact orbias the current evaluation. While coding of chemicals is recommended for prospective
validation studies, the retrospective evaluations in the draft Addendum were based on
existing data, most of which were not generated using coded chemicals. However, the Panelagreed that the lack of chemical coding was not likely to bias the evaluation since this study
was retrospective. This is supported by the fact that many of the chemical structures includedin the analyses did not contain known structural motifs associated with allergic contact
dermatitis/chemical hypersensitivity and therefore there was no a priori expectation that the
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chemical tested would be a sensitizer. Furthermore, many of the substances tested wereapparently evaluated for hazard assessment purposes rather than to test the predictive abilityof the traditional LLNA. Thus, there does not appear to be any bias in chemical selection for
the expanded dataset considered in the study of applicability domain for the traditional
LLNA.
For some substances submitted using the traditional LLNA test method, it was not possible toconfirm whether the data were generated based on pooled lymph nodes among animals
within a dose group, as allowed in OECD TG 429 (OECD 2002), or individual animal
responses, as recommended by ICCVAM (1999) and required by EPA (2003). ICCVAM(1999) and EPA (2003) both recommend the use of statistical analyses to aid in theinterpretation of traditional LLNA study results; such analyses necessitate data collectedfrom individual animals. Additionally, Cockshott et al. (2006) reported that using individual
animal data allowed for outlier animal results within a dose group to be identified. The Panelwas asked whether the analysis of the performance of the traditional LLNA for testing
mixtures, metal compounds, and substances in aqueous solutions should be limited to datafrom studies that collected individual animal data, and then to comment on the potential
impact on the accuracy analysis of including results from studies in which pooled animal data
were collected. The Panel concluded that, although individual animal data were preferred,pooled animal data should not be excluded automatically from this retrospective analysis.
2.2.2

Test Method Accuracy

The Panel was asked whether the relevance (e.g., accuracy/concordance, sensitivity,
specificity, false positive and false negative rates) of the traditional LLNA for testing
mixtures, metal compounds, and substances in aqueous solutions had been adequately
evaluated and compared to the human and guinea pig test results. The Panel agreed that thecomparative assessment of the relevance of the traditional LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions appeared to be as comprehensive as was
feasible. However, because of the limited number of comparisons available, the accuracy
statistics probably do not give a complete picture of the usefulness and limitations of the
traditional LLNA for identifying skin sensitizers among these types of substances or whenusing an aqueous vehicle.
When multiple traditional LLNA studies were available for the same substance, the “majority
call” (among studies using the same vehicle and generally tested over the same concentration
range) was used by ICCVAM to assign an overall classification for the purposes of theaccuracy analysis. For example, if chemical X was tested five times and was positive in threestudies and negative in two, the overall classification was positive. The Panel was asked
whether they agreed with this approach. They expressed their concern about the approach in
the following way; if all nickel-containing compounds in the analysis were viewed as agroup, there were four positive calls and four negative calls (see Appendix C2 of the draft
Addendum). Using the “majority call” approach, the overall call would be determined by thenext available study, which may not provide the correct call. More data would be needed toconfirm whether the classification was appropriate. For this dataset, most of the “negative
calls” had SI values that approached 3.0. Thus, a more suitable method might be to base theoverall call on the SI data, while giving greater positive call consideration/weight to SI
values just below 3.0. It may also be useful to perform a meta-analysis. It is important for the

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Addendum to mention the potential impact of using the “majority call” decision, rather thanrelying on a weight-of-evidence approach, on the accuracy analyses.
2.2.3

Data Quality

For some studies included in the draft Addendum, it was not possible to determine whether
or not they had been conducted in accordance with GLP guidelines. Original records forsome of the non-GLP studies included in this evaluation could not be obtained. As a result,an independent audit could not be conducted to confirm that the reported data was the sameas the data recorded in laboratory notebooks. Neither was it possible to obtain the results of
GLP audits for all studies conducted in accordance with GLP guidelines. The Panel was
asked to discuss what impact this lack of information might have on the evaluation of the
traditional LLNA for testing mixtures, metal compounds, and substances in aqueous
solutions and whether such studies should be excluded from an analysis of test method
accuracy. The Panel considered it important to note if the data were obtained from studies
conducted according to international GLP guidelines, since ideally this should be the process
followed. However, the Panel viewed that data from studies that could not be confirmed as
being GLP-compliant were still appropriate to include in the accuracy analysis, provided thatthe data were from the peer-reviewed literature or from sources with high quality laboratory
management practices. Much of the value for this draft Addendum was the potential tosupplement the data available at the time of the ICCVAM (1999) analysis. Additional
information on test substance identification would clearly be useful in the continued
evaluation of the applicability domain of the traditional LLNA, but omitting data on
mixtures, metals, or use of aqueous solutions based solely on the lack of GLP compliancewould lessen the impact of the current retrospective analysis and did not seem warranted.
However, if the original data were not available, it would be appropriate to note this in the
final version of the Addendum.
2.2.4

Consideration of All Available Data and Relevant Information

The Panel was asked whether the draft Addendum included all of the relevant data for studies
conducted using the traditional LLNA for testing mixtures, metal compounds, and substancesin aqueous solutions. The Panel considered that, although it was possible that there might bea few studies in the literature to augment the analysis, it seemed that the relevant data had
been identified and the response to the FR notice and the literature citations examined had
included the most relevant studies.
2.3

Comments on the Draft ICCVAM Test Method Recommendations on the
Traditional LLNA for Testing Aqueous Solutions, Metals, and Mixtures

2.3.1

Test Method Usefulness and Limitations

ICCVAM stated that more data would be needed before a recommendation on the usefulness
and limitations of the traditional LLNA for testing mixtures could be made, due to thelimitations associated with the available mixtures database (i.e., unknown formulae, lack ofhuman data). The Panel was asked whether they agreed that the available data supported the
ICCVAM draft recommendations for the traditional LLNA with regard to testing mixtures interms of the proposed test method usefulness and limitations. The Panel agreed thatICCVAM’s draft recommendation with respect to the traditional LLNA testing of mixturesappeared valid based on the limitations inherent in the available data set. Still, the Panel
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May 2008

urged that the ICCVAM recommendation indicate that the approach may be viable. The
Panel further recommended that the test method recommendations summary should indicatethat the limitations include relatively poor concordance of traditional LLNA outcomes for
mixtures with to those obtained in guinea pig tests. Routine comparisons of accuracy
according to classification criteria may not be sufficient to evaluate the concordance for
mixtures, and furthermore, the guinea pig tests are not necessarily valid for mixtures. The
Panel also indicated that the term “mixtures” was used too broadly (i.e., can represent an
infinite number of materials) and it would be more beneficial to specify types or formulationsof mixtures that are being examined.
ICCVAM recommended that, based on the available data for metals, the traditional LLNA
was useful for the testing of metal compounds, with the exception of nickel. The Panel was
asked whether they agreed that the available data supported the ICCVAM draft
recommendations for the traditional LLNA with regard to testing metals in terms of the
proposed test method usefulness and limitations. Based on the available information, thePanel agreed that the draft recommendations with regard to testing metals appeared to bevalid. In particular, the evidence for most metals (e.g., accuracy of 86% (12/14), sensitivityof 100% (9/9), specificity of 60% (3/5), 0% (0/9) false negatives) when comparing traditional
LLNA results to those obtained from evaluations in humans supported the use of the
traditional LLNA as a hazard identification tool for metals, excluding nickel. However, thePanel recommended that it would be worthwhile to study further the variable results obtained
for nickel since there is a wealth of literature on allergic contact dermatitis of nickel inhumans.
In a minority opinion, Dr. Dagmar Jírová stated that it should not be concluded that thetraditional LLNA was not suitable for testing nickel compounds, because the different
vehicles used may have had a significant impact on the ability of nickel to penetrate the skinand be bioavailable. She noted that nickel chloride and nickel sulfate were both positive inaqueous solutions, and negative only when non-aqueous vehicles were used. In human
exposures, nickel compounds were applied in aqueous solutions. Thus, this may serve as
sufficient justification to use aqueous vehicles when nickel, and perhaps also othersubstances, are tested and evaluated in the traditional LLNA. When DMSO was used as thevehicle, the SI value increased with increasing nickel concentration. Unfortunately, no data
were available for concentrations over 5% for either nickel compound in DMSO. Nickel
chloride as 10% in aqueous solution reached an SI of 6.6. Inconsistent test results due to thevehicle have also occurred in other in vitro studies (e.g., phototoxicity). Thus, Dr. Jírová
concluded that the traditional LLNA could be used even for testing nickel compounds whenother vehicles (in particular aqueous) are used.
Due to the limited number of substances tested in aqueous solutions, ICCVAM
recommended that more data would be needed before a recommendation on the usefulness
and limitations of the traditional LLNA for testing substances in aqueous solutions could be
made. The Panel was asked whether they agreed that the available data supported thisICCVAM draft recommendation for the traditional LLNA with regard to the testing of
substances in aqueous solutions. The Panel agreed that the draft ICCVAM recommendation
was appropriate and that more data were required before an adequate evaluation of the use of
the traditional LLNA with aqueous solutions could be conducted.

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2.3.2

May 2008

Test Method Protocol

The Panel was asked whether they agreed that the available data supported the ICCVAM
draft recommendations for the traditional LLNA in terms of the proposed test method
standardized protocol. The Panel agreed that, in general, the results of the assessment in thedraft Addendum supported the proposals for standardized conduct of the traditional LLNA.
However, this conclusion depended on a side-by-side reading of the draft Addendum and the
ICCVAM (1999) protocol. The Panel suggested expanding the brief section of the draft test
method recommendations dealing with test method protocol for the traditional LLNA
(Section 2.0) to specifically point out how the conclusions of the applicability domain
evaluation may affect the standard traditional LLNA protocol. For example, the evaluation of
aqueous solutions apparently resulted in the methodological recommendation that aqueous
test solutions be avoided and the further recommendation of a hierarchy of organic solvents
to be considered as dosing vehicles. The emphasis might be on using a vehicle to whichhumans may actually be exposed.
2.3.3

Future Studies

The Panel was asked whether they agreed that the available data support the ICCVAM draft
recommendations for the traditional LLNA in terms of the proposed future studies. The Panelagreed that the ICCVAM recommendation for continued accrual of information fromtraditional LLNA evaluations of mixtures, metals, and aqueous solutions with comparative
data for guinea pig and human tests was appropriate. The traditional LLNA accuracy formetals of 86% and sensitivity of 100% (0% false negative) was excellent; a specificity of
60% (40% false positive) was considered acceptable as over-classification maintains safe
human use. The Panel encouraged the use of the traditional LLNA to acquire further
information on mixtures, metals, and aqueous solutions. However, the Panel suggested that,given resource limitations, it would be important to prioritize the recommendations in orderto focus on what is most important.

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May 2008

3.0

Non-Radioactive LLNA Protocol - The LLNA: Daicel Adenosine
Triphosphate (LLNA: DA) Test Method

3.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

3.1.1

General Comments

The Panel was asked if there were any errors in the draft LLNA: DA BRD that should becorrected, if omissions of existing relevant data had been identified, or if there was additional
information that should be included. As a general comment, the Panel noted that the draft
BRD clearly and succinctly provided an overview of the LLNA: DA test method and therelevant validation study data. The draft BRD indicated that the LLNA: DA differs from thetraditional LLNA in the method of measuring proliferation (measures levels of adenosine
triphosphate [ATP] instead of radioactivity), substance treatment (pretreating the test sitewith 1% sodium lauryl sulfate [SLS] prior to test substance application and an additional
treatment on day 7), and sampling time (draining auricular lymph nodes are collected on day
8 rather than on day 6). Because the traditional LLNA evaluates the induction phase only, therelevance of results with the LLNA: DA (and any other LLNA protocol) should always be
considered in the context of human experimental sensitization data, human epidemiologic
data, and elicitation in the clinical setting.
3.1.2

Comments with Specific References to the Text

The Panel noted the following text that should be clarified or corrected in the final version of
the LLNA: DA BRD:•

Line 428: The text should read “1% SLS”, not “1% SDS”; the same
terminology should be used throughout rather than going back and forth
between SLS and sodium dodecyl sulfate (SDS).

•

Line 449: The text and formula in lines 448-450 appear misplaced and instead
seem to belong in Section 7.0. Additionally, X and Y should be defined andthe “Var (ln SI)” formula should be clarified.

•

Table 3-1: There were 33 substances in the table, yet the discussion of thetable in the text referred to 31 substances. Although the reason for thisapparent discrepancy becomes more evident later on, this should be discussed
up front.

•

Table 3-2 and 3-3: The interlaboratory distribution and testing of thesensitizers versus non-sensitizers should be indicated here.

•

Table 6-1: Although the table clearly provided a comparison of the different
methods, it would be useful if the footer for this table also indicated the basis
for the differences in substances included in each analysis (i.e., n=25, 26, or29) as stated in the text.

•

Table 7-1 was only moderately helpful because the standard deviations (SD)were not calculated on a log scale. Given the skewness in the data, the ranges
given were misleading indicators of increases due to the vehicles. Without the
samples sizes, an analysis of variance was impossible to calculate, and that

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May 2008

would have been the appropriate measure of differences between theexperiments. In order to make recommendations regarding needed
reproducibility experiments, it would have been helpful to have a power
analysis for this situation. That is, for each vehicle with each chemical, using
two, three, four and five animals per dose group per experiment, how many
experiments need to be run to detect significant differences between theexperiments?
3.2

Comments on the Validation Status of the LLNA: DA

3.2.1

Test Method Protocol

Based on its 1998 evaluation of the traditional LLNA procedure, ICCVAM recommended
that at least five animals be used in each test group (ICCVAM 1999). The LLNA: DA
validation studies presented in the draft BRD were performed using four animals per dosegroup. Thus, the Panel was asked to comment on the potential impact of using fewer than
five mice per dose group. The Panel noted that supplemental statistical information they were
provided with indicated that the power for detecting a three-fold increase in the SI value inthe treatment group for the LLNA: DA dataset evaluated in the draft BRD was estimated tobe 95% for a sample size of three mice per dose group (see Table 3-1). Since an increase offalse negatives may not be an issue, the potential opportunity exists for utilizing this smallergroup size. The Panel cautioned, however, that using less than five animals per group might
result in a less precise estimate of the mean response, which, in turn, will impact accuracy.
Also, if technical errors further reduce the sample size, accuracy is further reduced. Thus, thePanel recommended that all initial validation studies adhere to the ICCVAM-recommended
protocol (ICCVAM 1999; Dean et al. 2001) of five animals per dose group until sufficient
information is generated to indicate that the use of fewer animals per dose group is
statistically valid.

3-2Independent Peer Review Panel Report

Table 3-1

May 2008

Power Calculations for the LLNA: DA
3.0-fold
Increase

2.5-fold
Increase

2.0-fold
Increase

1.5-fold
Increase

1.3-fold
Increase

Mean Rx response

8835

7362.55890

4417.53828.5Log (mean Rx
response)

9.086

8.904

8.681

8.393

8.250

Difference fromcontrol (log scale)

1.098

0.916

0.693

0.405

0.262

Difference/SD

3.95

3.29

2.49

1.46

0.94

Power for N=5

99%

99%

95%

50-80%<50%

Power for N=499%

95-99%

90%

50%

<50%

Power for N=395%

90-95%

80%

<50%

<50%

Other power

–

–

–

95% (N=11)95% (N=25)

Other power

–

–

–

90% (N=9)90% (N=20)

Parameter

The power calculations above are based on a one-sided p<0.05 Student’s t-test applied to log-transformed datafrom vehicle control LLNA: DA tests.
Abbreviations: Fold-increase=Required increase above the vehicle control for a positive response;
Rx=Treatment; N=Number; SD=Standard deviation.

The data generated for the substances analyzed in the LLNA: DA interlaboratory validation
studies came from auricular lymph nodes that were pooled across animals in each treatmentgroup. The Panel was asked to comment on the potential impact of including pooled animal
data on the accuracy analysis of the LLNA: DA. The Panel noted that a statistical analysis ofdifferences between treatment-related and vehicle control ATP levels could not be
determined without measures of variability. Individual animal data highlights technical issues
and allows for consideration of dose-response information and statistical analyses.
The LLNA: DA differs from the traditional LLNA in the treatment schedule and by including
a pretreatment step with 1% SLS just prior to application of the test substance. The Panel was
asked to comment on the appropriateness of these protocol differences. The Panel did notconsider these differences to be significant, as long as it could be demonstrated that the 1%
SLS pretreatment step and the additional test substance treatment on day 7 did not induce askin reaction indicative of the elicitation phase of skin sensitization. Although it was being
used at a lower concentration than the estimated concentration needed to produce a
stimulation index of 3 (EC3), the Panel expressed concern about pretreating the mouse ear
with 1% SLS since SLS is an irritant and positive in the traditional LLNA. Consequently, theinherent sensitivity of the LLNA may be modified by the 1% SLS pretreatment step. To
demonstrate that these concerns are not justified, the Panel concluded that weak irritants andweak sensitizers needed to be tested in the LLNA: DA assay with and without pretreatment
with 1% SLS. The test method developer might also consider using decision criteria otherthan SI ≥3.0 such that 1% SLS pretreatment is no longer necessary.

3-3Independent Peer Review Panel Report

3.2.2

May 2008

Substances Used for the Validation Studies

The Panel was asked if they considered the substances tested in the LLNA: DA to be
representative of a sufficient range of chemical classes and physicochemical properties thatthe test method would be applicable to any of the types of chemicals and products that are
typically tested for skin sensitization potential. The Panel considered the database of test
substances tested in the LLNA: DA representative of a sufficient range of chemicals. The
selected substances included solids and liquids and a range of solvents/vehicles. The databasealso represented a range of sensitizing potency, a variety of different chemical classes andsubstances with differing requirements for metabolic activation. However, it might have been
useful to have also included substances with clearly different protein reaction mechanisms(protein binding), as well as dyes, natural extracts, and mixtures.
3.2.3

Test Method Accuracy

The accuracy analysis in the draft LLNA: DA BRD was based on overall concordance withthe traditional LLNA. Accuracy statistics compared to the guinea pig tests and humandata/experience were also provided. The Panel, when asked if they considered thesecomparisons appropriate for assessing the accuracy of the LLNA: DA, agreed that thecomparisons to the traditional LLNA performance and also to the guinea pig and humansensitization data were important. The Panel also stressed that, because the traditional LLNA
only evaluates the induction phase, the relevance of the LLNA: DA results should always be
considered in the context of human experimental sensitization data, human epidemiologic
data, and elicitation in the clinical setting.
The Panel was asked if they considered the evaluation of the relevance of the LLNA: DA andthe comparison to the traditional LLNA to be adequate. The Panel noted that Table 6-1 of thedraft LLNA: DA BRD clearly provided a comparison of the different reference methods (i.e.,
traditional LLNA, human tests, and guinea pig tests). Thus, the Panel concluded that therelevance of the LLNA: DA had been adequately evaluated. However, including data on
more substances is likely to further strengthen confidence in the concordance data.
One substance, 2-mercaptobenzothiazole, produced a false negative response compared tothe traditional LLNA when tested using the LLNA: DA. The Panel was asked if they could
identify any characteristics associated with this or similar substances, compared to thecorrectly identified sensitizers, that might signal that this type of discordant response would
occur, and therefore using the LLNA: DA to test such substances would not be appropriate(or that negative results for substances with such properties may warrant additional testing).
The Panel could not identify specific characteristics that might explain the false negative
response using the LLNA: DA. Although understanding the solubility and stability of the test
substance in different vehicles is important, the differences in response did not seem to be
explained by the vehicle differences (AOO and dimethylformamide [DMF]) between the two
tests. In addition, the impact of 1% SLS pretreatment on the negative response in the LLNA:
DA is not known but should be considered.
One substance, benzalkonium chloride, produced a false positive response compared to the
traditional LLNA and guinea pig test when tested using the LLNA: DA. The Panel was asked
if they could identify any characteristics associated with this or similar substances, compared

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May 2008

to the correctly identified non-sensitizers, that might signal that this type of discordant
response would occur, and therefore using the LLNA: DA to test such substances would not
be appropriate (or that positive results for substances with such properties may warrant
additional testing). The Panel could not identify specific characteristics that might explain thefalse positive response for this substance in the LLNA: DA. The Panel viewed that it was
important to note, however, that this chemical is a well-known skin irritant, and on occasion
it had also been considered a human sensitizer, typically on the basis of positive diagnostic
patch test data. Thus, the Panel reiterated that the relevance of LLNA: DA results should
always be considered in the context of human experimental sensitization data, humanepidemiologic data, and elicitation in the clinical setting. The actual impact of the 1% SLS
pretreatment step on the LLNA: DA has not been well established, although van Och et al.
(2000) and De Jong et al. (2002) have reported that 1% SLS pretreatment enhances the
response in the traditional LLNA.
3.2.4

Test Method Reliability

The Panel was asked if they considered the intralaboratory reproducibility of the LLNA: DA
to have been adequately evaluated and compared to the traditional LLNA (refer to Table 7-1of the draft LLNA: DA BRD). The Panel noted that only eugenol and isoeugenol, twosensitizers with similar chemical structures, were tested. The Panel recommended testing a
positive control commonly used in the traditional LLNA (e.g., HCA) for a more complete
evaluation of intralaboratory reproducibility. In addition, it was unclear if the tests were truly
independent. Factors that might indicate independence should have been documented (e.g.,time interval between experiments, different animal shipment, different reagents, different
operator).
The Panel was also asked if they considered the interlaboratory reproducibility of the LLNA:
DA to have been adequately evaluated and compared to the traditional LLNA. The Panelnoted that the interlaboratory reproducibility of the assay could not be adequately evaluatedgiven the lack of original study data and limitations in the study design. Study design
limitations included:•

Pooled lymph nodes were used from mice within a dose group. This precluded
an analysis of variation between laboratories.

•

The lead laboratory established the dose levels to be tested by the otherlaboratories participating in the interlaboratory validation effort. In a minority
opinion, Drs. Nathalie Alépée and Michael Woolhiser asserted that for aneffective and efficient interlaboratory evaluation, it seemed reasonable to set
dose levels for all laboratories based on results from the lead laboratory.

In addition, the Panel considered that the interlaboratory studies could benefit by performing
more than one test on two commonly used positive controls (i.e., HCA and DNCB).
The draft LLNA: DA BRD contained an analysis of data from two interlaboratory
reproducibility validation studies that used coded substances, as well as an intralaboratory
accuracy validation study with 31 substances that were not coded. The Panel was asked ifthey considered the lack of coding of the test substances to have adversely impacted orbiased the intralaboratory accuracy evaluation. The Panel commented that, in the validationof a new assay, it is better to avoid the potential for bias by testing coded substances.

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May 2008

However, the Panel concluded the data already generated for the LLNA: DA test methodshould be considered and not be rejected in the current validation evaluation.
The lead laboratory established the dose levels tested by the participating laboratories in thetwo interlaboratory validation studies. The Panel was asked if this adversely impacted orbiased the evaluation. The Panel considered that the choice of the maximum test substanceconcentration is crucial for the proper performance of the traditional LLNA as well as any
modified LLNA. Thus, predetermining the dose levels to be tested for each substance might
have reduced variability between the two interlaboratory studies. In a minority opinion, Drs.
Nathalie Alépée and Michael Woolhiser asserted that for an effective and efficientinterlaboratory evaluation, it seemed reasonable to set dose levels for all laboratories basedon results from the lead laboratory.
3.2.5

Data Quality

The studies evaluated in the draft BRD for the LLNA: DA were not conducted in accordancewith GLP guidelines although they were reportedly done in laboratories that conduct GLP
studies, and were conducted "in the spirit" of GLP (K. Idehara, personal communication).
Furthermore, the original records for the interlaboratory studies were requested but have notyet been obtained. As a result, an independent audit could not be conducted to confirm thatthe reported data was the same as the data recorded in laboratory notebooks. The Panel was
asked to comment on the potential impact this might have had on the evaluation of the
LLNA: DA. The Panel commented that, ideally, GLP compliance is recommended forvalidation studies, but the current studies should not be rejected based on the lack of GLP
compliance alone. However, all the raw data obtained through the validation process should
be made available and audited for accuracy. The Panel further commented that since theoriginal records for the interlaboratory studies have not yet been provided, recommendations
from ICCVAM should be contingent upon receiving these data. Obtaining original laboratory
records is a necessary step to confirm that all data generated during the validation studies
have been provided, and that the reported data are the same as the data recorded in laboratory
notebooks.
3.2.6

Consideration of All Available Data and Relevant Information

The Panel was asked to comment on whether all of the relevant data identified in publishedor unpublished studies that employed the LLNA: DA had been adequately compared. The
Panel viewed that, generally, it seemed that all of the relevant results had been adequately
identified and considered. However, as mentioned above, all of the original data supportingthese results have not been provided. The Panel again expressed concern related to the effectof pretreating the mouse ear with 1% SLS and the Panel therefore recommended that theresults from van Och et al. (2000) and De Jong et al. (2002) should be considered.
3.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: DA

3.3.1

Test Method Usefulness and Limitations

The Panel was asked to comment on whether the available data supported the ICCVAM draft
recommendations for the LLNA: DA procedure in terms of the proposed test method
usefulness and limitations. The Panel agreed with ICCVAM’s recommendation, which statedthat the LLNA: DA might be useful for identifying substances as potential skin sensitizers
3-6Independent Peer Review Panel Report

May 2008

and non-sensitizers, but this recommendation was contingent upon the receipt of additional
data and information. The Panel further added that information on the possibility of skinreactions suggestive of the onset of the elicitation phase and the impact of the 1% SLS
pretreatment step on the performance of the LLNA: DA should be evaluated. The Panel also
considered that the ICCVAM proposed limitations needed to be more clearly defined, as it
was not clear from the draft recommendations what points were considered as limitations.
For instance, limitations that are known for the traditional LLNA would likely apply to thismodified protocol as well and these should be noted.
The Panel was asked whether restrictions on using radioactive materials would warrant that
the LLNA: DA be routinely recommended for hazard identification of skin sensitizing
substances in lieu of having to possibly use guinea pig tests. The Panel noted that, based on
gaps in the currently available dataset and information described in this report, the LLNA:
DA could not yet be recommended for the routine use for hazard identification of skinsensitizing substances, regardless of whether restrictions on using radioactive materials were
present or not. Generally, non-radioactive LLNA test methods are preferred in lieu of using
guinea pig tests because fewer animals are used and animal pain and distress is reduced.
However, policy issues regarding restrictions on radioactivity should have no impact on thisscience-based conclusion.
The Panel was asked if, from a public health perspective, the recommended guidance for
evaluating negatives were sufficient to address concerns associated with the false negative
rate of 5% (1/19 substances) calculated for the LLNA: DA. The Panel noted that this was nota scientific question, rather a risk characterization issue, and could not be answered without
considering other factors such as intended use, target population, etc. The Panel was also
asked if, from a testing strategy perspective, the ICCVAM guidance addressed concerns
associated with the false positive rate of 10% (1/10 substances) calculated for the LLNA: DA
and/or if they had other suggestions for additional guidance or limitations. The Panel again
commented that this was not a scientific question but a risk characterization issue and could
not be answered without considering other factors such as intended use, target population,etc. Furthermore, the Panel noted that it would be difficult to generalize the finding of one
test substance being a “false” result. Instead, they considered it better to identify reasons why
a substance was a “false” result. Certainly, if a “false” result is suspected, confirmatory
testing with another mouse LLNA method was not recommended. It might be important tofollow a suspected inaccuracy with an investigation of the mechanistic basis for the
discordance.
3.3.2

Test Method Protocol

The Panel was asked if they agreed that the available data supported the ICCVAM draft
recommendations for the LLNA: DA procedure in terms of the proposed test method
standardized protocols or what recommendations they would make. The Panel noted thatavailable data did not support all of the ICCVAM draft recommendations in the LLNA: DA
standardized protocol. First, the ICCVAM protocol (ICCVAM 1999; Dean et al. 2001) for
the traditional LLNA recommends using at least five animals per dose group. Although thePanel agreed that five animals per dose group should be recommended for validation studies,
they suggested that power calculations would be useful in determining if subsequent use of
the modified test method could use fewer animals per dose group. For the LLNA: DA test
method, the Panel noted that based on statistical power calculations that were provided as
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Independent Peer Review Panel Report

May 2008

supplemental information, using four animals per group instead of five did not appear to be alimitation (i.e., detecting a 3.0-fold increase in the SI with four animals per group was
estimated to have a 99% confidence level). In addition, the Panel generally agreed with therecommendation in the ICCVAM protocol (ICCVAM 1999; Dean et al. 2001) that individual
animal data should be collected. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel,
Dagmar Jírová, Raymond Pieters, and Michael Woolhiser stated that if laboratories wereoperating under OECD guidance (OECD 2002) and a reliable validation dataset had been
generated, then pooled data from at least four animals could be considered acceptable.
Of greater importance, the Panel concluded that pretreatment with 1% SLS should not be
accepted until its impact on the performance of the LLNA: DA has been adequately
characterized. Although used at a concentration below its EC3, the Panel was concerned
about pretreating the mouse ear with an irritant reported as positive in the traditional LLNA.
To demonstrate that these concerns are not justified, the Panel recommended that substancesthat are weak irritants and weak sensitizers be tested in the LLNA: DA with and without
pretreatment with 1% SLS. It also needed to be demonstrated that the 1% SLS pretreatments,
as well as the additional test substance treatment on day 7, did not induce a skin reaction that
could be indicative of the onset of the elicitation phase of skin sensitization.
The Panel was asked to comment on whether the limit dose procedure could be applied to the
LLNA: DA. The Panel concluded that if the limit dose procedure is considered applicable tothe traditional LLNA, then it should also be applicable to the LLNA: DA, in order to reduce
the number of animals used. This would require adherence to the LLNA: DA test method
protocol, with the exception that the middle and low dose groups would be omitted in thelimit dose version.
3.3.3

Future Studies

The Panel was asked if they agreed that the available data support the ICCVAM draft
recommendations for the LLNA: DA in terms of the proposed future studies or, if not, what
recommendations they would make. The Panel stated that the available data supported the
ICCVAM draft recommendations for the LLNA: DA in terms of a more comprehensive
evaluation using more non-sensitizers within and across laboratories. In a minority opinion,
Dr. Thomas Gebel stated that although testing of more non-sensitizers might be warranted forinterlaboratory validation studies, a sufficient number of non-sensitizers had been tested
within the same laboratory (Table 6.3 in the draft BRD).
However, the Panel viewed that there were additional studies that ICCVAM might consider.
As previously mentioned, the Panel recommended that the 1% SLS pretreatment step should
not be accepted until its impact on the performance of the LLNA: DA had been adequately
characterized. Furthermore, it should be demonstrated that such pretreatments did not inducea skin reaction that could be indicative of the onset of the elicitation phase of skinsensitization. It might also be of interest to evaluate ATP as a marker of lymph node
proliferation using the traditional LLNA dosing scheme and lymph node collection schedule.
Lastly, the Panel considered that studies on the reliability of outlier analysis in small sample
sizes and the effects of reduced sample size on the power of the LLNA: DA test methodshould be proposed.

3-8Independent Peer Review Panel Report

3.3.4

May 2008

Performance Standards

The draft LLNA: DA BRD indicated that the LLNA: DA protocol differed from the
ICCVAM-recommended protocol for the traditional LLNA (ICCVAM 1999; Dean et al.
2001) in the method used to assess lymphocyte proliferation in the auricular lymph nodes. In
addition, there are differences between the two protocols that relate to how and when the test
substance is applied and when the lymph nodes are collected (Table 2-1 and Appendix A in
the draft LLNA: DA BRD). According to the proposed draft ICCVAM LLNA PerformanceStandards for the traditional LLNA, any change to the LLNA protocol other than the method
used to assess lymphocyte proliferation would be considered a “major” change. The Panelwas asked if they agreed that these should be considered “major” changes and therefore theusefulness and limitations of the LLNA: DA should not be assessed using the draft ICCVAM
LLNA Performance Standards. The Panel commented that answering this question depended
on having a clear definition of what constitutes a “major” versus a “minor” change, and what
may constitute a different protocol altogether. Depending on the goal of the assay, whether achange is “major” versus “minor” may not be relevant. Ultimately, if a test method is able to
make the correct prediction with regard to the dermal sensitization potential of a test
substance, then the issue of “major” versus “minor” modifications might not apply.
Considering the robust nature of the current draft ICCVAM LLNA Performance Standards, it
is difficult to identify the need for additional requirements for methods like the LLNA: DA.
Thus, the draft ICCVAM LLNA Performance Standards could be used to evaluate the
LLNA: DA as a mechanistically and functionally similar test method.
The Panel was asked, even if the draft ICCVAM LLNA Performance Standards were notfound applicable to the LLNA: DA, whether an analysis based on 13 of the 18 proposed
required reference substances in the performance standards would impact the overall
evaluation of the test method accuracy. The Panel commented that the accuracy analysis
based on 13 of the 18 proposed required reference substances in the performance standards(with one false negative substance) should have no impact on the overall evaluation of test
method accuracy as 31 substances have been tested. However, given the concern regarding
pretreatment with 1% SLS, the Panel stated that testing of substances with and without 1%
SLS was needed to characterize the effect of this pretreatment on the performance of theassay. The Panel concluded that as described above, the idea of “major” versus “minor”
changes might be reconsidered, thus the current draft ICCVAM LLNA PerformanceStandards could be applicable to the LLNA: DA as a mechanistically and functionally similar
test method.

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4.0

Non-Radioactive LLNA Protocol – The LLNA:
Bromodeoxyuridine Detected by Flow Cytometry (BrdU-FC) Test
Method

4.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

4.1.1

General Comments

The Panel was asked if there were any errors in the draft LLNA: BrdU-FC BRD that should
be corrected, if omissions of existing relevant data had been identified, or if there wasadditional information that should be included. The Panel noted that overall, errors andomissions in the draft LLNA: BrdU-FC BRD were few. The majority of omissions relating to
the data records were identified in the text, and all reasonable efforts to obtain additional
information from MB Research Labs, the developer of the LLNA: BrdU-FC, appear to havebeen made.
The following describes the identified errors, omissions, and/or information gaps in the draft
LLNA: BrdU-FC BRD that should be addressed:

4.1.2

•

Data are available in the peer-reviewed literature on the application of BrdU
in the LLNA with histochemical or enzyme-linked immunosorbent assay(ELISA) detection. This could be briefly mentioned in the final BRD for the
LLNA: BrdU-FC method, simply as a means of indicating the utility of nonradiolabeled tracer methods in the LLNA.

•

It should be noted that a potential reason why nickel chloride was negative inthe LLNA: BrdU-FC may be due to oral tolerance in the mice that was
induced by nickel-containing nipples of drinking bottles and nickel cages (Van
Hoogstraten et al. 1993).

•

The vehicle(s) used with the test substances should be stated.

•

Information on experience of the inter-laboratory transferability of othertechnologies that depend upon flow cytometry technology as the key dataread-out should be included.

•

All raw data for the LLNA: BrdU-FC and the enhanced LLNA (eLLNA):
BrdU-FC should be made available.

Comments with Specific References to the Text

The Panel stated that the following comments and/or suggested corrections relevant tospecific parts of the draft LLNA: BrdU-FC BRD text should be addressed:•

Line 226: Citation was made to a reference dated 2001 by MB Research Labs
which established their development of the LLNA: BrdU-FC; however, noreference was included in Section 12.0 (References) of the draft BRD.

•

Lines 232-233: For a sensitizer, the SI should be greater than or equal to three.

•Line 246: “i.e., positive” should be explained.

•Line 254: 11% should be 17%.
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Line 263: For purposes of completeness, it may be worthwhile to add a brief
description of the comparative accuracy of the available traditional LLNA,
LLNA: BrdU-FC, and eLLNA: BrdU-FC results versus humanmaximization/patch test data. This information is of importance and displayedin Tables 6-1 and 6-2.

•

Lines 286-288: The issue of the refinement/reduction in animal use that might
follow the availability of a scientifically validated non-radioactive variant of
the LLNA was mentioned in the draft BRD as a benefit but it was notquantified, and no authoritative reference was cited in support.

•

Line 288: The final LLNA: BrdU-FC BRD should explain why the BrdU
method would result in less pain and distress to the animals (i.e., does theroute of injection of BrdU vs. 3H-methyl thymidine produce less discomfort?).

•Line 335: Reference was made to a citation dated 2001 by MB Research Labs
which established their development of the LLNA: BrdU-FC; however, nocitation was included in Section 12.0 (References) of the draft BRD.

•

Line 356: The sentence starting “To evaluate excessive skin…” implies that
evaluation of excessive skin irritation by measuring ear thickness isrecommended by the ICCVAM LLNA protocol although it is only
recommended in the LLNA: BrdU-FC protocol.

•

Lines 365-366: Consider supplementing the list of abbreviations for Figure 21 with B220+, B:T, CD69+, and IAk+. Also, the figure shows I-Ak+ while allother text uses IAk+. The MB Research Labs protocol shows I-Ak+.

•

Figure 2-1: Should be redrawn to show the SI decision point lines coming offof the “Analyze Proliferating LNC (lymph node cells)” box rather than the“Inject BrdU and Excise…” box.

•

Lines 500-503: Classification of “equivocal results” was unclear without datacomparison (i.e., benzocaine produced divergent results in both tests). Werethese results unlike what was expected from human data? What were the data
for salicylic acid and mercaptobenzothiazole?

•Lines 552-558: It may be useful for comparative purposes to add summary
accuracy data for the traditional LLNA versus human maximization/patch test
data from the larger data set reported in the 1999 ICCVAM LLNA report tothe section of the final BRD which discusses performance of the LLNA:
BrdU-FC method.

•

Table 6-3: Benzocaine was missing and it seems that salicylic acid was thesame in both traditional LLNA and LLNA: BrdU-FC. Mercaptobenzothiazole
was not reported for the LLNA: BrdU-FC. This needs to be corrected or
explained.

•

Table 6-5: The human outcome for benzalkonium chloride and ethylene
glycol methacrylate should be negative. See also Table 6-6.

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4.2

Comments on the Validation Status of the LLNA: BrdU-FC

4.2.1

Test Method Protocol

The LLNA: BrdU-FC protocol includes routine measurements of ear swelling as an indicatorof excessive dermal irritation. The Panel was asked if they considered this procedure to be an
appropriate approach and if this measurement should be recommended for routine inclusion
into all LLNA protocols. The Panel stated that, as a quantitative parameter associated with
inflammatory cell influx and fluid retention near the site of test substance application, ear
swelling (or other quantitative measurements) should be carefully considered for inclusion
into all LLNA protocols. This might assist in differentiating between sensitizers and irritants,
assist in the interpretation of equivocal results, and possibly detect other procedure-related
problems that might require further exploration/consideration.
The LLNA: BrdU-FC protocol also includes optional quantification of immunophenotypicmarkers as an additional mechanism for distinguishing irritants from sensitizers. The Panelwas asked if they considered this to be an appropriate approach to reduce false positives, andif the correct markers were being considered. The Panel was also asked if thesemeasurements should be recommended for routine inclusion in the LLNA: BrdU-FC. The
Panel agreed that the use of immunological markers would be appropriate for detailed
studies, as it might reduce the frequency of false positives (irritants) and improvecomparisons with human data. However, since the primary use of the LLNA is fordiscrimination of human hazard from direct chemical contact, it could be argued that somefalse positives are acceptable (especially for methods which have relatively lower rates offalse negatives). Given this dominant use, application of immunological markers would
likely be too detailed and costly for routine LLNA use. Thus, the Panel suggested that resultsof ear swelling measurements be compared with the more technically complex flow
cytometry markers to determine if similar results might be obtained. Furthermore, alternative
immunological markers for discriminating between irritants and sensitizers may be available,
although the draft LLNA: BrdU-FC BRD did not contain information allowing any informed
decision on whether other markers might be more predictive. Thus, based on current
knowledge, the current markers suggested in the draft LLNA: BrdU-FC BRD seemed
acceptable to the Panel. Two other possibilities suggested were a surface marker relating to
CD4 T-helper cells (Th) or Th1 cells (interferon-γ).
The Panel was also asked to comment on the appropriateness of the "sequential strategy"
used in the eLLNA: BrdU-FC (see Figure 2-1 of the draft LLNA: BrdU-FC BRD).
Generally, the Panel viewed that incorporation of immune parameters improved the value ofa predictive assay and may also help explain mechanisms, which is important. Still, the“sequential strategy” used in the eLLNA: BrdU-FC for discriminating irritation fromsensitization might be more sensible for research studies because of resource and costconsiderations, and may not be appropriate for routine use of the LLNA in hazard
identification. For human hazard detection, more simplified methods should be available fordiscrimination of irritants.
4.2.2

Substances Used for the Validation Studies

The Panel was asked to consider if the substances tested in the LLNA: BrdU-FC wererepresentative of a sufficient range of chemical classes and physicochemical properties such

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that the test method would be applicable to any of the types of chemicals and products thatare typically tested for skin sensitization potential. The Panel agreed that if the proviso thatthe applicability domain limitations published for the traditional LLNA remained in force,the substances tested in the LLNA: BrdU-FC seemed representative of a sufficient range ofchemical classes and physical chemical properties and it would likely be applicable to manyof the types of chemicals and products that are typically tested for skin sensitization
potential. However, the available LLNA: BrdU-FC database was relatively small comparedto the large number of substances assessed in the traditional LLNA and this implied somecaution in assuming that assay performance was concordant with the traditional LLNA.
4.2.3

Test Method Accuracy

The accuracy analysis presented in the draft LLNA: BrdU-FC BRD was based on overall
concordance with the traditional LLNA. Accuracy statistics compared to the guinea pig tests
and human data/experience were also provided. The Panel was asked if these comparisonswere appropriate for assessing the accuracy of the LLNA: BrdU-FC. The Panel viewed thatsince the traditional LLNA is used to provide human hazard identification and information
relevant to human health, the accuracy statistics compared to human data/experience were
important. Since the LLNA: BrdU-FC is fairly similar to the traditional LLNA, guinea pig
comparisons might not have been necessary. However, taken together, the availability of bothhuman data/experience and guinea pig data allowed additional insights that might have
expanded the applicability domain of the LLNA: BrdU-FC, or indicated improved
performance with respect to LLNA false negatives and positives.
The Panel was then asked if the relevance (e.g., accuracy/concordance, sensitivity, specificity,
false positive and false negative rates) of the LLNA: BrdU-FC had been adequately evaluatedand compared to the traditional LLNA. The Panel agreed that the relevance of the LLNA:
BrdU-FC was adequately evaluated and compared to the traditional LLNA, and supported
the inclusion of accuracy analyses with and without equivocal materials.
Three substances (benzalkonium chloride, resorcinol, and Tween 80) produced a false
positive response compared to the traditional LLNA and guinea pig test when tested using
the LLNA: BrdU-FC (based on immunophenotyping, benzalkonium chloride wassubsequently classified as an irritant rather than a sensitizer). The Panel was asked if they
could identify any characteristics associated with these or similar substances that mightsuggest that using the LLNA: BrdU-FC to test such substances would not be appropriate orthat positive results for substances with such properties may warrant additional testing.
Overall, the Panel stated that there were not any patterns or unifying concepts that explained
the three false positive results in the available data set. They noted that only a single
laboratory is using the LLNA: BrdU-FC method and recommended that the raw data on
which the reports were prepared be made available in order to allow further investigation.
The Panel also suggested that additional studies be conducted to determine whether LLNA:
BrdU-FC results with these three substances are repeatable.
Dr. Raymond Pieters stated that benzalkonium chloride and Tween 80 are consideredaggressive irritants, but both published data (Manetz and Meade 1999; Varani et al. 2008)
and unpublished data from his laboratory has shown that benzalkonium chloride (5%) ismore potent than SLS in the stimulation of lymph node cell proliferation and may therefore
may actually be considered a sensitizer. However, in the traditional LLNA these compounds
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did not increase the SI above the threshold for a positive response (i.e., SI ≥3), so they wereidentified as non-sensitizers.
4.2.4

Test Method Reliability

The Panel was asked if the intralaboratory reproducibility of the LLNA: BrdU-FC had beenadequately evaluated and compared to the traditional LLNA and if any limitations wereapparent based on this assessment. The draft LLNA: BrdU-FC BRD analyzed data fromrepeat testing of HCA in six different vehicles and intralaboratory reproducibility wasassessed by a coefficient of variation (CV) evaluation. The calculated CVs ranged from 30%
to 53%. The Panel agreed that the relatively large SD and associated CV values raisedquestions about the extent of experiment-to-experiment variability. There was less concern
about vehicle choice and effects on the range of group means than about the CVs greater than
50% for the group means of HCA tested in DMSO and AOO. The large number of repeatedexperiments for these tests would have been expected to dramatically reduce variability. The
Panel concluded that the results suggested that key elements of assay standardization were
not yet developed. Further evaluation using other positive control substances would havebeen valuable to more adequately characterize reproducibility.
4.2.5

Data Quality

The studies evaluated in the draft BRD for the LLNA: BrdU-FC were not all conducted inaccordance with GLP guidelines although they were done in a laboratory that routinely
conducts GLP studies (G. DeGeorge, personal communication). The Panel was asked to
discuss what impact this might have on the evaluation of the LLNA: BrdU-FC. The Panelconsidered that, even without formal GLP compliance, the current LLNA: BrdU-FC resultsappeared to reflect a sincere attempt to perform work of high quality. The only area in whicha lack of full GLP compliance may have been a source of assay variability was in thequantitative analysis of dosing solutions. For instance, failure to appreciate differences incomposition of dosing solutions between experiments caused by test article instability orother phenomena may account for the relative large variability in intralaboratory data andpossibly of some of the discordant results (i.e., false negatives and differences in LLNA:
BrdU-FC results between repeat studies for the same substance). Thus, the Panel viewed thatany additional studies undertaken to validate the test method should ideally be GLPcompliant.
Furthermore, the original records for these studies were requested but had not yet beenobtained at the time of the Panel review. As a result, an independent audit could not be
conducted to confirm that the reported data was the same as the data recorded in laboratory
notebooks. The Panel was asked if they agreed that any recommendations from ICCVAM
should be contingent upon the completion of such an audit and findings that there were no
significant errors in data transcription. The Panel agreed that, although a request for original
data had been made, it was good practice to hold final recommendations until an independent
audit could be performed. While it would be expected that no serious errors would be
uncovered which would alter the current findings, an audit would confirm assay performanceto date and position ICCVAM for further consideration of the LLNA: BrdU-FC.

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4.2.6

May 2008

Consideration of All Available Data and Relevant Information

The Panel was also asked if, based on the draft LLNA: BrdU-FC BRD, all the relevant data
identified in published or unpublished studies that employ this test method had beenadequately considered. Furthermore, they were asked that if there were other comparative
test method data that were not considered in the draft BRD, how such data might be obtained.
Overall, the Panel considered that all the relevant data identified in published or unpublishedstudies that employed this test method had been adequately considered in the draft LLNA:
BrdU-FC BRD. However, some additional information was available in the peer-reviewed
literature on application of BrdU in the LLNA with other methods of detection (e.g.,histochemistry, ELISA). The Panel felt that these could have been briefly mentioned in thedraft BRD for the LLNA: BrdU-FC method, simply as a means of indicating the utility of
non-radiolabeled tracer methods in the LLNA. Furthermore, if an analysis of the CV for the
traditional LLNA was undertaken, a more direct comparison with the LLNA: BrdU-FC could
have been performed.
4.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-FC

4.3.1

Test Method Usefulness and Limitations

The Panel was asked if they agreed that the available data and test method performance
support the ICCVAM draft recommendations for the LLNA: BrdU-FC in terms of the
proposed test method usefulness and limitations (i.e., that it may be useful for identifying
substances as potential skin sensitizers and non-sensitizers, but that more information anddata are needed before a recommended use of the LLNA: BrdU-FC can be made). The Panelagreed that the available data and test method performance of the LLNA: BrdU-FC support
the draft ICCVAM recommendations. They considered the proposed test method usefulness
and limitations to have well summarized the limits of the information supplied and the
additional information that would need to be generated or provided for further considerationof this test method. As a result, the LLNA: BrdU-FC could not at this stage be consideredscientifically validated as a replacement alternative to the traditional LLNA. Still, the test
method recommendation should clearly state that the test method was not “invalid” butsimply that there was currently not sufficient evidence and information to affirm that it had
been adequately validated by ICCVAM. Instead, the Panel considered that the LLNA: BrdUFC could be recommended in instances where mechanistic information about a sensitizer isrequired.
The Panel was asked if restrictions on using radioactive materials were or were not present,
whether or not the LLNA: BrdU-FC should be routinely recommended for hazard
identification of skin sensitizing substances in lieu of having to possibly use guinea pig tests.
The Panel agreed that it is preferable to use alternative methods for the LLNA (i.e., ELISA
detection of BrdU or histochemical detection of BrdU-labeled cells), as opposed toapplication of guinea pig test methods, if a limitation on radioisotope use exists (e.g., the lackof a radioactivity use license). This rationale is based on avoidance of the less quantitative
guinea pig test methods, which may employ adjuvant treatment with associated animal stressand harm. Still, at this time, the Panel considered that data gaps in the LLNA: BrdU-FC
method precluded recommending it for routine hazard identification of skin sensitizing
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substances in lieu of the traditional LLNA, whether or not limitations on using radioactive
materials exist. Policy issues regarding restrictions on radioactivity should have no impact on
this science-based conclusion.
The Panel was asked if the ICCVAM recommendations adequately addressed concerns
associated with the false positive rate of 17% (3/18 substances) calculated for the LLNA:
BrdU-FC and if there were other suggestions for additional guidance or limitations thatshould be considered. The Panel agreed that the relatively high false positive rate wasadequately identified and discussed, and that no mechanistic reason could be identified forthese results based on available information. The Panel noted that it might be worthwhile topoint out in the final BRD the impact on human health of false positive results versus false
negative results in the context of hazard screening and identification. Consideration of factors
such as intended use and target population of the false positive substances would further aid
in characterizing human risk for these substances.
4.3.2

Test Method Protocol

The Panel was asked whether or not they agreed that the available data supported the
ICCVAM draft recommendations for the LLNA: BrdU-FC procedure in terms of the
proposed test method standardized protocol and if not, what recommendations would they
make. The Panel noted that the draft ICCVAM recommendations for conduct of astandardized method for the LLNA: BrdU-FC variant were relatively brief and stated onlythat all applicable portions of the 1999 ICCVAM procedure be carefully followed. The Panelagreed that the available data supported the ICCVAM draft recommendations for the LLNA:
BrdU-FC procedure in terms of adhering to the ICCVAM LLNA protocol (ICCVAM 1999;
Dean et al. 2001). In particular, the Panel agreed that at least five animals per dose groupshould be used, particularly in light of Dr. Haseman's power analysis (see Table 4-1). The
Panel did note however that power calculations could be undertaken to determine if fewer
animals per dose group might be adequate for post-validation studies, though Dr. Haseman's
power analyses suggest that this is unlikely. The majority of the Panel also agreed with the
ICCVAM-recommended protocol to use individual animal data although a minority opinion
by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, Raymond Pieters, and Michael
Woolhiser stated that if laboratories were operating under OECD TG 429 guidance (OECD2002) and a reliable validation dataset had been generated, then pooled data from at least fouranimals per dose group could be considered acceptable.
Further, the Panel considered the methodological description of the LLNA: BrdU-FC
procedure supplied by MB Research Labs (Appendix A to the draft BRD) to becomprehensive. The utility of ear swelling or other methods to detect inflammation/excessive
local irritation appear to be warranted in every variation of the LLNA (including the
traditional LLNA), but should be further investigated before routine inclusion in any protocol
is recommended.
The Panel was asked whether the LLNA limit dose procedure could be applied to the LLNA:
BrdU-FC. The Panel agreed that the LLNA limit dose procedure could be applied to the
LLNA: BrdU-FC as long as the limitations associated with the limit dose procedure wereappreciated. Furthermore, application of the limit dose procedure to the LLNA: BrdU-FC
would require adherence to a validated LLNA: BrdU-FC test method protocol with the
exception that the middle and low dose groups would be excluded. Furthermore, it would

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need to be confirmed that the number/pattern of sensitizers that would have been
identified/missed from the “high dose” group would mirror that of the traditional LLNA.
Table 4-1

Power Calculations for the LLNA: BrdU-FC
3.0-fold
Increase

2.5-fold
Increase

2.0-fold
Increase

1.5-fold
Increase

1.3-fold
Increase

Mean Rx response

30279

25232.5

20186

15139.5

13120.9

Log (Mean Rx response)

10.31810.136

9.913

9.625

9.482

Difference from control(log scale)

1.098

0.916

0.693

0.405

0.262

Difference/SD

1.75

1.46

1.10

0.65

0.42

Power for N=580%

50-80%

<50%

<50%

<50%

Power for N=450-80%

50%

<50%

<50%

<50%

Power for N=3

50%

<50%

<50%

<50%

<50%

Other Power

95% (N=9)95% (N=12)

95% (N=19)

95% (N=52)

95% (N>100)

Other Power

90% (N=7)

90% (N=10)

90% (N=15)

90% (N=42)

90% (N>100)

The power calculations above are based on a one-sided p<0.05 Student’s t-test applied to log-transformed datafrom vehicle control LLNA: BrdU-FC tests.
Abbreviations: Fold-increase=Required increase above the vehicle control for a positive response; N=Number;
Rx=Treatment; SD=Standard deviation.

4.3.3

Future Studies

Finally, the Panel was asked whether they agreed that the available data supported the
ICCVAM draft recommendations for the LLNA: BrdU-FC in terms of the proposed futurestudies. The Panel agreed that the ICCVAM draft recommendations for future studies
highlighted the unanswered questions raised by the available database. Specifically,
conducting interlaboratory studies as a part of the validation process was considered
important. As mentioned previously, the Panel viewed that the immunological markers
suggested for the LLNA: BrdU-FC in the draft BRD were acceptable but that additional
immunological markers for discrimination of irritant versus sensitization phenomena mightalso be identified. A suggestion for a future study was to use the surface marker relating to
CD4 Th cells or internal marker relating to Th1 cells (interferon-γ).
In general, for any future work, the Panel considered that efforts should be made to decrease
the variability and thereby increase the power of the test in order to ensure that more animals
were not needed relative to the traditional LLNA or other alternative LLNA protocols. For
instance, further optimization of the LLNA: BrdU-FC method should include kinetic studies
to demonstrate that the optimal protocol was being used.
4.3.4

Performance Standards

The draft BRD indicated that the LLNA: BrdU-FC protocol differs from the ICCVAMrecommended protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes. According to
the proposed draft ICCVAM LLNA Performance Standards for the traditional LLNA, anychange to the LLNA protocol other than the method used to assess lymphocyte proliferation

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was considered a “major” change. According to this criterion, the Panel considered theprotocol differences between the LLNA: BrdU-FC and the traditional LLNA to be “minor”
changes, and therefore considered that the validity of this test method could be based on thedraft ICCVAM LLNA Performance Standards. However, the Panel also recognized that thisdepended on a clear definition of what constituted a “major” versus a “minor” change, or adifferent protocol altogether. Thus, further consideration of this topic could be addressed
once the recommendations for the current draft ICCVAM LLNA Performance Standards
were finalized. The Panel found it difficult to identify any additional requirements formethods like the LLNA: BrdU-FC.
Even if the draft ICCVAM LLNA Performance Standards were not found to apply to the
LLNA: BrdU-FC, the Panel considered that the impact of the LLNA: BrdU-FC accuracy
analysis based on 13 of the 18 proposed required reference substances in the draft ICCVAM
LLNA Performance Standards should not have a major impact on the overall evaluation oftest method accuracy, as 45 substances, representative of an appropriate range, were tested.
However, based on consideration for development of LLNA performance standards, it would
be desirable for validation purposes that the substances missing from the range of 18 standard
materials be assessed in the LLNA: BrdU-FC protocol.
The draft LLNA: BrdU-FC BRD also indicated that three out of six sensitizers for which
EC3 data were available had EC3 values that were outside of the proposed 0.5x to 2.0x EC3
acceptability range, which was developed based on the traditional LLNA. The Panel viewedthat the primary concern seemed to have less to do with the variation in the response thanwith a concern that the range of response would skew the interpretation of any LLNA: BrdUFC results used for sensitization potency estimates. Furthermore, it was not known if thesame vehicle was used to derive both EC3 values/ranges. The proposed 0.5 x to 2.0 x range
seemed to be based upon empirical/goodness of fit rather than any biological constant. The
appropriateness of this range should be considered further when the finalized ICCVAM
LLNA Performance Standards document is considered. In general, if the vehicles weredifferent the question is irrelevant.
4.3.5

Comments with Specific References to the Text

The Panel made the following comment with specific reference to the text in the draft
ICCVAM test method recommendations on the LLNA: BrdU-FC and suggested that it beaddressed:•

Lines 39-41: For parallel construction of this sentence with the preceding
sentence, suggest substituting the following "One of the other equivocal
substances, salicylic acid, is one of the recommended reference standard
materials used as a non-sensitizer in the draft ICCVAM LLNA PerformanceStandards…". This is based on the assumption that salicylic acid was thesubstance intended for discussion and that it was used in the draft ICCVAM
LLNA Performance Standards as a model non-sensitizer.

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5.0

Non-Radioactive LLNA Protocol - The LLNA:
Bromodeoxyuridine Detected by ELISA (BrdU-ELISA) Test
Method5.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

5.1.1

General Comments

The Panel was asked if there were any errors in the draft LLNA: BrdU-ELISA BRD thatshould be corrected, if omissions of existing relevant data had been identified, or if there wasadditional information that should be included. The Panel noted that, in general, all of thedata included were relevant, and that it was apparent that considerable effort had been
involved in carefully developing the comprehensive database. The Panel noted that theywould have preferred to have the original papers by Dr. Takeyoshi included in the review
materials, but they were easily retrieved from the journal websites. The Panel indicated thatraw data (i.e., the actual optical density at 370 nm [OD370] readings for the triplicates and the
SD of the triplicates) are necessary for a thorough evaluation. Additionally, the Panel notedthat only a relatively small number of substances had been tested in the LLNA: BrdUELISA.
When considering the animal welfare impact of implementing the LLNA: BrdU-ELISA, thePanel agreed that it would be less painful than guinea pig tests in those circumstances where
the use of radioactive materials are restricted. Thus, the Panel agreed that the test represents a
potential refinement. The Panel further stated that, if there is not an option to replace theguinea pig test with a non-animal test, decreasing the extent of pain and distress should be
the first animal welfare priority. The Panel cautioned that at some point, however, thenumbers of animals being utilized must be considered. An eventual recommendation that the
LLNA: BrdU-ELISA be routinely used instead of guinea pig test methods where the use ofradioactive substances are restricted would apparently require a significant increase in thenumber of mice killed per test (to increase the statistical power of the test method - see
Section 5.1.2 below) if an SI ≥1.3 is deemed the appropriate criterion to use for determining
a positive response. The Panel stated that it would be helpful to know how many guinea pigs
are currently being used nationally and internationally for skin sensitization tests, and how
many mice would be used in the LLNA: BrdU-ELISA with the SI ≥1.3 criterion. Even anorder of magnitude estimate would help the Panel judge whether the increase in numbers ofmice needed is justified as the quest to relieve pain in guinea pigs is pursued.
5.1.2

General Statistical Comments

The Panel was concerned about using an SI of ≥1.3 to optimize the performance of the
LLNA: BrdU-ELISA method. One Panel member’s extensive experience with ELISA
protocols was cited as evidence that the difference between the OD370 of the vehicle and thepositive test at 1.3 would not likely be statistically significant. The Panel recommended thatthe raw data must be reviewed to evaluate this. In addition, based on Dr. Joseph Haseman’s
power analysis (see Table 5-1), the Panel stated that it was difficult to justify using a SI ≥1.3
as the decision criterion since it would result in a significant increase in the number ofanimals needed to obtain an acceptable confidence level. In this regard, the Panel

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recommended that power calculations should be routinely recommended to ensure that theappropriate number of animals per dose group is being analyzed.
Table 5-1

Power Calculations for the LLNA: BrdU-ELISA
3.0-fold
Increase

2.0-fold
Increase

1.3-fold
Increase

Mean Rx response

0.399

0.266

0.173

Log (mean Rx
response)

-0.92

-1.32

-1.75

Difference fromcontrol (log scale)

1.10

0.70

0.27

Difference/SD

3.64

2.32

0.89

Power for N=499%

80-90%<50%

Other power

95% (N=3)

95% (N=5)

50% (N=8)

Other power–

50-80% (N=3)

80% (N=16)

Other power––

90% (N=22)

Parameter

The power calculations above are based on a one-sided p<0.05 Student’s t-test applied to log-transformed datafrom vehicle control LLNA: BrdU-ELISA tests.
Abbreviations: Fold-increase=Required increase above the vehicle control for a positive response; N=Number;
Rx=Treatment; SD=Standard deviation.

5.1.3

Comments with Specific References to the Text

The Panel also identified the following minor formatting and grammatical errors, as well as
information gaps, in the draft BRD:•

The Panel noted a discrepancy between the draft LLNA: BrdU-ELISA BRD
and the draft ICCVAM LLNA Performance Standards in the vehicle used fortesting 2-mercaptobenzothiazole. Table 6-2 of the draft LLNA: BrdU-ELISA
BRD indicated that the vehicle was AOO but the revised draft ICCVAM
LLNA Performance Standards indicated that the vehicle was DMF (see pageC15, C22 of September 7, 2007, draft and page B-6 of January 7, 2008,
revised draft). Additionally, Table 1 on page C-7 of the revised draft
ICCVAM LLNA Performance Standards listed AOO as the vehicle for 2mercaptobenzothiazole. For both vehicles, the revised draft ICCVAM LLNA
Performance Standards indicated that the EC3 value is 2.5%, although the text
on page 10 of the draft LLNA: BrdU-ELISA BRD stated “the NICEATM
database of traditional LLNA studies indicates that 2-mercaptobenzothiazole
has a higher EC3 value when tested in AOO (mean EC3=9.8%) comparedwith DMF (mean EC3=2.5%)...”•The Panel noted that Table 6-1 of the draft LLNA: BrdU-ELISA BRD
indicated that, when compared to the guinea pig and human test data, thesensitivity and specificity of the LLNA: BrdU-ELISA is lower than that of the
traditional LLNA. In fact, depending on the SI threshold value used, the5-2Independent Peer Review Panel Report

May 2008

sensitivity and specificity of the LLNA: BrdU-ELISA can be higher than thatof the traditional LLNA.
The Panel recommended that the draft LLNA: BrdU-ELISA BRD be updated to rectify theseerrors and omissions.
5.2

Comments on the Validation Status of the LLNA: BrdU-ELISA

5.2.1

Test Method Protocol

The data generated for the substances analyzed in the LLNA: BrdU-ELISA test method camefrom auricular lymph nodes from four individual mice in each dose group. The ICCVAMrecommended LLNA protocol (ICCVAM 1999; Dean et al. 2001) and OECD TG 429(OECD 2002) recommend a minimum of five animals per dose group when collecting
individual animal data. The Panel was asked what impact might the use of four animals perdose group have on the accuracy and reliability of the LLNA: BrdU-ELISA, and if the Panelagreed with the ICCVAM recommendation that future use of this test method protocol
should include five animals per dose group. The Panel majority agreed with the ICCVAM
recommendation that future use of this test method should use five animals per dose groupand collect individual animal data, as per the ICCVAM-recommended protocol. A minority
opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, Raymond Pieters, andMichael Woolhiser stated that if laboratories were operating under OECD guidance (OECD2002) and a reliable validation dataset had been generated, then pooled data from at least fouranimals could be considered acceptable. Based on the supplemental data provided by Dr.
Haseman, the power to detect a three-fold increase with a sample size of four was determined
to be 99%. These calculations, however, assume that a sample size of four is always
obtained. If a sample size of four was planned and fewer usable data values were obtained,
then the experiment might be compromised. Furthermore, the Panel concluded that testing
for and eliminating “outliers” from experiments with small sample sizes is questionable. A
reduction in sample size from five to four was not recommended unless data was provided on
the frequency with which “outliers” occurred and an analysis is performed that establishes
that a reduction in the nominal sample size from five to four would not compromise theperformance of the test method. The Panel stated that the handling of suspected “outliers”
and the use of robust statistics are issues that need to be addressed in such an analysis. For
example, robust procedures may compensate for apparent “outliers” and eliminate theimpulse to discard data. An example is calculating the mean values used in the SI on a log
scale and then exponentiating the result to construct the SI.
The Panel also indicated that it was important to routinely include a positive control group in
test method validation experiments (e.g., HCA), which was likely not the case for most of the
LLNA: BrdU-ELISA validation experiments. Although the Panel did not reach consensus,
they did consider the suggestion that for laboratories in which the LLNA is “routinely”
performed, positive controls (e.g., HCA or a substance that matches the chemical class of thetest substances) could be run at intervals for quality control purposes rather than concurrent
with each experiment in which substances are tested. The Panel also discussed that omitting
the concurrent positive control should not be recommended for laboratories that perform the
LLNA only “occasionally”. In their discussions, the Panel was not able to conclude whatshould constitute “routine” or “occasional” LLNA use or what would be an appropriate
interval between positive control testing when a concurrent positive control is not used.
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5.2.2

May 2008

Substances Used for the Validation Studies

The Panel was asked whether the LLNA: BrdU-ELISA database was representative of a
sufficient range of chemical classes and physicochemical properties such that the test method
would be applicable to any of the types of chemicals and products typically tested for skinsensitization potential. The Panel indicated that the ratio of solids to liquids was notcomparable; more solids should be included. The Panel further indicated that more
substances for which traditional LLNA data are available should be tested, and thatcompounds including metals (e.g., nickel, cobalt), mixtures, and substances in aqueous
solutions should be included.
5.2.3

Test Method Accuracy

The current accuracy analysis using an SI ≥3.0 or SI ≥1.3 to identify sensitizers is based on
overall concordance with the traditional LLNA. Accuracy statistics compared to the guinea
pig tests and human data/experience were also provided to the Panel. The Panel was asked
whether these comparisons were appropriate for assessing the accuracy of the LLNA: BrdUELISA. The Panel indicated that comparing the LLNA: BrdU-ELISA performance to the
traditional LLNA and the guinea pig tests were appropriate. Comparisons between the
LLNA: BrdU-ELISA and human data were considered particularly valuable because thetraditional LLNA doesn’t match human data with 100% accuracy. For this reason, the Panelconsidered comparing the performance of the LLNA: BrdU-ELISA with that of the
traditional LLNA with respect to predicting the human outcomes to be the best method ofcomparing these two LLNA protocols. The Panel concluded that in moving forward with any
test method recommendation, key importance should be placed on interpreting the test results
and making them clinically applicable to humans.
Takeyoshi et al. (2007) performed an accuracy analysis using decision criteria other than an
SI ≥3.0 to classify substances as sensitizers. Maximal accuracy for the LLNA: BrdU-ELISA
occurred when an SI ≥1.3 was used to distinguish between sensitizers and non-sensitizers.
Using this decision criterion, the LLNA: BrdU-ELISA achieved an accuracy of 91% (21/23),
with a sensitivity of 100% (16/16) and a specificity of 71% (5/7) (i.e., there were no false
negatives and two false positives). The Panel was asked whether this analysis supported arecommendation that the decision criteria be based on an SI ≥1.3, and if there were concernswith using such a small increase (i.e., 1.3-fold) above the vehicle control response as thebasis for identifying a positive response. The Panel did not support using an SI ≥1.3 as thecriterion for positive results. An SI=1.0 means there was no difference between the vehicle
control and the test substance. An SI=1.3 represents a 30% increase from the vehicle control.
The difference between the OD370 of the vehicle and the positive test at 1.3 may not be
statistically significant. An SI=3.0, which represents a three-fold difference between thevehicle and a positive test, would be a more believable positive difference. If the positive test
criteria must be reduced to 1.3, then the Panel questioned whether the protocol is useful in its
current state. The supplemental information that provided power calculations indicated that it
would not be realistic to expect to detect a 1.3 fold increase in the control response without asignificant addition of animals. Although using SI ≥1.3 increases the accuracy of the test, itcomes at an increased cost to animals, which merits consideration. Furthermore, the
ICCVAM (1999) report stated that an irritating chemical might induce proliferation, but thatthe response seldom exceeds an SI ≥3.0 (page 6). The Panel concluded that this might

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provide further justification against using a low SI (e.g., 1.3) as a threshold for a positive
response.
The Panel was asked if the relevance (e.g., accuracy/concordance, sensitivity, specificity,
false positive and false negative rates) of the LLNA: BrdU-ELISA, using the SI ≥3.0
criterion, had been adequately evaluated and compared to the traditional LLNA (refer also toTable 6-1 of the draft LLNA: BrdU-ELISA BRD). If not, the Panel was asked what otheranalyses should be performed. The Panel agreed that the relevance of the LLNA: BrdUELISA, using the SI ≥3.0 criterion, had been adequately evaluated. The Panel further statedthat a better evaluation could be performed, however, if the database for the LLNA: BrdUELISA included more substances with traditional LLNA, guinea pig, and human data. The
Panel considered the false negative rate of the test method to be excessive when results arecompared with that obtained in the traditional LLNA (29/33/27% for the various datasets) orwith human data (39%) – the results should be at least comparable with the traditional
LLNA.
Using the SI ≥3.0 criterion, there were four substances (aniline, 4-chloroaniline, 2mercaptothiazole, and hydroxycitronellal) that produced false negative responses when tested
using the LLNA: BrdU-ELISA compared to the traditional LLNA. The Panel was asked
whether it could identify any characteristics associated with these or similar substances,compared to the correctly identified sensitizers, that might indicate that such substances
should not be tested in the LLNA: BrdU-ELISA or that negative results for such substances
should indicate a need for confirmatory testing. The Panel could not identify any
characteristics associated with these substances that might allow the identification of thesesubstances as false negatives prior to testing. The Panel stated that the LLNA: BrdU-ELISA
test, using the standard SI ≥3.0 to indicate positive results, simply does not perform well for
identifying sensitizers.
5.2.4

Test Method Reliability

The Panel was asked whether the intralaboratory reproducibility of the LLNA: BrdU-ELISA
had been adequately evaluated and compared to the traditional LLNA, and whether any
limitations were apparent based on this intralaboratory reproducibility assessment. The Panelindicated that the number of substances evaluated for intralaboratory reproducibility was too
few and, in some cases, there was a wide variation in repeat test results for the same
substance. Only six substances (five sensitizers and only one non-sensitizer) were tested
multiple times. The non-sensitizer, propylene glycol, was tested only twice and opposite
results were obtained. The Panel considered the results of an intralaboratory reproducibility
evaluation that was based on two discordant results only to be unacceptable. The numbers
calculated in Table 7-1 of the draft LLNA: BrdU-ELISA BRD are correct, but the Panelquestioned the dependability of the data since only two to three values were available forcalculating the mean and CV. The Panel considered the CV values (over 30%) high,compared to the traditional LLNA (draft LLNA: BrdU-ELISA BRD Tables 7-1 to 7-3). The
Panel stated that at least four independent tests with three concentrations tested represent asolid basis for calculation. The Panel considered the number of tests for intralaboratory
concordance analysis to be insufficient, and stated that more intralaboratory testing is needed.
The Panel recommended an evaluation of the intralaboratory reproducibility of the EC ≥1.3
and that the analysis of the variability of the ECt be conducted on a log scale.

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The substances evaluated for intralaboratory reproducibility of the LLNA: BrdU-ELISA
study were not coded. The Panel was asked whether the lack of coding of test substances
adversely impacts or biases the current evaluation. The Panel stated that, although coding of
substances is preferred for independent testing and evaluation of test results, the current data
should not be rejected from consideration because the substances tested were not coded.
The Japanese Center for Validation of Alternative Methods (JaCVAM) has implemented amulti-laboratory validation study of the LLNA: BrdU-ELISA. The Panel was asked whether
the study design was appropriate to adequately determine the extent of interlaboratory
reproducibility for the LLNA: BrdU-ELISA. If not, the Panel was asked what other studies
should be performed. The Panel stated that they had insufficient time to evaluate the study
design and that they could not evaluate interlaboratory reproducibility because the study data
were not available at the time of their evaluation.
5.2.5

Data Quality

The studies evaluated in the draft BRD for the LLNA: BrdU-ELISA were not conducted instrict accordance with GLP guidelines, although there were reportedly performed inlaboratories that conduct GLP studies (M. Takeyoshi, personal communication). In otherwords, an audit report was not available. Also, the raw data were unavailable for an
independent audit. The Panel was asked to discuss what impact this might have on theevaluation of the LLNA: BrdU-ELISA. The Panel concluded that ideally, validation studies
should be performed in accordance with GLP guidelines. Although the systems employed fortests (i.e., test facilities, staff, reagents, etc.) were identical to those for GLP-compliant
studies, the data quality may be questioned and therefore should at least be available for a
retrospective independent audit. However, in this case, the Panel concluded that the lack of
GLP compliance was not likely the reason for the poor results obtained with the LLNA:
BrdU-ELISA.
The original records for these studies were requested but had not been received by the time
the Panel convened. As a result, an independent audit could not be conducted to confirm thatthe reported data in peer reviewed publications and a poster presentation is the same as theraw data. The Panel was asked whether any recommendations from ICCVAM should becontingent upon the completion of such an audit and findings that there were no significant
errors in data transcription. The Panel concluded that, to have confidence in data quality,
ICCVAM recommendations should be contingent upon the completion of an independent
audit. Moreover, if an SI ≥1.3 is used as the criterion for positive results, review of the raw
data is necessary to confirm statistically significant differences. The Panel concluded that thistest, as described, had poor accuracy, poor sensitivity, and poor specificity. The Panel statedthat changing the SI decision criterion from 3.0 to improve test performance, especially tosuch a drastic change as SI ≥1.3, is a mistake and sets a dangerous precedent.
5.2.6

Consideration of All Available Data and Relevant Information

Based on the draft LLNA: BrdU-ELISA BRD, the Panel was asked whether all the relevant
data identified in published or unpublished studies that employ this test method had beenadequately considered, and if other comparative test method data that were not consideredwere available. If yes, the Panel was asked to suggest how to obtain such data. The Panelbelieved that all of the relevant data, with the exception of the interlaboratory reproducibility
study, were presented and that they were not aware of any omissions.
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5.3

Comments on the Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-ELISA

5.3.1

Test Method Usefulness and Limitations

The Panel was asked whether the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA in terms of the proposed test method
usefulness and limitations. The Panel agreed with the ICCVAM recommendation that the
LLNA: BrdU-ELISA may be useful for identifying substances as potential skin sensitizers
and non-sensitizers but that, at this time, more information and data are needed before arecommended use of the LLNA: BrdU-ELISA can be made. The Panel also stated that adetailed protocol is needed, in addition to sufficient quantitative data for a morecomprehensive analysis based on a larger set of balanced reference substances with regard tophysicochemical properties and sensitization potency, as well as an evaluation ofinterlaboratory reproducibility.
The Panel was asked whether the LLNA: BrdU-ELISA should be routinely recommended forhazard identification of skin-sensitizing substances in lieu of using guinea pig tests if
restrictions on using radioactive materials are present, due to the fact that fewer animals
might be used and because pain and distress would be avoided. The Panel stated that if theaccuracy of the test method was at least similar to the traditional LLNA, the LLNA: BrdUELISA might be routinely recommended for hazard identification of skin-sensitizing
substances in terms of reduction of animals and refinement of the pain and distress associatedwith guinea pig tests. Clearly, using the LLNA: BrdU-ELISA instead of the traditional
LLNA or guinea pig test methods would also offer advantages for the environment due to theuse of a non-radioactive probe chemical. However, the Panel stated that the accuracy of thecurrent LLNA: BrdU-ELISA dataset at SI ≥3.0 was inadequate and not equivalent to the
traditional LLNA. The Panel also noted that if an SI ≥1.3 was used because of its apparent
increased accuracy, additional mice (over and above the number needed in the standard
LLNA test) would apparently be needed (see Table 5-1). Thus, the Panel stated that
reduction of animals would not be achieved. In this regard, the Panel noted that somequantification of the total animal use numbers would be useful as it is not clear whether theincreased number of mice used would outweigh the avoidance of pain and distress in guinea
pigs.
The Panel was asked whether the LLNA: BrdU-ELISA procedure or other valid and accepted
non-radioactive method could be routinely recommended for hazard identification of skinsensitizing substances instead of the traditional LLNA if limitations in using radioactive
materials are not present. The Panel stated that the LLNA: BrdU-ELISA procedure could not
be routinely recommended for hazard identification of skin sensitizing substances instead of
the traditional LLNA, because the accuracy of this test at SI ≥3.0 was inadequate. In otherwords, the current dataset available for the LLNA: BrdU-ELISA did not predict the guinea
pig or human outcomes as accurately as the traditional LLNA. Thus, the Panel acknowledgedthat there is the possibility that additional data might impact on the accuracy statistics andeliminate this concern. The Panel stated that factors that weigh on a decision of replacementof the LLNA with a non-radioactive method would include:•

Are more animals needed?

•

Is the replacement test safer and less complex?
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May 2008•

Is the replacement test more efficient?

•

Is the replacement test less costly?

The Panel stated that additional factors to consider might exist, but overall recommended thatwhether or not restrictions on radioactivity exist, a test that causes the least pain and uses the
fewest number animals should be preferred, as long as adequate test method performance ismaintained. Clearly, policy issues regarding restrictions on radioactivity should have no
impact on this science-based conclusion.
The Panel was asked whether using a decision criterion of SI ≥1.3 instead of SI ≥3.0 resolved
any concerns with respect to potential false positives or false negatives that may occur in thistest method. The Panel was also asked for other suggestions for additional guidance orlimitations that should be considered. The Panel stated that using a decision criterion of SI≥1.3 instead of SI ≥3.0 would not itself resolve any concerns; more raw data are needed for abroader set of reference positive and negative sensitizers, including metals, mixtures, andaqueous solutions. The Panel also stated that a detailed protocol is needed, as is an evaluationof interlaboratory reproducibility. The Panel considered the current database to beinadequate, but based on the limited database, concluded that it might be more appropriate touse a statistically based decision criteria than a stimulation index.
5.3.2

Test Method Protocol

The ICCVAM draft recommendations state that the LLNA: BrdU-ELISA protocol should
adhere to the ICCVAM LLNA protocol (ICCVAM 1999; Dean et al. 2001), except formeasurement of lymphocyte proliferation. The Panel was asked whether the available data
support the ICCVAM draft recommendations for the LLNA: BrdU-ELISA procedure interms of the proposed test method standardized protocols. In general, the Panel agreed thatthe available data support the ICCVAM draft recommendations for this test method in termsof the standardized protocol. As stated previously, the Panel majority agreed with the
ICCVAM recommendation that future studies should use five animals per dose group andcollect individual animal data, as per the-ICCVAM-recommended protocol. A minority
opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, Raymond Pieters, andMichael Woolhiser stated that if laboratories were operating under OECD guidance (OECD2002) and a reliable validation dataset had been generated, then pooled data from at least fouranimals could be considered acceptable. The Panel further noted that using an SI <3.0 would
require more animals to achieve adequate statistical power (Table 5-1) and therefore any
considerations of reducing the SI to improve test method accuracy should include this point.
The Panel was asked whether the traditional LLNA limit dose procedure could be applied to
the LLNA: BrdU-ELISA. The Panel stated that, if the LLNA: BrdU-ELISA was considered
equivalent to the traditional LLNA, then it would be appropriate to apply the LLNA limit
dose procedure to this test method. The Panel explained that, as in the case of the traditional
LLNA, the protocol would be the same except for testing the maximum dose only, so
applying the limit dose procedure would appear to have the same opportunity to reduce thenumber of animals needed to perform the test. However, using an SI ≥3.0 would not beappropriate because of the associated low accuracy in identifying sensitizers.

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5.3.3

May 2008

Future Studies

The Panel was asked whether the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA in terms of the proposed future studies. The
Panel stated that the proposed future studies were justified. The Panel concluded that it isimportant to consider non-radioactive methods because, in some laboratories, it is difficult or
not permissible to use radioactivity. The Panel also stated that, if more data were available
and there was less variability in this test method, it might warrant re-evaluation. The Panelconcluded that more data are needed, especially for determination of the appropriate
threshold value for the decision criterion, and that interlaboratory reproducibility should bealso evaluated (which presumably will occur once the Japanese interlaboratory validation
effort is complete).
5.3.4

Performance Standards

The LLNA: BrdU-ELISA protocol differs from the ICCVAM-recommended protocol for the
traditional LLNA (ICCVAM 1999; Dean et al. 2001) in the method used to assess
lymphocyte proliferation in the auricular lymph nodes. According to the proposed draft
ICCVAM LLNA Performance Standards for the traditional LLNA, any change to the LLNA
protocol other than the method used to assess lymphocyte proliferation is considered a“major” change. The Panel was asked whether protocol differences between the LLNA:
BrdU-ELISA and the traditional LLNA should be considered only “minor” changes andtherefore if the validity of this test method should be based on the draft ICCVAM LLNA
Performance Standards. In general, the Panel agreed that the LLNA: BrdU-ELISA protocol
differs only in the method used to assess lymphocyte proliferation. Thus, based on the current
draft ICCVAM LLNA Performance Standards, it should be considered as having only
“minor” changes and therefore the validity of this test method could be based only on thedraft ICCVAM LLNA Performance Standards.
However, the Panel concluded also that the answer to this question might differ depending on
what the draft ICCVAM LLNA Performance Standards ultimately constitutes as a “major”
change, a “minor” change, or a different protocol altogether. The Panel further stated that,
depending on the goal of the assay, these distinctions may not be relevant. Ultimately, if atest method is able to make the correct prediction with regard to the sensitization potential ofa test substance, then the issue of “major” versus “minor” changes in the protocol should not
apply.
The Panel was asked, even if the draft ICCVAM LLNA Performance Standards do not apply
to the LLNA: BrdU-ELISA, what impact should the accuracy analysis based on eight of the
18 proposed required reference substances in the draft ICCVAM LLNA PerformanceStandards (only one false negative and no false positives) have on the overall evaluation oftest method accuracy. The Panel concluded that the accuracy analysis based only on eight of
the 18 proposed required reference substances had a significant impact on the evaluation oftest method accuracy. The low number of experiments provided data that resulted inunacceptable test method performance.
The Panel was asked whether there were concerns that 4/4 sensitizers, for which EC3 data
were available, had EC3 values that were outside of the proposed recommended 0.5x to 2.0x
EC3 acceptability range developed based on the traditional LLNA. The Panel concluded that

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the EC3 values outside the recommended 0.5x to 2.0x EC3 acceptability range raised
concerns related to test reproducibility and reliability.2
The Panel was asked whether separate performance standards should be developed for the
LLNA: BrdU-ELISA. The Panel concluded that separate performance standards for the
LLNA: BrdU-ELISA were not needed because the test principles are identical to the
traditional LLNA.

2

During their public meeting on March 4-6, 2008, the Panel's discussion, conclusions and recommendations on
the LLNA: BrdU-ELISA took place prior to the discussion, conclusions and recommendations on the draft
ICCVAM LLNA performance standards. Following their discussion of the draft LLNA performance standards,
the Panel concluded that that an evaluation of test method accuracy should be based on overall accuracy
statistics when compared to the traditional LLNA, and not on a chemical-by-chemical match that is based on
obtaining an EC3 value within a specified range of EC3 values.

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6.0

Draft ICCVAM LLNA Performance Standards

6.1

Comments on the Proposed Purpose and Applicability

ICCVAM proposed that these performance standards should only be applicable to versions of
the LLNA that incorporate “minor” modifications to the traditional LLNA. Currently, this islimited to the use of non-radioactive reagents to measure lymphocyte proliferation. It isconsidered essential that the modified LLNA should otherwise adhere to all other aspects ofthe traditional LLNA protocol, as defined by ICCVAM (1999) and Dean et al. (2001). This
includes aspects such as: the sex and strain of mouse used, the number of mice per dosegroup, the timing and site of test article treatment, the duration between the last treatment andlymph node collection, the inclusion of concurrent negative and positive control groups, themeasured endpoint (i.e., lymphocyte proliferation in the draining auricular lymph node), andthe collection of data at the level of the individual mouse. The Panel was asked if they agreed
that the use of non-radioactive reagents for measuring cell proliferation in the lymph nodes
constitutes a “minor” modification to the traditional LLNA protocol.
The Panel noted that the draft ICCVAM LLNA Performance Standards are proposed forevaluating the acceptability of test methods that are functionally and mechanistically similar
to the traditional LLNA (i.e., measuring the same biological effect), and understood thatICCVAM proposed that these performance standards should only be applicable to protocols
that incorporate “minor” modifications to the traditional LLNA, as defined above. The Panelunanimously agreed that based on ICCVAM’s definition, the use of non-radioactive reagentsfor measuring cell proliferation is a “minor” modification of the traditional LLNA protocol.
However, the Panel also agreed that other modifications may be considered “minor” and thata better strategy for the performance standards might be to define criteria that need to besatisfied to insure that the method is mechanistically and functionally similar (see criteria
listed under essential test method components). Examples of potentially acceptable
modifications identified by the Panel include sex, strain, the use of rats rather than mice,
number of animals per group, and timing of test article treatment.
Regardless of the modification, the Panel stated that the modified test method should bedesigned to measure only the induction phase of the immune response. This is crucial, since
the traditional LLNA is intended for hazard identification with the underlying principle that
stimulation of cell proliferation in the draining lymph node suggests that sensitization (i.e.,
induction) is occurring. Using only the induction phase as the method to identify hazardous
substances involves a short time frame, and reduces pain and distress in treated animals (i.e.,
no dermatitis response). Furthermore, the Panel stated that the performance standards should
not imply that the traditional LLNA, or any alternative LLNA protocol, is capable ofspecifically distinguishing a type IV hypersensitivity reaction (as might be inferred from thetext beginning with line 342 of the draft ICCVAM LLNA Performance Standards document).
Therefore, reference to type IV hypersensitivity reaction should be removed from the
document.
The Panel was asked if they considered it necessary that a modified LLNA keep the samedecision criteria for distinguishing between sensitizers and non-sensitizers as the traditional
LLNA (i.e., an SI ≥3.0). The Panel considered it unnecessary for a modified LLNA to keep
this same decision criteria as a different method for measuring cellular proliferation might

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have better concordance with the human data at a SI different than 3.0. Thus, with any
modified LLNA, the SI threshold defining a sensitizer would need to be established (i.e., it isimportant to consider if the results are biologically relevant to humans).
The Panel was asked if other procedural modifications could be identified as “minor”, basedon the description in the draft ICCVAM LLNA Performance Standards document, andtherefore could be evaluated for equivalence to the traditional LLNA using the proposed
performance standards. The Panel reiterated that sex, strain, the use of rats rather than mice,animals per group, and timing of test article treatment are also potentially “minor”
modifications. Furthermore, the proposed performance standards appear robust; therefore,
regardless of the modification (i.e., “major” or “minor”), there is the same expectation fortest method performance. Dr. James McDougal offered a minority opinion to express his
concern about the potential impact that allowing alternative LLNA protocols withmodifications other than the method by which lymphocyte proliferation was measured would
have.
The Panel was asked if they considered the draft ICCVAM LLNA Performance Standards
applicable to the LLNA limit dose procedure. The Panel noted that the current draft
ICCVAM LLNA Performance Standards could be applicable to the LLNA limit dose
procedure as long as it is recognized that this procedure can only be used for a yes/no hazard
classification (i.e., an ECt estimate is not feasible).
6.2

Comments on the Essential Test Method Components

The essential test method components are based on the ICCVAM-recommended protocol(ICCVAM 1999; Dean et al. 2001), which is the basis for the current EPA (2003) test
guideline. There are some notable differences between these protocols and OECD TG 429(OECD 2002) for the LLNA. The Panel was asked to comment on, when evaluations of nonradioactive versions of the traditional LLNA are conducted using these performancestandards, whether it is necessary that the validation studies follow the ICCVAMrecommended protocol. The Panel indicated that ideally, there would be one globally
recognized set of performance standards (ICCVAM, ECVAM, JaCVAM). However, whenvalidating versions of the traditional LLNA where the only difference is in the use of a nonradioactive method to measure cell proliferation, the ICCVAM-recommended protocol
should be used. If more extensive changes to the protocol are being considered, the following
requirements should be considered during modifications of the LLNA:•

Application of the test substance should be to the skin, with sampling of thelymph nodes draining that site.

•

Cell proliferation should be measured in the draining lymph node.

•No skin reaction should be present, since presence of a skin reaction might
indicate the onset of the elicitation phase of skin sensitization.

•

Data should be collected at the level of the individual animal to allow for an
estimate of the variance within control and treatment groups. Using thisvariance, a power analysis needs to be conducted to demonstrate that themodified method is utilizing a sufficient number of animals per treatmentgroup to permit hazard identification with at least 95% power.

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If dose response information is needed, there should be an adequate number ofdose groups (n ≥3) with which to adequately characterize the dose response
for a given test substance.

The Panel was asked to comment on whether validation studies should include a concurrent
positive control with each test substance and if so, whether the concurrent testing of thepositive control and test substance should be conducted in the same vehicle or if different
vehicles were acceptable. The Panel noted that a concurrent positive control should be
included in each validation study to ensure that the test system was operating as expected andtechnical errors were not occurring. A concurrent positive control would be especially usefulwhen an unknown test material was being tested or when a laboratory was collecting adataset to serve as historical control data. However, if a known sensitizer was being tested, aconcurrent positive control might not be needed, thus reducing animal use. Finally, the Panelconcluded that the positive control should be tested in the same vehicle as the test substance.
Using a different vehicle for the positive control would require an additional set of vehicle
control animals.
The Panel was also asked whether the validation studies should use a minimum of fiveanimals per dose group and collect lymph node data from individual animals. The Panelcommented that until sufficient data were collected to enable a reliable power calculation to
be conducted to determine the optimal number of animals per dose group, at least fiveanimals per dose group should be used. The Panel also agreed that when validating amodified LLNA protocol, lymph node proliferation should be evaluated at the level of theindividual animal within each dose group. Variance is only measurable if lymph nodes fromindividual animals are assessed. If the variability within a dose group of a modified LLNA
protocol was substantially less than the traditional LLNA, reducing the number of animals
per dose group might yield similar results. A minority opinion by Drs. Nathalie Alépée,Thomas Gebel, Dagmar Jírová, Raymond Pieters, and Michael Woolhiser stated that if
laboratories were operating under OECD guidance (OECD 2002) and a reliable validation
dataset had been generated, then pooled data from at least four animals could be considered
acceptable.
6.3

Comments on the Proposed Reference Substances

The Panel was asked if they agreed with the selection and prioritization criteria used to selectthe performance standards reference substances. The Panel noted that the rationale forselection of the reference substances included in the draft ICCVAM LLNA PerformanceStandards was well documented (taking into account the physicochemical characteristics, thepurity, the stability, the quality of the in vivo data, and the chemical classes covered). The
substances also appeared to be distributed over a wide range of EC3 values. However, theavailable database for some of the substances was insufficient. Among the 13 sensitizers in
the “required” list, only five appear to have a robust database (i.e., have been tested in at least
three independent studies). Thus, consideration should be given to revising the list of
substances and/or making the data for the substances on the current list more robust. Ideally,
the reference list should be based only on substances with robust data for LLNA, human, andguinea pig tests.
The rationale for the number of substances included on the "required" list of substances(n=18) was provided in the draft ICCVAM LLNA Performance Standards. In addition, there
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were four additional substances that were described as problematic in the traditional LLNA
(i.e., false negatives and false positives). The Panel was asked if they considered 18“required” substances to be an adequate number upon which to evaluate the performance of
non-radioactive LLNA test methods, where the only protocol modification is the method forassessing cell proliferation in the auricular lymph nodes, and if not, how many reference
chemicals should be tested. The Panel commented that ideally, one would like to be able todemonstrate that an assay is equivalent to the traditional LLNA. However, with the small
number of reference substances available, establishing equivalence will be extremely
difficult. Therefore, the Panel recommended that, for use in hazard identification, a modified
method should be evaluated with all 22 substances (including false negatives and false
positives) and accuracy statistics calculated. To the extent possible, rationale for any
discordant results should be provided, but the most potent sensitizers (e.g., DNCB) should
always be identifiable. There also should be considerable weight given to the balance
between animal welfare and human safety when considering the adequacy of test method
accuracy.
The panel considered it noteworthy that 19 of the 22 substances on the draft ICCVAM list
are in common with the ECVAM performance standards list. The Panel also considered it
important that substances be coded during validation studies.
It is also relevant to note that the Panel discussed the value of GLP procedures on several
occasions during the meeting. In each instance, the Panel agreed that data collected under
GLP conditions would be greatly preferred, particularly for reasons of data quality and theassociated reliability of any interpretations. However, they noted that GLP compliance would
not be considered a requirement that would automatically exclude data from consideration.
The Panel concluded that other factors could be used to identify high quality data. Examples
would include published in a peer-reviewed journal or obtained from a study conducted in alaboratory that routinely conducts GLP studies. Data generated under non-GLP conditions
would be subject to a critical quality review, and as such the Panel considered it important toobtain the original records in order to confirm the reported data.
The Panel was asked if they considered the types of substances included in the reference
substance list, with regard to relative sensitization potency, physicochemical characteristics,
and vehicles, to be representative of the overall diversity of substances that are likely to betested for skin sensitization. The Panel concluded that although the list should not be
considered all-inclusive, it was sufficiently representative.
The Panel was also asked if there were other types of information relevant to skinsensitization that should be considered in order to demonstrate an adequately diverse
reference list. The Panel commented that identifying concentrations of each of the substancesthat are known to cause excessive local irritation or overt systemic toxicity would be useful.
The Panel was asked if there were other substances that they considered to be more
appropriate for assessing the sensitivity (i.e., ability of the test method to correctly identify
sensitizing substances) and specificity (i.e., ability of the test method to correctly identify
non-sensitizing substances) of non-radioactive LLNA test methods, and for which there isavailable LLNA, guinea pig, and human data. The Panel could not identify such substances
given the time frame for consideration but reiterated that substances in the reference list
should have robust data.

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A subset of "discordant chemicals" (i.e., false negative or two false positive compared toguinea pig tests or human data) were included as “optional” substances that could be studied
to evaluate if the proposed modifications might provide improved performance relative to the
traditional LLNA. The Panel was asked to comment on the appropriateness of includingthese specific substances in the reference list, whether they should be required, whether
different substances should be included, and if more false negative/positive substances
should be tested. As mentioned previously, the Panel commented that it was appropriate toinclude such substances in the reference list and that they should be required and evaluatedduring the validation of alternative LLNA assays that are functionally and mechanisticallysimilar to the traditional LLNA assay. The Panel noted that the substances that wereconsidered discordant depended on the species to which comparisons are made (i.e., LLNA
vs. guinea pig or LLNA vs. human). Still, since the “discordant compounds” were false
negatives or positives in the traditional LLNA, they would provide an opportunity todetermine if modifications to the traditional LLNA may even have increased accuracy.
Finally, the Panel was asked if "correct" results with these discordant chemicals would be
sufficient to consider the alternative test method to be more predictive of skin sensitization
than the traditional LLNA. The Panel concluded that correct results with the “discordantchemicals” would not be sufficient to consider the alternative test method to be morepredictive of skin sensitization, but it could provide supporting evidence to indicate further
testing with additional compounds would be of value.
6.4

Comments on the Test Method Accuracy Standards

The draft ICCVAM LLNA Performance Standards state that the non-radioactive proposed
LLNA test method should exactly match the accuracy of the traditional LLNA whenevaluated with the minimum set of 18 reference substances. The Panel was asked if theyagreed that test method accuracy should be based on a chemical-by-chemical match withregard to identifying the chemicals as sensitizers or non-sensitizers. The Panel commented
that although an assay that is able to predict the same hazard classification for the reference
substances as the traditional LLNA is desired, with the small number of reference substances
available (n=18), clearly establishing equivalence will be extremely difficult. Furthermore,even with this small number, there is a statistical multiple comparisons3 problem because
more than one chemical is being tested. The likelihood that a modified LLNA will fail todemonstrate equivalence to the traditional LLNA will increase with the number of chemicals
that must be identified correctly. A statistical measure of concordance should be calculated
so that accuracies can be compared between methods.
The Panel reiterated their recommendation that, for use in hazard identification, a modified
test method should be evaluated with all 22 substances (including false negatives and false
positives) and accuracy statistics calculated. A statistical measure of concordance should be
calculated so that accuracies can be compared between methods. To the extent possible,
3

When multiple experiments are conducted and multiple observations, comparisons or hypothesis tests are
conducted, the chance of observing rare events increases. Suppose, for example, that an interval is establishedsuch that 5% of observations from a particular population of data are outside that interval. Then if k independent
experiments generate data from this population (e.g., a standard normal distribution), the chances that all 20
results will lie inside the interval is (1.0 - 0.05)k (N. Flournoy, personal communication).

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rationale for any discordant results should be provided. However, the most potent sensitizers(e.g., DNCB) should always be identifiable. Considerable weight should be given to thebalance between animal welfare and human safety when considering the adequacy of test
method accuracy.
The draft ICCVAM LLNA Performance Standards recommend that, for each sensitizer, thethreshold concentration that induces a positive SI response should be within 0.5x to 2.0x of
the concentration obtained for the EC3 in the traditional LLNA. As described in the draft
ICCVAM LLNA Performance Standards, statistical approaches have been used in an attempt
to identify an appropriate range, but these calculated ranges do not appear to be the mostpractical. The Panel was asked to comment on the appropriateness of using this criterion tojudge the equivalency of a non-radioactive version of the traditional LLNA and, if thisapproach was not acceptable, to suggest an alternative along with the basis for this approach.
The Panel commented that the usefulness and limitations of the traditional LLNA for
determining relative potency have not been definitively established, and therefore
equivalence should not be based strictly on potency. Furthermore, the current database does
not support the inclusion of EC3 values as a component of the accuracy evaluation. The
range of 0.5x to 2x EC3 value suggested in the draft ICCVAM LLNA PerformanceStandards document are based on the experience with a range of known skin sensitizers
tested in the standard LLNA. However, based on the available data provided, the 0.5x to 2.0x
EC3 range may be too restrictive if a strict interpretation of equivalence is applied. The
chances of a failure to achieve an EC3 within this range would vary from chemical tochemical depending upon the inherent underlying variability and robustness in the estimationof the EC3. For those chemicals for which the EC3 can be accurately estimated, the failure
rate may be close to zero. For other, more variable chemicals, perhaps with fewer data points,
the failure rate for a single chemical will be much higher.
The Panel reiterated their concern with regard to EC3 values (i.e., the statistical multiple
comparisons problem). The likelihood that a modified LLNA will fail to demonstrateequivalence to the traditional LLNA will increase with the number of chemicals tested, theextent to which the new test must obtain the same EC3 value, and how independent theresults are for different chemicals in the same lab.
For five of the 13 sensitizers on the draft ICCVAM reference substances list, the reference
EC3 value was based on a single LLNA study (Table C1 of the draft ICCVAM LLNA
Performance Standards). The Panel was asked to comment on the appropriateness of
including such chemicals in the list of recommended reference substances and whether or not
the 0.5x to 2.0x criteria should be applied to such substances. The Panel concluded that theappropriateness of the 0.5x to 2.0x ECt range had not been adequately justified. It was
inappropriate to include chemicals represented by only one LLNA study on a list ofrecommended reference substances, as there was insufficient data by which to calculate arobust mean ECt value. Thus, those compounds should either (1) be exchanged forcompounds with sufficient EC3 data (i.e., have been tested in at least three independentstudies using the same solvent), or (2) retained but not considered to be part of the ECt
criterion until such data has been collected.

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Comments on the Test Method Reliability Standards

The draft ICCVAM LLNA Performance Standards state that acceptable intralaboratory
reproducibility will be indicated by a laboratory obtaining, in each of four independent
experiments conducted with at least one week between each experiment, ECt values (theestimated concentration needed to produce an SI of a defined threshold [e.g., EC3]) for HCA
that are generally within 0.5x to 2.0x (i.e., 5% to 20%) of the historical mean EC3concentration (10%) for this substance, based on existing available traditional LLNA data.
The Panel was asked if they considered four repeat experiments to be adequate. The Panelconcluded that four experiments would be adequate, as requiring four independent
experiments is similar to the original LLNA submission, as is a one-week interval between
experiments. Therefore, these requirements were appropriate for a comparison of modified
methods to the traditional LLNA. However, it would be useful to have this number evaluatedstatistically (see Section 6.7).
The Panel was asked if they considered testing HCA adequate for demonstrating
intralaboratory reproducibility and if not, which substance(s) should be tested. The Panelconcluded that HCA testing would be adequate for demonstrating intralaboratory
reproducibility and would allow an effective comparison to the traditional LLNA.
The Panel was asked to comment on whether the required one-week interval between
independent tests was adequate and/or appropriate. The Panel concluded that the minimum
one-week interval seemed logical and that the more important clarifying information might
be the elements that define independent tests (e.g., different animal shipment, different
reagents, different operator, blind testing).
The Panel was asked to comment on the appropriateness of the criteria for acceptability(generally within 0.5x to 2.0x EC3 for HCA), or to describe another criteria and explain thebasis for their recommendation. The Panel concluded that the criteria for acceptability
appeared to be appropriate because the statistical multiple comparisons issue does not exist.
However, given that there is so much data and experience with HCA and the fact that only
one compound is being tested (not 18), it is reasonable to evaluate reproducibility using themean ± 3 standard deviations rather than the 0.5x to 2.0x EC3 range to account for a single
comparison (see Section 6.7 regarding data transformation recommendations). The Panelnoted that historical control data using HCA in the same vehicle could be used todemonstrate adequate intralaboratory reproducibility.
The draft ICCVAM LLNA Performance Standards state that acceptable interlaboratory
reproducibility will be indicated by each of three laboratories obtaining ECt values for HCA
and DNCB from a single experiment that are generally within 0.5x to 2.0x (5% to 20% and0.025 to 0.1%, respectively) of the mean historical EC3 concentration (10% and 0.05%,
respectively) obtained for these two substances in the traditional LLNA. The Panel was asked
if they considered the single experiment per substance in each laboratory to be adequate. The
Panel concluded that, considering the overall validation plan for a given laboratory, multiple
experiments (n=3) within each laboratory should be conducted.
The Panel was asked if they considered testing HCA and DNCB to be adequate fordemonstrating interlaboratory reproducibility and if not, which substance(s) should be tested.
The Panel concluded that, since there is a great deal of data and experience with HCA and6-7

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DNCB, and many laboratories have successfully worked with them in the traditional LLNA,
they should be considered adequate for this purpose.
The Panel was asked if they considered the criteria for acceptability to be appropriate. The
Panel concluded that the criteria for acceptability (i.e., generally within 0.5x to 2.0x ECt for
HCA and DNCB) appeared to be appropriate because the statistical multiple comparisonsproblem was relatively minor given that only two substances are being tested. However,given that there is so much data and experience with HCA and DNCB and the fact that twocompounds are being tested (not 18), it is reasonable to evaluate reproducibility using themean ± 4.5 standard deviations to account for statistical multiple comparisons (see Section
6.7 regarding data transformation recommendations). The Panel also noted that historicalcontrol data using HCA and DNCB in the same respective vehicle could be used todemonstrate adequate interlaboratory reproducibility.
6.6

Summary

The Panel was asked what criteria should be used to evaluate the equivalence of a radioactive
or non-radioactive LLNA method to the traditional LLNA, if one were proposed with a“major” change, as defined in the draft ICCVAM LLNA Performance Standards (e.g.,different mouse strain or use of male mice, change in the schedule for test article
administration, change in schedule for lymph node excision, etc.). The Panel commented thatthe idea of what is a “major” and a “minor” change should be re-considered (refer to
Question 2 regarding essential test components). The final version of the performancestandards should be adequate to evaluate any protocol modifications.
The Panel was asked if a new set of performance standards would be required for a modified
version of the LLNA that incorporated one or more “major” protocol changes. Based on theabove response, the Panel concluded that a new set would not be required.
The Panel was asked to comment on how many reference substances might be consideredadequate for evaluating the validity of a modified version of the LLNA with a “major”
protocol change; specifically, if the 18 minimum reference substances in the draft ICCVAM
LLNA Performance Standards would be sufficient. The Panel concluded that additional
substances should not be considered necessary. However, since eight of the proposed
sensitizers had limited data (i.e., EC3 values based on ≤2 LLNA studies), other substanceswith more robust data should be considered as replacements. Furthermore, if the goal is toevaluate a specific applicability domain, additional test substances might be needed.
The Panel was asked to comment, regardless of the number of reference substances, whether
the alternative LLNA with a “major” change should be required to obtain the same “call”(and potency for sensitizers) as the traditional LLNA for the 18 minimum reference
substances in the draft ICCVAM LLNA Performance Standards. The Panel reiterated that anassay that is equivalent to the traditional LLNA is desired, but with the small number ofreference substances available, clearly establishing equivalence will be extremely difficult.
They also reiterated their concern regarding the statistical multiple comparisons problem.
For use in hazard identification, a proposed modified LLNA should be evaluated with all 22
substances (including false negatives and false positives) and accuracy statistics calculated sothat accuracies can be compared between the modified test method and the traditional LLNA.
To the extent possible, rationale for any discordant results should be provided. However, the
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most potent sensitizers (e.g., DNCB) should always be identifiable. Considerable weightshould be given to the balance between animal welfare and human safety when considering
the adequacy of test method accuracy.
The Panel was asked to identify any additional specific substances that should be used. The
Panel concluded that while additional substances should not be needed, it would be useful toidentify replacements for the eight proposed sensitizers with limited test data. If the goal is toevaluate a specific applicability domain, additional test substances might be needed.
6.7

Additional Statistical Comments

During the evaluation of the draft ICCVAM LLNA Performance Standards, the Panel noted anumber of statistical issues that should be addressed. They suggested that in order to achievea normal distribution of the data and to reduce differences between groups, a suitablevariance stabilizing transformation (e.g., log transformation, square root transformation)
should be applied in all statistical analyses and in reporting summary standard deviations.
The Panel also suggested that there should be a more rigorous evaluation of what would beconsidered an appropriate range of ECt values to include as a requirement. This would be astatistical evaluation that takes into consideration the variability of ECt values generated
among the sensitizers included on the performance standards reference substances list and thestatistical multiple comparisons problem and the fact that sample sizes that are less than 30
invalidate statistics based on the normal distribution (Young 2007).
Furthermore, bioequivalence models have been developed (Berger and Hsu 1996) and should
be applied to the LLNA. Probability values can be used as descriptive statistics and as suchprovide a summary measure of weight-of-evidence that would be useful for comparison of
performance standards across test methods. In this context, it would be informative to havestatistical tests of data generated for these purposes. A test of concordance for measuring theaccuracy of classification should be done.
Intralaboratory tests should include analysis of variance (ANOVA)-like tests with a test forno trend, with the null hypothesis being that there is a difference and the alternative beingthat the difference is bioequivalent. Interlaboratory tests should include ANOVA-like tests
with the null hypothesis being that there is a difference and the alternative hypothesis beingthat the difference is bioequivalent. The reliability tests require “bioequivalence” to be
defined (i.e., what is acceptable to be considered equivalent).
It is not known whether these specific statistical tests can be identified in the literature or ifthey need to be developed. If they do need to be developed, this should be given a priority.
Prior to running reliability studies, these statistical methods should be used to determine theappropriate number of substances and the number of times each substance needs to be tested
within and among laboratories in the study design (see also ISO 5725 [ISO 1994] and ASTM
Standard E691 [ASTM 2005]). The power for the traditional LLNA should be established forcomparison purposes.

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7.0

Use of the LLNA for Potency Determinations

7.1

Comments on the Draft BRD for Completeness, Errors, and Omissions

The Panel was asked if there were any errors in the draft BRD on the use of the LLNA for
potency determinations that should be corrected, if omissions of existing relevant data had
been identified, or if there was additional information that should be included. The Panelnoted alternative analyses that would better help evaluate the use of the traditional LLNA forskin sensitization potency (see the discussion of the categorization scheme in Section 7.2 andthe discussion of future studies in Section 7.3).
7.2

Comments on the Validation Status of the Traditional LLNA to Determine
Skin Sensitization Potency

7.2.1

Substances Used for the Validation Studies

The Panel was asked to consider whether the validation status of the traditional LLNA for
potency categorization (i.e., “strong” vs. “weak” sensitizers) has been adequately
characterized, and if the traditional LLNA is sufficiently accurate and reliable to be used as astand-alone assay for characterizing the potency of sensitizing substances, based on thecomparison to human and guinea pig responses. The Panel agreed that the LLNA database of
170 substances with comparative guinea pig (i.e., Guinea Pig Maximization Test or Buehler
Test) and/or human data (i.e., Human Maximization Test [HMT] and/or Human Repeat
Insult Patch Test [HRIPT], but not human clinical observations) is sufficient in number andwell balanced for this evaluation. The database included 112 substances (97 sensitizers, 15
non-sensitizers) with comparative human data and 105 substances (52 sensitizers, 53 nonsensitizers) with comparative guinea pig data. Known contact sensitizers of public healthconcern from various chemical groups are included. The Panel further agreed that thesesubstances were representative of a sufficient range of chemical classes and physicalchemical properties so that it would be applicable to the types of chemicals and products
typically tested for skin sensitization potential.
While coding of chemicals to reduce bias is recommended for validation studies, thisevaluation was based on a retrospective evaluation of existing data, most of which weregenerated using chemicals that were not coded. The Panel was asked whether the lack ofcoding of test substances adversely impacted or biased the current evaluation. Given thenature of the studies (i.e., the testing was not conducted to demonstrate the ability of the
LLNA to be used for potency characterization), the Panel stated that the lack of coding likely
had no impact on the current evaluation.
For some substances tested for sensitization using the traditional LLNA, it was not possible
to determine whether the data were generated using pooled or individual animal lymph node
samples within a dose group (the former allowed in OECD TG 429 [OECD 2002]; the latteras recommended in the ICCVAM 2001 protocol and required in the EPA 2003 skinsensitization test guideline). Cockshott et al. (2006) reported that using individual animal
data allowed for technical problems during an experiment and outlier animals within a dosegroup to be identified. Considering this, the Panel was asked whether the analysis of theperformance of the traditional LLNA for potency determinations should be limited to datafrom studies that can be confirmed as using individual animal data collection procedures.
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A majority of the Panel agreed that, ideally, future traditional LLNA potency determinations
should be based on data from studies that use individual data collection procedures, as thiswould allow for the identification of outliers that might skew the average group stimulationindex. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, RaymondPieters, and Michael Woolhiser stated that if laboratories were operating under OECD
guidance (OECD 2002) and a reliable validation dataset had been generated, then pooled datafrom at least four animals could be considered acceptable.
7.2.2

Test Method Accuracy

The Panel was further asked what impact the inclusion of pooled animal data might have on
the accuracy analysis included in Section 6.0 of the draft ICCVAM LLNA potency BRD.
With regard to this retrospective dataset, the Panel agreed that pooled data should not be
excluded from the current analysis to assess potency determinations for the traditional
LLNA. The Panel stated that it is impossible to assess the impact of using pooled data
without a separate analysis of the ability of the traditional LLNA to be used forcharacterizing skin sensitization potency using pooled vs. individual data, which the Panelrecommended be done (see the discussion of future studies in Section 7.3).
A minority opinion from Dr. Dagmar Jírová stated that, since OECD TG 429 (OECD 2002)
allows the use of both pooled and individual animal data, the analysis that includes bothtypes of data is appropriate. Even with the diversity of data sources (the vehicle is not known
for 43% of substances tested in the traditional LLNA; human data were obtained by different,
even undefined methods, etc.), the outcome of the evaluation was good, which documents thestrength and robustness of the traditional LLNA.
The Panel was asked whether the correct classification, as well as the over- and underclassification, rates of the traditional LLNA for sensitization potency determinations had
been adequately compared and appropriately evaluated based on the corresponding humanand guinea pig data (refer also to Section 6.0 of the draft ICCVAM LLNA potency BRD).
The Panel agreed that the two approaches used in the draft BRD for analyzing the ability ofthe traditional LLNA to discriminate between strong and weak skin sensitizers wereappropriate and correct. In these two approaches, the traditional LLNA was evaluated, after
identifying the optimal EC3 value, for its ability to correctly classify strong and weaksensitizers as defined by human or guinea pig threshold values based on: (1) sensitizers only,and (2) sensitizers combined with false positives, false negatives, and non-sensitizers.
A minority opinion from Dr. Howard Maibach stated that the relevance of the traditional
LLNA to human clinical observations has not been sufficiently determined and should be.
The accuracy analysis (see Section 6.0 of draft ICCVAM BRD) focuses on a proposed twolevel categorization scheme (weak sensitizers vs. strong sensitizers) for both human andguinea pig data. The Panel was asked whether this was an appropriate categorization scheme,
or if other categorization schemes should be considered. The Panel agreed that the two-level
categorization scheme was appropriate, especially considering the fact that, for humansituations, risk assessment should be performed, and therefore more categories are notneeded. Even a weak sensitizer under heavy exposure and individual circumstances may
reach a comparable risk level as a strong sensitizer under conditions of low exposure.

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A minority opinion from Drs. Raymond Pieters and Michael Woolhiser recommended the
addition of at least a moderate category since certain compounds will always be on theborder between weak and strong. Dr. Pieters specifically recommended the categorization
scheme of Kimber et al. (2003), which is based on five categories if non-sensitizers areincluded.
Of the two human threshold concentrations that are proposed in this two-category
categorization scheme (i.e., <250 µg/cm2 or <500 µg/cm2), the Panel was asked whichthreshold was the most appropriate for categorizing sensitizing substances as strong vs. weakfor humans, or if another threshold was more appropriate for this purpose. The Panel notedthat this validation was based on comparison to guinea pig and HMT/HRIPT information.
These data relate only to induction and do not permit an assessment of risk in humans for
elicitation.
For the data provided, the Panel concluded that the best results were obtained using thedecision criterion of 250 µg/cm2 and the corresponding optimal traditional LLNA EC3 valueof 9.4%. Using this cut-off when traditional LLNA false negative and false positive
substances are included in the analysis, in addition to sensitizers in both the traditional LLNA
and in humans using the HRIPT and/or HMT, correct classification of strong sensitizers was
79% and underclassification was 21%. Underclassification of substances in this context
means classification as weak instead of strong sensitizers (i.e., they are not missed assensitizers regarding the labeling and safety of consumers). The Panel stated that more data
are needed to determine if another threshold is more appropriate.
When the potency categorization analysis was based on sensitizers only, the guinea pig tests
predicted weak sensitizers with higher accuracy than did the LLNA (89% vs. 75% for the
250 µg/cm2 cutoff and 83% vs. 60% for the 500 µg/cm2 cutoff), which is logical because theguinea pig test methodology involves all phases of the sensitization process and usually
involves adjuvants. However, the guinea pig tests were less accurate for the prediction ofstrong sensitizers compared to LLNA (48% vs. 71% for the 250 µg/cm2 cutoff and 42% vs.
63% for the 500 µg/cm2 cutoff), which represents a higher risk for consumers. For theprotection of public health, it is more important to correctly identify strong sensitizers thanweak sensitizers.
The Panel was asked whether the draft BRD adequately characterized the usefulness andlimitations of the LLNA for potency categorizations. If not, the Panel was asked what
additions or changes should be made to the description of usefulness and limitations in thedraft BRD. The Panel stated that additional evaluations should be conducted to determine theimpact on potency categorization if the human threshold data are evaluated differently (e.g.,alternative lowest observed effect level [LOEL] safety factors other than 10, using LOEL
data only, using no observed effect level [NOEL] data only), and if this might improve thecorrelation between the LLNA and the human results. According to the Panel, the approachof directly comparing the LOEL values without using a safety factor compares values ofsimilar significance in humans and in the LLNA. In other words, the LOEL in humans
describes the threshold induction area dose in humans and the EC3 value in the traditional
LLNA is the threshold induction area dose and thus could be the analogous value to thehuman LOEL. The Panel further stated that traditional LLNA tests based on pooled orindividual lymph nodes for a dose group should be evaluated independently to assess the

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impact of using pooled data on the accuracy analysis for skin sensitization potency. Finally,
the Panel stated that the effect of different vehicles should be recognized as a limitation in thedata analysis given the demonstrated variability of results.
7.2.3

Test Method Reliability

The Panel was asked whether the reliability (e.g., intralaboratory repeatability, intra- andinter-laboratory reproducibility) of the traditional LLNA for potency determinations had beenadequately evaluated. If not, the Panel was asked what other analyses should be performed.
Similar to their recommendations for test method accuracy, the Panel stated that additional
evaluations of reliability should be conducted based on using different approaches for humanthreshold data (e.g., using alternative LOEL safety factors other than 10, using LOEL data
only, using NOEL data only). The Panel further stated that the reliability of LLNA based onusing pooled or individual animal data should be evaluated independently. Finally, the Panelstated that the effect of different vehicles should be recognized as a limitation in the data
analysis, as a source of increased variability.
7.2.4

Data Quality

It was not possible to determine whether or not all studies included in the draft LLNA
potency BRD had been conducted in accordance with GLP guidelines, nor was it possible toobtain the results of GLP audits for all studies determined to be GLP-compliant. The Panelwas asked to discuss what impact this might have on the evaluation of the LLNA for potency
determinations and whether any of the non-GLP studies should be excluded from theanalyses. The Panel concluded that it was important to note if the data were obtained fromstudies conducted according to GLP guidelines, as ideally this should be the case. However,the Panel concluded that data from studies that could not be confirmed as being GLPcompliant, but that were from peer-reviewed literature or other sources with high-quality
laboratory management practices were still appropriate to include in this retrospective
analysis.
As described in the draft BRD, original records for some of the non-GLP studies included inthis evaluation could not be obtained. As a result, an independent audit could not be
conducted to confirm that the reported data is the same as the data recorded in laboratory
notebooks. Considering this, the Panel was asked whether the results of these studies (all ofwhich are currently included) be excluded from any of the performance analyses. The Panelconsidered the data to have been generated by repeatedly published and reliable laboratories
and therefore did not question the adequacy/quality of the retrospective data analysis. Thus,
although data should be checked when available, exclusion of data was not deemednecessary, in this case.
7.2.5

Consideration of All Available Data and Relevant Information

Based on the draft BRD, the Panel was asked whether all the relevant data identified inpublished or unpublished studies conducted using the traditional LLNA had been adequately
considered. If not, the Panel was asked what other studies should be considered. The Panelrecommended that the LOELs from Akkan et al. (2003) be used instead of the DSA05 values
from Schneider and Akkan (2004) in all of the potency analyses. A minority opinion by Dr.
Thomas Gebel stated that it was acceptable to use the DSA05 values from Akkan et al. (2003)
as LOEL values in the evaluation. Dr. Gebel mentioned that the DSA05 value is a LOEL

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value adjusted to 5% incidence of induction. Akkan et al. (2003) used the DSA05 value tocorrect for different human studies leading to different inductions. Dr. Gebel further statedthat as the DSA05 is corrected for an induction rate of 5%, it would be better to compare withthe traditional LLNA EC3 than to use the default uncorrected LOEL.
7.3

Comments on the Draft ICCVAM Test Method Recommendations for the Useof the LLNA for Potency Determination

7.3.1

Test Method Usefulness and Limitations

With regard to the use of the LLNA for potency categorization (i.e., strong vs. weaksensitizers), the ICCVAM draft recommendation is that the traditional LLNA should not be
considered as a stand-alone test method for predicting sensitization potency, but must instead
be used as part of a weight-of-evidence evaluation to discriminate between strong and weaksensitizers. This is based on the fact that, although there is a significant positive correlation
between traditional LLNA EC3 values and human sensitization threshold doses, thiscorrelation is not strong [see detailed discussion in the draft ICCVAM recommendations].
The Panel agreed that the traditional LLNA should not be considered a stand-alone assay for
categorization of skin sensitization potency, but it could be used in a weight-of-evidence
evaluation (e.g., along with quantitative structure-activity relationship [QSAR], peptide
reactivity, human evidence) to discriminate between strong and weak sensitizers. The Panelfurther stated that there are additional studies proposed that may provide a better correlationand improve prediction of potency categorization (see the discussion of future studies
below).
A minority opinion from Drs. Thomas Gebel and Dagmar Jírová stated that there is asignificant positive correlation between EC3 values and human threshold values. It is likelythat limitations in estimating human threshold values and the inclusion of human NOEL
values in the current evaluation contributed negatively to the resulting R2 value of 0.405(when LLNA EC3 data vs. human threshold data were compared, see Table 6-2 of the draft
ICCVAM BRD). Thus, the R2 value may improve when the additional analyses that havebeen suggested by the Panel are conducted.
The Panel stated that the effect of different vehicles should be recognized as a limitation in
the data analysis and a likely source of within and between laboratory variability.
7.3.2

Test Method Protocol

The Panel was asked whether the ICCVAM-recommended LLNA protocol (ICCVAM 1999;
EPA 2003) should be used when generating data that will or might be considered forsensitization potency categorization decisions. The Panel agreed that this protocol should be
used. A minority opinion by Drs. Nathalie Alépée, Thomas Gebel, Dagmar Jírová, RaymondPieters, and Michael Woolhiser stated that if laboratories were operating under OECD
guidance (OECD 2002) and a reliable validation dataset had been generated, then pooled datafrom at least four animals could be considered acceptable.
The Panel was asked whether the relevant testing guidelines for the traditional LLNA should
be updated to include the calculation of an EC3 value. The Panel agreed with thisrecommendation. The calculation of an EC3 value is briefly described in the draft ICCVAM

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LLNA Performance Standards for specific situations with references to Basketter et al.
(2000) and Ryan et al. (2007).
7.3.3

Future Studies

The Panel was asked whether the available data support the ICCVAM draft
recommendations for the traditional LLNA in terms of the proposed future studies. The Panelagreed and concluded that more data are needed to determine the optimal threshold in
humans for distinguishing between strong and weak sensitizers. However, the Panel
discouraged conducting new animal studies unless it was likely that results from such studies
would lead to an overall reduction in animal use. The Panel stated further that the traditional
LLNA appears to be a robust rodent assay for the quantification of the induction of cellmediated immunity. Thus, use of the traditional LLNA for potency determination can beused in conjunction with QSAR information, guinea pig assays, HRIPT/HMT, and thequantitative data of elicitation and frequency of positive response in humans in a weight-ofevidence approach. The Panel further stated that additional evaluations should be conducted
to determine the impact on potency categorization if the human threshold data are evaluateddifferently (e.g., alternative LOEL safety factors other than 10, using LOEL data only, using
NOEL data only). This might improve the correlation between LLNA and human data. The
Panel further stated that LLNA tests based on pooled or individual animal data should be
evaluated independently to assess the impact of using pooled data on the accuracy for
determining skin sensitization potency.
The Panel recommended a statistical analysis to determine where an appropriate cutoff value
between weak or strong sensitizers might be best defined for traditional LLNA data. For
example, receiver operating characteristic curves could be used to identify the optimum cutoff for determining the difference between weak and strong sensitizers.
Finally, the Panel stated that the effect of different vehicles should be recognized as alimitation in the current data analysis, that this was a source of variability within and between
laboratories, and that its impact should be considered in future analyses.

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References

Akkan Z, Kalberlah F, Oltmanns J, Schneider K. 2004. Beurteilung der Wirkst€arkehautsensibilisierender Chemikalien anhand des Local Lymph Node Assay (LLNA) [inGerman]. Bremerhaven, Germany: Federal Institute for Occupational Safety and Health.
ASTM. 2005. ASTM Standard E691-05 Standard Practice for Conducting an Interlaboratory
Study to Determine the Precision of a Test Method. In: Annual Book of ASTM Standards.
West Conshohocken: ASTM International. www.astm.org.
Basketter DA, Blaikie L, Dearman RJ, Kimber I, Ryan CA, Gerberick GF, et al. 2000. Use of
the local lymph node assay for the estimation of relative contact allergenic potency. Contact
Dermatitis 42:344-348.
Berger RL, Hsu JC. 1996. Bioequivalence trials, intersection-union tests and equivalence
confidence sets. Stat Sci 11:283-319.
Cockshott A, Evans P, Ryan CA, Gerberick GF, Betts CJ, Dearman RJ, et al. 2006. The local
lymph node assay in practice: a current regulatory perspective. Hum Exp Toxicol 25:387394.
Dean JH, Twerdok LE, Tice RR, Sailstad DM, Hattan DG, Stokes WS. 2001. ICCVAM
evaluation of the murine local lymph node assay: II. Conclusions and recommendations of an
independent scientific peer review panel. Regul Toxicol Pharmacol 34:258-273.
De Jong WH, van Och FM, Den Hartog Jager CF, Spiekstra SW, Slob W, Vandebriel RJ, etal. 2002. Ranking of allergenic potency of rubber chemicals in a modified local lymph node
assay. Toxicol Sci 66:226–232.
EPA. 2003. Health Effects Test Guideline OPPTS 870.2600. Skin Sensitization. Research
Triangle Park, NC: Environmental Protection Agency.
Gaspari AA. 2007. Mechanism of action and other potential roles of an immune response
modifier. Cutis 79(4 SUPPL.):36-45.
Gerberick GF, Ryan CA, Kern PS, Dearman RJ, Kimber I, Patlewicz GY, et al. 2004. A
chemical dataset for evaluation of alternative approaches to skin-sensitization testing.
Contact Dermatitis 50:274-288.
Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP. 2007.
Quantification of chemical peptide reactivity for screening contact allergens: a classification
tree model approach. Toxicol Sci 97:417-27.
ICCVAM. 1999. The Murine Local Lymph Node Assay: A Test Method for Assessing theAllergic Contact Dermatitis Potential of Chemicals/Compounds. The Results of an
Independent Peer Review Evaluation Coordinated by the Interagency Coordinating
Committee on the Validation of Alternative Toxicological Methods (ICCVAM) and the
National Toxicology Program Center for the Evaluation of Alternative Toxicological
Methods (NICEATM). NIH Publication No. 99-4494. Research Triangle Park, NC: National
Institute of Environmental Health Sciences.

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ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised,
and Alternative Test Methods. NIH Publication No: 03-4508. Research Triangle Park, NC:
National Toxicology Program.
ISO. 1994. ISO 5725. Accuracy (trueness and precision) of measurement methods andresults. Geneva: International Organization for Standardization.
ISO. 2002. ISO 10993-10: Tests for Irritation and Sensitization. Geneva: International
Organization for Standardization.
Jowsey IR, Clapp CJ, Safford R, Gibbons BT, Basketter D. 2008. The impact of vehicle on
the relative potency of skin sensitising chemicals in the local lymph node assay. In Press.
Kimber I, Basketter DA, Butler M, Gamer A, Garrigue JL, Newsome C, et al. 2003.
Classification of allergens according to potency: proposals. Food Chem Toxicol 41:17991809.
Kimber I, Dearman RJ, Betts CJ, Gerberick GF, Ryan CA, Kern PS, et al. 2006. The local
lymph node assay and skin sensitization: a cut-down screen to reduce animal requirements?
Contact Dermatitis 54:181-185.
Manetz TS, Meade BJ. 1999. Development of a flow cytometry assay for the identificationand differentiation of chemicals with the potential to elicit irritation, IgE-mediated, or T cellmediated hypersensitivity responses. Toxicol Sci. 48:206-17.
OECD. 2002. Guideline for the Testing of Chemicals 429 - Skin Sensitisation: Local Lymph
Node Assay. Paris: OECD.
Ryan CA, Chaney JG, Gerberick GF, Kern PS, Dearman RJ, Kimber I, Basketter DA. 2007.
Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency.
Cutan Ocul Toxicol 26:135-145.
Schneider K, Akkan Z. 2004. Quantitative relationship between the local lymph node assay
and human skin sensitization assays. Regul Toxicol Pharmacol 39:245-55.
Sailstad DM, Hattan D, Hill RN, Stokes WS. 2001. ICCVAM evaluation of the murine local
lymph node assay: I. The ICCVAM review process. Regul Toxicol Pharmacol 34:249-257.
Takeyoshi M. 2007. Promising endpoint and assay performance of non-radioisotopic local
lymph node assay based on BrdU incorporation. Sixth World Congress on Alternatives andAnimal Use in the Life Sciences. August 21-25, 2007. Tokyo, Japan.
Van Hoogstraten IM, Boos C, Boden D, Von Blomberg ME, Scheper RJ, Kraal G. 1993.
Oral induction of tolerance to nickel sensitization in mice. J Invest Dermatol 101:26-31.
Van Och FMM, Slob W, De Jong WH, Vandebriel RJ, Van Loveren H. 2000. A quantitative
method for assessing the sensitizing potency of low molecular weight chemicals using a local
lymph node assay: employment of a regression method that includes determination of theuncertainty margins. Toxicology 146:49-59.
Varani J, Perone P, Spahlinger DM, Singer LM, Diegel KL, Bobrowski WF, et al. 2008.
Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) inmonolayer culture for assessment of chemically induced skin damage. Toxicol Pathol
35:693-701.

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Young SS. 2007. Gaming the system: chaos from multiple testing. Institute of Mathematical
Statistics Bulletin 36:13

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Appendix A
Peer Review Panel Member Biosketches

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Panel Member Biosketches
Nathalie Alépée, Ph.D.
Dr. Alépée performed research leading to a Ph.D. in Medical Virology and Microbiology at
the Centre National de la Recherché Scientifique research institute, Gif sur Yvette, France.
She is currently the Global Pfizer Leader for photosafety, including the global portfolio
support and Associate Research Fellow in Investigative Toxicology, at Pfizer Global
Research and Development, Amboise, France. As a laboratory manager in the Molecular andCellular Toxicology Group with Pfizer, she implemented the Local Lymph Node Assay(LLNA) in the laboratory. She serves on the European Centre for the Validation of
Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC), representing theEuropean Federation of Pharmaceutical Industries Associations (EFPIA). She is also the
Pfizer representative to the European Partnership on Alternative to Animal Testing (EPAA),in two working groups; Identification of Opportunities, Including R&D (working group 2),
and Validation and Acceptance (working group 5). She served as a peer reviewer of the
reduced LLNA test protocol and prediction model for ESAC in 2007 and has been designatedas an ESAC peer reviewer for ECVAM's performance standards for the standard LLNA.
Anne Marie Api, Ph.D.
Dr. Api received a Ph.D. from Aston University in Birmingham, England and is currently
Vice President of Human Health Sciences at the Research Institute for Fragrance Materials(RIFM), as well as the Scientific Director. She is responsible for the human health scientificprogram, and the investigation and initiation of new research and testing projects for RIFM.
She is also Adjunct Assistant Professor at the University of Medicine and Dentistry of New
Jersey. She is a member of 10 professional organizations, including the American Contact
Dermatitis Society, the European Society of Contact Dermatitis, and the Society ofInvestigative Dermatology. She participated in the World Health Organization (WHO)International Workshop in Skin Sensitization in Chemical Risk Assessment held in Berlin,
Germany in 2006. She is author of over 100 publications and presentations relevant todermatology and dermatotoxicology.
Nancy Flournoy, M.S., Ph.D.
Dr. Flournoy received a M.S. degree in Biostatistics from the University of California at Los
Angeles, and a Ph.D. in Biomathematics from the University of Washington. She is Professor
and Chair of the Department of Statistics at the University of Missouri-Columbia. Her
research interests include adaptive designs, bioinformatics, chemometrics, clinical trials, andenvironmetrics. She has an extensive list of edited volumes and papers on statistical theory,
statistical genetics and immunology, epidemiology in immune suppressed subjects, clinical
trials for prevention and treatment of viral infection, transplantation biology and its effects ondigestion, lungs, eyes, mouth, and central nervous system, optimization of statisticalprocessing, and additional papers, interviews, and technical reports. She has editorial
responsibilities for numerous statistical journals, serves on numerous advisory boards, andnominating committees. She is a member and past Chair of the Council of Sections of theAmerican Statistical Association, and served in various other statistical, medical andtoxicological societies or programs as Chair or as a member of the Board of Directors. She isa former member of the Scientific Advisory Committee on Alternative ToxicologicalA-3

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Methods. She also served on the Expert Panels for the National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)
and the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) that evaluated the Revised Up-and-Down Procedure; the Current Validation
Status of In Vitro Test Methods for Identifying Ocular Corrosives and Severe Irritants; andFive In Vitro Pyrogen Test Methods.
Thomas Gebel, Ph.D.
Dr. Gebel received a Ph.D. in Toxicology from the University of Mainz and is certified as a
toxicologist by the German Society of Toxicology. His scientific interests are inbiomonitoring, genetic toxicology, environmental hygiene, and occupational toxicology. He
has published over 40 papers in peer-reviewed scientific journals. He is employed by theGerman Federal Institute for Occupational Safety and Health, and is an Associate Professor
at the University of Goettingen. Dr. Gebel is currently a member of the Organisation for
Economic Co-operation and Development (OECD) Globally Harmonized System ofClassification and Labeling of Chemicals (GHS) expert group on sensitization and head of
the German advisory committee on classification and labeling of existing substances andbiocides. Dr. Gebel also is head of the German Delegations to the United Nations Economic
and Social Council Sub-Committee of Experts on the GHS, and to the OECD Task Force on
Harmonisation of Classification and Labeling. He participated in the WHO International
Workshop in Skin Sensitization in Chemical Risk Assessment held in Berlin, Germany in2006.
Sidney Green Ph.D., F.A.T.S.
Dr. Green received a Ph.D. in Biochemical Pharmacology from Howard University. His
research interests include toxicology, mutagenic assay systems, and alternatives to animals in
toxicology. He is currently Graduate Professor of Pharmacology at Howard University and afaculty member at the Centers for Alternatives to Animal Testing at the Johns Hopkins
University School of Public Health. Previously, he has been Director of the Department of
Toxicology at Covance Laboratories Inc. and the Director of the Division of ToxicologicalResearch at the U.S. Food and Drug Administration (FDA). Dr. Green is a Fellow of the
Academy of Toxicological Sciences (F.A.T.S.). He has served on numerous expert panels
and committees. He was a participant in an International Workshop organized by ICCVAM
and NICEATM on In Vitro Methods for Assessing Acute Systemic Toxicity in 2000. He
served on the ICCVAM/NICEATM Expert Panels that evaluated the Corrositex® Test
Method for Assessing Dermal Corrosivity Potential of Chemicals, and In Vitro Test Methods
for Identifying Ocular Corrosives and Severe Irritants. He is a former member of the
ICCVAM Advisory Committee on Alternative Toxicological Methods (ACATM) and of
SACATM. He has authored over 60 publications for peer-reviewed journals.
Kim Headrick, B.Admin., B.Sc.
Kim Headrick received Bachelor of Administration and B.Sc. degrees from the University ofOttawa, Canada. She is currently International Harmonization and Senior Policy Advisor for
Health Canada, and Chair of the UN Sub-Committee of Experts on GHS. She manages theoverall strategy for the implementation of the GHS in Canada. She was awarded the Queen
Elizabeth Commemorative Golden Jubilee Medal in 2002, which focuses on theachievements of people who, over the past 50 years, have created the Canada of today. She is
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a member of the OECD Task Force on Harmonization of Classification and Labelling and theOECD Expert Group Meeting on Sensitization Hazards.
Dagmar Jírová, M.D., Ph.D.
Dr. Jírová received a Ph.D. from the Medical Faculty of Hygiene at Charles University in
Prague. She is currently the Head of the Reference Center for Cosmetics, and Head of
National Reference Laboratory for Experimental Immunotoxicology at the National Instituteof Public Health in the Czech Republic. Her main responsibilities include safety assessmentof consumer products, particularly cosmetics and their ingredients, performance of
toxicological methods in vivo in animals, human patch testing for local toxicity assessment,
and introduction of in vitro techniques for screening of toxicological endpoints using cell andtissue cultures. She represents the Czech Republic in the Standing Committee on Cosmeticsof the European Commission. She is an ESAC-ECVAM member and was involved in Peer
Review Panel for Skin Irritation Validation Study and LLNA test protocol and prediction
model. She is author of more than 100 publications and presentations relevant todermatotoxicology including a recent presentation at the 6th World Congress on Alternatives& Animal Use in the Life Sciences, held in Tokyo, 2007, titled “Comparison of Human Skin
Irritation and Photoirritation Patch Test Data with Cellular in vitro Assays and Animal invivo data”.
David Lovell, Ph.D., B.Sc. (Hons), F.S.S., FIBiol, CStat, CBiol
Dr. Lovell received a Ph.D. from the Department of Human Genetics and Biometry,
University College, London. He is currently Reader in Medical Statistics at the Postgraduate
Medical School at the University of Surrey. Previously, he was Associate Director and Headof Biostatistics support to Clinical Pharmacogenomics at Pfizer Global Research andDevelopment in Sandwich, Kent providing data management and statistical support topharmacogenetics and genomics. He joined Pfizer in 1999 as the Biometrics Head of ClinicalPharmacogenetics. Before joining Pfizer, Dr. Lovell was the Head of the Science Division at
BIBRA International, Carshalton, which included Molecular Biology, Genetic Toxicology,
Biostatistics and Computer Services. At BIBRA, Dr. Lovell managed the statistical andcomputing group providing specialized statistical support to BIBRA’s Clinical Unit andcontract research work. He conducted and managed research programs on genetics, statistics
and quantitative risk assessment for the European Union (EU) and U.K. Government
Departments. His research interests at BIBRA were in the use of mathematical and statisticalmethods together with genetic models in the understanding of toxicological mechanisms andrisk assessment problems. Dr. Lovell had previously been a Senior Research Officer with the
U.K. Medical Research Council (MRC) Experimental Embryology and Teratology Unit, avisiting Postdoctoral Fellow at the National Institute of Environmental Health Sciences(NIEHS) in North Carolina, U.S., a Geneticist at the MRC Laboratories, Carshalton, and a
Research Assistant in Cytogenetics at Birmingham University. He has acted as a consultant toa number of organizations, has considerable experience of working with Regulatory Authorities,
has many publications related to his work and has wide experience of making presentations to awide range of audiences. He is a member of the U.K. Government’s advisory Committee on
Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM) andthe Independent Scientific Advisory Committee for Medicines and Healthcare Products
Regulatory Agency database research. He served on the NICEATM-ICCVAM Expert Panels

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that evaluated the Frog Embryo Teratogenesis Assay - Xenopus, In Vitro Test Methods for
Identifying Ocular Corrosives and Severe Irritants, and Five In Vitro Pyrogen Test Methods.
Michael Luster, Ph.D.
Dr. Luster received a Ph.D. in Immunology from Loyola University of Chicago. He wasformerly Chief, Toxicology and Molecular Biology Branch, Health Effects Laboratory
Division, National Institute for Occupational Safety and Health (NIOSH), and currently
serves as a senior advisor to the Director of the Health Effects Laboratories and the staff of
Toxicology and Molecular Biology Branch at NIOSH. Program areas include neuroscience,
dermatology, molecular carcinogenesis, molecular epidemiology, molecular toxicology,
molecular epidemiology, and inflammation/immunotoxicology. In addition, Dr. Luster
conducts basic and applied research in immunotoxicology including its application in risk
assessment. Current research activities include molecular epidemiology studies of genetic
polymorphism involved in workplace-related diseases and experimental studies involving
occupational allergic rhinitis. Dr. Luster is also working with various staff at the U.S.
Environmental Protection Agency (EPA) through the Risk Assessment Forum to develop
immunotoxicity testing guidelines. He also directed two studies for the NTP on the
Toxicology and the Carcinogenesis of Promethazine and Ortho-phenylphenol, in 1990 and
1986, respectively. He is a co-author of over 300 publications in peer-reviewed journals.
Howard Maibach, M.D.
Dr. Maibach received an M.D. from Tulane University. He is currently a professor in the
Department of Dermatology at the University of California, San Francisco (USCF), where heis also Chief of the Occupational Dermatology Clinic. In his 35 years at UCSF, Dr. Maibach
has written and lectured extensively on dermatotoxicology and dermatopharmacology. His
current research programs include defining the chemical-biologic faces of irritant dermatitis
and the study of percutaneous penetration. Dr. Maibach served on the 1998 ICCVAM Peer
Panel that evaluated the Murine LLNA. Dr. Maibach has been on the editorial boards of over
30 scientific journals and is a member of 19 professional societies including the American
Academy of Dermatology, San Francisco Dermatological Society, and the International
Commission on Occupational Health. He has co-authored over 1500 publications related todermatology.
James McDougal, Ph.D., F.A.T.S.
Dr. McDougal earned a Ph.D. in Pharmacology/Toxicology at the University of Arizona. He
is currently Professor and Director of Toxicology Research in the Department ofPharmacology and Toxicology at Wright State University’s Boonshoft School of Medicine.
Prior to his appointment at Wright State, he worked in the Air Force toxicology researchorganization for about 17 years. He has active skin research programs related to dermal
pharmacokinetics, molecular biology of skin irritation, dermal risk assessment, andbiologically-based mathematical modeling. He has served on many national committees,
published more than 75 manuscripts, and consults for a wide variety of government andindustry organizations. Dr. McDougal is a member of the National Academy of Sciences(National Research Council) Committee on Toxicology and the American Congress ofGovernmental Industrial Hygienists Threshold Limit Value Committee for Chemical
substances. Dr. McDougal is also past president of the Dermal Toxicology Specialty Sectionof the Society of Toxicology.
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Michael Olson, Ph.D., A.T.S.
Dr. Olson received a Ph.D. in Toxicology from the University of Arkansas for Medical
Sciences, with dissertation research conducted at the FDA National Center for ToxicologicalResearch. Following graduate training, he served as NIEHS National Research Service
Award Post-doctoral Fellow in the Department of Pharmacology, School of Medicine University of North Carolina. Currently he is Director, Occupational Toxicology, Corporate
Environment Health and Safety for GlaxoSmithKline. Dr. Olson is a Fellow of the Academyof Toxicological Sciences (A.T.S.). His research interests include mechanisms of chemicallyinduced toxicity; genetic toxicity; xenobiotic metabolism; alternative methods in toxicology;
hazard evaluation, risk assessment, and communication. Dr. Olson has authored a number ofpeer-reviewed manuscripts and book chapters in these areas as well as preparing manyoccupational health effects reviews for pharmaceutical active ingredients, isolated
intermediates, and associated chemicals. He has served as an editorial board member and ad
hoc referee for numerous toxicology and biosciences journals. In addition, he has worked asa Visiting Scientist, EPA, as well as advisor to EPA Risk Assessment Forum, U.S. National
Institutes of Health (NIH) (Toxicology Study Section I), U.S. Air Force, Transportation
Research Board, and the National Research Council - National Academy of Sciences. A
member of several biomedical professional societies, Dr. Olson has served in elective andappointed positions in the Society of Toxicology, including Chairman of the Society of
Toxicology (SOT) Occupational Health Specialty Section.
Raymond Pieters, Ph.D.
Dr. Pieters received a Ph.D. at Utrecht University and is currently an Associate Professor at
the Institute for Risk Assessment Sciences, and Group Leader for Immunotoxicology at that
institution. In 2007, he presented a paper on Development of Strategies to Assess Drug
Hypersensitivity at the Congress of the European Societies of Toxicology. He was involvedin the development of the Reporter Antigen Popliteal Lymph Node Assay, an assay to assess
the immunomodulating potential of chemicals, which enables differentiation between
immunosensitizing chemicals (sensitizers), immunostimulating chemicals (irritants), andchemicals that have no apparent immunological effects. He has published over 70 papers on
sensitization and other subjects in immunotoxicology in peer-reviewed journals, including areview article, Murine Models of Drug Hypersensitivity, in 2005.
Jean Regal, Ph.D.
Dr. Regal received a Ph.D. in Pharmacology from the University of Minnesota. She iscurrently a Professor in the Department of Pharmacology, Department of Biochemistry &Molecular Biology and Associate Dean of Faculty Affairs, Medical School Duluth,
University of Minnesota. Her current research is focused on respiratory allergy, especiallyasthma. She has served on multiple NIH review panels regarding asthma, as an
immunotoxicologist in 2000 for an Institute of Medicine Committee on Health Effects
Associated with Exposures Experienced during the Persian Gulf War, as well as on the 1998
ICCVAM Peer Panel that evaluated the Murine LLNA. In 2007 she served as an ad hoc
reviewer for the NTP Board of Scientific Counselors for two nominations: Artificial Butter
Flavoring Mixture & O-phthalaldehyde, at NIEHS. Also in 2007, she served on an NIEHS
Center in Environmental Toxicology pilot project program for the University of Texas
Medical Branch at Galveston. She is currently Vice-President-elect of the Immunotoxicology

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Specialty Section of SOT and Associate Editor of the Journal of Immunotoxicology. Dr.
Regal has authored over 50 research articles and reviews in peer-reviewed journals and holds
two patents on pulmonary administration of sCR1 and other complement inhibitory proteins.
Jonathan Richmond, B.Sc. (Hons) Med.Sci., MB ChB, FRCSEd, FRMS
Dr. Richmond received a Bachelor of Science in Medical Science with Honors (B.Sc. [Hons]Med.Sci.) and Bachelor of Medicine and Bachelor of Surgery (MB ChB) degrees with
Distinction in Medicine and Therapeutics from Edinburgh University. Presently, he is headof the Animals Scientific Procedures Division at the Home Office. He is a Fellow of theRoyal College of Surgeons of Edinburgh (FRCSEd) and a Fellow of the Royal Society of
Medicine (FRMS). Other appointments include convener of the U.K. interdepartmentalgroup on the 3Rs, board member U.K. National Centre for the 3Rs, convener of theInternational Standards Organization Technical Corrigendum 194/Working Group 3(Biocompatibility of Medical Device Materials), and member of related expert working
groups. He is a former member of the EU Committee on Scientific and Technical Progress
and past Chairman of the European Commission Technical Expert Working Group on ethicalreview. He served as chair of the peer review panel for the reduced LLNA test protocol andprediction model for ESAC in 2007 and has been designated as an ESAC peer reviewer for
ECVAM's performance standards for the standard LLNA. He served on the
ICCVAM/NICEATM Expert Panel that evaluated Five In Vitro Pyrogen Test Methods. He
has a variety of publications in peer-reviewed journals and national and international
meetings, on the principles and practice of surgery, regulation of biomedical research,principles of humane research, bioethics, and public policy.
Peter Theran, V.M.D.
Dr. Theran holds a Doctor of Veterinary Medicine degree from the University ofPennsylvania. He has had many years of experience both as a veterinary internal medicine
specialist at the Massachusetts Society for Prevention of Cruelty to Animals’ Angell
Memorial Animal Hospital in Boston, and as the director of Boston University Medical
Center's Laboratory Animal Science Center. He presently serves on a number of government
committees as an animal welfare member, and is a member of the Board of Directors of theInstitute for In Vitro Sciences in Gaithersburg, MD and Chimp Haven in Shreveport,
Louisiana. He served on the NICEATM-ICCVAM Expert Panels that evaluated the In Vitro
Test Methods for Identifying Ocular Corrosives and Severe Irritants, and Five In Vitro
Pyrogen Test Methods. He is a former member of ACATM and SACATM. He is presently
working as a consultant.
Stephen Ullrich, Ph.D.
Dr. Ullrich received a Ph.D. in Microbiology from Georgetown University. He is currently
the Dallas/Fort Worth Living Legends Professor, and Professor of Immunology at the
University of Texas, M.D. Anderson Cancer Center, where he is also Associate Director, The
Center for Cancer Immunology Research. He is also a member of the Animal Research
Strategic Advisory Committee. He has served numerous national review committees andpanels, including the 1998 ICCVAM Peer Panel that evaluated the Murine LLNA. Dr.
Ullrich has authored over 75 peer-reviewed publications, over 30 invited articles, and heholds four patents in the U.S., E.U., and Australia for a UV-induced Immunosuppressive
Substance. He is the past President of the American Society for Photobiology.
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Michael Woolhiser, Ph.D.
Dr. Woolhiser received a Ph.D. in Pharmacology and Toxicology from the Medical College
of Virginia at Virginia Commonwealth University. He is a specialist in immunotoxicology
and is currently a toxicologist for the Dow Chemical Company where he serves as aTechnical Leader for Immunotoxicology, and Polyurethane Business Toxicology Consultant.
Dr. Woolhiser is also an Adjunct Professor at the Center for Integrative Toxicology,
Michigan State University. He is a member of the Program Committee of the Society of
Toxicology's Immunotoxicology Specialty Section. He has served on numerous working
groups, including an LLNA Expert Working Group under the European Crop Protection
Agency's Toxicology Expert Group, a European Centre for Ecotoxicology and Toxicology ofChemicals LLNA Task Force. He has authored 29 peer-reviewed publications.
Takahiko Yoshida, M.D., Ph.D.
Dr. Yoshida earned his M.D. and a Ph.D. in Medical Science from Tokai University. He iscurrently Professor in the Department of Health Science at Asahikawa Medical College.
Prior to this appointment, he held the posts of Instructor, Assistant Professor and Associate
Professor at the Tokai University School of Medicine. He has also been a Guest Researcher
at NIEHS. He has also worked as an occupational physician for major Japanese corporations,
including Toyota and Sony. Dr. Yoshida’s research interests include occupational health,public health, environmental health and preventative medicine. He is a member of theInternational Congress of Occupational Health, the Japanese Society of Hygiene, theJapanese Society of Immunotoxicology, the Japanese Society of Clinical Ecology, and the
SOT.

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Appendix B
Questions for the Peer Review Panel
B1
B2
B3
B4
B5
B6
B7

LLNA Limit Dose Procedure ..............................................................................B-3
LLNA for Testing Aqueous Solutions, Metals, and Mixtures..........................B-11
Non-Radioactive LLNA Protocol - LLNA: DA Test Method ..........................B-17
Non-Radioactive LLNA Protocol - LLNA: BrdU-FC Test Method ................B-25
Non-Radioactive LLNA Protocol - LLNA: BrdU-ELISA Test Method ..........B-33
Draft ICCVAM Performance Standards for the LLNA ..................................B-41
Use of the LLNA for Potency Determinations..................................................B-47

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Appendix B1
Questions for the Peer Review Panel: LLNA Limit Dose Procedure

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Instructions for the Peer Review Panel: LLNA Limit Dose Procedure
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the murine local lymph node assay (LLNA) limit dose procedure as a substitute forthe traditional LLNA for assessing the allergic contact dermatitis potential of chemicals andother substances.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA limit dose procedure. You are first asked to review the information in the draft
ICCVAM LLNA limit dose procedure Background Review Document (BRD) forcompleteness, and to identify any errors or omissions of existing relevant data or informationthat should be included. You are then asked to evaluate the information in this BRD todetermine the extent to which each of the applicable criteria for validation and acceptance of
toxicological test methods (ICCVAM 20031) have been appropriately addressed for the
proposed use of the LLNA limit dose procedure. Adequate validation2 is a prerequisite for atest method to be considered for use in regulatory decision-making by U.S. Federal agencies.
This validation process characterizes the usefulness and limitations of a test method for its
intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA limit dose procedure (i.e., the proposed test method use, the proposed recommended
standardized protocol, the proposed test method performance standards, and any proposed
additional studies) and comment on whether the recommendations are supported by the
information provided in the draft LLNA limit dose procedure BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA limit dose procedure.
These include questions prepared by the ICCVAM IWG to ensure that the assessment
provides adequate information to facilitate U.S. Federal agency decisions on the regulatory
acceptability of this test method, and/or adequate guidance for organizations that may be
involved in conducting or supporting further development, standardization, and/or validation.
The overall question to consider is whether the validation status of the LLNA limit dose
procedure has been adequately characterized for its intended purpose, and is it sufficiently
accurate and reliable to be used for the identification of sensitizing substances and nonsensitizing substances in place of the traditional LLNA procedure when there is not a needfor dose response information, in order to reduce the number of animals required for suchtesting.
I.

Questions to the Panel: Review for Errors and Omissions

1

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
2
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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1. Are there any errors or omissions of existing relevant data or information thatshould be included in the draft BRD?
II.

Questions to the Panel: LLNA Limit Dose Procedure Draft BRD
1. For the proposed LLNA limit dose procedure, ICCVAM recommends that the
number of animals used in each group should be the same as that recommended
by ICCVAM for the traditional LLNA based on its 1998 evaluation of the LLNA,
and that individual animal data should be collected and reported (ICCVAM,
1999). Do you agree that these are appropriate provisions for the limit dose
procedure? Please explain your answer.
2. Do you consider the traditional LLNA database representative of a sufficient
range of chemical classes and physical chemical properties that it would beapplicable to any of the types of chemicals and products that are typically tested
for skin sensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using thelimit dose procedure? What chemicals or products should be evaluated to fill thisdata gap? Please explain your answer.
3. While coding of chemicals is recommended for prospective studies, thisevaluation is based on a retrospective evaluation of existing data, most of which
was not generated using coded chemicals to reduce the potential for bias. Does thelack of coding of test substances adversely impact or bias the current evaluation?
Please explain your answer.
4. For some substances submitted using the traditional LLNA test method, it was notpossible to confirm whether the data were generated using pooled animal data foreach dose group (as allowed in Test Guideline [TG] 429 of the Organisation for
Economic Co-operation and Development [OECD]). ICCVAM (1999), Dean etal. (2001), and EPA (2003) recommend the use of statistical analyses to help
interpret LLNA study results, which necessitates data collected at the level of theindividual animal, while Cockshott et al. (2006) reported that using individual
animal data allowed for technical problems during an experiment to be identified.
Considering this, should the analysis of the performance of the LLNA limit dose
procedure against the traditional LLNA be limited to data from studies that can be
confirmed as using individual animal data collection procedures? What impact
might the inclusion of pooled animal data have on the accuracy analysis of the
LLNA limit procedure? Please explain your answer.
5. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA limit dose procedure beenadequately evaluated and compared to the traditional LLNA (refer also to Table
6-1 of the draft ICCVAM BRD)? If not, what other analyses should be
performed?
6. There were five substances for which the highest concentration tested produced
an SI of less than 3.0, while lower concentrations of these substances produced an
SI of greater than 3.0 (see Table 6-2 of the draft ICCVAM BRD). These

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substances are classified as “false negatives” compared to what was obtained in
the traditional LLNA. Can you identify any characteristics associated with theseor other substances that might signal that this type of abnormal dose response
might occur, and therefore using the LLNA limit dose procedure would not beappropriate? Please explain your answer.
7. Does the BRD adequately characterize the usefulness and limitations of the
LLNA limit dose procedure based on the accuracy analyses? If not, what
additions or changes should be made to the current usefulness and limitations?
Please explain your answer.
8. Is it appropriate to assume that the intra- and inter-laboratory reproducibility of
the LLNA limit dose procedure and the traditional LLNA will be similar, basedon the fact that they use identical protocols with the exception of the number ofdoses used? Do you agree? Does reducing the number of test substances dosegroups from three to one reduce the reliability of the assay? Please explain youranswer.
9. For some studies included in the draft BRD, it was not possible to determine
whether or not they had been conducted in accordance with Good Laboratory
Practice (GLP) guidelines. Original records for some of the non-GLP studies
included in this evaluation could not be obtained. As a result, an independent
audit could not be conducted to confirm that the reported data is the same as thedata recorded in laboratory notebooks. Neither was it possible to obtain the resultsof GLP audits for all studies conducted in accordance with GLP guidelines.
Considering this, should the results of these studies (all of which are currently
included) be excluded from any of the performance analyses? Please explain youranswer.
10. Based on the draft BRD, have all the relevant data identified in published or
unpublished studies conducted using the traditional LLNA been adequately
considered? If not, what other traditional LLNA data needs to be considered andhow can it be obtained?

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III.

May 2008

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA Limit Dose Procedure
1. Do the available data support the ICCVAM draft recommendations for the LLNA
limit dose procedure in terms of the proposed test method usefulness andlimitations? If not, what recommendations would you make? Please explain youranswer.
•Should the LLNA limit dose procedure be routinely recommended for thehazard identification of skin sensitizing chemicals when potency information
is not required? Please explain your answer.

•

If potency information is required, should the LLNA limit dose procedure be
routinely recommended as the initial test to identify sensitizers before
conducting the traditional LLNA as a way to further reduce animal use, sincenegative results would not require further testing? Please explain your answer.

•

Based on the existing database, there is a false negative rate of 1.6% (5/313
positive compounds) for the LLNA limit dose approach compared to theresults obtained in the traditional LLNA. Do you consider that this isadequately addressed by the proposed cautionary language and weight ofevidence consideration for negative substances? Please explain your answer.

2. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA limit dose procedure in terms of the proposed test method
standardized protocol? If not, then what recommendations would you make?
Please explain your answer.
•

The recommended ICCVAM protocol (ICCVAM 1999; Dean et al. 2001;
EPA 2003), as well as OECD TG 429, specifies that the highest dose tested
should be the highest soluble concentration that does not induce systemic
toxicity and/or excessive skin irritation. However, Kimber et al. (2006)
concluded that negative results obtained from studies where the highest
concentration tested was below 10% should be considered invalid, andadopted a 10% application concentration as a threshold of confidence forcategorization of a chemical as being negative while noting that the figure
should not be considered as inviolable. Are the data presented in the draft
BRD (i.e., 5/313 positive substances in the NICEATM database were negative
at concentrations ≤10%, but were positive at higher concentrations) adequate
to conclude that this threshold concentration is not appropriate? If a negative
result was obtained for a test substance in a study where the highest
concentration that could be tested (based on systemic toxicity or excessive
local irritation, as described in ICCVAM [1999], Dean et al. [2001], and EPA[2003]) was <10%, should additional testing be required? Do you agree thatthe current approach for selecting the “limit” dose is appropriate or do you
conclude that there is a threshold concentration for the LLNA at which a
negative result could always be considered as an acceptable result? If so, whatis that concentration? Please explain your answer.

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3. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA limit dose procedure in terms of the proposed future studies? If not,then what recommendations would you make? Please explain your answer.

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Appendix B2
Questions for the Peer Review Panel:
LLNA for Testing Aqueous Solutions, Metals, and Mixtures

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Instructions for the Peer Review Panel: LLNA for Testing Aqueous Solutions, Metals,
and Mixtures
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is currently updating theoriginal validation report of the LLNA (ICCVAM 1999) based on a comprehensive review ofavailable data and information regarding the current validity of the LLNA for assessing theskin sensitizing potential of mixtures, metal compounds, and substances tested in aqueous
solutions. The information is based on a retrospective review of LLNA data derived from a
database of over 500 substances (including mixtures) tested in the LLNA and builds on theprevious ICCVAM evaluation of the LLNA, which was based on 209 substances (ICCVAM
1999). In the original ICCVAM report, the performance of the LLNA was compared to 1) the
results from guinea pig tests and 2) information about sensitizers in humans (e.g., humanmaximization test [HMT] results, substances used in human repeat insult patch test [HRIPT],
clinical data), where available. This addendum updates the LLNA performance analyses for
mixtures, metal compounds, and substances tested in aqueous solutions when compared tohuman and guinea pig results.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA. You are first asked to review the information in the draft Addendum to the
ICCVAM (1999) report for completeness, and to identify any errors or omissions of existing
relevant data or information that should be included. You are then asked to evaluate theinformation in this Addendum to determine the extent to which each of the applicable criteria
for validation and acceptance of toxicological test methods (ICCVAM 20033) have been
appropriately addressed for the proposed use of the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions. Adequate validation4 is a prerequisite for atest method to be considered for use in regulatory decision-making by U.S. Federal agencies.
This validation process characterizes the usefulness and limitations of a test method for its
intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA (i.e., the proposed test method use, the proposed recommended standardized protocol,
the proposed test method performance standards, and any proposed additional studies) andcomment on whether the recommendations are supported by the information provided in thedraft Addendum.
The questions relating to the draft Addendum that must be addressed are provided in
Sections I and II of this guidance, while Section III contains questions relating to the draft
ICCVAM test method recommendations on the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions.

3

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
4
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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These include questions prepared by the ICCVAM IWG to ensure that the assessment
provides adequate information to facilitate U.S. Federal agency decisions on the regulatory
acceptability of this test method, and/or adequate guidance for organizations that may be
involved in conducting or supporting further development, standardization, and/or validation.
The overall question to consider is whether the validation status of the LLNA for testing
mixtures, metal compounds, and substances in aqueous solutions has been adequately
characterized, and is it sufficiently accurate and reliable to be used for the identification ofsensitizing substances based on a comparison to either human or guinea pig responses.
I.

Questions to the Panel: Review for Errors and Omissions
1. In the draft Addendum, are there any errors that need to be corrected or omissionsof existing relevant data or information that should be included?

II.

Questions to the Panel: Updated LLNA Applicability Domain Addendum
1. Do you consider the database of substances evaluated representative of a
sufficient range of mixtures, metal compounds, and substances in aqueous
solutions that are typically tested for skin sensitization potential? Please explain
your answer.
2. For the purpose of this evaluation, aqueous solutions were defined by the
proportion of water (at least 20%) (i.e., substances or mixtures that were tested in
an aqueous or an organic:aqueous vehicle were labeled as aqueous solutions). Do
you consider this to be an appropriate criterion for defining aqueous solutions? If
not, what would be more appropriate? Please explain your answer.
3. While coding of chemicals is recommended for prospective studies, thisevaluation is based on a retrospective evaluation of existing data, most of which
was not generated using coded chemicals to reduce bias. Does the lack of codingof test substances adversely impact or bias the current evaluation? Please provide
a rationale for your answer.
4. For some substances submitted using the LLNA, it was not possible to confirm
whether the data were generated using pooled animal data for each dose group (asallowed in Test Guideline [TG] 429 of the Organisation for Economic Cooperation and Development [OECD]) rather than individual animal data (asrecommended in the ICCVAM 2001 protocol)? Cockshott et al. (2006) reported
that using individual animal data allowed for technical problems during anexperiment to be identified. Considering this, should the analysis of theperformance of the LLNA for testing mixtures, metal compounds, and substancesin aqueous solutions be limited to data from studies that can be confirmed asusing individual animal data collection procedures? What impact might theinclusion of pooled animal data have on the accuracy analysis included in Section
5.0 of the draft Addendum? Please explain your answer.
5. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions been adequately evaluated andcompared to the human and guinea pig (refer also to Section 5.0 of the draft

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Addendum)? If not, what other analyses should be performed? Please explain
your answer.
6. When multiple LLNA studies were available for the same substance, the majority
call (where all studies used the same vehicle and the same concentration range)
was used to assign an overall classification for the purposes of the accuracy
analysis. For example, if chemical X was tested 5 times and was positive in 3
studies and negative in two, the overall classification was positive. Do you agreewith the approach to assigning overall classifications? If not, how would you
propose that this be accomplished? Please explain your answer.
7. Does the Addendum adequately characterize the usefulness and limitations of the
LLNA for testing mixtures, metal compounds, and substances in aqueous
solutions based on the accuracy analyses? If not, what additions or changes
should be made to the current usefulness and limitations? Please explain youranswer.
8. For some studies included in the draft Addendum, it was not possible to determine
whether or not they had been conducted in accordance with Good Laboratory
Practice (GLP) guidelines. Neither was it possible to obtain the results of GLP
audits for all studies conducted in accordance with GLP guidelines. Please discuss
what impact this lack might have on the evaluation of the LLNA for testing
mixtures, metal compounds, and substances in aqueous solutions and whether
such studies should be excluded from any analysis.
9. As described in the draft Addendum, original records for some of the non-GLP
studies included in this evaluation could not be obtained. As a result, an
independent audit could not be conducted to confirm that the reported data is thesame as the data recorded in laboratory notebooks. Considering this, should theresults of these studies (all of which are currently included) be excluded from anyof the performance analyses? Please explain your answer.
10. Based on the draft Addendum, have all the relevant data identified in published or
unpublished studies conducted using the LLNA for testing mixtures, metalcompounds, and substances in aqueous solutions been adequately considered? If
not, what other studies should to be considered?
III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA for Testing Aqueous Solutions, Metals, and Mixtures
1. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA with regard to testing mixtures, metal compounds, and substancesin aqueous solutions in terms of the proposed test method usefulness andlimitations? Please explain your answer. If not, what recommendations would you
make?
2. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed test method standardized protocol? Please
explain your answer. If not, then what recommendations would the Panel make?

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3. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed future studies? Please explain youranswer. If not, then what recommendations would you make?

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Appendix B3
Questions for the Peer Review Panel:
Non-Radioactive LLNA Protocol - LLNA: DA Test Method

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Instructions for the Peer Review Panel: Non-Radioactive LLNA Protocol: LLNA: DA
Test Method
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program (NTP) Interagency Center for
the Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating thevalidation status of the LLNA: DA (Local Lymph Node Assay-Daicel adenosine triphosphate[ATP]) for assessing the allergic contact dermatitis (ACD) potential of chemicals and othersubstances. This test method, developed by Daicel Chemical Industries, Ltd. (Tokyo, Japan),is a non-radiolabeled version of the traditional LLNA, and is based on measuring levels of
ATP in the auricular lymph nodes as an indicator of increased cell proliferation.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA: DA. You are first asked to review the information in the draft ICCVAM
LLNA: DA Background Review Document (BRD) for completeness, and to identify any
errors or omissions of existing relevant data or information that should be included. You arethen asked to evaluate the information in the BRD to determine the extent to which each of
the applicable criteria for validation and acceptance of toxicological test methods (ICCVAM
20035) have been appropriately addressed for the proposed use of the LLNA: DA. Adequate
validation6 is a prerequisite for a test method to be considered for use in regulatory decisionmaking by U.S. Federal agencies. The validation process characterizes the usefulness andlimitations of a test method for a specific intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA: DA (i.e., the proposed test method use, the proposed recommended standardized
protocol, the proposed test method performance standards, and any proposed additional
studies) and comment on whether the recommendations are supported by the information
provided in the draft LLNA: DA BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA: DA.
These include questions prepared by the ICCVAM Immunotoxicity Working Group (IWG)
to ensure that the assessment provides adequate information to facilitate U.S. Federal agency
decisions on the regulatory acceptability of this test method. The questions are also intended
to obtain guidance that will be helpful to federal agencies and other organizations that may
be involved in conducting or supporting further development, standardization, and/or
validation studies.
The overall question to consider is whether the validation status of the LLNA: DA has beenadequately characterized for its intended purpose, and is it sufficiently accurate and reliable

5

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
6
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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to be used for the identification of sensitizing substances and non-sensitizing substances inplace of the traditional LLNA procedure.
I.

Questions to the Panel: Review of the Draft LLNA: DA BRD for Errors andOmissions
1. In the draft LLNA: DA BRD, are there any errors in the BRD that should becorrected, or omissions of existing relevant data or information that should be
included?

II.

Questions to the Panel: Draft LLNA: DA BRD
1. Test Method Protocol
i. The traditional LLNA protocol (ICCVAM 1999; Dean et al. 2001)
recommends a minimum of five successfully treated animals per dose group.
Current validation of the LLNA: DA was performed using four animals perdose group. What impact might using fewer mice have on the accuracy
analysis of the LLNA: DA? Please explain your answer.
ii. The data generated for the substances analyzed in the LLNA: DA test methodcome from auricular lymph nodes that were pooled across mice in each dosegroup rather than being analyzed on an individual animal data. What impact
might the inclusion of pooled animal data have on the accuracy analysis of the
LLNA: DA? Please explain your answer.
iii. The LLNA: DA differs from the traditional LLNA in the treatment scheduleand by including a pre-treatment with 1% SLS prior to application of the test
substance. Do you consider these changes to be appropriate? Please explain
your answer.
2. Substances Used for the Validation Studies
i. Do you consider the LLNA: DA database representative of a sufficient rangeof chemical classes and physicochemical properties that it would be applicable
to any of the types of chemicals and products that are typically tested for skinsensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using
the LLNA: DA? What chemicals or products should be evaluated to fill thisdata gap? Please explain your answer.
3. Test Method Accuracy
i. The current accuracy analysis is based on overall concordance with thetraditional LLNA. Accuracy statistics compared to the guinea pig tests andhuman data/experience have also been provided. Are these comparisonsappropriate for assessing the accuracy of the LLNA: DA? Please explain youranswer.
ii. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: DA been adequately evaluatedand compared to the traditional LLNA (refer also to Table 6-1 of the draft

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ICCVAM BRD)? If not, what other analyses should be performed? Please
explain your answer.
iii. There was one substance (2-mercaptobenzothiazole) that produced a “false
negative” response compared to the traditional LLNA when tested using the
LLNA: DA. The mean EC3 in the traditional LLNA for this substance is 2.5(n=2), it is positive in both the guinea pig and human, and has "high" peptide
reactivity as per Gerberick et al. (2007). Can you identify any characteristics
associated with this or similar substances, compared to the correctly identified
sensitizers, that might signal that this type of discordant response might occur,and therefore using the LLNA: DA to test such substances would not beappropriate or that negative results for such substances should indicate a needfor confirmatory testing? Please explain your answer.
iv. There was one substance (benzalkonium chloride) that produced a “false
positive” response compared to the traditional LLNA and guinea pig testwhen tested using the LLNA: DA. Can you identify any characteristics
associated with this or similar substances, compared to the correctly identified
non-sensitizers that might signal that this type of discordant response mightoccur, and therefore using the LLNA: DA to test such substances would not
be appropriate, or that positive results for substances with such properties may
warrant additional testing? Please explain your answer.
4. Test Method Reliability (Intra- and Inter-laboratory reproducibility)
i. Has the intralaboratory reproducibility of the LLNA: DA been adequately
evaluated and compared to the traditional LLNA (refer also to Table 7-1 ofthe draft LLNA: DA BRD)? If not, what other analyses should be performed?
Are any limitations apparent based on this intralaboratory reproducibility
assessment? Please explain your answer.
ii. Has the interlaboratory reproducibility of the LLNA: DA been adequately
evaluated and compared to the traditional LLNA (refer also to Tables 7-2 and7-3 of the draft LLNA: DA BRD)? If not, what other analyses should be
performed? Are any limitations apparent based on this interlaboratory
reproducibility assessment? Please explain your answer.
iii. The draft LLNA: DA BRD analyzes data from two interlaboratory validation
studies that used coded substances, as well as an intralaboratory validation
study with 31 substances that were not coded. Does the lack of coding of test
substances adversely impact or bias the current evaluation? In addition, it
appears that the lead laboratory established the dose levels tested in the twointerlaboratory validation studies and the participating laboratories did not
determine their own dose levels for testing. Does this adversely impact or bias
the current evaluation? Please explain your answer.
5. Data Quality
i. The studies evaluated in the draft BRD for the LLNA: DA were not conductedin accordance with Good Laboratory Practices (GLP) guidelines although
there were reportedly done in laboratories that conduct GLP studies and were

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conducted "in the spirit" of GLP (K. Idehara, personal communication).
Please discuss what impact this might have on the evaluation of the LLNA:
DA.
ii. The original records for these studies were requested but have not yet beenobtained. As a result, an independent audit could not be conducted to confirm
that the reported data is the same as the data recorded in laboratory notebooks.
Should any recommendations from ICCVAM be contingent upon thecompletion of such an audit and findings that there were no significant errors
in data transcription? Please explain your answer.
6. Consideration of all available data and relevant information
i. Based on the draft LLNA: DA BRD, have all the relevant data identified inpublished or unpublished studies that employ this test method been adequately
considered? Are there other comparative test method data that were notconsidered in the draft BRD, but are available for consideration? If yes, please
explain how to obtain such data.
IV.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA: DA
1. Test Method Usefulness and Limitations
i. Do you agree that the available data and test method performance (accuracy
and reliability) support the ICCVAM draft recommendations for the LLNA:
DA in terms of the proposed test method usefulness and limitations? Please
explain your answer.
ii. If restrictions on using radioactive materials are present, should the LLNA:
DA be routinely recommended for hazard identification of skin sensitizing
substances in lieu of having to possibly use guinea pig tests? Please explain
your answer.
iii. Even if limitations in using radioactive materials are not present, should the
LLNA: DA procedure or other valid and accepted non-radioactive method beroutinely recommended for hazard identification of skin sensitizing substances
instead of the traditional LLNA? Please explain your answer.
iv. From a public health perspective, is the recommended guidance for evaluating
negatives sufficient to address concerns associated with the false negative rateof 5% (1/19 substances) calculated for the LLNA: DA? Do you havesuggestions for additional guidance or limitations? Please explain youranswer.
v. From a testing strategy perspective, does the ICCVAM guidance addressconcerns associated with the false positive rate of 10% (1/10 substances)calculated for the LLNA: DA? Are there other suggestions for additional suchguidance or limitations? Please explain your answer.
2. Test method protocol

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i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: DA procedure in terms of the proposed test
method standardized protocols? If not, what recommendations would you
make? Please explain your answer.
ii. Can the traditional LLNA limit dose procedure be applied to the LLNA: DA?
Please explain your answer.
3. Future Studies
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: DA in terms of the proposed future studies?
If not, then what recommendations would you make? Please explain youranswer.
4. Performance Standards
i. The LLNA: DA protocol differs from the ICCVAM-recommended protocol
for the traditional LLNA (ICCVAM 1999; Dean et al. 2001; EPA 2003) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes.
In addition, there are differences between the two protocols that relate to how
and when the test substance is applied and when the lymph nodes arecollected (Table 2-1 and Appendix A in the draft LLNA: DA BRD).
According to the proposed draft ICCVAM Performance Standards for the
traditional LLNA
(http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm), anychange to the LLNA protocol other than the method used to assess
lymphocyte proliferation is considered a major change. Do you agree thatthese should be considered major changes and therefore the usefulness andlimitations of the LLNA: DA should not be assessed using the draft ICCVAM
Performance Standards? Please explain your answer.
ii. Even if the draft ICCVAM Performance Standards do not apply to the LLNA:
DA, what impact should the accuracy analysis based on 13 of the 18 required
performance standards substances (only one false negative and no false
positives) have on the overall evaluation of test method accuracy? Please
explain your answer.
iii. Should separate performance standards be developed for the LLNA: DA?
Please explain your answer.

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Appendix B4
Questions for the Peer Review Panel:
Non-Radioactive LLNA Protocol - LLNA: BrdU-FC Test Method

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Instructions for the Peer Review Panel: Non-Radioactive LLNA Protocol: LLNA:
BrdU-FC Test Method
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the LLNA: BrdU-FC (Local Lymph Node Assay: Bromodeoxyuridine detected byflow cytometry) for assessing the allergic contact dermatitis potential of chemicals and othersubstances. This test method, developed by MB Research Labs (Spinnerstown, PA), is a nonradiolabeled version of the traditional LLNA, and is based on measuring the incorporation of
bromodeoxyuridine (BrdU) into the DNA of dividing lymphocytes using flow cytometry as
in indicator of cell proliferation.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA: BrdU-FC. You are first asked to review the information in the draft ICCVAM
LLNA: BrdU-FC Background Review Document (BRD) for completeness, and to identify
any errors or omissions of existing relevant data or information that should be included. You
are then asked to evaluate the information in the BRD to determine the extent to which eachof the applicable criteria for validation and acceptance of toxicological test methods(ICCVAM 20037) have been appropriately addressed for the proposed use of the LLNA:
BrdU-FC. Adequate validation8 is a prerequisite for a test method to be considered for use inregulatory decision-making by U.S. Federal agencies. The validation process characterizes
the usefulness and limitations of a test method for a specific intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA: BrdU-FC (i.e., the proposed test method use, the proposed recommended
standardized protocol, the proposed test method performance standards, and any proposed
additional studies) and comment on whether the recommendations are supported by the
information provided in the draft LLNA: BrdU-FC BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA: BrdU-FC.
These include questions prepared by the ICCVAM Immunotoxicity Working Group (IWG)
to ensure that the assessment provides adequate information to facilitate U.S. Federal agency
decisions on the regulatory acceptability of this test method. The questions are also intended
to obtain guidance that will be helpful to federal agencies and other organizations that may
be involved in conducting or supporting further development, standardization, and/or
validation studies.
The overall question to consider is whether the validation status of the LLNA: BrdU-FC has
been adequately characterized for its intended purpose, and is it sufficiently accurate and

7

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
8
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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reliable to be used for the identification of sensitizing substances and non-sensitizing
substances in place of the traditional LLNA procedure.
I.

Questions to the Panel: Review of the Draft LLNA: BrdU-FC BRD for Errors
and Omissions
1. In the draft LLNA: BrdU-FC BRD, are there any errors in the BRD that should becorrected, or omissions of existing relevant data or information that should be
included?

II.

Questions to the Panel: Draft LLNA: BrdU-FC BRD
1. Test Method Protocol
i. The LLNA: BrdU-FC includes routine measurements of ear swelling as anindicator of excessive dermal irritation. Do you consider this procedure to bean appropriate approach? Do you think that this measurement should berecommended for routine inclusion into all LLNA protocols? Please explain
your answers.
ii. The LLNA: BrdU-FC also includes optional quantification of
immunophenotypic markers as an additional mechanism for distinguishingirritants from sensitizers. Do you consider this to be an appropriate approachto reduce false positives? Are the correct markers being considered or do you
recommend other/additional markers? Should these measurements berecommended for routine inclusion in the LLNA: BrdU-FC? Please explain
your answers.
iii. Please comment on the appropriateness of the "sequential strategy" used in theeLLNA: BrdU-FC (see Figure 2-1 of the draft BRD).
2. Substances Used for the Validation Studies
i. Do you consider the LLNA: BrdU-FC database representative of a sufficient
range of chemical classes and physicochemical properties that it would beapplicable to any of the types of chemicals and products that are typicallytested for skin sensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using
the LLNA: BrdU-FC? What chemicals or products should be evaluated to fillthis data gap? Please explain your answers.
3. Test Method Accuracyi.

The current accuracy analysis is based on overall concordance with thetraditional LLNA. Accuracy statistics compared to the Guinea Pig tests andhuman data/experience have also been provided. Are these comparisonsappropriate for assessing the accuracy of the LLNA: BrdU-FC? Please explain
your answer.

ii.

Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: BrdU-FC been adequately
evaluated and compared to the traditional LLNA (refer also to Table 6-1 of

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the draft ICCVAM BRD)? If not, what other analyses should be performed?
Please explain your answer.
iii.

Three substances (benzalkonium chloride, resorcinol, and Tween 80)
produced a “false positive” response compared to the traditional LLNA andguinea pig test when tested using the LLNA: BRDU-FC (Based on
immunophenotyping, benzalkonium chloride was subsequently classified as
an irritant rather than a sensitizer). Can you identify any characteristics
associated with these or similar substances, compared to the correctly
identified non-sensitizers that might signal that this type of discordant
response might occur, and therefore using the LLNA: BrdU-FC to test such
substances would not be appropriate, or that positive results for substanceswith such properties may warrant additional testing? Please explain youranswer.

4. Test Method Reliability (Intra- and Inter-laboratory reproducibility)i.

Has the intralaboratory reproducibility of the LLNA: BrdU-FC beenadequately evaluated and compared to the traditional LLNA (refer also toTable 7-1 of the draft LLNA: BrdU-FC BRD)? If not, what other analyses
should be performed? Are any limitations apparent based on this intralaboratory reproducibility assessment? Please explain your answer.

ii.

The draft LLNA: BrdU-FC BRD analyzes data from repeat testing of hexyl
cinnamic aldehyde (HCA) in six different vehicles and intralaboratory
reproducibility is assessed by coefficient of variation (CV). The calculated
CVs ranged from 30% to 53%. Based on these data, are there concerns with
the intralaboratory reproducibility of the LLNA: BrdU-FC? Please explain
your answer.

5. Data Qualityi.

The studies evaluated in the draft BRD for the LLNA: BrdU-FC were not all
conducted in accordance with Good Laboratory Practices (GLP) guidelines
although there were done in a laboratory that routinely conducts GLP studies(G. DeGeorge, personal communication). Please discuss what impact thismight have on the evaluation of the LLNA: BrdU-FC.

ii.

The original records for these studies were requested but have not yet beenobtained. As a result, an independent audit could not be conducted to confirm
that the reported data is the same as the data recorded in laboratory notebooks.
Do you agree that any recommendations from ICCVAM should be contingent
upon the completion of such an audit and findings that there were no
significant errors in data transcription? Please explain your answer.
Consideration of all available data and relevant informationiii.

Based on the draft LLNA: BrdU-FC BRD, have all the relevant data identified
in published or unpublished studies that employ this test method beenadequately considered? Are there other comparative test method data thatwere not considered in the draft BRD, but are available for consideration? If
yes, please explain how to obtain such data.

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III.

May 2008

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-FC
1. Test Method Usefulness and Limitations
i. Do you agree that the available data and test method performance (accuracy
and reliability) support the ICCVAM draft recommendations for the LLNA:
BrdU-FC in terms of the proposed test method usefulness and limitations?
Please explain your answer.
ii. If restrictions on using radioactive materials are present, should the LLNA:
BrdU-FC be routinely recommended for hazard identification of skinsensitizing substances in lieu of having to possibly use guinea pig tests?
Please explain your answer.
iii. Even if limitations in using radioactive materials are not present, should the
LLNA: BrdU-FC procedure be routinely recommended for hazard
identification of skin sensitizing substances instead of the traditional LLNA?
If not, then why? Please explain your answer.
iv. Do the ICCVAM recommendations adequately address concerns associatedwith the false positive rate of 17% (3/18 substances) calculated for the LLNA:
BrdU-FC? Are there other suggestions for additional such guidance orlimitations that should be considered? Please explain your answer.
2. Test Method Protocol
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-FC procedure in terms of the proposed
test method standardized protocol? If not, then what recommendations would
you make? Please explain your answer.
ii. Can the traditional LLNA limit dose procedure be applied to the LLNA:
BrdU-FC? Please explain your answer.
3. Future Studies
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-FC in terms of the proposed futurestudies? What other recommendations would you make? Please explain youranswer.
4. Performance Standards
i. The LLNA: BrdU-FC protocol differs from the ICCVAM-recommended
protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes.
According to the proposed draft ICCVAM Performance Standards for the
traditional LLNA
(http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm), anychange to the LLNA protocol other than the method used to assess
lymphocyte proliferation is considered a major change. Do you agree that
protocol differences between the LLNA: BrdU-FC and the traditional LLNA

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should be considered only minor changes and therefore the validity of this test
method should be based only on the draft ICCVAM Performance Standards?
Please explain your answer.
ii. Even if the draft ICCVAM Performance Standards do not apply to the LLNA:
BrdU-FC, what impact should the accuracy analysis based on 13 of the 18
required performance standards substances have on the overall evaluation oftest method accuracy? Please explain your answer.
iii. Are there concerns that 3/6 sensitizers, for which EC3 data were available,
had EC3 values that were outside of the proposed 0.5x to 2.0x EC3
acceptability range developed based on the traditional LLNA? Please explain
your answer.

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Appendix B5
Questions for the Peer Review Panel:
Non-Radioactive LLNA Protocol - LLNA: BrdU-ELISA Test Method

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Instructions for the Peer Review Panel: Non-Radioactive LLNA Protocol: LLNA:
BrdU-ELISA Test Method
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the LLNA: BrdU-ELISA (Local Lymph Node Assay with bromodeoxyuridine[BrdU] detected by ELISA) for assessing the allergic contact dermatitis potential ofchemicals and other substances. This test method, developed by Dr. Masahiro Takeyoshi(Tokyo, Japan), is a non-radiolabeled version of the traditional LLNA based on measuringlevels of incorporated BrdU in the auricular lymph nodes as an indicator of increased cellproliferation.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA: BrdU-ELISA. You are first asked to review the information in the draft
ICCVAM LLNA: BrdU-ELISA Background Review Document (BRD) for completeness,and to identify any errors or omissions of existing relevant data or information that should be
included. You are then asked to evaluate the information in the BRD to determine the extent
to which each of the applicable criteria for validation and acceptance of toxicological test
methods (ICCVAM 20039) have been appropriately addressed for the proposed use of the
LLNA: BrdU-ELISA. Adequate validation10 is a prerequisite for a test method to beconsidered for use in regulatory decision-making by U.S. Federal agencies. The validation
process characterizes the usefulness and limitations of a test method for a specific intended
use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA: BrdU-ELISA (i.e., the proposed test method use, the proposed recommended
standardized protocol, the proposed test method performance standards, and any proposed
additional studies) and comment on whether the recommendations are supported by the
information provided in the draft LLNA: BrdU-ELISA BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA: BrdU-ELISA.
These include questions prepared by the ICCVAM Immunotoxicity Working Group (IWG)
to ensure that the assessment provides adequate information to facilitate U.S. Federal agency
decisions on the regulatory acceptability of this test method. The questions are also intended
to obtain guidance that will be helpful to federal agencies and other organizations that may
be involved in conducting or supporting further development, standardization, and/or
validation studies.

9

ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
10
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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The overall question to consider is whether the validation status of the LLNA: BrdU-ELISA
has been adequately characterized for its intended purpose, and is it sufficiently accurate andreliable to be used for the identification of sensitizing substances and non-sensitizing
substances in place of the traditional LLNA procedure.
I.

Questions to the Panel: Comments on the Draft LLNA: BRDU-ELISA BRD forErrors and Omissions
1. In the draft LLNA: BrdU-ELISA BRD, are there any errors in the BRD thatshould be corrected, or omissions of existing relevant data or information thatshould be included?

II.

Questions to the Panel: Draft LLNA: BrdU-ELISA BRD
1. Test Method Protocol
i. The data generated for the substances analyzed in the LLNA: BrdU-ELISA
test method come from auricular lymph nodes from four individual mice ineach dose group. The recommended ICCVAM LLNA protocol and OECD
Test Guideline 429 recommend a minimum of five animals per dose group forcollecting individual animal data. What impact might the use of four animals
per dose group have on the accuracy and reliability of the LLNA: BrdUELISA? Do you agree with the ICCVAM recommendation that future use ofthis test method protocol should include five animals per dose group? Please
explain your answer.
2. Substances Used for the Validation Studies
i. Do you consider the LLNA: BrdU-ELISA database representative of a
sufficient range of chemical classes and physicochemical properties that thetest method would be applicable to any of the types of chemicals and productsthat are typically tested for skin sensitization potential? If not, what are therelevant chemical classes/properties (other than those that are identified aslimitations in the traditional LLNA) that should be tested with caution, or not
evaluated using the LLNA: BrdU-ELISA? What chemicals or products shouldbe evaluated to fill this data gap? Please explain your answers.
3. Test Method Accuracyi.

The current accuracy analysis using a Stimulation Index (SI) ≥3 or SI ≥1.3 toidentify sensitizers is based on overall concordance with the traditional
LLNA. Accuracy statistics compared to the Guinea Pig tests and humandata/experience have also been provided. Are these comparisons appropriate
for assessing the accuracy of the LLNA: BrdU-ELISA? Please explain youranswer.

ii.

Takeyoshi et al. (2007) performed an accuracy analysis using decision criteriaother than SI ≥3 to classify substances as sensitizers. Maximal accuracy for
the LLNA: BrdU-ELISA occurred when an SI ≥1.3 was used to distinguishbetween sensitizers and non-sensitizers. Using this decision criteria, they
achieved an accuracy of 91% (21/23), with a sensitivity of 100% (16/16) and a
specificity of 71% (5/7) (i.e., there were no false negatives and two false

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positives). Does this analysis support a recommendation that the decision
criteria be based on an SI ≥1.3? Are there concerns with using a small
increase (i.e., 1.3-fold) above the vehicle control response as the basis for
identifying a positive response? Please explain your answers.
iii.

Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA: BrdU-ELISA, using the SI ≥3
criterion, been adequately evaluated and compared to the traditional LLNA
(refer also to Table 6-1 of the draft ICCVAM BRD)? If not, what otheranalyses should be performed? Please explain your answer.

iv.

Using the SI ≥3 criterion, there were four substances (aniline, 4-chloroaniline,
2-mercaptothiazole, and hydroxycitronellal) when tested using the LLNA:
BrdU-ELISA that produced “false negative” responses compared to the
traditional LLNA. 4-Chloroanline and aniline are amines. 2Mercaptobenzothiazole is a heterocylic compound and hydroxycitronellal is ahydrocarbon. 2-Mercaptobenzothiazole is a liquid, but the other threesubstances are solids. Can you identify any characteristics associated withthese or similar substances, compared to the correctly identified sensitizers,that might signal that this type of discordant response might occur, andtherefore using the LLNA: BrdU-ELISA to test such substances would not beappropriate or that negative results for such substances should indicate a needfor confirmatory testing? Please explain your answer.

4. Test Method Reliability (Intra- and Inter-laboratory reproducibility)
i. Has the intralaboratory reproducibility of the LLNA: BrdU-ELISA beenadequately evaluated and compared to the traditional LLNA (refer also toTables 7-1 through 7-3 of the draft LLNA: BrdU-ELISA BRD)? If not, what
other analyses should be performed? Are any limitations apparent based on
this intra-laboratory reproducibility assessment? Please explain your answers.
ii. The substances evaluated for intralaboratory reproducibility of the LLNA:
BrdU-ELISA study were not coded. Does the lack of coding of test substances
adversely impact or bias the current evaluation? Please explain your answer.
iii. The Japanese Center for the Validation of Alternative Methods (JaCVAM)
has implemented a multi-laboratory validation study of the LLNA: BrdUELISA. Although the results from this study have yet to be reported, we arehoping to obtain information on the study design (i.e., with regard to number
and types of chemicals tested and the number of laboratories involved). If wedo, do you consider the design appropriate to adequately determine the extent
of interlaboratory reproducibility for the LLNA: BrdU-ELISA? If not, what
other analyses should be performed? Are any limitations apparent based on
this study design? Please explain your answer.
5. Data Quality
i. The studies evaluated in the draft BRD for the LLNA: BrdU-ELISA were notconducted in strict accordance with all provisions of the Good Laboratory
Practice (GLP) guidelines, although there were reportedly performed in

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laboratories that conduct GLP studies (M. Takeyoshi, personalcommunication). Please discuss what impact this might have on the evaluationof the LLNA: BrdU-ELISA.
ii. The original records for these studies were requested but were not available.
As a result, an independent audit could not be conducted to confirm that thereported data in peer reviewed publications and a poster presentation is thesame as the data recorded in laboratory notebooks. Should any
recommendations from ICCVAM be contingent upon the completion of suchan audit and findings that there were no significant errors in datatranscription? Please explain your answer.
6. Consideration of all available data and relevant information
i. Based on the draft LLNA: BrdU-ELISA BRD, have all the relevant data
identified in published or unpublished studies that employ this test method
been adequately considered? Are there other comparative test method datathat were not considered in the draft BRD, but are available for consideration?
If yes, please explain how to obtain such data.

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III.

May 2008

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA: BrdU-ELISA
1. Test Method Usefulness and Limitations
i. Do you agree that the available data and test method performance (accuracy
and reliability) support the ICCVAM draft recommendations for the LLNA:
BrdU-ELISA in terms of the proposed test method usefulness and limitations?
Please explain your answer.
ii. If restrictions on using radioactive materials are present, should the LLNA:
BrdU-ELISA be routinely recommended for hazard identification of skinsensitizing substances in lieu of using guinea pig tests due to the advantagesof fewer animals and the avoidance of pain and distress? Please explain youranswer.
iii. Even if limitations in using radioactive materials are not present, should the
LLNA: BrdU-ELISA procedure or other valid and accepted non-radioactive
method be routinely recommended for hazard identification of skin sensitizing
substances instead of the traditional LLNA? Please explain your answer.
iv. Does using a decision criterion of SI ≥1.3 instead of SI ≥3.0 resolve any
concerns with respect to potential false positives or false negatives that may
occur in this test method? Are there other suggestions for additional suchguidance or limitations that should be considered? Please explain youranswer.
2. Test Method Protocol
i. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA procedure in terms of the
proposed test method standardized protocols? If not, then what
recommendations would you make? Please explain your answer.
ii. Can the traditional LLNA limit dose procedure be applied to the LLNA:
BrdU-ELISA? Please explain your answer.
3. Future Studies
a. Do you agree that the available data support the ICCVAM draft
recommendations for the LLNA: BrdU-ELISA in terms of the proposed futurestudies? If not, then what recommendations would you make? Please explain
your answer.
i.

Performance Standards
i. The LLNA: BrdU-ELISA protocol differs from the ICCVAM-recommended
protocol for the traditional LLNA (ICCVAM 1999; Dean et al. 2001) in themethod used to assess lymphocyte proliferation in the auricular lymph nodes.
According to the proposed draft ICCVAM Performance Standards for the
traditional LLNA
(http://iccvam.niehs.nih.gov/methods/immunotox/llna_PerfStds.htm), anychange to the LLNA protocol other than the method used to assess

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lymphocyte proliferation is considered a major change. Do you agree that
protocol differences between the LLNA: BrdU-ELISA and the traditional
LLNA should be considered only minor changes and therefore the validity ofthis test method should be based only on the draft ICCVAM PerformanceStandards? Please explain your answer.
ii. Even if the draft ICCVAM Performance Standards do not apply to the LLNA:
BrdU-ELISA, what impact should the accuracy analysis based on eight of the
18 required performance standards substances (only one false negative and nofalse positives) have on the overall evaluation of test method accuracy? Please
explain your answer.
iii. Are there concerns that 4/4 sensitizers, for which EC3 data were available,
had EC3 values that were outside of the recommended 0.5x to 2.0x EC3
acceptability range developed based on the traditional LLNA? Please explain
your answer.
iv. Should separate performance standards be developed for the LLNA: BrdUELISA? Please explain your answer.

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Appendix B6
Questions for the Peer Review Panel:
Draft ICCVAM Performance Standards for the LLNA

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Instructions for the Peer Review Panel: Draft ICCVAM Performance Standards for the
LLNA
ICCVAM has developed draft LLNA performance standards consisting of essential test
method components, a minimum list of reference substances, and expected accuracy andreliability values. These are proposed for evaluating the acceptability of proposed test
methods that are mechanistically and functionally similar to the traditional LLNA test
method protocol previously recommended by ICCVAM.
The overall question for the Panel is whether these performance standards are consideredadequate for assessing the accuracy and reliability of test method protocols that are based on
similar scientific principles and that measure the same biological effect as the traditional
LLNA.
1) Purpose and Applicabilitya) ICCVAM proposes that these performance standards should only be applicable toversions of the LLNA that incorporate minor modifications to the traditional LLNA.
Currently, this is limited to the use of non-radioactive reagents to measurelymphocyte proliferation. It is considered essential that the modified LLNA should
otherwise adhere to all other aspects of the traditional LLNA protocol, as defined by
ICCVAM (1999) and Dean et al. (2001). This includes aspects such as: the sex andstrain of mouse used, the number of mice per dose group, the timing and site of test
article treatment, the duration between the last treatment and lymph node collection,
the inclusion of concurrent negative and positive control groups, the measured
endpoint (i.e., lymphocyte proliferation in the draining auricular lymph node), and thecollection of data at the level of the individual mouse. Do you agree that the use of
non-radioactive reagents for measuring cell proliferation in the lymph nodes, if that is
the only difference, constitutes a minor modification to the traditional LLNA
protocol? Is it necessary to keep the same decision criteria for distinguishing between
sensitizers and non-sensitizers (i.e., an SI of 3)? Please explain your answer.
b) Are there other procedural modifications that you consider minor and therefore could
be evaluated for equivalence to the traditional LLNA using the proposed performancestandards? If yes, please explain what they are and why.
c) Do you consider these performance standards to also be applicable to the LLNA limit
dose procedure? Please explain your answer.
2) Essential Test Method Componentsa) The essential test method components are based on the ICCVAM recommended
protocol (ICCVAM 1999; Dean et al. 2001), which is the basis for the current U.S.
Environmental Protection Agency (EPA 2003) test guideline (TG). There are somenotable differences between these protocols and the Organisation for Economic Cooperation and Development TG 429 for the LLNA (OECD 2002). When evaluationsof non-radioactive versions of the traditional LLNA are conducted using these
performance standards, is it necessary that the validation studies follow the ICCVAM
recommended protocol? Specifically, should the studies include: 1) a concurrent
positive control with each test substance; 2) using a minimum of five animals perdose group; and 3) measuring proliferation in lymph nodes from individual animals
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rather than pooling lymph nodes across all animals in a dose group? Please explain
your answers.
b) Should the concurrent testing of the positive control and test substance be conductedin the same vehicle or can different vehicles be used? Please explain your answer.
3) Proposed Reference Substancesa) Do you agree with the selection and prioritization criteria used to select theperformance standards reference substances? Please explain your answer.
b) The rationale for the number of substances included on the "required" list of
substances (n=18) is provided in Appendix C of the draft ICCVAM PerformanceStandards. Do you consider this to be an adequate number upon which to evaluate theperformance of non-radioactive LLNA test methods, where the only protocol
modification is the method for assessing cell proliferation in the auricular lymph
nodes? If not, how many reference chemicals should be tested? Please explain youranswer.
c) Do you consider the types of substances included in the reference substance list, withregard to relative sensitization potency, physicochemical characteristics, and vehicles,
to be representative of the overall diversity of substances that are likely to be tested
for skin sensitization? Please explain your answer.
d) Are there other types of information relevant to skin sensitization that should beconsidered in order to demonstrate an adequately diverse reference list? If yes, please
explain what additional information should be included.
e) Are there other substances that you consider to be more appropriate for assessing thesensitivity (ability of the test method to correctly identify sensitizing substances) andspecificity (ability of the test method to correctly identify non-sensitizing substances)of non-radioactive LLNA test methods, and for which there is available LLNA,
guinea pig, and human data? If yes, please name the substances and explain why.
f) Four "discordant chemicals" (i.e., two LLNA false negatives and two false positives
compared to guinea pig tests or human data) are included as optional substances that
could be studied to evaluate if the proposed modifications might provide improved
performance relative to the traditional LLNA.
•

Please comment on the appropriateness of including these specific substances in
the reference list. Should different substances be included? Should more false
negative/positive substances be tested? If so, what are they? Please explain youranswers.

•

Do you consider their "optional" status appropriate, or should testing thesesubstances be required? Please explain your answer.

•

Would "correct" results with these four discordant chemicals be sufficient toconsider the alternative test method to be more predictive of skin sensitization
than the traditional LLNA? Please explain your answer.

4) Test Method Accuracy Standards

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May 2008a) The draft ICCVAM Performance Standards state that the non-radioactive proposed
LLNA test method should exactly match the accuracy of the traditional LLNA whenevaluated with the minimum set of 18 reference substances. Do you agree that test
method accuracy should be based on a chemical-by-chemical match with regard to
identifying the chemicals as sensitizers or non-sensitizers? Please explain youranswer.
b) The draft ICCVAM LLNA Performance Standards recommend that, for eachsensitizer, the threshold concentration that induces a positive SI response should bewithin 0.5x to 2.0x of the concentration obtained for the EC3 in the traditional LLNA.
As described in Appendix D of the draft ICCVAM Performance Standards, statistical
approaches have been used in an attempt to identify an appropriate range, but thesecalculated ranges do not appear to be the most practical. In contrast, the NICEATM
LLNA database demonstrates that EC3 values from replicate tests for a sensitizing
chemical when tested using the same solvent are rarely outside of this proposed 0.5x
to 2.0x acceptability EC3 range. Please comment on the appropriateness of using thiscriterion to judge the equivalency of a non-radioactive version of the traditional
LLNA. If this approach is not acceptable, please explain why, and present analternative approach along with the basis for this approach.
c) For five of the 13 sensitizers on the draft ICCVAM reference substances list, thereference EC3 value is based on a single LLNA study (see Table C1 of the draft
ICCVAM Performance Standards). Please comment on the appropriateness of
including such chemicals in the list of recommended reference substances andwhether or not the 0.5x to 2.0x criteria should be applied to such substances. Please
explain your answer.
5) Test Method Reliability Standardsa) The draft ICCVAM Performance Standards state that acceptable intralaboratory
reproducibility will be indicated by a laboratory obtaining, in each of four
independent experiments conducted with at least one week between each experiment,
ECt values (the estimated concentration needed to produce an SI of a defined
threshold [e.g., EC3]) for hexyl cinnamic aldehyde (HCA) that are generally within
0.5x to 2.0x (i.e., 5% to 20%) of the historical mean EC3 concentration (10%) for thissubstance, based on existing available traditional LLNA data.
•

Do you consider the number of repeat experiments (n=4) to be adequate? Please
explain your answer.

•

Do you consider testing HCA adequate for demonstrating intralaboratory
reproducibility? If not, which substance(s) should be tested? Please explain youranswer.

•Is the required one-week interval between independent tests adequate and/or
appropriate? If not, please provide an alternative schedule and explain the basis
for your recommendation.

•

Do you consider the criteria for acceptability to be appropriate? If not, please
describe another criteria and explain the basis for your recommendation.

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May 2008b) The draft ICCVAM Performance Standards state that acceptable interlaboratory
reproducibility will be indicated by each of three laboratories obtaining ECt valuesfor HCA and 2,4-dinitrochlorobenzene (DNCB) from a single experiment that are
generally within 0.5x to 2.0x (5% to 20% and 0.025 to 0.1%, respectively) of themean historical EC3 concentration (10% and 0.05%, respectively) obtained for these
two substances in the traditional LLNA.
•

Do you consider the single experiment per substance in each laboratory to beadequate? If not, please provide an alternative approach and explain why.

•

Do you consider testing HCA and DNCB to be adequate for demonstratinginterlaboratory reproducibility? If not, which substance(s) should be tested?
Please explain your answer.

•

Do you consider the criteria for acceptability to be appropriate? If not, please
describe another criteria and explain the basis for your recommendation.

6) Summary Questiona) If a radioactive or non-radioactive LLNA method were proposed with a “majorchange” (e.g., different mouse strain or use of male mice, change in the schedule fortest article administration, change in schedule for lymph node excision, etc.), whatcriteria should be used to evaluate the equivalence of this method to the traditional
LLNA?
•

Would a new set of performance standards be required for this method? Please
explain your answer.

•

How many reference substances might be considered adequate? Would the 18
minimum reference substances in the draft ICCVAM LLNA PerformanceStandards be sufficient? If more substances are considered necessary, how many
should there be tested and what should their characteristics be? Please explain
your answer.

•

Regardless of the number of reference substances, should the alternative LLNA
be required to obtain the same call (and potency for sensitizers) as the traditional
LLNA for the 18 minimum reference substances in the draft ICCVAM LLNA
Performance Standards? Please explain your answer.

•

Are there additional specific substances that should be used? If yes, what are
they? Please explain your answer.

•

What, if any, additional information would be considered necessary and why?

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May 2008

Appendix B7
Questions for the Peer Review Panel: Use of the LLNA for Potency Determinations

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Instructions for the Peer Review Panel: Use of the LLNA for Potency Determinations
The Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) in conjunction with National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) is evaluating the validation
status of the murine local lymph node assay (LLNA) for assessing the potential potency of
substances to cause allergic contact dermatitis.
The following materials are intended to guide you in your review of the ICCVAM evaluationof the LLNA for potency determinations. You are first asked to review the information in thedraft ICCVAM LLNA for potency determinations Background Review Document (BRD) forcompleteness, and to identify any errors or omissions of existing relevant data or informationthat should be included. You are then asked to evaluate the information in this BRD todetermine the extent to which each of the applicable criteria for validation and acceptance of
toxicological test methods (ICCVAM 200311) have been appropriately addressed for the
proposed use of the LLNA for potency determinations. Adequate validation12 is aprerequisite for a test method to be considered for use in regulatory decision-making by U.S.
Federal agencies. This validation process characterizes the usefulness and limitations of a test
method for its intended use.
Lastly, you are asked to consider the ICCVAM draft test method recommendations for the
LLNA for potency determinations (i.e., the proposed test method use, the proposed
recommended standardized protocol, the proposed test method performance standards, andany proposed additional studies) and comment on whether the recommendations aresupported by the information provided in the draft BRD.
The questions relating to the draft BRD that must be addressed are provided in Sections I
and II of this guidance, while Section III contains questions relating to the draft ICCVAM
test method recommendations on the LLNA for potency determinations.
These include questions prepared by the ICCVAM IWG to ensure that the assessment
provides adequate information to facilitate U.S. Federal agency decisions on the regulatory
acceptability of this test method, and/or adequate guidance for organizations that may be
involved in conducting or supporting further development, standardization, and/or validation.
The overall question to consider is whether the validation status of the LLNA for potency
determinations has been adequately characterized, and is it sufficiently accurate and reliable
to be used for the identification of sensitizing substances according to their relative potencyclassification based on a comparison to either human or guinea pig responses.
I.

Questions to the Panel: Review for Errors and Omissions
1. Are there any errors or omissions of existing relevant data or information thatshould be included in the draft BRD?

11ICCVAM. 2003. ICCVAM Guidelines for the Nomination and Submission of New, Revised, andAlternative Test Methods. NIH Publication No. 03-4508. Research Triangle Park, NC. NIEHS(Available: http://iccvam.niehs.nih.gov/docs/guidelines/subguide.htm).
12
Validation is the process by which the reliability and accuracy of a test method are established for a
specific purpose (ICCVAM 2003).
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II.

May 2008

Questions to the Panel: LLNA for Potency Determinations Draft BRD
1. Do you consider the database of substances evaluated representative of a
sufficient range of chemical classes and physical chemical properties that it would
be applicable to any of the types of chemicals and products that are typicallytested for skin sensitization potential? If not, what are the relevant chemicalclasses/properties (other than those that are identified as limitations in the
traditional LLNA) that should be tested with caution, or not evaluated using thefor potency determinations? What chemicals or products should be evaluated tofill this data gap? Please explain your recommendation.
2. While coding of chemicals is recommended for prospective studies, thisevaluation is based on a retrospective evaluation of existing data, most of which
was not generated using coded chemicals to reduce bias. Does the lack of codingof test substances adversely impact or bias the current evaluation? Please provide
a rationale for your answer.
3. For some substances submitted using the LLNA, it was not possible to confirm
whether the data were generated using pooled animal data for each dose group (asallowed in Test Guideline [TG] 429 of the Organisation for Economic
Co-operation and Development [OECD]) rather than individual animal data (asrecommended in the ICCVAM 2001 protocol)? Cockshott et al. (2006) reported
that using individual animal data allowed for technical problems during anexperiment to be identified. Considering this, should the analysis of theperformance of the LLNA for potency determinations be limited to data fromstudies that can be confirmed as using individual animal data collection
procedures? What impact might the inclusion of pooled animal data have on theaccuracy analysis included in Section 6.0 of the draft ICCVAM BRD? Please
explain your answer.
4. Has the relevance (e.g., accuracy/concordance, sensitivity, specificity, false
positive and false negative rates) of the LLNA for potency determinations beenadequately evaluated and compared to the human and guinea pig (refer also to
Section 6.0 of the draft ICCVAM BRD)? If not, what other analyses should be
performed?
5. The accuracy analysis (see Section 6.0 of the draft ICCVAM BRD) focuses on the
two-level categorization scheme proposed by the United Nations Globally
Harmonized System for Classification and Labelling for both human and guinea
data. Should other categorization schemes be considered?
6. Does the BRD adequately characterize the usefulness and limitations of the
LLNA for potency determinations based on the accuracy analyses? If not, what
additions or changes should be made to the current usefulness and limitations?
7. Has the reliability (e.g., intralaboratory repeatability, intra- and inter-laboratory
reproducibility) of the LLNA for potency determinations been adequately
evaluated (refer also to Section 7.0 of the draft ICCVAM BRD)? If not, what
other analyses should be performed?

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8. For some studies included in the draft BRD, it was not possible to determine
whether or not they had been conducted in accordance with Good Laboratory
Practice (GLP) guidelines. Neither was it possible to obtain the results of GLP
audits for all studies conducted in accordance with GLP guidelines. Please discuss
what impact this lack might have on the evaluation of the LLNA for potency
determinations and whether such studies should be excluded from any analysis.
9. As described in the draft BRD, original records for some of the non-GLP studies
included in this evaluation could not be obtained. As a result, an independent
audit could not be conducted to confirm that the reported data is the same as thedata recorded in laboratory notebooks. Considering this, should the results ofthese studies (all of which are currently included) be excluded from any of theperformance analyses? If yes, please explain.
10. Based on the draft BRD, have all the relevant data identified in published or
unpublished studies conducted using the LLNA for potency determinations beenadequately considered? If not, what other studies should to be considered?
III.

Questions to the Panel: Draft ICCVAM Test Method Recommendations on the
LLNA for Potency Determinations
1. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA for potency determinations in terms of the proposed test method
usefulness and limitations? Please explain your answer. If not, why
recommendations would you make?
•Should the LLNA be routinely recommended for the hazard classification of
the skin sensitization potency of chemicals?

2. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed test method standardized protocol? Please
explain your answer. If not, then what recommendations would you make?
3. Should the relevant testing guidelines for the LLNA be updated to include thecalculation of an EC3 value?
4. Do you agree that the available data support the ICCVAM draft recommendations
for the LLNA in terms of the proposed future studies? Please explain youranswer. If not, then what recommendations would you make?

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