新規試験法提案書

皮膚感作性試験代替法
ARE-Nrf2 luciferase LuSens test method(LuSens test method)

令和元年11月

国立医薬品食品衛生研究所

新規試験法提案書
令和元年 11 月 18 日
No.

皮膚感作性試験代替法

2019-02

ARE-Nrf2 luciferase LuSens test method (LuSens test method)
に関する提案

令和元年 11 月 14 日に国立医薬品食品衛生研究所にて開催された新規試験法評価会議（通称：JaCVAM 評価会議）において以下の提案がなされた。
提案内容： 本試験法は、JaCVAM 皮膚感作性試験資料編纂委員会により、KeratinoSensTM 試験法とほぼ同等の予測性と評価されているため、本試験法で陽性の結果が得られた場合、その化学物質を強い感作性物質に分類することは行政上可能である。しかしながら、
KeratinoSensTM 試験法同様、稀に偽陽性の結果が生じることに留意しなければならな
い。一方、本試験法で陰性の結果が得られても偽陰性の可能性もあり、本試験法単独
でその皮膚感作性を評価することは難しい。本試験法は、その特性を十分に理解した上で、その他の情報源と組み合わせて適切に評価することが、行政的な受け入れに必要である。
この提案書は、皮膚感作性試験資料編纂委員会によりまとめられた文書を用いて、JaCVAM評
価会議が評価および検討した結果、その有用性が確認されたことから作成された。
以上の理由により、行政当局の安全性評価方法として皮膚感作性試験代替法

LuSens test

method の使用を提案するものである。

大野泰雄
JaCVAM 評価会議

平林容子
議長

JaCVAM 運営委員会

委員長

i

JaCVAM 評価会議
大野泰雄

（公益財団法人 木原記念横浜生命科学振興財団）： 座長

五十嵐良明（国立医薬品食品衛生研究所）

石井雄二（国立医薬品食品衛生研究所 安全性生物試験研究センター）

稲若邦文（日本化学工業協会）

井上智彰（日本免疫毒性学会）

今井教安（日本動物実験代替法学会）

岩瀬裕美子（日本製薬工業協会）

久保文宏（独立行政法人 医薬品医療機器総合機構）

杉山真理子（日本化粧品工業連合会）*

中村るりこ（独立行政法人 製品評価技術基盤機構）

西川秋佳（国立医薬品食品衛生研究所 病理部 / 済生会宇都宮病院）

西村次平（独立行政法人 医薬品医療機器総合機構）

沼澤（日本毒性学会）

聡

平林容子（国立医薬品食品衛生研究所 安全性生物試験研究センター）

廣田衞彦（日本化粧品工業連合会）**

増村健一（日本環境変異原学会）

横関博雄（日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日*：平成 30 年 4 月 1 日～平成 31 年 3 月 31 日**：平成 31 年 4 月 1 日～令和 2 年 3 月 31 日

ii

JaCVAM 運営委員会
平林容子（国立医薬品食品衛生研究所 安全性生物試験研究センター）：委員長

池田三恵（独立行政法人 医薬品医療機器総合機構）

石井孝司（国立感染症研究所）

小川久美子（国立医薬品食品衛生研究所 安全性生物試験研究センター 病理部）

奥田晴宏

（国立医薬品食品衛生研究所）

諫田泰成（国立医薬品食品衛生研究所 安全性生物試験研究センター 薬理部）

北嶋

（国立医薬品食品衛生研究所 安全性生物試験研究センター 毒性部）

聡

高木篤也

（国立医薬品食品衛生研究所 安全性生物試験研究センター 毒性部 動物管理室）

高畑正浩（厚生労働省 医薬・生活衛生局 医薬品審査管理課）

束野正明（厚生労働省 医薬・生活衛生局 医薬品審査管理課 化学物質安全対策室）

野坂佳伸（厚生労働省 医薬・生活衛生局 医薬品審査管理課 化学物質安全対策室）

広瀬明彦（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 安全性予測評価部）

笛木修（独立行政法人 医薬品医療機器総合機構）

渕岡学（厚生労働省 医薬・生活衛生局 医薬品審査管理課 化学物質安全対策室）

本間正充（国立医薬品食品衛生研究所 安全性生物試験研究センター 変異遺伝部）

足利太可雄（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 安全性予測評価部第二室）：事務局

小島肇（国立医薬品食品衛生研究所 安全性生物試験研究センター 安全性予測評価部第二室）：事務局

iii

JaCVAM statement on the
ARE-Nrf2 luciferase LuSens test method for skin sensitization(LuSens test method)
At a meeting held on 14 November 2019 at the National Institute of Health Sciences (NIHS)
in Kanagawa, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: A review of the LuSens test method by the JaCVAM Skin Sensitization Editorial
Committee has determined that the LuSens test method exhibits a predictive
capacity roughly equivalent to that of the KeratinoSens™ test method and that test
chemicals yielding positive results in LuSens testing should in a regulatory context
be considered strong sensitizers belonging to Category 1 of the United Nations
Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS). At the same time, however, it should be remembered that, as with the
KeratinoSens™ test method, LuSens testing does in rare cases yield false positive
results. Conversely, LuSens testing also yields false negatives, which means that it
would be unreasonable to use it as a standalone test for predicting skin sensitization
potential. We therefore conclude that the use of the LuSens test method in a
regulatory context requires a thorough understanding of the assay’s strengths andweaknesses as a prerequisite to its application within an integrated approach totesting and assessment (IATA) that will also take into account information fromother sources.
This statement was prepared to acknowledge that the results of a review and study by the
JaCVAM Regulatory Acceptance Board have confirmed the usefulness of this assay.
Based on the above, we propose the LuSens test method as a useful means for estimating skinsensitization by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson
JaCVAM Steering Committee

November 18, 2019v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee and is composed of nominees from industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Mr. Yasuo Ohno (Kihara Memorial Yokohama Foundation for the Advancement ofLife Sciences): Chairperson
Ms. Yoko Hirabayashi (Center for Biological Safety and Research: CBSR, National
Institute of Health Sciences: NIHS)
Mr. Morihiko Hirota (Japan Cosmetic Industry Association)**Mr. Yoshiaki Ikarashi (NIHS)
Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Kunifumi Inawaka (Japan Chemical Industry Association)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology)
Mr. Yuji Ishii (CBSR, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Fumihiro Kubo (Pharmaceuticals and Medical Devices Agency)
Mr. Kenichi Masumura (Japanese Environmental Mutagen Society)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (CBSR, NIHS/ Saiseikai Utsunomiya Hospital)
Mr. Jihei Nishimura (Pharmaceuticals and Medical Devices Agency)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)*
Mr. Hiroo Yokozeki (Japanese Society for Cutaneous Immunology and Allergy)
Term: From 1st April 2018 to 31st March 2020*: From 1st April 2018 to 31st March 2019**: From 1st April 2019 to 31st March 2020

vi

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

Ms. Yoko Hirabayashi (CBSR, NIHS): Chairperson
Mr. Manabu Fuchioka (Ministry of Health, Labour and Welfare)
Mr. Osamu Fueki (Pharmaceuticals and Medical Devices Agency)
Mr. Akihiko Hirose (Division of Risk Assessment, CBSR, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, CBSR, NIHS)
Ms. Mie Ikeda (Pharmaceuticals and Medical Devices Agency)
Mr. Koji Ishii (National Institute of Infectious Diseases)
Mr. Yasunari Kanda (Division of Pharmacology, CBSR, NIHS)
Mr. Satoshi Kitajima (Division of Toxicology, CBSR, NIHS)
Mr. Yoshinobu Nosaka (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, CBSR, NIHS)
Mr. Haruhiro Okuda (NIHS)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology,
CBSR, NIHS)
Mr. Masahiro Takahata (Ministry of Health, Labour and Welfare)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Takao Ashikaga (Division of Risk Assessment, CBSR, NIHS): Secretary
Mr. Hajime Kojima (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

皮膚感作性試験代替法
ARE-Nrf2 luciferase LuSens test method (LuSens test method)目次

評価会議報告書 ------------------------------------------------------------------------------------ 1
評価報告書 ------------------------------------------------------------------------------------------ 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 442D, IN VITRO SKINSENSITISATION ASSAYS ADDRESSING THE AOP KEY EVENT ON KERATINOCYTE
ACTIVATION ---------------------------------------------------------------------------------------------25

評価会議報告書

皮膚感作性試験代替法
ARE-Nrf2 luciferase LuSens test method (LuSens test method)

JaCVAM 評価会議

令和元年（2019 年）10 月 2 日

11

JaCVAM 評価会議
大野泰雄

（公益財団法人 木原記念横浜生命科学振興財団）：座長

五十嵐良明（国立医薬品食品衛生研究所）

石井雄二（国立医薬品食品衛生研究所 安全性生物試験研究センター）

稲若邦文（日本化学工業協会）

井上智彰（日本免疫毒性学会）

今井教安（日本動物実験代替法学会）

岩瀬裕美子（日本製薬工業協会）

久保文宏（独立行政法人 医薬品医療機器総合機構）

杉山真理子（日本化粧品工業連合会）*

中村るりこ（独立行政法人 製品評価技術基盤機構）

西川秋佳（国立医薬品食品衛生研究所 病理部 / 済生会宇都宮病院）

西村次平（独立行政法人 医薬品医療機器総合機構）

沼澤（日本毒性学会）

聡

平林容子（国立医薬品食品衛生研究所 安全性生物試験研究センター）

廣田衞彦（日本化粧品工業連合会）**

増村健一（日本環境変異原学会）

横関博雄（日本皮膚免疫アレルギー学会）
任期：平成 30 年 4 月 1 日～令和 2 年 3 月 31 日*：平成 30 年 4 月 1 日～平成 31 年 3 月 31 日**：平成 31 年 4 月 1 日～令和 2 年 3 月 31 日

22

ARE1-Nrf22 luciferase LuSens test method（以下、本試験法）は、既に経済協力開発機構（OECD）
試験法ガイドライン（TG）442D に登録されている ARE-Nrf2 luciferase KeratinoSensTM test
method（KeratinoSensTM 試験法）と同様に、化学物質の皮膚感作性を予測する試験法である。
本試験法は、ケラチノサイトにおける炎症性応答の一つである Keap1-Nrf2-ARE pathway
を利用したレポーターアッセイである。本試験法は、開発者（BASF 社）によるインハウス試験および外部 4 機関の参加による TG 422D の Performance standards（性能標準）に基づく
多施設バリデーション研究が行われ、その結果を EURL ECVAM Scientific Advisory Committee（ESAC）が第三者評価し、2018 年 6 月に OECD TG 442D Appendix 1B として追記された。
JaCVAM 評価会議は、皮膚感作性試験資料編纂委員会により作成された皮膚感作性試験評
価報告書 ARE-Nrf2 luciferase LuSens test method （LuSens test method）1）を基に本試験法の妥当性について検討した。
１．試験法の定義
名称：ARE-Nrf2 luciferase LuSens test method
代替する対象毒性試験：モルモットを用いる皮膚感作性試験（OECD TG 406）およびマウス
を 用 い る 局 所 リ ン パ 節 試 験 [LLNA (OECD TG 429) 、 LLNA:DA (OECD TG 442A) 、
LLNA:BrdU-ELISAor -FCM (OECD TG 442B)]
試験法の概略：本試験法 2)は、既に OECD TG 442D に登録されている KeratinoSensTM 試験法と同様、Keap1-Nrf2- ARE pathway を利用したレポーターアッセイである。Keap1-Nrf2- ARE
pathway は、転写因子 Nrf2 の抑制因子である Keap1 および ARE が関係する遺伝子発現経路である。Nrf2 と Keap1 の結合は、ARE に依存して発現する遺伝子群の発現量を制御している。Keap1 のシステイン残基に求電子性の化学物質が結合すると、Nrf2 は Keap1 から解離し、核内へ移行して DNA 上の ARE に結合する。その結果、下流の遺伝子群の発現が誘導され、化学物質による障害から細胞を保護するために機能する。多くの皮膚感作性物質が Keap1-Nrf2-ARE pathway を活性化することが知られており 3, 4)、in vitro 感作性試験法の開発に利用されている。
KeratinoSensTM 試験法 5)では、Aldo-Keto Reductase Family 1 Member C2（AKR1C2）遺伝子の ARE をエンハンサーとするルシフェラーゼレポーター遺伝子を安定的に導入した
HaCaT 細胞（ヒトケラチノサイト系培養細胞）を用いるのに対し、本試験法 6)ではラットの NADPH:quinone oxidoreductase 1（NQO1）遺伝子由来の ARE をエンハンサーとするルシフェラーゼレポーター遺伝子を安定的に導入したヒトケラチノサイト系培養細胞（株名は不詳）を試験に使用する。いずれの方法も化学物質による Keap1-Nrf2-ARE pathwayの活性化に伴って誘導されるルシフェラーゼの活性を、基質を添加して発光強度を測定することにより、化学物質の皮膚感作性を評価する方法である。

1
2

ARE: Antioxidant response element
Nrf2: Nuclear factor (erythroid-derived 2)-like 2
3
3

２．評価に用いた資料および評価内容の科学的妥当性
本試験法は、EURL ECVAM によるバリデーション研究は行われず、BASF 社によるインハウス試験および外部 4 機関の参加による性能標準に基づくバリデーション研究が行われ 6-8)、
その結果が ESAC によって評価されている 9)。それらの公表資料を基に、JaCVAM 皮膚感作性試験資料編纂委員会は、本試験法の皮膚感作性試験代替法としての科学的妥当性について、ESAC の評価結果と同様に、本試験法と KeratinoSensTM 試験法の予測性はほぼ同等と評価している 1）。また、同様の原理に基づく試験法である KeratinoSensTM 試験法が科学的に妥当であることは、平成 27 年に JaCVAM 評価会議において既に認められており 10）、本試験
法も科学的に妥当であると考える。
３．本試験法の有用性と適用限界
本試験法はケラチノサイトにおける ARE 活性化による遺伝子発現を指標にした試験法であることから、同様に Keap1-Nrf2-ARE pathway を利用したレポーターアッセイである
KeratinoSensTM 試験法と基本的な操作は同様である。本試験法における LuSens は開発者より無償提供される。
本試験法は、さまざまな構造や物理化学的性質を有する化学物質に適用可能であることが示されている 6）。また、本試験法に用いる溶媒に溶解する、もしくは安定に分散する化合物は適用可能であるが、適用可能最高濃度においても細胞毒性がみられない場合（細胞生存率 70%以上）の陰性結果は「評価不能」とされる。
本試験法は、KeratinoSensTM 試験法と同様、リジン残基特異的に結合する物質は偽陰性と判定されることが考えられ、注意が必要である。本試験法に使用する細胞株の代謝能は限定的であり、プロハプテンおよびプレハプテンは偽陰性となる可能性がある。一方、感作性のないケミカルストレッサー（例えば酸化ストレスを誘導する物質）は KeratinoSensTM 試験法と同様、偽陽性となる可能性がある。さらに、ルシフェラーゼに干渉する化学物質も評価に影響する可能性がある。陰性対照 8 物質および陽性対照 12 物質を用いて 5 施設で実施されたバリデーション研究では、感度 92%、特異度 75%、正確度 85%であり、特異度において性能標準の基準（80%以上）を満たさなかった。従って、本試験法において陽性の結果が得られた場合にも、偽陽性の可能性があることに留意しなければならない。開発者によるインハウス試験においては、ヒトデータ（69 物質）と比較した場合、感度は 83%、特異度は 78%、
正確度は 81%、LLNA データ（72 物質）と比較した場合、感度は 75%、特異度は 71%、正確度は 74%であった 6)。偽陰性となった 13 物質中 2 物質（Phthalic anhydride 及び Propyl
gallate）は UN GHS 1A に区分される物質であった。以上より、本試験法単独では皮膚感作性の予測性は不十分であり、証拠の重み付けや他の試験法と組み合わせでの評価を推奨する 。
本試験法は、Integrated Approaches to Testing and Assessment（IATA）において他の試験法と組み合わせることにより、感作性物質と非感作性物質との区別に使用することが可能である。しかし、本試験法単独での感作性強度分類や UN Globally Harmonized System of44Classification and Labeling of Chemicals（UN GHS）のサブカテゴリー分類への応用には適さない。
上記の様々な限界を勘案し、本試験法は単独での皮膚感作性の判定には不十分であるが、証拠の重み付けや他の試験法との組合せで用いることにより、化学物質の感作性を判断する上で重要な情報を与える。
４．目的とする物質又は製品の毒性を評価する試験法としての、社会的受け入れ性および行
政上の利用の可能性
社会的受け入れ性：
本試験法は、KeratinoSensTM 試験法同様、細胞培養の技術と 96 ウェル対応のルミノメー
ターがあれば容易に実施可能である。実施に必要なランニングコストも KeratinoSensTM 試験法と同様、約 1.5 万円と見積もられ、LLNA（同約 10 万円）より低額であり、h-CLAT（同約 2 万円）や U-SENSTM（同約 1.8 万円）とほぼ同程度であった。さらに本試験法は細胞の入手にあたりライセンス契約を結ぶ必要がなく、輸送費実費負担により細胞株の入手が可能である。本試験法の実施と結果の解釈に当たっては、化学物質の性質と本試験法の適用限界を見極める必要があるが、本試験法は皮膚感作性を有する多くの化学物質が ARE により制御されている遺伝子の発現を誘導するという皮膚感作性発現機序における重要なイベントを検出しており、化学物質の皮膚感作性を考える上で重要な情報を与える。さらに生きた動物を用いないという点で 3Rs の精神に合致していることから、本試験法の社会的受け入れ性は高い。
行政上の利用性：
本試験法は、JaCVAM 皮膚感作性試験資料編纂委員会により、KeratinoSensTM 試験法とほぼ同等の予測性と評価されているため、本試験法で陽性の結果が得られた場合、その化学物
質を強い感作性物質である UN GHS 区分 1 に分類することは行政上可能である。しかしながら、KeratinoSensTM 試験法同様、稀に偽陽性の結果が生じることに留意しなければならな
い。一方、本試験法で陰性の結果が得られても偽陰性の可能性もあり、本試験法単独でその皮膚感作性を評価することは難しい。本試験法は、その特性を十分に理解した上で、IATAを構成するその他の情報源と組み合わせて適切に評価することが、行政的な受け入れに必
要である。

5
5

引用文献
1) JaCVAM 皮膚 感 作 性 試 験 資 料 編 纂 委 員 会 ： 皮 膚 感 作 性 試 験 評 価 報 告 書 ARE-Nrf2
luciferase LuSens test method (LuSens test method) （令和元年 9 月 30 日）
2) OECD (2018) Key event based test guideline. In vitro skin sensitization assay addressing the AOP
key event on keratinocyte activation. OECD Guideline for the Testing of Chemicals. No.442D,
OECD, Paris. Accessible at: https://www.oecd-ilibrary.org/docserver/9789264229822-en.pdf?
expires=1540286232&id=id&accname=guest&checksum=F9DC614E0BBF02D46223C8FCAB
CFBA5E
3) Ade N, Leon F, Pallardy M, Peiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I, Ourlin
JC; HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells
and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol Sci. 2009; 107(2):451-60.
4) Vandebriel RJ, Pennings JL, Baken KA, Pronk TE, Boorsma A, Gottschalk R, Van Loveren H;
Keratinocyte gene expression profiles discriminate sensitizing and irritating compounds. Toxicol
Sci. 2010; 117(1):81-9.
5) Emter R, Ellis G, Natsch A; Performance of a novel keratinocyte-based reporter cell line to screen
skin sensitizers in vitro, Toxicol Applied Pharmacol, 2010; 245: 281-290.
6) Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, vanRavenzwaay B, Landsiedel R; LuSens: a keratinocyte based ARE reporter gene assay for use inintegrated testing strategies for skin sensitization hazard identification, Toxicol In Vitro, 2014;
28(8):1482-97.
7) Kolle SN; Corrigendum to “LuSens: a keratinocyte based ARE reporter gene assay for use inintegrated testing strategies for skin sensitization hazard identification.” by Ramirez, et al.,
Toxicol In Vitro. 2014; Dec; 28(8):1482–97. doi: 10.1016/j.tiv.2014.08.002
8) Ramirez T., Stein N., Aumann A., Remus T., Edwards A., Norman K.G., Ryan C., Bader J.E., Fehr
M., Burleson F., Foertsch L., Wang X., Gerberick F., Beilstein P., Hoffmann S., Mehling A., vanRavenzwaay B., Landsiedel R.; Intra- and inter-laboratory reproducibility and accuracy of the
LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers,
Toxicol. In Vitro, 2016; 32: 278-286.
9) ESAC (2016). ESAC Opinion on the BASF-coordinated Performance Standards-based validationof the LuSens test method for skin sensitization testing. Available at: http://publications.jrc.ec.
europa.eu/repository/bitstream/JRC103706/esac_opinion_2016-04_lusens_final.pdf10) JaCVAM 評価会議

評価会議報告書 ARE-Nrf2 Luciferase Test Method（角化細胞株レポー

ターアッセイ）（平成 27 年 4 月 30 日）

66評価報告書皮膚感作性試験代替法
ARE-Nrf2 luciferase LuSens test method (LuSens test method)

皮膚感作性試験資料編纂委員会

令和元年（2019 年）9 月 30 日

7

皮膚感作性試験資料編纂委員会

8

小島幸一

（一般財団法人

食品薬品安全センター）

足利太可雄（国立医薬品食品衛生研究所）

安達玲子（国立医薬品食品衛生研究所）

佐藤一博（国立大学法人

瀬崎拓人（三井化学株式会社）

武吉正博（一般財団法人

福山朋季（麻布大学）福井大学）
化学物質評価研究機構）

要旨
皮膚感作性は化学物質の安全性評価において重要な評価項目であり、モルモットやマウスを用いた動物実験によって評価されてきた。近年 EU における欧州化学品規制では、コン
ピューターを用いた定量的構造活性相関（Quantitative Structure-Activity Relationship、以下、
QSAR と記す）モデルや in vitro 試験法による安全性評価が推奨されており、動物実験によって安全性が評価された成分を含む化粧品の販売が禁止（2013 年 3 月全面施行）されたことから、in vitro 試験法の開発が強く望まれている。
ARE-Nrf2 luciferase LuSens test method（以下、LuSens test method と記す）は、既に経済協力開発機構（以下、OECD と記す）試験法ガイドライン（以下、TG と記す）TG 442D に登録されている ARE-Nrf2 luciferase KeratinoSensTM test method（以下、KeratinoSensTM と記す）と同様に、感作性発現機序における第 2 の Key event（「2．試験法の原理」参照）に対応する試験法であり、基本原理は Keap1-Nrf2-ARE pathway を利用したレポーターアッセイである。
LuSens test method は、開発者（BASF 社）によるインハウス試験および外部 4 機関の参加による TG 442D の Performance standards（以下、性能標準と記す）に基づくバリデーション研
究が行われ、その結果を EURL ECVAM Scientific Advisory Committee（以下、ESAC と記す）
が第三者評価し、2018 年 6 月に OECD の TG に追記された（OECD TG 442D Appendix 1B）。
本報告書は、OECD TG 442D Appendix 1B および関連資料などをもとに、試験手順をまとめ、有用性や限界などについて評価したものである。
性能標準に記載されている 20 物質のうちの 12 物質を用いた 3 機関による施設内再現性
は 100%であった。性能標準に記載されている 20 物質を用いて開発者を含む 5 施設において、少なくとも 1 物質につき 3 施設以上で実施した施設間再現性は 100%であった。いずれも性能標準で要求している 80%以上であることという基準を満たしていた。
開発者によるインハウス試験においては、ヒトデータ（69 物質）と比較した場合、感度は 83%、特異度は 78%、正確度は 81%、LLNA データ（72 物質）と比較した場合、感度は75%、特異度は 71%、正確度は 74%であった。
性能標準に記載されている 20 物質を用いて行った LuSens test method のバリデーション研
究と KeratinoSensTM のバリデーションデータを比較したところ、両試験法とも 20 物質中 17
物質を正しく判定しており、正確度は 85%であった。両試験法の試験結果の詳細は、ESACで評価され、両試験法についてどちらか一方を推奨する科学的な根拠はないとされており、両試験法の予測性はほぼ同等と本委員会では評価した。
LuSens test method は、KeratinoSensTM と同様、リジン残基特異的に結合する物質は偽陰性と判定されることが考えられ、注意が必要である。本試験法に使用する細胞株の代謝能は限定的であり、プロハプテンおよびプレハプテンは陰性となる可能性がある。一方、感作性のないケミカルストレッサー（例えば酸化ストレスを誘導する物質）も KeratinoSensTM と同様、偽陽性となる可能性がある。さらに、ルシフェラーゼに干渉する化学物質も評価に影響する可能性がある。
以上の結果から、LuSens test method は、KeratinoSensTM と同様、試験の実施と評価のた
めの戦略的統合方式（Integrated Approaches to Testing and Assessment, 以下、IATA と記す）において他の試験法と組み合わせることにより、感作性物質と非感作性物質との区別に使用

9することが可能である。しかし、本試験法単独での感作性強度分類や UN GHS（UN Globally
Harmonized System of Classification and Labeling of Chemicals）のサブカテゴリー分類への応用には適さない。
本委員会は、上記の本試験法の様々な限界を勘案すると、本試験法単独では皮膚感作性の判定は不十分であり、証拠の重み付けや他の試験法との組合せで用いることを推奨する。
1．緒言
皮膚感作性を評価することは化学物質の安全性評価において重要である。化学物質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皮膚感作性試験（OECD TG 406）やマウスを用いる局所リンパ節試験（以下、LLNA と記す。OECD TG
429）がある。この[3H-Methyl]-thymidine 取込量を測定する LLNA 以外に、ATP 量を測定す
る LLNA:DA（OECD TG 442A）や Bromodeoxyuridine 量を測定する LLNA: BrdU-ELISA お
よび BrdU-FCM（OECD TG 442B）がある。
EU における欧州化学品規則（REACH: Registration, Evaluation, Authorisation and Restrictionof Chemicals）では、安全性評価においてはコンピューターを用いた QSAR モデルや in vitro
試験等による代替法の活用が推奨されており、動物実験により安全性が評価された成分を含んだ化粧品の販売が禁止された（2013 年 3 月全面施行）。そのため、化学物質の皮膚感作性を評価する代替法の開発が強く求められた。
現在、ペプチドとの結合反応を利用した Direct Peptide Reactivity Assay（DPRA, OECD TG
442C）、多くの皮膚感作性物質が Antioxidant response element (ARE）に制御される遺伝子の発現を誘導することを利用した ARE-Nrf2 Luciferase Test Method（KeratinoSensTM, OECD TG
442D）、単球系細胞の活性化を利用した human Cell Line Activation Test（h-CLAT, OECD TG
442E）、U-SENSTM（OECD TG 442E）および IL-8 Luc assay（OECD TG 442E）などの皮膚感作性試験の in vitro 法が EURL ECVAM 等においてバリデーション研究が行われガイドライ
ン化されている。
LuSens test method 1) は、ヒトケラチノサイト由来細胞株にルシフェラーゼレポーター遺伝子を安定的に導入した細胞系を使用する試験法である。EURL ECVAM によるバリデーシ
ョン研究は行われず、BASF 社を中心に TG 442D の性能標準に基づくバリデーション研
究が行われた 2, 3)。その結果について、ESAC による第三者評価が行われ、2018 年 6 月にOECD の TG 442D に追記された（Appendix 1B）1)。一方、KeratinoSensTM は、OECD TG 442D
Appendix 1A となった。
JaCVAM 皮膚感作性試験資料編纂委員会（以下、委員会）は、LuSens test method の皮膚感作性試験代替法としての科学的妥当性について、BASF 社主導で行われたバリデーション研
究の結果および現在までに公開されている情報等をもとに評価したので、その結果を報告する。
2．試験法の原理
皮膚感作性は、ヒトでは接触皮膚炎、動物（齧歯類）では接触過敏症として知られる化学物質の毒性の一つである。OECD がまとめた Adverse Outcome Pathway（有害性発現経10

路、AOP）では、化学物質による皮膚感作は次の 4 つの Key event（KE）からなるとされ
ている。
KE1:

化学物質とタンパク質のシステイン残基あるいはリジン残基との共有結合

KE2:

ケラチノサイト応答（細胞防御関連遺伝子の活性化等）

KE3:

樹状細胞応答（特異的細胞表面マーカー、炎症性サイトカインの発現等）

KE4:

リンパ節応答（T 細胞の活性化、増殖等）

LuSens test method は既に OECD TG 442D に登録されている KeratinoSensTM と同様に、上記の KE2 に対応する試験法であり、基本原理は Keap1-Nrf2- ARE pathway（図 1）を利用したレポーターアッセイである。
Keap1-Nrf2- ARE pathway は、転写因子 Nrf2（Nuclear factor-erythroid 2-related factor 2）の抑制因子である Keap1（Kelch-like ECH-associated protein 1）および ARE が関係する遺伝子発現経路である。Nrf2 は Keap1 と結合し、ARE に依存して発現する遺伝子群の発現量を制御している。Keap1 のシステイン残基に求電子性の化学物質が結合すると、Nrf2 は Keap1 から解離し、核内へ移行して DNA 上の ARE に結合する。その結果、下流の遺伝子群の発現が誘導され、化学物質による障害から細胞を保護するために機能する。多くの皮膚感作性物
質が Keap1-Nrf2-ARE pathway を活性化することが知られており 4, 5)、in vitro 感作性試験法の開発に利用されている。

図 1．Keap1-Nrf2-ARE pathway の模式図
KeratinoSensTM 6) では、Aldo-Keto Reductase Family 1 Member C2（AKR1C2）遺伝子の AREをエンハンサーとするルシフェラーゼレポーター遺伝子を安定的に導入した HaCaT 細胞（ヒトケラチノサイト系培養細胞）を用いるのに対し、LuSens test method 7) ではラットのNADPH:quinone oxidoreductase 1（NQO1）遺伝子由来の ARE をエンハンサーとするルシフェラーゼレポーター遺伝子を安定的に導入したヒトケラチノサイト系培養細胞（株名は不詳）を試験に使用する。

11

いずれの方法も化学物質による Keap1-Nrf2-ARE pathway の活性化に伴って誘導されるルシフェラーゼの活性を、基質を添加して発光強度を測定することにより、化学物質の皮膚感
作性を評価する方法である。
3．試験手順／判定

LuSens test method を実施するうえでのプロトコルは EURL ECVAM のデータベース（DB-ALM）より、遺伝子組み換え培養細胞株（LuSens）は BASF 社よりそれぞれ提供

されている。ルシフェラーゼレポーター遺伝子検査は、既に同 TG に収載されている
KeratinoSensTM と同様に、①発光検出試薬は Promega 社から購入し、②レポーター遺伝子として利用されている改変ルシフェラーゼ遺伝子（Luc2）の使用に際しては、特許権者である Promega 社とライセンス契約を必要とする。

3−1．遺伝子改変細胞の準備および調製
ARE 制御下のルシフェラーゼレポーター遺伝子を安定的に取り込んだトランスジェ
ニック細胞系を用いる。細胞の受領後、標準プロトコルで指定された継代数の細胞（1から 3 代）を増殖し、主ストック細胞として凍結保存する。主ストック細胞から増殖させた細胞は、指定された継代数（20 代）以内で試験に使用する。細胞培養培地は標準プ
ロトコルに記載されているように基礎培地（例：Dulbecco’s Modified Eagle’s medium（DMEM））に成長因子と抗生物質として Penicillin/Streptomycin および Puromycin を加えたものを使用する。しかし、検査実施中の細胞には抗生物質は使用しない。実験には、継代の時も含め 1 度も 100%コンフルエントになった事がない細胞を、80～90%コンフ
ルエントの状態で用いる。実験前日に、培養フラスコから調製した均一な細胞懸濁液を96 ウェルプレートに播種（10,000 cells / well）する。96 ウェルプレートへの播種時には、ウェル間の細胞数に偏りが出ないように、調製した細胞懸濁液が常に均一となるように留意する。ルシフェラーゼ活性測定および細胞生存性検査では、濃度毎に 3 ウェルを実施する。
3−2．被験物質の調製および濃度設定手順
被験物質および対照物質ともに原則として実験当日の調製とする（安定的に凍結保存されている液体は実験当日に溶解する）。被験物質はジメチルスルホキシド（DMSO,CAS No. 67-68-5）等の適切な媒体に溶解して、最高濃度（例：200 mM）の溶液を調製する（DMSO はそれ自体が滅菌状態にあると考える）。DMSO に不溶の場合は、滅菌精製水あるいは培養液にて同様に調製し、その溶液は、滅菌（例えば濾過）する。分子量が不明の被験物質の場合、200 mg/mL あるいは 20%（w/v）の溶液とする。DMSO、滅菌
水あるいは培養液以外の媒体を使用した場合は、その科学的な妥当性を明記する。
これらの溶液を DMSO（不溶の場合は滅菌水または滅菌培養液）で倍々希釈して 12
段階の濃度（0.098～200 mM）溶液を調製する。これを血清含有培養液で 25 倍希釈する。これらの調製溶液を各ウェルに加え、最終濃度を 0.98～2000 µM とする。分子量が不明の被験物質の場合、段階希釈は DMSO もしくは適切な媒体で実施し、最終濃度を12

0.98～2000 μg/mL とする。
濃度設定のための細胞毒性検査は上記に記載された濃度を基に実施し、細胞生存率
が 75%となる濃度（CV75）を決定するために実施する。CV75 はルシフェラーゼ活性測
定および並行して実施される細胞毒性検査における濃度設定の際に使用される（例：公比 1.2 で設定した濃度群において、CV75 より 1 濃度高い濃度（CV75×1.2）、CV75、CV75 の下 4 濃度（CV75/1.2, CV75/1.44, CV75/1.73, CV75/2.07））。細胞毒性が強すぎる、弱すぎる、もしくは溶解性が著しく低い被験物質の場合は、正当な理由に基づき代替の濃度が使用される。
媒体対照（例：DMSO）は、検査毎に 1 プレートあたり十分なウェル数を同様に調製する（例：標準プロトコルに準じ、細胞毒性検査では 12 ウェル、ルシフェラーゼ活性測定では 24 ウェル）
。媒体は被験物質や陽性対照物質と同様に希釈を行い（例：1%）、
細胞生存率に影響を及ぼさないようにする。
陰性対照も検査毎に 1 プレートあたり十分なウェル数を同様に調製する（例：標準プロトコルに準じ、細胞毒性検査では 3 ウェル、ルシフェラーゼ活性測定では 6 ウェル）。
LuSens test method では、陰性反応が得られる事が既知の非感作性物質の DL-Lactic acid
（CAS No. 50-21-5, ≥ 99%）を 5000 μM（もしくは 450 μg/mL）にて用いる。上記以外の陰性対照物質についても十分な背景データがある場合には使用可能とする。また、十分なウェル数の培養液のみを細胞に添加した無処理区画を同様に調製する（例：標準プロ
トコルに準じ、細胞毒性検査では 6 ウェル、ルシフェラーゼ活性測定では 12 ウェル）。
LuSens test methodが適切に実施されているかを確認する目的で、陽性対照を検査毎に1プレートあたり十分なウェル数を同様に調製する（例：標準プロトコルに準じ、細胞毒性検査では 2 ウェル、ルシフェラーゼ活性測定では 5 ウェル）。LuSens test methodでは、120 μM Ethylene Glycol Dimethacrylate（EGDMA, CAS No. 97-90-5, ≥ 99%）を被験物
質と同様の希釈方法にて適用する。120 μM にて細胞毒性がみられる、もしくは陽性反応がみられない場合は、細胞毒性がみられない（細胞生存率が70%以上）もしくは陽性反応がみられる濃度を使用する。上記以外の陽性対照物質についても十分な背景データがある場合には使用可能とする。
3−3．被験物質および対照物質の適用
各被験物質および陽性対照物質について、1 プレート毎に 3 ウェルずつ、独立した少なくとも 2 回の繰り返し検査（各被験物質および陽性対照物質について n = 6）を実施
する。独立した 2 回の検査結果が不一致の場合、3 回目の検査を実施し、それぞれ合計
で n = 9 とする。各検査は別日に同じ継代細胞を用いるが、各実験は新たな被験物質調製液および細胞を用いて別日に行う。
細胞を播種した 24 時間培養後のプレートの培養液を捨て、1 ウェルあたり 150 µL の血清含有培地（抗生物質不含）で置き換える。調製した被験物質溶液等を 50 µL ずつ各
ウェルに加え、48 時間、37± 1℃、5%CO2 インキュベータ内で培養する。ただし、1 ウ
ェルは無処置（無細胞、空ウェル）とする。ウェルからの蒸発や交差汚染を避けるためにプレートごとに遮蔽する。

13

3−4．ルシフェラーゼ活性の測定
適切なルシフェラーゼ活性の測定には、1）感度の良いルミノメーター、2）光の交差による測定の妨害を防ぐに十分なウェルの高さを持ったプレート、3）十分な感度とばらつきの低い測定値を得るためのルシフェラーゼ基質の選択、および 4）適切かつ安定した背景値が重要である。これらを確認するために TG 442D Appendix 1B-Annex 3 に示されたセットアップ方法を試験前に確認することを勧める。
培養終了後、上清を捨て、リン酸緩衝化生理食塩水で一度洗う。細胞溶解用緩衝液を各ウェルに加え、暗所で十分な時間処理する（例えば5～10分）。細胞溶解物を含むプ
レートを標準プロトコルに従って処理し、ルミノメーターで測定する。
3−5．細胞生存率の測定
培養終了後に培地を 0.5 mg/ml の MTT
LuSens test method における細胞生存率の測定は、（3-（4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium
bromide; CAS No. 298-93-1）含有新鮮培地に交換する。細胞は 2 時間、37± 1℃、5%CO2 イ
ンキュベータ内で培養する。 培養後、MTT 含有培地を捨て、10% SDS （Sodium Dodecyl
Sulphate）溶液などで一晩細胞溶解後、570 nm の吸光度を測定する。
3−6．データの解析
LuSens test method では、以下の指標を算出する。
・被験物質および陽性対照で観察されたルシフェラーゼ活性の最大誘導倍率
・被験物質の 75%生存率（CV75）
ルシフェラーゼ活性が 1.5 倍以上の被験物質各濃度について、ルシフェラーゼ活性の誘導が統計学的に陰性対照に対して有意（p＜0.05、two-tailed Student’s t-test）であるかを検証する。さらに、上記の濃度において細胞毒性がみられないことを確認する。グラフを作成して視覚的に確認することも推奨される。明らかな濃度依存性が認められない
場合や濃度反応曲線が二相性を示す場合には、測定を繰り返し、被験物質特異的か、測定エラーなのか、などを確認する。二相性であることが確認できた場合は、低い方のEC1.5 値を選択する。
3−7．試験成立の条件
LuSens test method においては、以下の 3 条件をすべて満たす場合に成立する。
・陽性対照物質の 120 μM EGDMA（もしくはそれと同等の濃度）において、平均のルシフェラーゼ活性が 2.5 以上かつ細胞生存率が媒体対照群と比較して 70%以上でなくてはならない。
・陰性対照物質（例：5000 μM DL-Lactic acid）および無処置区画において平均のルシ
フェラーゼ活性が媒体対照と比較して 1.5 より低くなければならない。
・プレート間の媒体対照の平均変動係数が 20%以下でなければならない。
・少なくとも被験物質 3 濃度の細胞生存率が媒体対象群と比較して 70%以上でなけ

14

ればならない。さらに、結果が陰性の場合、少なくとも 1 濃度の細胞生存率は 70%より低くなければならない。
3−8．陽性の判定
2 回の繰り返し実験の 2 回あるいは 3 回の繰り返し実験の 2 回で、以下の条件に合致した場合に被験物質は感作性物質と判断する。そうでない場合は、陰性と判断する。
・連続した細胞毒性のない（細胞生存率が 70%以上）被験物質 2 濃度においてルシフ
ェラーゼ活性が媒体対照と比較して 1.5 倍以上で、媒体対照と比較して統計学的に有意であること。上記を満たすためには少なくとも被験物質 3 濃度において細胞毒性がない必要がある。
上記に加え、2000 µM（分子量未知の場合は 2000 mg/mL）でも検査間のばらつきなく陰性で、細胞毒性もみられない場合、その結果は評価不能とする。
4．精度
本試験法の技術移転性、施設内再現性、施設間再現性については、BASF 社によるインハ
ウス試験、および外部 4 施設が参加したバリデーション研究において検討され、EURLECVAM によって評価されている 2, 3, 7, 8)。
4−1．技術移転性 2, 8)
8 物質を用いて、主導施設の BASF 社から Burleson Research Technologies 社、DSM
Nutritional Products 社、Institute for In Vitro Sciences（IIVS）および The Procter & Gamble 社の4 施 設 へ の技 術 移 転性に つ い て評価 が 行 われた （ Phase I）。 4 施 設の う ち 2 施 設 はKeratinoSensTM のバリデーション研究にも参加していた。技術移転性の基準は、（1）8 物質中 6 物質以上で正しい判定結果が得られること（2）Validity criteria を満たす試験結果の割合が全体の 70%以上であることとされた。1 施設は開発者である BASF 社で試験法を習得し、3 施設は SOP の提供だけで試験を実施した。
結果を表 1-1 に示す。全ての施設で上記（1）、（2）の基準を満たしており、技術移転性に問題は無いと考えられた。但し、陽性対照物質である EGDMA に関しては、その細胞毒性により細胞生存率が 60%未満となる場合があったため、以降のバリデーション研究（Phase
II）では、Validity criteria として陽性対照物質を添加した際の細胞生存率が 70%以上である
ことが追加された。

15

表 1-1.

技術移転性に関する試験結果

Lead lab

Total repetitions
Valid repetitions (%)

Results
Positive Negative
5
0
4
1
0
4
2
0
0
2
3
0
2
0
2
2
30
90

Total repetitions
Valid repetitions (%)

Results
Positive Negative
2
2
3
0
0
2
4
0
0
4
3
0
2
0
0
2
24
1 00

Total repetitions
Valid repetitions (%)

Results
Positive Negative
2
0
3
0
2
2
2
0
0
2
2
0
2
1
0
2
21
95

Total repetitions
Valid repetitions (%)

Results
Positive Negative
2
1
3
1
0
3
4
0
1
3
3
0
3
0
0
3
34
79

Total repetitions
Valid repetitions (%)

Results
Positive Negative
2
1
4
0
0
4
4
0
0
5
3
0
0
3
0
3
40
73

Human/LLNA data
α-Hexyl-cinnamic aldehyde
1-Chloro-2,4-dinitrobenzene
Chlorobenzene
CitralDL-Lactic acid
Ethylene glycol dimethacrylate
Methyl salicylate
Sulfanilamide

+
+
+
+
-

invalid
0
0
0
1
1
0
1
0

Final
+
+
+
+
+
?
6/ 8

Lab 1
Human/LLNA data
α-Hexyl-cinnamic aldehyde
1-Chloro-2,4-dinitrobenzene
Chlorobenzene
CitralDL-Lactic acid
Ethylene glycol dimethacrylate
Methyl salicylate
Sulfanilamide

+
+
+
+
-

invalid
0
0
0
0
0
0
0
0

Final
?
+
+
+
+
6/ 8

Lab 2
Human/LLNA data
α-Hexyl-cinnamic aldehyde
1-Chloro-2,4-dinitrobenzene
Chlorobenzene
CitralDL-Lactic acid
Ethylene glycol dimethacrylate
Methyl salicylate
Sulfanilamide

+
+
+
+
-

invalid
1
0
0
0
0
0
0
0

Final
+
+
?
+
+
+
6/ 8

Lab 3
Human/LLNA data
α-Hexyl-cinnamic aldehyde
1-Chloro-2,4-dinitrobenzene
Chlorobenzene
CitralDL-Lactic acid
Ethylene glycol dimethacrylate
Methyl salicylate
Sulfanilamide

+
+
+
+
-

invalid
2
1
1
0
0
1
1
1

Final
+
+
+
+
+
7/ 8

Lab 4
Human/LLNA data
α-Hexyl-cinnamic aldehyde
1-Chloro-2,4-dinitrobenzene
Chlorobenzene
CitralDL-Lactic acid
Ethylene glycol dimethacrylate
Methyl salicylate
Sulfanilamide

16

+
+
+
+
-

invalid
2
1
1
1
0
2
2
2

Final
+
+
+
+
8/8

4−2．施設内再現性 2, 7, 8)
BASF 社によるインハウス試験では、74 物質中 69 物質（93%）で再現性がみられた 2, 7)。
3 機関によるバリデーション研究では、性能標準 9) に記載されている 20 物質の内から 12物質が選択され、施設内再現性が評価された。3 機関とも施設内再現性は 100%であり、性能標準に記載されている 80%以上という基準を満たしていた（表 1-2）2, 8)。ただし、ヒト/
LLNA データと比較すると、4-Methoxy-acetophenone は偽陽性、Phenyl benzoate は偽陰性である。なお本委員会では、用いられた 20 物質および 12 物質の Chemical class、Mechanism、
感作性カテゴリー、Pro/ prehapten に関する情報から、これらの物質は適切に選択されたものと判断した。
表 1-2．施設内再現性に関する試験結果 2)+: positive/skin sensitiser prediction; -: negative/non-sensitiser prediction; #experiments conductedin the transferability phase; *experiments with three repetitions.

4−3．施設間再現性 2, 8)性能標準 9) に記載されている 20 物質を用いて施設間再現性が評価された。12 物質に関しては、前述の 3 施設（Lead lab、Lab 1 および Lab 2）の施設内再現性評価の際の結果が用いられた。残りの 8 物質に関しては Lead lab、Lab 3 および Lab 4 の 3 施設で試験を実施した。また Lab 3、Lab 4 の 2 施設はさらに 5 物質の試験を実施した。
結果を表 1-3 に示す。全ての物質で施設間再現性は 100%であり、性能標準に記載されている 80%以上という基準を満たしていた。ただし、ヒト / LLNA データと比較すると、4Methoxy-acetophenone および Methyl salicylate は偽陽性、Phenyl benzoate は偽陰性である。

17

表 1-3．5 施設によるバリデーション研究結果 2)

NS: non-sensitiser; S: sensitiser; na: not applicable; +: positive/skin sensitiser prediction; -:negative/non-sensitiser prediction; nt: not tested; #experiment conducted in the transferability phase.

5．正確度（感度および特異度）2, 3, 7, 8, 9)
BASF 社による 74 物質を用いたインハウス試験の結果 2, 7, 8)、および 5 施設によるバリデーション研究の結果（表 1-3）2, 9)から、感度、特異度および正確度が評価された。
BASF 社によるインハウス試験においては、ヒトデータ（69 物質）と比較した場合、感度は 83%、特異度は 78%、正確度は 81%、LLNA データ（72 物質）と比較した場合、感度は75%、特異度は 71%、正確度は 74%であった（なおインハウス試験結果 7) およびその改訂
結果 8)とも本文中の数値が表の内容と一致しなかったため、これらの正確度の数値は表の記載から本委員会により計算された）。本試験法の感度は 83%（ヒト）、75%（LLNA）であり、陰性の結果が得られた場合は偽陰性の可能性を考慮し、本試験法のみで皮膚感作性を陰性と判定することはできない。さらに偽陰性となった 13 物質中 2 物質（Phthalic anhydride 及び Propyl gallate）は UN GHS 1A に区分される物質であったことに特に注意する必要がある。

18

また、特異度も 78%（ヒト）と 71%（LLNA）であることから、本試験法で陽性の結果が得られた場合にも、偽陽性の可能性があることに留意しなければならない。
Phthalic anhydride はアシル基転移剤であり 7)、リジン残基特異的に結合することが知られているため、Keap1-Nrf2-ARE pathway の活性化を指標とする本試験法では偽陰性になったと考えられる。Propyl gallate については Prohapten の一種である Pro Michael acceptor であり
7)、代謝されないとタンパクと結合しないため偽陰性になったと考えられる。その他の偽陰性物質については、アシル基転移剤（Benzoyl peroxide および Phenyl benzoate）、Pro/ prehapten（4-Allylanisole, Ethylene diamine および Resorcinol）、システイン残基と共有結合しない金属
イオン（Nickel chloride）などの理由が考えられた。一方、偽陽性物質については、光感作性物質として知られている 6-Methlycoumarin が Michael acceptor としてタンパク質と結合する可能性がある以外、不明であった。
5 施設によるバリデーション研究（表 1-3）においては、感度は 88-100%、特異度は 7586%、正確度は 83-92%であった。ただし、試験に用いた検体のサブセットは施設によって異なっている。Lab 3 および Lab 4 の感度および特異度が他施設より高いのは、他施設の試験において偽陰性となった Phenyl benzoate および偽陽性となった 4-Methoxy-acetophenone の試験を実施していないためと考えられる。20 物質全ての試験結果をまとめると、感度は 92%、
特異度は 75%、正確度は 85%であった。性能標準 10) では、これら 20 物質を用いた評価において、感度、特異度および正確度が 80%以上という基準が示されている。本試験法の感度および正確度は 80%以上であったが、特異度はこの基準を満たさなかった。特異度が 80%未満だったのは非感作性物質 8 品のうち 2 品が偽陽性になったためであり、本試験法の特異度を判断するためにはこの被験物質数では不十分と考えられたものの、本試験法において陽性の結果が得られた場合には、偽陽性の可能性があることに留意しなければならない。
上記 20 物質の本試験法と KeratinoSensTM の VRM（Validated Reference Method）による比較試験結果を表 2-1 に示す。両試験法とも 20 物質中 17 物質を正しく判定しており、正確度
は 85%であった。本試験法で偽陽性となった Methyl salicylate は、KeratinoSensTM では陰性と判定され、本試験法で陽性と判定された Eugenol は KeratinoSensTM では偽陰性となった。
ESAC opinion 3) では、両試験法のバリデーション研究結果の詳細が確認されており、この 2物質については両試験法ともに陽性と判定された個別の実験（run）と陰性と判定された個別の実験があったこと、すなわちこの 2 物質は判定の難しい物質であると記載されている（表 2-2）。両試験法で偽陰性となった Phenyl benzoate は Weak sensitizer である。また 4Methoxy-acetophenone は両試験法で偽陽性となった。これらの結果から、ESAC opinion では両試験法についてどちらか一方を推奨する科学的な根拠はないと評価されており、両試験
法の予測性はほぼ同等と考えられる。
以上より、本試験法単独では皮膚感作性の予測性は不十分であり、証拠の重み付けや他の試験法と組み合わせでの評価を推奨する。

19

表 2-1．LuSens test method と KeratinoSensTM との比較-12)

NS: non-sensitiser; S: sensitiser; +: positive/skin sensitiser; -: negative/non-sensitiser.

20

表 2-2．LuSens test method と KeratinoSensTM との比較-2 3)

P = Positive run
N = Negative run( ) = Tested in addition to what is rerequested in the performance standards (PS)( ) brackets correspond to an experiment, e.g., for experiment (N-P-P) the final conclusion is P.
In bold face are the 12 reference substances for which, according to the PS, WLR information needs
to be generated. BLR information in turn, is required for all 20 reference substances.
6．評価可能な物質の範囲および有用性と限界
本試験法は、さまざまな構造や物理化学的性質を有する化学物質に適用可能であることが示されている 7)。また、本試験法に用いる溶媒に溶解する、もしくは安定に分散する化合物は適用可能であるが、適用可能最高濃度においても細胞毒性がみられない場合（細胞生存率 70%以上）の陰性結果は「評価不能」とされる。
本試験法は KE2 を評価する in vitro 試験法である KeratinoSensTM と同様、システイン残基との反応が必要な Keap1-Nrf2-ARE pathway の活性化を指標としており、リジン残基特異的に結合し、感作性を示す物質は偽陰性と判定されることが考えられるため、そのような物質（例えば、UN GHS 1A の Phthalic anhydride）の陰性結果は注意が必要である 7)。本試験法

21

に使用する細胞株の代謝能は限定的であり 11)、本試験条件からも、プロハプテンおよびプレハプテン（例えば、UN GHS 1A の Propyl gallate）は陰性となる可能性がある。一方、感作性のないケミカルストレッサー（例えば酸化ストレスを誘導する物質）は KeratinoSensTMと同様、偽陽性となる可能性がある 12)。さらに、ルシフェラーゼ活性に干渉する化学物質も評価に影響する可能性がある 13)。
本試験法は、細胞培養の技術と 96 ウェル対応のルミノメーターの使用技術があれば容易に実施可能である。LuSens test method はケラチノサイトにおける ARE-dependent pathway による遺伝子発現を指標にした検査法であることから、同様に Keap1- Nrf2-ARE pathway を利用したレポーターアッセイである KeratinoSensTM と基本的な操作は同様である。本試験法の実施に必要なランニングコストは KeratinoSensTM と同様約 1.5 万円と見積もられ、LLNA（同約 10 万円）より低額であり、h-CLAT（同約 2 万円）や U-SENSTM（同約 1.8 万円）とほぼ同程度であった。本試験法の場合、開発元の BASF 社より細胞株の入手が無償で可能である（輸送費など実費別）。一方、KeratinoSensTM では現在は細胞株の入手に関して販売を委託された acCELLerate 社より 500 ユーロで購入可能である（実費別）。両試験法とも細胞の入手に関して開発元とのライセンス契約は不要である。
本試験法は、IATA において他の試験法と組み合わせることにより、感作性物質と非感作性物質との識別に使用することが可能である。しかし、本試験法単独での感作性強度分類やGHS のサブカテゴリー分類への応用には適さない。
7．結論
LuSens test method は、既に OECD TG 442D に登録されている KeratinoSensTM と同様に、感作性発現機序における KE2 に対応する試験法である。
KeratinoSensTM では、AKR1C2 遺伝子の ARE をエンハンサーとするルシフェラーゼレポ
ーター遺伝子を安定的に導入した HaCaT 細胞（ヒトケラチノサイト系培養細胞）を用いるのに対し、LuSens test method ではラットの NQO1 遺伝子由来の ARE をエンハンサーとする
ルシフェラーゼレポーター遺伝子を安定的に導入したヒトケラチノサイト系培養細胞（株名不詳）を使用する。いずれも化学物質による Keap1-Nrf2-ARE pathway の活性化に伴って誘導されるルシフェラーゼの活性を、基質を添加して発光強度を測定することにより、化学物質の皮膚感作性を評価する方法である。
LuSens test method は、EURL ECVAM によるバリデーション研究は行われず、BASF 社によるインハウス試験および外部 4 機関が参加した性能標準に基づくバリデーション研究が行われ、その結果を EURL ECVAM が評価して、2018 年 6 月に OECD TG 442D に追記された（Appendix 1B）。
本試験法は、細胞培養の技術と 96 ウェル対応のルミノメーターの使用技術があれば容易に実施可能である。また、本試験法に用いる細胞株は BASF 社から入手可能である。
BASF 社による 74 物質を用いた施設内再現性は 93%であり、3 機関による施設内再現性（12 物質について）は 100%であった。20 物質を用いて BASF 社を含む 5 施設において少なくとも 1 物質につき 3 施設以上が実施した施設間再現性試験の結果は 100%であった。いずれも性能標準で要求している 80%以上の基準を満たしていた。

22

BASF 社によるインハウス試験においては、ヒトデータ（69 物質）と比較した場合、感度は 83%、特異度は 78%、正確度は 81%、LLNA データ（72 物質）と比較した場合、感度は75%、特異度は 71%、正確度は 74%であった。用いた検体のサブセットは施設によって異なっているが、5 施設によるバリデーション研究においては、感度は 88-100%、特異度は 7586%、正確度は 83-92%であった。
性能標準に記載されている 20 物質を用いて行われた LuSens test method と KeratinoSensTMの比較試験の結果では、両試験法とも 20 物質中 17 物質を正しく判定しており、正確度は85%であった。両試験法の試験結果の詳細から、ESAC による評価では両試験法についてど
ちらか一方を推奨する科学的な根拠はないとしている。本委員会は両試験法の予測性はほぼ同等と評価した。
LuSens test method は、KeratinoSensTM と同様にシステイン残基との反応が必要な Keap1Nrf2-ARE pathway の活性化を指標としており、リジン残基特異的に結合し、感作性を示す
物質は偽陰性と判定されることが考えられるので注意が必要である。本試験法に使用する細胞株の代謝能は限定的であり、プロハプテンおよびプレハプテンは陰性となる可能性がある。一方、感作性のないケミカルストレッサー（例えば酸化ストレスを誘導する物質）はKeratinoSensTM と同様、偽陽性となる可能性がある。さらに、ルシフェラーゼに干渉する化学物質も評価に影響する可能性がある。
以上の結果から、LuSens test method は、IATA において他の試験法と組み合わせることにより、感作性物質と非感作性物質との区別に使用することが可能である。しかし、本試験法
単独での感作性強度分類や UN GHS のサブカテゴリー分類への応用には適さない。
本委員会は、上記の本試験法の様々な限界を勘案すると、本試験法単独では皮膚感作性の判定は不十分であり、証拠の重み付けや他の試験法との組合せで用いることを推奨する。
引用文献
1)

OECD (2018). Key event based test guideline. In vitro skin sensitization assay addressing the
AOP key event on keratinocyte activation. OECD Guideline for the Testing of Chemicals. No.
442D, OECD, Paris. Accessible at: https://www.oecd-ilibrary.org/docserver/9789264229822-en.
pdf?expires=1540286232&id=id&accname=guest&checksum=F9DC614E0BBF02D46223C8F
CABCFBA5E

2)EURL ECVAM (2018). The LuSens Test Method Validation Study Report. Accessible at;https://tsar.jrc.ec.europa.eu/test-method/tm2011-10

3)

ESAC (2016). ESAC Opinion on the BASF-coordinated Performance Standards-based validationof the LuSens test method for skin sensitization testing. Available at: http://publications.jrc.ec.
europa.eu/repository/bitstream/JRC103706/esac_opinion_2016-04_lusens_final.pdf

4)

Ade N, Leon F, Pallardy M, Peiffer JL, Kerdine-Romer S, Tissier MH, Bonnet PA, Fabre I, Ourlin
JC; HMOX1 and NQO1 genes are upregulated in response to contact sensitizers in dendritic cells
and THP-1 cell line: role of the Keap1/Nrf2 pathway. Toxicol Sci. 2009; 107(2):451-60.

5)Vandebriel RJ, Pennings JL, Baken KA, Pronk TE, Boorsma A, Gottschalk R, Van Loveren H;
Keratinocyte gene expression profiles discriminate sensitizing and irritating compounds. Toxicol23

Sci. 2010; 117(1):81-9.
6)

Emter R, Ellis G, Natsch A; Performance of a novel keratinocyte-based reporter cell line to screen
skin sensitizers in vitro. Toxicol Applied Pharmacol, 2010; 245: 281-290.

7)

Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, vanRavenzwaay B, Landsiedel R; LuSens: a keratinocyte based ARE reporter gene assay for use inintegrated testing strategies for skin sensitization hazard identification. Toxicol In Vitro. 2014;
28(8):1482-97.

8)

Kolle SN; Corrigendum to “LuSens: a keratinocyte based ARE reporter gene assay for use inintegrated testing strategies for skin sensitization hazard identification.” by Ramirez et al., Toxicol
In Vitro. 2014; Dec; 28(8):1482–97. doi: 10.1016/j.tiv.2014.08. 002

9)Ramirez T., Stein N., Aumann A., Remus T., Edwards A., Norman K.G., Ryan C., Bader J.E.,
Fehr M., Burleson F., Foertsch L., Wang X., Gerberick F., Beilstein P., Hoffmann S., Mehling A.,
van Ravenzwaay B., Landsiedel R; Intra- and inter-laboratory reproducibility and accuracy of the
LuSens assay: A reporter gene-cell line to detect keratinocyte activation by skin sensitizers.
Toxicol. In Vitro. 2016; 32: 278-286.

10) OECD (2015). Guidance Document No. 213. Performance Standards for assessment of proposed
similar or modified in vitro skin sensitisation ARE-NrF2 luciferase test methods. Series on
Testing and Assessment. Available at: http://www.oecd.org/env/ehs/testing/series-testingassessment-publications-number.htm
11) Fabian E., Vogel D., Blatz V., Ramirez T., Kolle S., Eltze T., van Ravenzwaay B., Oesch F.,
Landsiedel R. ; Xenobiotic metabolizn enzyme activities in cells used for testing skin sensitizationin vitro. Arch Toxicol. 2013; 87: 1683-1969.
12) EURL-ECVAM (2014). Recommendation on the KeratinoSensTM assay for skin sensitisation
testing, 42 pp. Available at: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvamrecomm
endations/recommendation-keratinosens-skin-sensitisation.
13) Thorne N., Inglese J., Auld D.S. ; Illuminating Insights into Firefly Luciferase and Other
Bioluminescent Reporters Used in Chemical Biology. Chemistry and Biology. 2010; 17: 646-657.

24

OECD/OCDE

442D

Adopted:
25June 2018

KEY EVENT BASED TEST GUIDELINE 442D
IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE AOP KEY
EVENT ON KERATINOCYTE ACTIVATIONGENERAL INTRODUCTION
Keratinocyte activation Key Event based Test Guideline
1.
A skin sensitiser refers to a substance that will lead to an allergic response
following repeated skin contact as defined by the United Nations Globally Harmonized
System of Classification and Labelling of Chemicals (UN GHS) (1). There is generalagreement on the key biological events underlying skin sensitisation. The current
knowledge of the chemical and biological mechanisms associated with skin sensitisation
has been summarised as an Adverse Outcome Pathway (AOP) (2), starting with themolecular initiating event through intermediate events to the adverse effect, namely
allergic contact dermatitis. This AOP focuses on chemicals that react with thiol (i.e.
cysteine) and primary amines (i.e. lysine) such as organic chemicals. In this instance, themolecular initiating event (i.e. the first key event) is the covalent binding of electrophilic
substances to nucleophilic centres in skin proteins. The second key event in this AOP
takes place in the keratinocytes and includes inflammatory responses as well as changes
in gene expression associated with specific cell signalling pathways such as theantioxidant/electrophile response element (ARE)-dependent pathways. The third key
event is the activation of dendritic cells, typically assessed by expression of specific cellsurface markers, chemokines and cytokines. The fourth key event is T-cell proliferation(3).
2.
This Test Guideline describes in vitro assays that address mechanisms describedunder the second Key Event of the AOP for skin sensitisation, namely keratinocyte
activation (2). The Test Guideline comprises test methods to be used for supporting thediscrimination between skin sensitisers and non-sensitisers in accordance with the
UN GHS (1). The test methods currently described in this Test Guideline are:


The ARE-Nrf2 luciferase KeratinoSens™ test method (Appendix IA), and

The ARE-Nrf2 luciferase LuSens test method (Appendix IB).

3.
These two in vitro ARE-Nrf2 luciferase test methods have been consideredscientifically valid. The KeratinoSens™ test method first underwent a validation study
followed by an independent peer-review by EURL ECVAM Scientific Advisory
Committee (ESAC) and positive recommendations by EURL ECVAM, and is consideredthe validated reference method (VRM) (3) (4) (5) (6). The LuSens test method later© OECD, (2018)
You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.
In accordance with the decision of the Council on a delegation of authority to amend Annex I of the decision of the council on the Mutual
Acceptance of Data in the assessment of chemicals [C(2018)49], this Guideline was amended by the OECD’s Joint Meeting of the Chemicals
Committee and the Working Party on Chemicals, Pesticides and Biotechnology by written procedure on 25 June 2018.

25

2│

442D

OECD/OCDE

underwent a Performance Standard-based validation study based on which it was also
reviewed and received positive opinion by ESAC (7) (8) (9) (10).
4.
The test methods included in this Test Guideline may differ in relation to theprocedure used to generate the data and the readouts measured but can be used
indiscriminately to address countries’ requirements for test results on the keratinocytes
activation Key Event of the AOP for skin sensitisation while benefiting from the Mutual
Acceptance of Data.

Background and principles of the test methods included in the Key Event based
Test Guidelines
5.
The assessment of skin sensitisation has typically involved the use of laboratory
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess
both the induction and elicitation phases of skin sensitisation. The murine tests, the
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
442B) (14), all assess the induction response exclusively, and have gained acceptancesince they provide an advantage over the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
6.
Mechanistically-based in chemico and in vitro test methods addressing the first
three key events of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; thepresent Test Guideline assesses keratinocyte activation addressing the second key eventand the OECD TG 442E addresses the activation of dendritic cells, the third key event of
the skin sensitisation AOP (16). Finally, the fourth key event representing T-cellproliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
7.
As keratinocyte activation represents only one key event of the skin sensitisation
AOP (2) (17), information generated with test methods developed to address this specific
key event may not be sufficient to conclude on the presence or absence of skinsensitisation potential of chemicals. Therefore data generated with the test methods
described in this Test Guideline are proposed to support the discrimination between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key eventsof the skin sensitisation AOP as well as non-testing methods, including read-across fromchemical analogues (17). Examples on the use of data generated with these methods
within Defined Approaches, i.e. approaches standardised both in relation to the set of
information sources used and in the procedure applied to derive predictions have been
published (17) and can be employed as useful elements within IATA.
8.
The test methods described in this Test Guideline cannot be used on their own,neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
GHS (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
framework, positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 201826

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

442D │ 3

9.
The term "test chemical" is used in this Test Guideline to refer to what is being
1
tested and is not related to the applicability of the test methods to the testing of monoconstituent substances, multi-constituent substances and/or mixtures. When testing insubmerged cultures, it should be determined that the test chemical is dissolved in the
exposure medium or at least forms a stable dispersion (e.g. by visual inspection of the test
chemical dissolved/prepared at the maximal final test concentration in the exposure
medium, showing that no undissolved residues remain and that no precipitate or phaseseparation forms if the solution is left to settle for several hours).
10.
Limited information is currently available on the applicability of the test methods
to multi-constituent substances/mixtures (18) (19) (20). Although not evaluated in thevalidation studies, the test methods may nevertheless be technically applicable to thetesting of multi-constituent substances and mixtures. When considering testing of
mixtures, difficult-to-test chemicals (e.g. unstable), or test chemicals not clearly within
the applicability domain described in this Guideline, upfront consideration should begiven to whether the results of such testing will yield results that are meaningful
scientifically. Moreover, when testing multi-constituent substances or mixtures,consideration should be given to possible interference of cytotoxic constituents with theobserved responses (e.g. the presence of a high content of non-sensitising cytotoxic
constituents may mask the response of weakly sensitising components or sensitising
components present at low concentration). It might, depending on the particular case, bescientifically justified to test either single main constituents forming the major fraction or
several fractions of the mixture to conclude on the sensitisation potential of the complex
mixture.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event of
Literaturepresenting T-cellay (LLNA) (12). 1) United Nations (UN) (2017). Globally Harmonized System of Classification and Labelling ofskin sensitisation
dress this specificabsence of skin 2)
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across from 3)th these methods
tion to the set of 4)
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own toChemicals (GHS). Seventh revised edition, New York and Geneva, United Nations Publications.
Available at: [https://www.unece.org/trans/danger/publi/ghs/ghs_rev07/07files_e0.html]
OECD (2012). Series on Testing and Assessment No. 168. The Adverse Outcome Pathway for
Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence.
Organisation for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/PART1&docLanguage=En
Emter R., Ellis G., Natsch A.(2010). Performance of a novel keratinocyte-based reporter cell line
to screen skin sensitizers in vitro. Toxicology and Applied Pharmacology 245, 281-290.
Natsch A., Bauch C., Foertsch L., Gerberick F., Normann K., Hilberer A., Inglis H., Landsiedel
R., Onken S., Reuter H., Schepky A., Emter R. (2011). The intra- and inter-laboratory
reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro:
results of a ring-study in five laboratories. Toxicol. In Vitro 25, 733-744.

1

In June 2013, the Joint Meeting agreed that where possible, a more consistent use of the term
"test chemical" describing what is being tested should be applied in new and updated Test
Guidelines.

©©OECD
OECD2018
2018

27

24 │

442D

OECD/OCDE

PerformanceStandard-based
study
on which
wasP.also5) underwent
Natsch A., aRyan
C.A., Foertsch
L., Emtervalidation
R., Jaworska
J., based
Gerberick
G.F., itKern(2013). A
reviewed
and145received
positivetested
opinionESAC (7) (8)(9) (10).
dataset on
chemicals
inbyalternative
assaysfor skin sensitization undergoingprevalidation. Journal of Applied Toxicology 33, 1337-1352.
4.
The test methods included in this Test Guideline may differ in relation to theused togeneratethe data andonthe
measuredassay
but forcan skinbe sensitisation
used6) procedure
EURL-ECVAM(2014).
Recommendation
thereadouts
KeratinoSensTM
indiscriminately
address countries’
requirements for test results on the keratinocytes
testing, 42 pp.to Available
at: [http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvamactivation
Key Event of the AOP for skin sensitisation while benefiting from the Mutual
recommendations/recommendation-keratinosens-skin-sensitisation].
Acceptance of Data.
7) Ramirez T., Mehling A., Kolle S.N., Wruck C.J., Teubner W., Eltze T., Aumann A., Urbisch D.,
van Ravenzwaay B., Landsiedel R. (2014). LuSens: a keratinocyte based ARE reporter gene
Backgroundand principles of the test methods included in the Key Event based
assay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol
Test
Guidelines
In Vitro
28, 1482-1497.
5.
Theof skin sensitisation
hasEdwards
typicallyA.,
involved
use Ryanof laboratory8) Ramirez T.,assessment
Stein N., Aumann
A., Remus T.,
Normanthe
K.G.,
C., Bader J.E.,
animals.
classical
use guinea-pigs,
the F.,
GuineaPig Maximisation
Fehr M.,The
BurlesonF., methods
Foertsch that
L., WangX., Gerberick
BeilsteinP., Hoffmann Test
S., Mehling(GPMT)of MagnussonB.,andKligman and
the Buehler
TG 406) (11),assessA., van Ravenzwaay
Landsiedel
R. (2016).
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and(OECD
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ToxicolVitro
32,and278-286.
(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
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ESAC (14),(2016).
on the BASF-coordinated
Performance
validation
allESACassessopinion
the induction
response exclusively,
and have Standards-based
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Available
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04_lusens_final.pdf].
6.
Mechanistically-based in chemico and in vitro test methods addressing the first10) threeOECD(2015).
Document
No 213.
Standards
assessmenttoofthe
proposed
key
eventsGuidanceof the skinsensitisation
AOPPerformance
have been adopted
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skin
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present
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and
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No.third
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Sensitisation.
theskinsensitisation
AOP
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Finally,thefourthkey
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representing
T-cellOrganisation for Economic Cooperation and Development, Paris. Available at:proliferation
is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
[http://www.oecd.org/env/testguidelines].
keratinocyte
only one
key eventof theSkin
skin sensitization:
sensitisation Local
12) 7.
OECD As(2010).
OECD activation
Guidelinesrepresents
for Chemical
Testing
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AOP
(2)
(17),
information
generatedwithtest
methods
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this Paris.
specific
Lymph Node assay. Organisation for Economic Cooperation and Development,
Available
key
event may not be sufficient to conclude on the presence or absence of skinat: [http://www.oecd.org/env/testguidelines].
sensitisation potential of chemicals. Therefore data generated with the test methods13) describedOECD (2010).
Guidelines
for Chemical
Testing
442A.Skin between
sensitization:in this OECD
Test Guideline
are proposed
to support
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assay:DA.
Organisationfor
Economic
Cooperation
andDevelopment,sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated Paris.
Available at: [http://www.oecd.org/env/testguidelines].
Approaches
to Testing and Assessment (IATA), together with other relevant
complementarye.g. derived
from in vitro
assaysaddressing
othersensitization:
key events Local
14) OECD (2010). information,
OECD Guidelines
for Chemical
TestingNo.
442B. Skinofthe skinsensitisation
AOP as wellOrganisation
as non-testing
includingread-across
from
Lymph
Node
assay: BrdU-ELISA.
formethods,
Economic
Cooperation
and Development,chemicalanalogues(17). Examples on the use of data generated with these methods
Paris. Available
at: [http://www.oecd.org/env/testguidelines].
within Defined Approaches, i.e. approaches standardised both in relation to the set of15) information
OECD (2015).
OECD
forprocedure
the Testingof Chemicals
No. 442C: have
In Chemicosources
usedGuideline
and in theappliedto derive predictions
been Skin
Sensitisation:
DirectPeptideReactivity
Assay(DPRA).
Organisationfor
Economic
Cooperation
published (17) and can be employed as useful elements within IATA.
and Development, Paris. Available at: [http://www.oecd.org/env/testguidelines].
8.
The test methods described in this Test Guideline cannot be used on their own,
16) neither
OECD to(2017).
OECD skin
Guideline
for theof Chemicals
442E: In
Vitro Skin
sub-categorise
sensitisers
into Testing
subcategories
1A and 1BNo.
as definedby UN
Sensitisation
assaysaddressing
the
AOP
Key
Event
on
ActivationofDendritic
GHS (1), for authorities implementing these two optional subcategories, nor to predict Cells.
Organisation
for Economic
CooperationHowever,and Development,
Available at:potency
for safetyassessment decisions.
depending on Paris.
the regulatory[http://www.oecd.org/env/testguidelines].
framework,
positive results generated with these methods may be used on their own toa chemical
intoonUN
GHS &category
1.
17) classify
OECD (2016).
SeriesTesting
Assessment
No. 256: Guidance Document On The Reporting
Of Defined Approaches And Individual Information Sources To Be Used Within Integrated© OECD 201828

OECD/OCDE

which it was also

442D │ 5

Approaches To Testing And Assessment (IATA) For Skin Sensitisation, Annex 1 and Annex 2.
ENV/JM/HA(2016)29. Organisation for Economic Cooperation and Development, Paris.
Available at: [https://community.oecd.org/community/iatass].

in relation to thebut can be used 18) Andres E., Sa-Rocha V.M., Barrichello C., Haupt T., Ellis G., Natsch A. (2013). The sensitivity
the keratinocytesof the KeratinoSensTM assay to evaluate plant extracts: A pilot study. Toxicology In Vitro 27,
from the Mutual
1220-1225.

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

19) Kolle, S.N., Mehling A., Teubner W., van Ravenzwaay B., Landsiedel R. (2013). Alternative
method in practice: Post-validation experience of the skin sensitization in vitro test strategy.
Toxicology Letters 221, S204.
20) Settivari RS, Gehen SC, Amado RA, Visconti NR, Boverhof DR, Carney EW (2015).
Application of the KeratinoSens™ assay for assessing the skin sensitization potential of
agrochemical active ingredients and formulations. Regul Toxicol Pharmacol; 72(2):350-60.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
2018

29

26 │

442D

OECD/OCDEa Performance Standard-based validation study based on which it was also
Annex underwent
I - DEFINITIONS

reviewed and received positive opinion by ESAC (7) (8) (9) (10).
Accuracy:closenessincludedof agreement
betweentest method
results
and accepted
4.
The Thetest methods
in this Test
Guideline
may differ
in relation
to thereferencevalues.
Itisameasureof
test
methodperformance
and
one
of
procedure used to generate the data and the readouts measured but can aspect
be used“relevance.”
The
term
is
often
used
interchangeably
with“concordance”,
to
mean
the
indiscriminately to address countries’ requirements for test results on the keratinocytes
proportionof correctof afortest
method(3).
activation Key
Event outcomesof the AOP
skinsensitisation
while benefiting from the Mutual
Acceptance of Data.
AOP (Adverse Outcome Pathway): sequence of events from the chemical structure of atarget chemical or group of similar chemicals through the molecular initiating event to an
Background and principles of the test methods included in the Key Event basedin vivo outcome of interest (2).

Test Guidelines

ARE: Antioxidant
response
element(also called
electrophile
responseis5.
The assessmentof skinsensitisation
has EpRE,
typically
involved theuse ofelement),laboratory
aanimals.
response
element
found
in
the
upstreampromoterregionofmanycytoprotective
andThe classical methods that use guinea-pigs, the Guinea Pig Maximisation Testphase
II genes.
When activatedby Nfr2,
mediatestheTesttranscriptional
these(GPMT)of Magnusson
and Kligman
andittheBuehler(OECD TGinduction406) (11),ofassess
genes.the induction and elicitation phases of skin sensitisation. The murine tests, theboth
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
CV: CellTG
viability(OECD442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
442B) (14), all assess the induction response exclusively, and have gained acceptance
Coefficientof variation:a measurea groupofsince they provide
an advantage
over of
thevariability
guinea pigthat
testsis incalculated
terms of foranimalwelfarereplicatedatabydividing
thestandard
deviation
by
the
mean.
It
can
be
multipliedby100
together with an objective measurement of the induction phase of skin sensitisation.
for expression as a percentage.
6.
Mechanistically-based in chemico and in vitro test methods addressing the firstthree
of theconcentrationskin sensitisation
AOP
adopted for contributing to theCV75:key
Theevents
estimated
resultingin have
75% beencell viability.
evaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes
the Directconcentration
Peptide Reactivity
Assay(15)foldaddressing
first key event; theEC1.5: Interpolated
resultingin a 1.5
luciferasethe
induction.
present Test Guideline assesses keratinocyte activation addressing the second key event
andOECD TG
442E induction:
addresses theactivationtheof ratio
dendritic
cells, the third
event
of
Foldtheluciferase
activity
Representsof luminescenceof keytreated
cells
the
skin
sensitisation
AOP
(16).
Finally,thefourthkey
event
representing
T-cell(minus blank) over the luminescence of the cells exposed to the concurrent
proliferation
is indirectly
assessedin the murine Local Lymph Node Assay (LLNA) (12).
solvent/vehicle
control (minus
blank).
7.
As keratinocyte activation represents only one key event of the skin sensitisation
IC30:
Concentration
effectinga reduction
viability
by 30%.
AOP (2)(17), information
generatedwith oftestcellularmethods
developed
to address this specific
key event may not be sufficient to conclude on the presence or absence of skin
IC50: Concentration effecting a reduction of cellular viability by 50%.
sensitisation potential of chemicals. Therefore data generated with the test methods
described in this Test Guideline are proposed to support the discrimination between skin
Hazard: Inherent property of an agent or situation having the potential to cause adverse
sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
effects when an organism, system or (sub) population is exposed to that agent.
Approaches to Testing and Assessment (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key events
IATA (Integrated Approach to Testing and Assessment): A structured approach usedof the skin sensitisation AOP as well as non-testing methods, including read-across fromfor hazard identification (potential), hazard characterisation (potency) and/or safetychemical analogues (17). Examples on the use of data generated with these methods
assessment (potential/potency and exposure) of a chemical or group of chemicals, which
within Defined Approaches, i.e. approaches standardised both in relation to the set ofstrategically integrates and weights all relevant data to inform regulatory decision
information sources used and in the procedure applied to derive predictions have been
regarding potential hazard and/or risk and/or the need for further targeted and therefore
published (17) and can be employed as useful elements within IATA.
minimal testing.
8.
The test methods described in this Test Guideline cannot be used on their own,
Imax: toMaximal
induction
factor of into
luciferase
activity1Acompared
the solvent
neither
sub-categoriseskin sensitisers
subcategories
and 1B as todefinedby UN(negative)
controlmeasuredat any test chemical
concentration.
GHS(1), forauthorities
implementing
these two
optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
Keap1: Kelch-like
1, is methods
a sensor may
proteinthat canregulate
framework,
positiveECH-associatedresults generatedproteinwith thesebe usedon theirownthetoNrf2
activity.
Under
un-induced
conditions
the
Keap1
sensorproteintargetstheNrf2
classify a chemical into UN GHS category 1.
transcription factor for ubiquitinylation and proteolytic degradation in the proteasome.
© OECD 201830

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to442D │ 7

Covalent modification of the reactive cysteine residues of Keap 1 by small molecules canlead to dissociation of Nrf2 from Keap1 (4) (5) (6).
Mixture: A mixture or a solution composed of two or more substances in which they do
not react (1).
Mono-constituent substance: A substance, defined by its quantitative composition, in
which one main constituent is present to at least 80% (w/w).
Multi-constituent substance: A substance, defined by its quantitative composition, in
which more than one main constituent is present in a concentration ≥ 10% (w/w) and< 80% (w/w). A multi-constituent substance is the result of a manufacturing process. The
difference between mixture and multi-constituent substance is that a mixture is obtainedby blending of two or more substances without chemical reaction. A multi-constituent
substance is the result of a chemical reaction.
Negative control: A sample containing all components of a test system and treated with asubstance known not to induce a positive response in the test system. This sample isprocessed with test chemical-treated samples and other control samples.
Nrf2: nuclear factor (erythroid-derived 2)-like 2, is a transcription factor involved in theantioxidant response pathway. When Nrf2 is not ubiquitinylated, it builds up in thecytoplasm and translocates into the nucleus, where it combines to the ARE in the
upstream promoter region of many cytoprotective genes, initiating their transcription (4)(5) (6).
Performance standards: Standards, based on a validated test method, that provide abasis for evaluating the comparability of a proposed test method that is mechanisticallyand functionally similar. Included are (i) essential test method components; (ii) a
minimum list of reference chemicals selected from among the chemicals used todemonstrate the acceptable performance of the validated test method; and (iii) thecomparable levels of accuracy and reliability, based on what was obtained for thevalidated test method, that the proposed test method should demonstrate when evaluatedusing the minimum list of reference chemicals (3).
Positive control: A replicate containing all components of a test system and treated witha substance known to induce a positive response. To ensure that variability in the positive
control response across time can be assessed, the magnitude of the positive response
should not be excessive.
Proficiency chemicals (substances): A subset of the Reference Chemicals included in the
Performance Standards that can be used by laboratories to demonstrate technicalcompetence with a standardised test method. Selection criteria for these substances
typically include that they represent the range of responses, are commercially available,
and have high quality reference data available.
Reference chemicals (substances): A set of chemicals to be used to demonstrate theability of a new test method to meet the acceptability criteria demonstrated by thevalidated reference test method(s). These chemicals should be representative of theclasses of chemicals for which the test method is expected to be used, and should©©OECD
OECD2018
2018

31

28 │

442D

OECD/OCDE

underwenta fullPerformanceStandard-based
validation
study
based
on which for
it wasalso
represent therange of responses
that may
be expectedfromthe chemicals
whichit
reviewed
and from
received
positive
opinionby ESAC (7) (8) (9) (10).
may be used,
strong,to weak,to negative.
4.
The test methods included in this Test Guideline may differ in relation to theRelevance: Description of relationship of the test to the effect of interest and whether it isprocedure used to generate the data and the readouts measured but can be usedmeaningful and useful for a particular purpose. It is the extent to which the test correctly
indiscriminately to address countries’ requirements for test results on the keratinocytes
measures or predicts the biological effect of interest. Relevance incorporates
activation Key Event of the AOP for skin sensitisation while benefiting from the Mutual
consideration of the accuracy (concordance) of a test method (3).
Acceptance of Data.
Reliability: Measures of the extent that a test method can be performed reproducibly
Backgroundand principles
the time,
test methods
includedin thebased
within and between
laboratoriesofoverwhen performed
using
the KeysameEvent
protocol.
It is
Test
Guidelines
assessedby calculating intra- and inter-laboratory reproducibility and intra-laboratory
repeatability(3).
5.
The assessmentof skin sensitisation has typically involved the use of laboratory
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
Reproducibility:
The agreementamongobtainedfromtesting
the406)same
substance(GPMT) of Magnusson
and Kligman
andresults
the Buehler
Test
(OECD
TG
(11),
assess
using
the
same
test
protocol(see
reliability)(3).
both the induction and elicitation phases of skin sensitisation. The murine tests, the
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
Sensitivity:
proportion
all aspositivechemicals
thatBrdU-FCM
are correctlyclassified(OECD TG The
442A)(13) as ofwell
LLNA:/ active
BrdU-ELISA
and
(OECD
TG
by the test
It isthea measureof accuracy
a test method
442B)(14),method.
all assess
induction
response for
exclusively,
and that
haveproduces
gained categoricalacceptanceresults,
and provide
is an importantconsiderationin assessing
thetests
relevanceof aoftestanimal
method(3).
since theyan advantage
over theguinea pig
in terms
welfaretogether with an objective measurement of the induction phase of skin sensitisation.
Solvent/vehicle control: A replicate containing all components of a test system except of
6.
vitro Ittest
methods
addressing
the first
the test Mechanistically-based
chemical, but includinginthechemicosolvent andthat inis used.
is usedto establish
the baseline
three
keyforeventsof the skinsensitisation
AOP
have been
adoptedresponse
the samples
treatedwith the test
chemicaldissolved
in forthe contributing
same solvent.to theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes
theTheDirectPeptide Reactivity
Assay /(15)addressing
the first
theSpecificity:
proportionof all negative
inactivechemicals
thatkey
are event;correctly
present
Test
Guideline
assesseskeratinocyte
activation
addressing
thesecondkey
eventclassified by the test method. It is a measure of accuracy for a test method that produces
andthe OECD
TG and442Eis addresses
the activationof dendritic
cells,thetherelevance
third key of
eventof
categoricalresults
an importantconsiderationin assessing
a testtheskinsensitisation
AOP
(16).
Finally,thefourthkey
event
representing
T-cellmethod (3).
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
Substance:
Chemical elements
andtheir compounds
naturalstate
or sensitisation
obtained by7.
As keratinocyte
activation
represents
only one in
keytheeventof theskin
anyproduction
process,
includingany
additivenecessarytopreserve
thestabilityof theAOP (2) (17), information generated with test methods developed to address this specificproduct
andany
impurities
deriving
fromtheprocessused,butexcludinganysolvent
key event may not be sufficient to conclude on the presence or absence of skinwhich
may bepotential
separatedofwithout
affectingthe stabilityof the substancechanging
its
sensitisation
chemicals.
Therefore
data generated
with theortest
methods
composition(1).
described in this Test Guideline are proposed to support the discrimination between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Testchemical:term "testis used(IATA),
to refer totogether
what is being
Approaches
toThe
Testingandchemical"
Assessmentwith tested.
other relevant
complementary information, e.g. derived from in vitro assays addressing other key events
United Nations Globally Harmonized System of Classification and Labelling ofof the skin sensitisation AOP as well as non-testing methods, including read-across from
Chemicals (UN GHS): A system proposing the classification of chemicals (substanceschemical analogues (17). Examples on the use of data generated with these methods
and mixtures) according to standardised types and levels of physical, health andwithin Defined Approaches, i.e. approaches standardised both in relation to the set ofenvironmental hazards, and addressing corresponding communication elements, such as
information sources used and in the procedure applied to derive predictions have been
pictograms, signal words, hazard statements, precautionary statements and safety data
published (17) and can be employed as useful elements within IATA.
sheets, so that to convey information on their adverse effects with a view to protect
8.
test methods
describedworkers,
in this Test
Guideline cannot
be usedtheir own,
people The(including
employers,transporters,
consumersandon emergency
neitherto sub-categoriseskin sensitisers
into subcategories 1A and 1B as defined by UN
responders)
and the environment(1).
GHS (1), for authorities implementing these two optional subcategories, nor to predict
UVCB: substances
unknown ordecisions.
variable composition,complex reactionor
potency
for safety ofassessment
However, dependingon the products
regulatory
biological materials.
framework,
positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 201832

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event based

442D │ 9

Validated Reference Method (VRM): the first method(s) endorsed as scientific valid
and used as a reference for performance-based validation studies.
Valid test method: A test method considered to have sufficient relevance and reliability
for a specific purpose and which is based on scientifically sound principles. A test method
is never valid in an absolute sense, but only in relation to a defined purpose (3).
Xeno-free: which does not contain any element that is not from the same species as thecells used, in this case, human.

use of laboratory
Literature
Maximisation Test
406) (11), assess
murine tests, the 1) United Nations (UN) (2017). Globally Harmonized System of Classification and Labelling ofChemicals (GHS). Seventh revised edition, New York and Geneva, United Nations Publications.
ions, LLNA: DA
Available at: [https://www.unece.org/trans/danger/publi/ghs/ghs_rev07/07files_e0.html]
FCM (OECD TG
ained acceptance 2) OECD (2012). Series on Testing and Assessment No. 168. The Adverse Outcome Pathway forof animal welfare
Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence.
ensitisation.
Organisation for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012ddressing the first)10/PART1&docLanguage=En
ontributing to theOECD TG 442C 3) OECD (2005). Guidance Document the Validation and International Acceptance of New or
st key event; theUpdated Test Methods for Hazard Assessment. OECD Environment, Health and Safety
second key event
publications, OECD Series on Testing and Assessment No.34. Organisation for Economic
third key event of
Cooperation and Development, Paris.
presenting T-cellay (LLNA) (12). 4) Natsch A. (2010). The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin
Sensitizers-Functional Relevance and Hypothesis on Innate Reactions to Skin Sensitizers.
skin sensitisation
Toxicological Sciences 113, 284-292.
dress this specificabsence of skin 5) Dinkova-Kostova A.T., Holtzclaw W.D., Kensler T.W. (2005). The role of Keap1 in cellular
protective responses. Chem. Res. Toxicol. 18, 1779-1791.
the test methods
tion between skin 6) Kansanen E., Kuosmanen S.M., Leinonen H., Levonen A.L. (2013). The Keap1-Nrf2 pathway:
within Integrated
Mechanisms of activation and dysregulation in cancer. Redox Biol. 1, 45-49.
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
2018

33

210││

442D
442D

OECD/OCDE

underwent
PerformanceStandard-based
validation
studyLuciferase
based on which it was also
Appendix
IA: InaVitro
Skin Sensitisation:
The
ARE-Nrf2
TM
reviewed andreceived
positive opinion by ESAC (7) (8) (9) (10).
KeratinoSens
Test
Method4.

The test methods included in this Test Guideline may differ in relation to the

INITIALCONSIDERATIONS,AND LIMITATIONS
procedure
used to generate APPLICABILITY
the data and the readouts
measured but can be used

indiscriminately to address countries’ requirements for test results on the keratinocytes
1.
TheKeytestEventmethodin thisto Test
Guideline
442D
addresses
theactivationof described
the AOP for
skinAppendix
sensitisation
while
benefitingfrom
the Mutual
second
key
event
of
the
skin
sensitisation
AOP(1),
namelykeratinocytes
activation,byAcceptance of Data.
assessing with the help of luciferase, the Nrf2-mediated activation of antioxidant response
element(ARE)-dependent
genes.
sensitisers
havebeen reported
inducegenes
that
Backgroundand principles
of Skinthe test
methods
includedin the to
Key
Event
based
are
regulated
by
the
ARE
(2)
(3).
Small
electrophilic
substances
such
as
skin
sensitisers
Test Guidelines
can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g.
5.
assessment ofof skinsensitisation
has typicallythe use of laboratory
covalentThe
modification
its cysteine
residue,resulting involved
in its dissociationfrom theanimals.
The factor
classical
methods
thatfactor-erythroid
use guinea-pigs,2-related
the Guinea
Pig2).
Maximisation
Test
transcription
Nrf2(nuclearfactor
The dissociated(GPMT)
Magnusson
Kligman and theBuehler
TG 406)for(11),assessNrf2 canofthen
activateand
ARE-dependent
genes
suchTestas (OECD
those coding
phase
II
both
the induction
elicitation
phases of skin sensitisation. The murine tests, thedetoxifying
enzymesand(2) (4)(5).
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
2.
vitro(13)ARE-Nrf2
test method(hereafter
called(OECD The
TG in
442A)
as wellluciferase
as LLNA:KeratinoSens™
BrdU-ELISA and
BrdU-FCM
(OECD
TG
the
KeratinoSens™
test
method)underwentvalidation
studies(3)(6)(7)
followedby an442B) (14), all assess the induction response exclusively, and have gained acceptanceindependent
peer review
conducted
bythe
theguinea
EuropeanLaboratory
for
since
they provide
an advantage
over
pig Union
tests inReferenceterms of animal
welfareAlternatives
toAnimalTesting(EURL
ECVAM)(8).
The
KeratinoSens™
test
methodtogether with an objective measurement of the induction phase of skin sensitisation.
was considered scientifically valid to be used as part of an IATA, to support the6.
Mechanistically-basedin chemicoandandnon-sensitisersin vitro test methods
first
discrimination
between skin sensitisers
for the addressing
purpose ofthehazardthree key events
identification(8).of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
3.
Basedon the Peptide
dataset fromthe validation
study
and in-house
testing
for thedescribes
the Direct
Reactivity
Assay (15)addressing
the first
keyusedevent;
theindependentpeer-review
of
the
test
method,theKeratinoSens™
test
methodprovedto bepresent Test Guideline assesses keratinocyte activation addressing the second key event
transferable
to TG
laboratories
experienced
in cell ofculture
techniquesofand the OECD
442E addresses
the activation
dendritic
cells, the (8).
thirdThekey level
event ofreproducibilityinpredictions
that
can
be
expectedfromtheKeratinoSens™
test
methodthe skin sensitisation AOP (16). Finally, the fourth key event representing T-cellisin the orderof 85% assessed
within andlaboratories
accuracy(77%
proliferation
is indirectlyin thebetween
murine Local
Lymph(8).
NodeTheAssay(LLNA)(12).155/201), sensitivity (78% - 71/91) and specificity (76% - 84/110) of the KeratinoSens™
7. method
As keratinocyte
activation
event
skin
sensitisation
test
for discriminating
skinrepresents
sensitisersonly(i.e.one
UN keyGHSCat. of1) thefromnon-sensitisers
AOP
(2)
(17),
information
generatedwithtest
methods
developedtoaddress
this
specific
when compared to LLNA results were calculated by considering all of the datasubmitted
keyEURL
eventECVAM
may notforbeevaluation
sufficientand
to peer-review
conclude onof thepresenceor (8).
absenceskintothe test
methodThese offiguressensitisationof chemicals.
data generated
withtest methods
aresimilar to potential
those publishedbased on Therefore
in-house testingof about 145
testthesubstances(77%
described 79%
in thissensitivity,
Test Guideline
are proposed
supportthe discrimination
betweenskin
accuracy,
72% specificity)(7).toThis
information
indicates theusefulness
sensitisers(i.e. UN GHS
and non-sensitiserswhen used
Integratedof
the KeratinoSens™
testCategory
method to1)contributeto the identificationof withinskin sensitisation
Approaches
to Testingand values
Assessment(IATA),
together with testotherrelevant
hazard.
However,
the accuracy
given here
for KeratinoSens™
methodas acomplementary
information,e.g.
derived
from
in
vitro
assaysaddressing
other
key
eventsstand-alone test method, are only indicative since the test method should be considered
inof the skin sensitisation
AOP asofwell
as non-testingfromcombination
with other sources
informationin the methods,
context ofincluding
a Definedread-acrossApproach oranchemical(17). Examples
on the use ofof paragraphs
data generated
with
these
IATA
andanalogues
in accordancewith the provisions
7 and
8 in
the methods
Generalwithin Defined
Approaches,
i.e. approaches
standardised
both inrelation tomethods
the set forofIntroductionof thisTest Guideline.
Furthermore
when evaluating
non-animal
information
sourcesused andtheinprocedure
to derive
haveanimal
been
skin
sensitisation,
it should
be in
kept
mind thatapplied
the LLNA
test aspredictions
well as otherpublished(17)fully
and reflect
can be theemployed
as in
usefulelements within IATA.
tests
may not
situation
humans.
8.
The thetestbasis
methods
described
thisTest Guidelinecannot be usedtheir own,
4.
Onof thecurrent in
data
available,
the KeratinoSens™
testonmethodwasneither toto besub-categorise
subcategories
and 1B functional
as defined groups,
by UN
shown
applicable toskintest sensitiserschemicals into
covering
a variety 1Aof organic
GHS (1),mechanisms,
for authorities
these twosubcategories,
nor
to predict
reactionskinimplementing
sensitisation potency(asoptional
determinedwith in vivo
studies)andpotencyforsafetyassessment
decisions.
However,dependingontheregulatory
physico-chemical properties (3) (6) (7) (8). The test method is applicable to test
framework,soluble
positivegeneratedthese methods
may bemedium
used on(i.e.
theira own
tochemicalsor resultsthat forma stablewith
dispersion
in the exposure
colloidclassify
a chemical
into theUNtestGHS
categorydoes
1. not settle or separate from the solvent intoorsuspension
in whichchemicaldifferent phases). Test chemicals that do not fulfil these conditions at the highest final© OECD 201834

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to442D │ 11

required concentration of 2 000 µM may still be tested at lower concentrations. In such acase, results fulfilling the criteria for positivity could still be used to support theidentification of the test chemical as a skin sensitiser. In cases where a negative result isobtained in a test with a maximal concentrations < 1000 µM and no cytotoxicity isreached, the result should be considered as inconclusive (see prediction model inparagraph 32). If cytotoxicity (< 70% viability) is reached at a maximal soluble test
concentration < 1000 µM, criteria for negativity can still be applied. In general monoconstituent substances with a LogP above 7 may be insoluble in the exposure medium,
however, if solubility or stable dispersion can be obtained and documented, testing may
still be conducted.
5.
Negative results should be interpreted with caution as substances with an
exclusive reactivity towards lysine-residues can be detected as negative by the test
method as the key mechanism leading to the activation of the Keap1-Nrf2-ARE pathway
appears to be the electrophilic reaction of stressors with nucleophilic thiols (cysteine
sulfhydryl groups) of Keap-1. Complementary information from peptide reactivity assays
may help addressing this uncertainty, in particular assays able to distinguish between
cysteine and lysine reactivity. Furthermore, because of the limited metabolic capability of
the cell line used (10) and because of the experimental conditions, pro-haptens (i.e.
chemicals requiring enzymatic activation for example via P450 enzymes) and pre-haptens(i.e. chemicals activated by auto-oxidation) in particular with a slow oxidation rate may
also provide negative results. However, it has been shown that the majority of pre-haptens(i.e. chemicals activated by auto-oxidation) and pro-haptens (i.e. chemicals requiring
enzymatic activation for example via P450 enzymes) are sufficiently well identified by a
combination of test methods covering key events 1, 2 and 3 on the AOP so that negative
results can in general be used to support classification (12) (20) (34). On the other hand,test chemicals that do not act as a sensitiser but are nevertheless chemical stressors maylead to false positive results (8). Finally, test chemicals that interfere with the luciferase
enzyme can confound the activity of luciferase in cell-based assays causing either
apparent inhibition or increased luminescence (13). For example, phytoestrogenconcentrations higher than 1 µM were reported to interfere with the luminescence signals
in other luciferase-based reporter gene assays due to over-activation of the luciferase
reporter gene (14) As a consequence, luciferase expression obtained at highconcentrations of phytoestrogens or similar compounds suspected of producing
phytoestrogen-like over-activation of the luciferase reporter gene needs to be examined
carefully (14). In cases where evidence can be demonstrated on the non-applicability of
the KeratinoSens™ test method to other specific categories of test chemicals, the test
method should not be used for those specific categories.
6.
In addition to supporting discrimination between skin sensitisers (i.e. UN GHS
Category 1) and non-sensitisers, the KeratinoSens™ test method also provides
concentration-response information that may potentially contribute to the assessment ofsensitising potency when used in integrated approaches such as IATA (11) (15).
Examples on how to use the KeratinoSens™ test method results in combination withother information sources are reported in the literature (7) (11) (16) (17) (18) (19) (20).
Specifically, the use of KeratinoSens™ test method dose-response data along withquantitative peptide reactivity data to assess potency in the LLNA and in human tests has
been described (21) and has been used in Bayesian integrated testing strategies on LLNA
potency (11) (22). Furthermore, evaluation has been conducted on how to specifically©©OECD
OECD2018
2018

35

212││

442D
442D

OECD/OCDE

underwenta Performance
study based on
it wasalso
address potency
in humans Standard-based(23). Finally, thevalidation
use of KeratinoSens™
testwhichmethodto assessreviewedofandreceived
positive
opinionESAC(8) (9) (10).
potency
specificchemicalclasseshasby
also
been(7)
described(21) (24).
4.
The test methods
includedin Annex
this Testmaydiffer in relation to the7.
Definitions
are provided
in the1 ofGuideline
the General
Introduction.
procedure used to generate the data and the readouts measured but can be used
indiscriminately
address countries’ requirements for test results on the keratinocytes
PRINCIPLE
OF THEtoTEST
activation Key Event of the AOP for skin sensitisation while benefiting from the Mutual8.
The ofKeratinoSens™
test method makes use of an immortalised adherent cell line
AcceptanceData.
derived from human keratinocytes stably harbouring a luciferase reporter gene under thecontrol of the antioxidant
response
elementof the human
AKR1C2This
gene
Backgroundand principlesof thetest methods
includedin thegene
Key(25).
Event
based
is
known
to
be
up-regulatedbyskin
sensitisers
(26)
(27).
The
cell
line
contains
theTest Guidelines
luciferase gene under the transcriptional control of a constitutive promoter fused with the5.
The assessmentof skinsignal
sensitisation
the useofofendogenous
laboratory
ARE element.
The luciferase
reflects has
the typically
activationinvolved
by sensitisers
animals.
The classicalmethods
that use guinea-pigs,
the Guinea
Maximisation
Nrf2 dependent
genes, andthe dependenceof the luciferase
signal Pig
in therecombinantTest
cell(GPMT)
ofMagnusson
andKligmanandtheBuehler
Test
(OECD
TG
406)
(11), assessline on Nrf2 has been demonstrated (28). This allows quantitative measurement(byboth
the inductionand elicitation
phasesof skinsensitisation.
The murine
tests,lightthe
luminescence
detection)of luciferase
geneinduction,using wellestablished
LLNA
(OECD
TG
429)
(12)
and
its
three
non-radioactive
modifications,
LLNA:
DA
producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription(OECD(13) exposure
as well to
as electrophilic
LLNA: BrdU-ELISA
and BrdU-FCM (OECD TG
factor in TG
cells442A)
following
test substances.
442B) (14), all assess the induction response exclusively, and have gained acceptance9.
Testprovide
chemicalsconsideredin themethodwelfare
if theysince
they
an areadvantage
overpositive
the guinea
pigKeratinoSens™
tests in terms testof animal
induceastatisticallysignificant
inductionoftheluciferase
activity
aboveatogether with an objective measurement of the induction phase of skin sensitisation. given
threshold (i.e.  1.5 fold, or 50% increase), below a defined concentration which does not
6.
Mechanistically-based in chemico and in vitro test methods addressing the firstsignificantly affect cell viability (i.e. below 1000 µM and at a concentration at which thethree key events of the skin sensitisation AOP have been adopted for contributing to thecellular viability is above 70% (3) (6). For this purpose, the maximal fold induction of theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
luciferase activity over solvent (negative) control (I ) is determined. Furthermore, since
describes the Direct Peptide Reactivity Assay (15)maxaddressing the first key event; thecells are exposed to series of concentrations of the test chemicals, the concentration
present Test Guideline assesses keratinocyte activation addressing the second key event
needed for a statistically significant induction of luciferase activity above the threshold
and the OECD TG 442E addresses the activation of dendritic cells, the third key event of(i.e. EC1.5 value) should be interpolated from the dose-response curve obtained from thethe skin sensitisation AOP (16). Finally, the fourth key event representing T-cell
series of tested concentrations of the test chemical (see paragraph 26 for calculations).
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
Finally, parallel cytotoxicity measurements should be conducted to assess whether
luciferase
occursat sub-cytotoxic
7.
Asinduction
keratinocyte
activation
representsconcentrations.
only one key event of the skin sensitisation
AOP (2) (17), information generated with test methods developed to address this specific10.
Prior to routine use of the KeratinoSens™ test method that adheres to this Test
key event may not be sufficient to conclude on the presence or absence of skin
Guideline, laboratories should demonstrate technical proficiency, using the ten
sensitisation potential of chemicals. Therefore data generated with the test methods
Proficiency Substances listed in Annex 1 of this Appendix.
described in this Test Guideline are proposed to support the discrimination between skin11.
Performancestandards(PS) (29)are available
to facilitate
validationof new or
sensitisers(i.e. UN GHS
Category
1) andnon-sensitiserswhen theusedwithin Integrated
modified in vitro
luciferase
test methods
similarto the KeratinoSens™
VRM
Approaches
to ARE-Nrf2
Testing andAssessment(IATA),
togetherwith other relevant
and allow for timely
amendmentof this
Test
Guideline
their inclusion.
complementary
information,e.g. derived
fromin vitro
assays foraddressing
other keyMutual
events
Acceptanceof Data (MAD)only asbe non-testingguaranteed methods,
for test methods
validatedaccordingofthe skin sensitisation
AOPwillas wellincludingread-across
from
to
the
PS,
if
these
test
methods
have
beenreviewed
andincluded
in
this
Test
Guidelinebychemical analogues (17). Examples on the use of data generated with these methods
the OECD.
withinDefined Approaches, i.e. approaches standardised both in relation to the set of
information sources used and in the procedure applied to derive predictions have been
PROCEDURE
published (17) and can be employed as useful elements within IATA.
8.
The test methods described in this Test Guideline cannot be used on their own,
12.
A DB-ALM protocol for the KeratinoSens™ test method is available and should
neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
be employed when implementing and using the test method in the laboratory (9).
GHS (1), for authorities implementing these two optional subcategories, nor to predict
Laboratories implementing the test method can obtain the recombinant cell line used in
potency for safety assessment decisions. However, depending on the regulatory
the KeratinoSens™ test method by signing a standard agreement with the test method
framework,
positive results generated with these methods may be used on their own todeveloper2 which includes the licence for the commercial use of the luciferase gene. The
classify a chemical into UN GHS category 1.
2

Givaudan Schweiz AG, CH-8310 Kemptthal© OECD 201836

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to442D │ 13

luciferase reporter gene assay is also subject to a Promega limited use licence that
requires the use of luminescent assay reagents purchased from Promega. The following
paragraphs provide with a description of the main components and procedures of the
KeratinoSens™ test method. Furthermore, an adaptation of the KeratinoSens™ test
method to xeno-free culture conditions using human reagents is described in Annex 2 ofthis Appendix (33). However, it is recommended that the relevant regulatory authorities
be consulted before deciding on the type of serum to be used in the KeratinoSens™ test
method.

Preparation of the keratinocyte cultures
13.
The KeratinoSens™ transgenic cell line having a stable insertion of the luciferase
reporter gene under the control of the ARE-element should be used. Upon receipt,
KeratinoSens™ cells are propagated as defined by the test method protocol (e.g. 2 to
4 passages) and stored frozen as a homogeneous stock. Cells from this original stock canbe propagated up to maximum 25 passages and are employed for routine testing using themaintenance/growth medium (Dulbecco's Modified Eagle's medium (DMEM) containing
serum and Geneticin to allow maintaining the gene) as described within the test method’s
DB-ALM protocol (9).
14.
For testing, cells should be 80-90% confluent, and care should be taken to ensurethat cells are never grown to full confluence. One day prior to testing cells are harvested,and distributed into 96-well plates at a cell density of 10,000 cells/well. Attention shouldbe paid to avoid sedimentation of the cells during seeding to ensure homogeneous cellnumber distribution across wells. If this is not the case, this step may give rise to highwell-to-well variability. For each repetition, three replicates are used for the luciferase
activity measurements, and at least one parallel replicate is used for the cell viability
assay.

Preparation of the test chemical and control substances
15.
The test chemical and control substances are prepared on the day of testing. Test
chemicals are dissolved in dimethyl sulfoxide (DMSO, CAS No. 67-68-5,  99% purity)
to the final desired concentration (e.g. 200 mM). The DMSO solutions can be consideredself-sterilising, so that no sterile filtration is needed. Test chemicals not soluble in DMSO
are dissolved in sterile water or culture medium, and the solutions sterilised by e.g.
filtration. For a test chemical which has no defined molecular weight (MW), a stock
solution is prepared to the default concentration of 40 mg/mL or 4% (w/v). In casesolvents other than DMSO, water or the culture medium are used, appropriate scientificrationale should be provided.
16.
Based on the stock solutions of the test chemical, serial dilutions are made using
DMSO or a suitable solvent (i.e. sterile water or culture medium) to obtain 12 masterconcentrations of the chemical to be tested (from 0.098 to 200 mM). Independent of thesolvent used, the master concentrations, are then further diluted 25 fold into culture
medium containing serum, and finally used for treatment with a further 4 fold dilution
factor so that the final concentrations of the tested chemical range from 0.98 to 2000 µM(based on a dilution factor of 2). Alternative concentrations may be used uponjustification (e.g. in case of cytotoxicity or poor solubility). For a test chemical which has
no defined MW, serial dilutions are made using DMSO or a suitable solvent to obtain the©©OECD
OECD2018
2018

37

214││

442D
442D

OECD/OCDE

underwenta Performance
validation
based on whichranging
it was fromalso
desired final
concentrationsStandard-basedof the test chemical(e.g.study
12 concentrations
reviewed
andµg/ml).
received positive opinion by ESAC (7) (8) (9) (10).
0.196 to 400
4.
The
test methods
included incontrol
this Test
Guidelinemaydiffereachin relation
to (i.e.
the17.
A concurrent
solvent/vehicle
shouldbe tested
withinrepetition
procedure
to agenerate
data andthe should
readouts
butplatecan (i.e.
be usedDMSO), forused
which
sufficientthenumber
of wells
be measured
prepared persix).
indiscriminately
to address
for test as
results
on thefor
keratinocytes
The solvent/vehicle
control countries’
undergoesrequirements
the same dilutions
described
the master
activation
Key inEventof the AOP
skinwhile benefiting
the Mutual
concentrations
paragraph16, sofor
that
thesensitisation
final solvent/vehicle
control fromconcentrationisAcceptance1%, known of
notData.
to affect cell viability and corresponding to the same concentration of
DMSO found in the tested chemical and in the positive control. For a test chemical not
soluble in DMSO,
for which thedilutionswere madein water,the Key
DMSO
levelbasedin allBackgroundand principlesof thetest methods
includedin the
Event
wells
of
the
final
test
solutionmustbeadjusted
to
1%
as
for
the
other
test
chemicalsandTest Guidelines
control substances. This solvent/vehicle control (i.e. DMSO) also represents the negative
5.
Thetheassessmentof skintest
sensitisation
control for
KeratinoSens™
method. has typically involved the use of laboratory
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
18.
concurrent
positive
control shouldbe testeda sufficientnumberwells(GPMT)Aof
Magnusson
and Kligman
and thealso
Buehler
Testin(OECDTG 406)(11),ofassess
withintheeach
repetition
describedphases
withinoftheskin
DB-ALM
protocolto demonstrate
both
inductionandaselicitation
sensitisation.
The(9)murine
tests, theappropriate
responseof thesystem.
Fornon-radioactive
example, five concentrations
cinnamic
LLNA(OECD
TG 429)(12)testandits threemodifications, of
LLNA:
DA
aldehydeTG(CAS
No.(13)14371-10-9,98% purity)
are used andwithineach replicate
in TG
the
(OECD
442A)
as well as LLNA:
BrdU-ELISA
BrdU-FCM(OECD
KeratinoSens™
test method,
for whicha seriesexclusively,of 5 master andconcentrations
rangingfrom
442B) (14), all assess
the induction
response
have gained
acceptance
0.4
6.4 mMare anpreparedin DMSO(froma 6.4pigmM
stocksolution)and diluted
assinceto they
provideadvantageover theguineatests
in termsof animal
welfaredescribed
for themaster concentrations
16, phaseso thatofthe
final
concentration of
together withan objective
measurement inof paragraphthe induction
skinsensitisation.
the positive control range from 4 to 64 µM. Other suitable positive controls, preferentially
6.
Mechanistically-based in chemico and in vitro test methods addressing the firstproviding EC1.5 values in the mid-range, may be used if historical data are available tothree key events of the skin sensitisation AOP have been adopted for contributing to thederive comparable run acceptance criteria.
evaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; theApplication of the test chemical and control substancespresent Test Guideline assesses keratinocyte activation addressing the second key event
19. the For
eachTG
test
chemicaland thepositive
control
one theexperimentneeded
and
OECD
442E
addresses
activationof substance,
dendritic cells,
third keyiseventof
to derivea prediction AOP(positive
negative),of event
at least
two independenttheskin sensitisation(16).or Finally,
theconsisting
fourth key
representing
T-cellrepetitions containing
threeinreplicatesn=6).
In casediscordant
proliferation
is indirectlyeachassessed
the murine(i.e.
Local
Lymph
Node of
Assay(LLNA)results(12).
between the two independent repetitions, a third repetition containing three replicates7.
As keratinocyte activation represents only one key event of the skin sensitisation
should be performed (i.e. n=9). Each independent repetition is performed on a different
AOP (2) (17), information generated with test methods developed to address this specificday with fresh stock solution of test chemicals and independently harvested cells. Cells
key event may not be sufficient to conclude on the presence or absence of skin
may come from the same passage however.
sensitisation potential of chemicals. Therefore data generated with the test methods
20.
After
seeding
described
paragraph
14, cellsthe
arediscrimination
grown for 24between
hours inskin
thedescribed
in thisTest asGuideline
areinproposed
to support
96-wells microtiterThe medium
is thenremoved andreplacedfresh
culturesensitisers(i.e. UN plates.
GHS Category
1) andnon-sensitisers
whenused withwithinIntegrated
medium (150 toµl culture
containing serum
but together
without Geneticin
as described
Approaches
Testing medium
and Assessment(IATA),
with otherrelevant
within the DB-ALM
protocole.g.
(9))derived
to which
50 inµl vitroof theassays
25 fold
diluted test
chemicalandcomplementary
information,fromaddressingotherkey events
control
substancesareadded.
At
least
one
well
per
plate
should
be
left
empty
(no
cells
of the skin sensitisation AOP as well as non-testing methods, including read-across fromand no treatment)
to assessbackgroundchemicalanalogues(17). Examples
onvalues.
the use of data generated with these methods
within Defined Approaches, i.e. approaches standardised both in relation to the set of21.
The treated plates are then incubated for about 48 hours at 37±1°C in the presence
information sources used and in the procedure applied to derive predictions have beenof 5% CO2. Care should be taken to avoid evaporation of volatile test chemicals andpublished (17) and can be employed as useful elements within IATA.
cross-contamination between wells by test chemicals by e.g. covering the plates with afoil during
withdescribed
the test chemicals.
8.
Theincubation
test methods
in this Test Guideline cannot be used on their own,neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
GHS (1), foractivity
authorities
implementing these two optional subcategories, nor to predict
Luciferase
measurements
potency for safety assessment decisions. However, depending on the regulatory
22.
The following factors are critical to ensure appropriate luminescence readings:
framework, positive results generated with these methods may be used on their own toclassify
a chemical
UN GHSluminometer,
category 1.
 thechoice ofinto
a sensitive© OECD 201838

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

442D │ 15

the use of a plate format with sufficient height to avoid light-cross-contamination,the use of a luciferase substrate with sufficient light output to ensure sufficientsensitivity and low variability; andan appropriate and stable background level.

Prior to testing, a control experiment setup as described in Annex 3 of this Appendix
should be carried out to ensure that these points are met.
23.
After the 48 hour exposure time with the test chemical and control substances,cells are washed with a phosphate buffered saline, and the relevant lysis buffer for
luminescence readings added to each well for a sufficient time (e.g. 20 min at room
temperature).
24.
Plates with the cell lysate are then placed in the luminometer for reading which is
programmed to: (i) add the luciferase substrate to each well (i.e. 50 µl), (ii) wait for
1 second, and (iii) integrate the luciferase activity for 2 seconds. In case alternative
settings are used, e.g. depending on the model of luminometer used, these should bejustified. Furthermore, a glow substrate may also be used provided that the quality control
experiment of Annex 3 of this Appendix is successfully fulfilled.

Cytotoxicity Assessment25.
For the KeratinoSensTM cell viability assay, medium is replaced after the 48 hour
exposure time with fresh medium containing 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 29893-1) and cells are incubated for 4 hours at 37±1°C in the presence of 5% CO2. The MTT
medium is then removed and cells are lysed by using an appropriate lysing agent for a
sufficient amount of time (e.g. 10% SDS overnight). After shaking, the absorption is thenmeasured at i.e. 600 nm with a photometer as described in the test method protocols (9).

skin sensitisation
DATA AND REPORTING
dress this specificabsence of skin
Data evaluation
the test methods
26.
The following parameters are calculated in the KeratinoSens™ test method:tion between skinwithin Integrated
 the maximal average fold induction of luciferase activity (Imax) value observed atother relevant
any concentration of the tested chemical and positive control;g other key events
 the EC1.5 value representing the concentration for which induction of luciferase
read-across from
activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) wasth these methods
obtained; andtion to the set of
ictions have been
 the IC50 and IC30 concentration values for which 50% and 30% reduction ofcellular viability occur respectively.
sed on their own,
Fold luciferase activity induction is calculated by Equation 1, and the overall maximalas defined by UN
fold induction (Imax) is calculated as the average of the individual repetitions.
es, nor to predict
−𝐿𝐿
(𝐿𝐿
)
n the regulatory
Equation 1:
𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝐹 𝐹 (𝐿𝐿𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 )−𝐿𝐿
𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠
𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏
d on their own to
where©©OECD
OECD2018
2018

39

216││

442D
442D

OECD/OCDE

underwenta Performance
validation
study
Lsample is theluminescenceStandard-based
reading in the test
chemicalwell based on which it was also
reviewed
positive reading
opinion by(7) (8)(9) (10).
Lblank isand
thereceived
luminescencein ESACthe blank
wellcontaining no cells and notreatment
4.
The test methods included in this Test Guideline may differ in relation to the
Lsolvent is the average luminescence reading in the wells containing cells and solvent
procedure
used to generate the data and the readouts measured but can be used(negative) control
indiscriminately to address countries’ requirements for test results on the keratinocytes
activation
Key Eventthe interpolation
AOP for skinaccording
sensitisation
while benefiting
the EC
Mutualby oflinearto Equation
2, and thefrom
overall
EC1.5 is calculated
1.5 is
AcceptanceData.
calculated asofthegeometric mean of the individual repetitions.
1.5− 𝐼𝐼𝑎𝑎(𝐶𝐶𝑏𝑏 − 𝐶𝐶included+ 𝐶𝐶Key
2: of the𝐸𝐸𝐸𝐸1.5𝑎𝑎 ) × ( 𝐼𝐼 − 𝐼𝐼in)the𝑎𝑎
Background andEquation
principles
test=methods
Event based𝑏𝑏𝑎𝑎
Test Guidelines

where
5.
The assessment of skin sensitisation has typically involved the use of laboratoryCtheclassicallowest concentrationµMguinea-pigs,
with > 1.5 foldaanimals. isThemethods thatinuse
the induction
Guinea Pig Maximisation TestCisthehighestconcentration
in
µM
with
<
1.5
fold
b
(GPMT) of Magnusson and Kligman and the Buehler Testinduction(OECD TG 406) (11), assessIa
the fold andinductionmeasured
lowestconcentration
with > tests,
1.5 foldboththeisinduction
elicitation
phasesatoftheskinsensitisation.
The murine
theinduction
(mean
of
three
replicatewells)
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
Ib
isfold induction
at theashighest
withfold induction
(mean
(OECD
TGthe
442A)
(13) as well
LLNA:concentration
BrdU-ELISA
and< 1.5
BrdU-FCM
(OECD
TG
of
three
replicatewells)
442B) (14), all assess the induction response exclusively, and have gained acceptancesince theyis provide
anby
advantage
Viability
calculated
Equation over
3: the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
(𝑉𝑉𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 −𝑉𝑉𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 )× 100addressing the firstEquation 3: in chemico
𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉)
6.
Mechanistically-based
and𝑉𝑉in(𝑉𝑉vitro test
−𝑉𝑉 methods
𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠

𝑏𝑏𝑏𝑏𝑎𝑎𝑎𝑎𝑎𝑎

three key events of the skin sensitisation AOP have been adopted for contributing to thewhereevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
isthe
the Direct
MTT-absorbancereading inAssay
the test
chemicalwell the first key event; theVdescribes
Peptide Reactivity(15)
addressing
sampleV
is theGuideline
MTT-absorbancereading in theblank wellcontaining
no cells
nopresent
Test
assesses keratinocyte
activation
addressing
the second
keyandevent
blank
treatment
andthe OECD TG 442E addresses the activation of dendritic cells, the third key event of
Vsolvent
the average MTT-absorbancereading
the wells
cells and solventtheskinissensitisation
AOP (16). Finally,
the infourth
key containing
event representing
T-cell(negative)
control
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
by linearinterpolation
Equation
4, and theIC
7. 50 andAsICkeratinocyte
activation
represents
only one according
key event toof theskin sensitisation
30 are calculated
overall
IC30 are calculated
aswiththe geometricmeanof the individual
repetitions.
AOP(2)IC(17),information
generated
test methods
developed
to address
this specific
50 andkey event may not be sufficient to conclude on thepresence or absence of skin(100−𝑥𝑥𝑥𝑥𝑥𝑥𝑥(𝐶𝐶𝑏𝑏 − 𝐶𝐶𝑎𝑎data) × (generated 𝑎𝑎)with
Equation
4:
𝐼𝐼𝐼𝐼𝑥𝑥 =Therefore+ 𝐶𝐶𝑎𝑎the test methods
sensitisation potentialof chemicals.
𝑉𝑉𝑏𝑏 − 𝑉𝑉𝑎𝑎
described in this Test Guideline are proposed to support the discrimination between skinwhere
sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches
Testing andtogether(50withother
relevant
X
is theto% reduction
at theAssessment
concentration(IATA),
to be calculated
and 30for IC
50 andcomplementary
information,e.g.
derived
from
in
vitro
assaysaddressing
other
key
eventsIC30)
ofsensitisation
AOP as wellread-across fromCa the skinis thelowest concentration
in asµMnon-testingwith > x%methods,
reductionincluding
in viability
chemicalExamplesinonµM
thewithuse< ofgeneratedwith these methods
Cbisanalogues
the highest(17).
concentration
x%datareductionin viability
withinDefinedi.e.lowest
approaches
standardisedin relation
the set ofV
is the %Approaches,
viability at theconcentration
with >both
x% reduction
in toviabilityainformation
sources
used
and
in
the
procedureappliedtoderivepredictions
haveis the % viability at the highest concentration with < x% reduction in viabilitybeenVbpublished (17) and can be employed as useful elements within IATA.
27.
For each concentration showing a luciferase activity induction equal or higher ()8.
The
teststatisticalmethodssignificancedescribed inisthis
Test Guidelinecannotbe used onStudent’s
their own,
than 1.5 fold,determined(e.g. using
a two-tailed
tneithertosub-categoriseskinsensitisers
into
subcategories
1A
and
1B
as
definedby UNtest) by comparing the luminescence values of the three replicate samples with
theGHS(1), for values
authorities
implementing
these
two optional
predict
luminescencein thesolvent/vehicle
control
wells to subcategories,
assess whether nor
the toluciferase
potencyforsafetyassessment
decisions.
However,dependingontheregulatory
activity induction is statistically significant (p <0.05). Furthermore, it should be checkedframework,
positive
results generated
withmethods
may be usedon theirownfold
tothat no significant
cytotoxic
effects occur
at thesethe lowest
concentration
leadingto 1.5classify a chemical
1.
luciferase
inductioninto
andUN
thatGHS
this category
concentrations
is below the IC30 value, indicating that
there is less than or equal to 30% reduction in cellular viability. In addition, at least two© OECD 201840

OECD/OCDE

442D │ 17

which it was also

consecutive concentrations should have > 70% viability, otherwise the concentration
range should be adjusted.

in relation to thebut can be used
the keratinocytes
from the Mutual

28.
It is recommended that data are visually checked with the help of graphs. If noclear dose-response curve is observed, or if the dose-response curve obtained is biphasic(i.e. crossing the threshold of 1.5 twice), the experiment should be repeated to verify
whether this is specific to the test chemical or due to an experimental artefact. In case thebiphasic response is reproducible in an independent experiment, the lower concentration,i.e. when the threshold of 1.5 is crossed the first time should be reported.

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to

29.
In the KeratinoSens™ test method, in the rare cases where a statistically nonsignificant luciferase induction equal or above 1.5 fold is observed followed by a higher
concentration with a statistically significant induction, results from this repetition are only
considered as valid and positive if the statistically significant induction equal or above thethreshold of 1.5 was obtained for a non-cytotoxic concentration.
30.
Finally, for test chemicals generating in the KeratinoSens™ test method a 1.5 foldor higher induction already at the lowest tested concentration (i.e. 0.98 µM), theEC1.5 value of <0.98 is set based on visual inspection of the dose-response curve.

Acceptance criteria
31.
The following acceptance criteria should be met when using the KeratinoSens™
test method.
The luciferase activity induction obtained with the positive control, cinnamic
aldehyde, should be statistically significant above the threshold of 1.5 (e.g. using
a t-test) in at least one of the tested concentrations (4 to 64 µM).

The EC1.5 value of the positive control should be within two standard deviationsof the historical mean of the testing facility (e.g. between 7 µM and 30 µM basedon the validation dataset) which should be regularly updated. In addition, theaverage induction in the three replicates for cinnamic aldehyde at 64 µM should
be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of
cinnamic aldehyde should be carefully checked, and tests may be accepted only ifthere is a clear dose-response with increasing luciferase activity induction atincreasing concentrations for the positive control.

The average coefficient of variation of the luminescence reading for thesolvent/vehicle control (i.e. DMSO) should be below 20% in each repetition. If
the variability is higher, results should be discarded.

Interpretation of results and prediction model
32.
A KeratinoSens™ prediction is considered positive if the following 4 conditions
are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™
prediction is considered negative (Figure 1):the Imax is equal or higher than () 1.5 fold and statistically significantly different
as compared to the solvent/vehicle control (as determined by a two-tailed,unpaired Student’s T-test);©©OECD
OECD2018
201841

218││

442D
442D

OECD/OCDE

underwenta Performance
validation
study
based
on which itwith
was also
 the cellularviabilityStandard-basedis higher than (>)
70% at thelowestconcentration
reviewed
and received
positiveactivity
opinion1.5
by ESAC(7) at(8)the(9)EC(10).
inductionof luciferase
fold (i.e.
1.5 determining
concentration);
4.
The test methods included in this Test Guideline may differ in relation to theprocedureused
to generate the data and the readouts measured but can be used
 the EC
1.5 value is less than (<) 1000 µM (or < 200 µg/mL for test chemicals withindiscriminately
address countries’ requirements for test results on the keratinocytes
no definedtoMW);
activation Key Event of the AOP for skin sensitisation while benefiting from the Mutual
 thereofisData.
a dose-dependent increase in luciferase induction (or a biphasic response
Acceptanceas mentioned under paragraph 28).

Backgroundand principles
ofthreethe test
includedin theKeydose-dependent
Event basedIfin a given repetition,
all of thefirstmethods
conditions
are met but
a clear
Test Guidelines
increasein luciferase induction cannot be observed, then the result of that repetition
shouldbeand further
be required1). In5.
Theconsideredassessmentinconclusiveof skin sensitisation
has testing
typicallymay
involved
the use(Figureof laboratory
addition,anegative
resultobtainedwithtestchemicals
testedatamaximaltest
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
concentration< 1000 µM(orKligman
200 µg/mL
for Buehler
test chemicals
with noMW)and(GPMT) of Magnusson
andand theTest (OECD
TGdefined406) (11),assess
which
do
not
reach
cytotoxicity(<
70%
viability)atthemaximaltestedconcentration
both the induction and elicitation phases of skin sensitisation. The murine tests, theshouldbe considered
inconclusive(seenon-radioactive
paragraph 4). modifications, LLNA: DA
LLNA also(OECD
TG 429) as(12)
and its three(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
442B) (14), all assess the induction response exclusively, and have gained acceptancesince they provide an advantage over the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
6.
Mechanistically-based in chemico and in vitro test methods addressing the first
three key events of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; thepresent Test Guideline assesses keratinocyte activation addressing the second key eventand the OECD TG 442E addresses the activation of dendritic cells, the third key event of
the skin sensitisation AOP (16). Finally, the fourth key event representing T-cellproliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
7.
As keratinocyte activation represents only one key event of the skin sensitisation
AOP (2) (17), information generated with test methods developed to address this specific
key event may not be sufficient to conclude on the presence or absence of skinsensitisation potential of chemicals. Therefore data generated with the test methods
described in this Test Guideline are proposed to support the discrimination between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key eventsof the skin sensitisation AOP as well as non-testing methods, including read-across fromchemical analogues (17). Examples on the use of data generated with these methods
within Defined Approaches, i.e. approaches standardised both in relation to the set of
information sources used and in the procedure applied to derive predictions have been
published (17) and can be employed as useful elements within IATA.
8.
The test methods described in this Test Guideline cannot be used on their own,neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
GHS (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
framework, positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 201842

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

442D │ 19

Figure 1. Prediction model used in the KeratinoSens

TM

test method.

A KeratinoSensTM prediction should be considered in the framework of a Defined Approach or of
an IATA and in accordance with the provisions of paragraphs 7 and 8 of the general introduction

Procedure for one repetition
Induction  1.5 fold ?
AND
Statistically significant
over solvent control?

NegativeNO

Perform at least two
independent repetitions

YES
Viability at lowest
concentration with > 1.5fold induction  70% ofsolvent control?

NegativeNO- If the two repetitions arenegative, final outcome is:
NEGATIVE

YES
EC1.5 < 1000 µM(or < 200g/ml if nodefined MW)?

NegativeNO

YES
Clear dose-response?

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

YES

NO

- If the two repetitions are
positive, final outcome is:
POSITIVE

In case the first two repetitions
are not concordant, perform athird repetition and conclude
on the basis of the mode of theoutcomes (i.e., 2 out of 3).

Inconclusive/
RepeatPositivesed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
2018

43

220││

442D
442D

OECD/OCDE

underwenta Performance
validation
study basedon which
it wasto also33.
In cases
when testStandard-based
chemicals induce
the luciferase
activity
very closethereviewed
received
opinion by(7) (8) (9) (10).
cytotoxic andlevels,they positive
can be positivein ESACsome repetitions
at non-cytotoxic levels (i.e.
EC1.5 determining concentration below (<) the IC30), and in other repetitions only at
4.
The test methods included in this Test Guideline may differ in relation to thecytotoxic levels (i.e. EC1.5 determining concentration above (>) the IC30). Such test
procedure used to generate the data and the readouts measured but can be usedchemicals shall be retested with more narrow dose-response analysis using a lower
indiscriminately to address countries’ requirements for test results on the keratinocytes
dilution(e.g. 1.33
orAOP
2 (=1.41)
dilution while
betweenwells), fromto determine
ifactivationfactor
Key Eventof thefor skin foldsensitisation
benefiting
the Mutual
induction
hasofoccurred
Acceptance
Data. at cytotoxic levels or not (3).

Testreport and principles of the test methods included in the Key Event based
Background34.
The test report should include the following information:
Test Guidelines
5.
The assessment of skin sensitisation has typically involved the use of laboratory
Test chemical
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
 Mono-constituent
substance(GPMT)of Magnusson andKligman and the Buehler Test (OECD TG 406) (11), assess
both the induction and elicitation phases of skin sensitisation. The murine tests, theo Chemical identification, such as IUPAC or CAS name(s), CAS number(s),LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
SMILES or InChI code, structural formula, and/or other identifiers, like batch/(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
lot number and expiry date;
442B) (14), all assess the induction response exclusively, and have gained acceptanceo provide
Physical anappearance,solubility,
molecular
weight,since they
advantagewater
oversolubility,
the guineaDMSO
pig tests
in termsof animal
welfareandphysicochemical
properties,
to skinthe extent
available;
together withanadditional
objective relevantmeasurementof the induction
phase ofsensitisation.
oStatement on (in)solubilityor stable
dispersion
exposureaddressing
media;
6.
Mechanistically-basedin chemicoand invitro testinmethods
the first
three key
the skin identity
sensitisation
AOP havebeen adopted
contributing
to theo events
Purity,ofchemicalof impurities
as appropriate
andforpracticallyfeasible,evaluation oftheskinsensitisation
hazardpotentialofchemicals:
the
OECD
TG
442C
etc;
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; theto testing,
if applicable(e.g. warming,present oTestTreatment
Guidelinepriorassesseskeratinocyte
activation
addressinggrinding);
the second key eventand the oOECD
TG
442E
addresses
theactivationofdendritic
cells,
thethird key event ofConcentration(s) tested;
the skin sensitisation AOP (16). Finally, the fourth key event representing T-cello Storage
conditions
and instability
to theLocal
extentavailable.
proliferation
is indirectly
assessed
the murine
Lymph
Node Assay (LLNA) (12).
substance,represents
UVCB andmixture:7.  Multi-constituent
As keratinocyte activation
only
one key event of the skin sensitisation
AOP (2)o (17),information
generatedwithtest
methods
developed
to address
this specificCharacterisation as far as possible by e.g. chemical
identity(see above),
key event purity,
may not
be sufficient
to conclude
on thepresence or absence
skinquantitative
occurrence
and relevant
physicochemical
propertiesof(seesensitisationabove)
potentialofchemicals.
Therefore
data
generated
with
the
test
methodsof the constituents, to the extent available;
described in this Test Guideline are proposed to support the discrimination between skino (i.e.
Physicalwater
DMSO solubility
and additional
sensitisers
UN appearance,
GHS Category1) solubility,and non-sensitiserswhen usedwithin Integrated
physicochemical
properties,(IATA),
to the extent
available;
Approachesrelevant
to Testingand Assessment
togetherwith other relevant
complementary
information,
derived fromin vitro
assaysother key eventso Molecularweighte.g.
or apparent
molecular
weightin addressing
case of mixtures/polymersof the skin sensitisation
AOP as well
non-testing
methods,
includingread-acrossfromof known compositions
or as
other
information
relevant
for the conductof thechemical analogues(17).
Examples
on
the
use
of
data
generated
with
these
methods
study;
within Defined Approaches, i.e. approaches standardised both in relation to the set ofo Statementon (in)solubilityor stable dispersion
exposure
media; have been
information
sources used
and in the procedure
applied toinderivepredictions
published
and canprior
be employedelements
IATA.
o (17)
Treatment
to testing,asifuseful
applicable(e.g.within
warming,
grinding);
8.
TheConcentration(s)test methods describedotested; in this Test Guideline cannot be used on their own,neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
o for
Storageconditions
and stability
to theavailable.
GHS (1),authorities
implementing
these
twoextent
optional
subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
Controls
framework, positive results generated with these methods may be used on their own toclassify
a chemical
into UN GHS category 1.
 Positive
control© OECD 201844

OECD/OCDE

442D │ 21

which it was alsoo Chemical identification, such as IUPAC or CAS name(s), CAS number(s),SMILES or InChI code, structural formula, and/or other identifiers;

in relation to thebut can be used
the keratinocytes
from the Mutualo Physical appearance, water solubility, DMSO solubility, molecular weight,
and additional relevant physicochemical properties, to the extent available andwhere applicable;

ey Event basedo Treatment prior to testing, if applicable (e.g. warming, grinding);o Purity, chemical identity of impurities as appropriate and practically feasible,
etc;
o Concentration(s) tested;

use of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

o Storage conditions and stability to the extent available;o Reference to historical positive control results demonstrating suitable run
acceptance criteria, if applicable.
o Chemical identification, such as IUPAC or CAS name(s), CAS number(s),and/or other identifiers;
o Purity, chemical identity of impurities as appropriate and practically feasible,
etc;

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

Solvent/vehicle/negative controlo Physical appearance, molecular weight, and additional relevant
physicochemical properties in the case other solvents/vehicles /negative
controls than those mentioned in this Appendix are used and to the extent
available;o Storage conditions and stability to the extent available;o Justification for choice of solvent/vehicle for each test chemical.
Test method conditions

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own toName and address of the sponsor, test facility and study director;

Description of test method used;

Cell line used, its storage conditions and source (e.g. the facility from which they
were obtained);

Passage number and level of confluence of cells used for testing;

Cell counting method used for seeding prior to testing and measures taken to
ensure homogeneous cell number distribution (cf. paragraph 14);

Luminometer used (e.g. model), including instrument settings, luciferase substrateused, and demonstration of appropriate luminescence measurements based on thecontrol test described in Annex 3 of this Appendix;The procedure used to demonstrate proficiency of the laboratory in performing
the test method (e.g. by testing of proficiency substances) or to demonstrate
reproducible performance of the test method over time.

©©OECD
OECD2018
2018

45

222││

442D
442D

OECD/OCDE

underwenta Performance Standard-based validation study based on which it was also
Test procedure
reviewed and received positive opinion by ESAC (7) (8) (9) (10).
 Number of repetitions and replicates used;
4.
The test methods included in this Test Guideline may differ in relation to the Testusedchemicalconcentrations,application
exposure
procedure
to generate
the data
and the procedure
readouts andmeasuredbuttimecanusedbe (ifuseddifferent
than
the
one
recommended)indiscriminately to address countries’ requirements for test results on the keratinocytes
activation
Key Eventthe AOPandfor decisionskin sensitisation
while benefiting from the Mutual Description
ofof
evaluation
criteria used;
Acceptance of Data.
 Description of study acceptance criteria used;

Backgroundand principlesof the testofmethods
included in the Key Event based Descriptionof any modifications
the test procedure.
Test Guidelines

Results
5.
The assessment of skin sensitisation has typically involved the use of laboratory
animals.
The classicalmethods
guinea-pigs,
the IC
GuineaPig
Maximisation
anduseviability
values (i.e.
) obtained
for the Test
test
 Tabulationof Imax,
EC1.5that
50, IC30(GPMT)chemicalof Magnusson
Kligmancontrol
and theforBuehler
Test (OECDTGas406)(11), assess
and forandthe positive
each repetition
as wellthe meanboth thevalues
induction
elicitationof skinsensitisation.
murinetests, the(Imax:andaverage;
EC1.5 phases
and viability
values:geometricThemean)
and SD
LLNA (OECDTG429)data(12)
andallitsindividual
three non-radioactive
LLNA:
DA
calculated
using
from
repetitions andmodifications,an indication of
the rating(OECD ofTG
442A)
(13)
as
well
as
LLNA:
BrdU-ELISA
andBrdU-FCM(OECDTG
the test chemical according to the prediction model;
442B) (14), all assess the induction response exclusively, and have gained acceptance theyCoefficientobtainedwithguinea
the luminescencethe welfare
since
provide ofanvariation
advantageover thepig tests inreadings
terms offoranimal
control foreachexperiment;
togethersolvent/vehicle/negativewith an objective measurementof theinductionphase of skin sensitisation.

A
graph
depictingdose-response
curvesforinductionof luciferase
activitythe
andfirst
6.
Mechanistically-based in chemico and in vitro test methods
addressing
viability;
three key
events of the skin sensitisation AOP have been adopted for contributing to theevaluationof the skinsensitisation
hazardpotential ofif chemicals: Descriptionof anyother relevant
observations,applicable. the OECD TG 442C
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; thepresent TestofGuideline
Discussion
the resultsassesses keratinocyte activation addressing the second key eventand the OECD TG 442E addresses the activation of dendritic cells, the third key event ofof theAOP
results
obtained
withthethe fourth
KeratinoSens™
testrepresenting
method;the skinDiscussion
sensitisation(16).
Finally,
key eventT-cellproliferation
is indirectly
assessedin the murine
Local Lymph
Node(LLNA)(12).
 Considerationof thetest method
results within
the context
ofAssayan IATA,if otherrelevant
information
is available.
7.
As keratinocyte
activation
represents only one key event of the skin sensitisation
AOP (2) (17), information generated with test methods developed to address this specificConclusion
key event may not be sufficient to conclude on the presence or absence of skinsensitisation potential of chemicals. Therefore data generated with the test methods
described in this Test Guideline are proposed to support the discrimination between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Literature
Approaches to Testing and Assessment (IATA), together with other relevant
e.g. derived
from in vitro
addressing
key events
1) complementary
OECD (2012). information,
Series on Testingand Assessment
No.assays
168. The
Adverseother
OutcomePathway forofthe Sensitisationskin sensitisation
AOP asas non-testing
including
Skin
Initiatedby well
Covalent
Binding methods,
to Proteins.
Part read-across
1: Scientificfrom
Evidence.
chemicalanalogues
Exampleson the use ofgenerated withParis.
these methods
Organisationfor (17).
Economic
Cooperation
anddataDevelopment,
Available at:withinDefined Approaches, i.e. approaches standardised both in relation to the set of
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012information
sources used and in the procedure applied to derive predictions have been
)10/PART1&docLanguage=En
published (17) and can be employed as useful elements within IATA.
2) Natsch A. (2010). The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin
8.
The test methodsRelevancedescribed andin thisTest GuidelinecannotReactions
be used on
Sensitizers-Functional
Hypothesis
on Innate
to their
Skin own,
Sensitizers.
neithertosub-categoriseskinsensitisers
into
subcategories
1A
and
1B
as
definedbyUN
Toxicological Sciences 113, 284-292.
GHS (1), for authorities implementing these two optional subcategories, nor to predict
3) potency
Emter R.,forEllissafety
G., NatschA.(2010).
Performanceof a novel
keratinocyte-based
reporter cell line
assessment
decisions.
However,dependingon the regulatory
toscreenskinsensitizers
invitro.
Toxicology
andAppliedPharmacology
245,281-290.
framework, positive results generated with these methods may be used on their own toa chemical A.T.,
into UNGHS category4) classify
Dinkova-Kostova
Holtzclaw
W.D., 1.
Kensler T.W. (2005). The role of Keap1 in cellular
protective responses. Chem. Res. Toxicol. 18, 1779-1791.
© OECD 201846

OECD/OCDE

442D │ 23

which it was also 5) Kansanen E., Kuosmanen S.M., Leinonen H., Levonen A.L. (2013). The Keap1-Nrf2 pathway:
Mechanisms of activation and dysregulation in cancer. Redox Biol. 1, 45-49.

in relation to the 6)
but can be used
the keratinocytes
from the Mutual

ey Event based

Natsch A., Bauch C., Foertsch L., Gerberick F., Normann K., Hilberer A., Inglis H.,
Landsiedel R., Onken S., Reuter H., Schepky A., Emter R. (2011). The intra- and interlaboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers invitro: results of a ring-study in five laboratories. Toxicol. In Vitro 25, 733-744.

7) Natsch A., Ryan C.A., Foertsch L., Emter R., Jaworska J., Gerberick G.F., Kern P. (2013). A
dataset on 145 chemicals tested in alternative assays for skin sensitization undergoingprevalidation. Journal of Applied Toxicology 33, 1337-1352.

8) EURL-ECVAM (2014). Recommendation on the KeratinoSensTM assay for skin sensitisation
use of laboratory
testing, 42 pp. Available at: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvamMaximisation Test
recommendations/recommendation-keratinosens-skin-sensitisation.
406) (11), assess
murine tests, the 9) DB-ALM (INVITTOX) (2013) Protocol 155: KeratinoSensTM., 17 pp. Available: [http://ecvamdbalm.jrc.ec.europa.eu/].
ions, LLNA: DA
FCM (OECD TG 10) Fabian E., Vogel D., Blatz V., Ramirez T., Kolle S., Eltze T., van Ravenzwaay B., Oesch F.,
ained acceptanceLandsiedel R. (2013). Xenobiotic metabolizin enzyme activities in cells used for testing skinof animal welfare
sensitization in vitro. Arch Toxicol 87, 1683-1969.
ensitisation.
11) Jaworska J., Dancik Y., Kern P., Gerberick F., Natsch A. (2013). Bayesian integrated testing
ddressing the firststrategy to assess skin sensitization potency: from theory to practice. J. Appl. Toxicol. 33,
ontributing to the1353-1364.
OECD TG 442C
st key event; the 12) Casati S., Aschberger K., Asturiol D., Basketter D., Dimitrov S., Dumont C., Karlberg A.T.,
Lepoittevin J.P., Patlewicz G., Roberts D.W., Worth A. (2016). Ability of non-animal methods
second key eventfor skin sensitisation to detect pre- and pro-haptens: Report and recommendations of an EURL
third key event of
ECVAM expert meeting. EUR 27752 EN. Available at : https://eurl-ecvam.jrc.ec.europa.eu/eurlpresenting T-cellecvam-status-reports/pre-prohapten-workshop-report.
ay (LLNA) (12).
skin sensitisation 13) Thorne N., Inglese J., Auld D.S. (2010). Illuminating Insights into Firefly Luciferase and Other
Bioluminescent Reporters Used in Chemical Biology. Chemistry and Biology 17, 646-657.
dress this specificabsence of skin 14) OECD (2012). BG1Luc Estrogen Receptor Transactivation Test Method for Identifying Estrogenthe test methods
Receptor Agonists and Antagonists. OECD Guidelines for Chemical Testing No. 457.
tion between skin
Organisation for Economic Cooperation and Development, Paris. Available at:within Integrated[http://www.oecd.org/env/testguidelines].
other relevant
g other key events 15) ECETOC (2003). Contact sensitization: Classification according to potency. European Centre for
Ecotoxicology & Toxicology of Chemicals (Technical Report No. 87).
read-across fromth these methods 16) Bauch C., Kolle S.N., Ramirez T., Eltze T., Fabian E., Mehling A., Teubner W., vantion to the set ofRavenzwaay B., Landsiedel R. (2012). Putting the parts together: combining in vitro methods toictions have been
test for skin sensitizing potentials. Regul. Toxicol. Pharmacol. 63, 489-504.

17) Natsch A., Emter R., Ellis G. (2009). Filling the concept with data: integrating data fromsed on their own,
different in vitro and in silico assays on skin sensitizers to explore the battery approach foras defined by UN
animal-free skin sensitization testing. Toxicol. Sci. 107, 106-121.
es, nor to predictn the regulatory 18) Ball N., Cagen S., Carrillo J.C., Certa H., Eigler D., Emter R., Faulhammer F., Garcia C.,
Graham C., Haux C., Kolle S.N., Kreiling R., Natsch A., Mehling A. (2011). Evaluating thed on their own tosensitization potential of surfactants: integrating data from the local lymph node assay, guinea©©OECD
OECD2018
2018

47

224││

442D
442D

OECD/OCDE

underwenta Performance
validation
study based on which
it wasalsoToxicol.
pig maximization
test, andStandard-basedin vitro methods
in a weight-of-evidence
approach.
Regulreviewed
and60,
received
positive opinion by ESAC (7) (8) (9) (10).
Pharmacol.
389-400.
methods
includedin this T.,
TestHonarvar
Guideline
in relationR.,toJaworska
the19) 4.
UrbischTheD., test
MehlingA., GuthK., RamirezN.,mayKollediffer
S., Landsiedel
J.,
procedure
used to generate
theA.,dataandR.,thereadouts
can be used
Kern P.S., GerberickF., Natsch
Emter
AshikagaT., measured
Miyazawa but
M., Sakaguchi
H. (2015).
indiscriminately
to address countries’
for test
on the test
keratinocytes
Assessing skin sensitization
hazard inrequirements
mice and men
usingresults
non-animal
methods. Regul.
activation
Key Event 71,
of the
AOP for skin sensitisation while benefiting from the Mutual
Toxicol. Pharmacol.
337–351.
Acceptance of Data.
20) Urbisch D., Becker M., Honarvar N., Kolle S.N., Mehling A., Teubner W., Wareing B.,
Landsiedel R. (2016). Assessment of Pre- and Pro-haptens Using Non-animal Test Methods for
Backgroundand principles of the test methods included in the Key Event based
Skin Sensitization. Chem. Res. Toxicol. 29, 901-13.

Test Guidelines21) Natsch A, Emter R, Gfeller H, Haupt T, Ellis G (2015). Predicting Skin Sensitizer Potency Based
5.
The Data
assessmentof skin sensitisation
has typicallyinvolvedGlobal
the useVersusof laboratory
on In Vitro
from KeratinoSens
and Kinetic
Peptide Binding:
Domain-Basedanimals.
The Toxicol
classicalSci
methods
that use guinea-pigs, the Guinea Pig Maximisation Test
Assessment.
143, 319-332.
(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess
22) both
Jaworska
JS, Natsch
Ryan C, phases
Strickland
J, Ashikaga
T, Miyazawa
M (2015).
Bayesian
the induction
andA,elicitationof skinsensitisation.
The murine
tests, theintegrated
testing(ITS)
potencyassessment: LLNA:
a decision
LLNA
(OECD
TGstrategy429) (12)andforitsskin
threesensitization
non-radioactive
modifications,
DA support
system forquantitative
weightand adaptiveand
testing
strategy.(OECDArch Toxicol
89,
(OECD
TG 442A)(13) aswell ofas evidence
LLNA: BrdU-ELISA
BrdU-FCM
TG
2355-2383.
442B)(14), all assess the induction response exclusively, and have gained acceptancethey J,
providean Paris
advantageover theDM,
guineain termsof animal
welfare23) since
Strickland
Zang Q,
M, Lehmann
AllenpigD,tests
ChoksiN, Matheson
J, Jacobs
A, Casey
togetherwithanobjective
measurementoftheinduction
phase
of
skin
sensitisation.
W, Kleinstreuer N (2017). Multivariate models for prediction of human skin sensitization hazard.
J Appl Toxicol
37, 347-360. in chemico and in vitro test methods addressing the first6.
Mechanistically-based
keyT,eventsof thesensitisation
AOPR,havebeen L,
adopted
forPO,
contributing
the24) three
DelainePontingDJ,skin
Niklasson
IB, Emter
Hagvall
NorrbyNatsch A,toLuthman
K,evaluation
of
the
skin
sensitisation
hazardpotentialofchemicals:
the
OECD
TG
442C
Karlberg AT (2014). Epoxyalcohols: bioactivation and conjugation required for skindescribes
the Chem
DirectRes
PeptideReactivity
Assay (15) addressing the first key event; thesensitization.
Toxicol
27, 1860-1870.
present Test Guideline assesses keratinocyte activation addressing the second key event25) andWasserman
W.W.,
Fahl addresses
W.E. (1997).
Comprehensive
analysisof theproteins
whichinteract
the OECD
TG 442E
the activationof dendritic
cells,
third key
eventof withthe antioxidant
responsive
element:
ARE-BP-1
therepresenting
chemoprotective
inductiontheskin sensitisation
AOP(16). correlation
Finally, theof fourth
key withevent
T-cellresponse.
Arch.
Biochem.
Biophys.
344,
387-396.
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
26) 7.
Gildea As
L.A.,
Ryan C.A.,activation
Foertsch L.M.,
Kennedy
DearmanR.J.,
I., Gerberick G.F.
keratinocyte
represents
only J.M.,
one key
event oftheKimberskin sensitisation(2006).
of geneexpression
changes
inducedby chemicalallergensin dendritic
AOP(2) Identification(17), information
generatedwith test
methods
developed
to address
this specificcells:
opportunitiesforskinsensitization
testing.
J.
Invest.
Dermatol.
126,
1813-1822.
key event may not be sufficient to conclude on the presence or absence of skinpotentialof Hulettechemicals.
Therefore
test methods27) sensitisation
Ryan C.A., Gildea
L.A.,
B.C.,
Dearmandata
R.J.,generated
Kimber I.withAndtheGerberick
G.F. (2004).
described
in
this
Test
Guideline
areproposedtosupportthediscrimination
betweenskin to a
Gene expressing changes in peripheral blood-derived dendritic cells following exposure
sensitisers
(i.e.
UN
GHS
Category1)
andnon-sensitisers
when
used
withinIntegrated
contact allergen. Toxicol. Lett. 150, 301-316.
Approaches to Testing and Assessment (IATA), together with other relevant28) complementary
Emter R., van derVeen J.W.,e.g.
AdamsonEzendamvan Loveren
H., other
NatschA.events(2013). Gene
information,
derivedG.,fromin vitroJ.,
assaysaddressing
key
expression
changes inducedin theKeratinoSens™
line: Discriminatingof
the skin sensitisation
AOP byas skinwell sensitizers
as non-testing
methods,
including cell
read-acrossfrom
Nrf2-dependentandNrf2-independent
events.
Toxicol.
In
Vitro
27,
2225-2232.
chemical analogues (17). Examples on the use of data generated with these methods
Approaches,
i.e. approaches
standardised Standards
both in relation
to the setofof29) within
OECD Defined(2015). GuidanceDocument
No 213. Performancefor assessment
proposed
information
sources
used
and
in
the
procedureappliedtoderivepredictions
have
been
similar or modified in vitro skin sensitisation ARE-NrF2 luciferase test methods. Series on
published(17) andcan be employed
as useful
within IATA.
Testing andAssessment.
Available
at: elements
http://www.oecd.org/env/ehs/testing/series-testingassessment-publications-number.htm
8.
The test methods described in this Test Guideline cannot be used on their own,neithertosub-categorise
sensitisers
subcategories
1A and 1B asAcceptancedefined by UN
30) OECD (2005).
Guidanceskin
Document
theinto
Validation
and Internationalof New or
GHS
(1),
for
authorities
implementing
these
two
optional
subcategories,
to predict
Updated Test Methods for Hazard Assessment. OECD Environment,nor
Healthand Safety
potency
for safety
decisions.
on the regulatory
publications,OECD assessment
Series on Testingand However,
Assessmentdepending
No.34. Organisation
for Economic
framework,
positive
results
generated
with
these
methodsmaybeused
on their own to
Cooperation and Development, Paris.
classify a chemical into UN GHS category 1.

© OECD 201848

OECD/OCDE

which it was also

442D │ 25

United nations (UN) (2017). Globally Harmonized System of Classification and Labelling ofChemicals (GHS). Seventh revised edition, New York and Geneva, United Nations
Publications.
Availableat:[https://www.unece.org/trans/danger/publi/ghs/ghs_rev07/07files_e0.html]

in relation to thebut can be used
the keratinocytes 31) Basketter D.A., Alépée N., Ashikaga T., Barroso J., Gilmour N., Goebel C., Hibatallah J.,
from the Mutual
Hoffmann S., Kern P., Martinozzi-Teissier S., Maxwell G., Reisinger K., Sakaguchi H., Schepky
A., Tailhardat M., Templier M. (2014). Categorization of chemicals according to their relative
human skin sensitizing potency. Dermatitis 25, 11-21.

ey Event based32) Belot N, Sim B, Longmore CL, Roscoe L, Treasure C. (2017) Adaptation of the KeratinoSens™
skin sensitisation test to animal-product-free cell culture.ALTEX. 2017 Mar 16. doi:
10.14573/altex.1701311
use of laboratory
Maximisation Test 33) Patlewicz G, Casati S, Basketter DA, Asturiol D, Roberts DW, Lepoittevin J-P, Worth A and406) (11), assess
Aschberger K (2016) Can currently available non-animal methods detect pre and pro haptens
murine tests, therelevant for skin sensitisation? Regul Toxicol Pharmacol, 82, 147-155.
ions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
201849

226││

442D
442D

OECD/OCDE

underwent
PerformanceStandard-based validation
study based on which it was also
APPENDIX
IA -aANNEX
1: PROFICIENCYSUBSTANCES
reviewed and received positive opinion by ESAC (7) (8) (9) (10).

InSkin
The in
ARE-Nrf2
KeratinoSensTM
Test
4. VitroThe
testSensitisation:
methods included
this Test Luciferase
Guideline may
differ in relation
to theMethodprocedure used to generate the data and the readouts measured but can be used
indiscriminately
to address
countries’
forthistest Appendix
results onoftheTest
keratinocytes
Prior to routine useof a test
method requirementsthat adheres to
Guideline
activation
Key Eventof thedemonstrate
AOP for skinsensitisation
while benefitingfrom
the Mutual
442D, laboratories
shouldtechnical
proficiency
by correctly
obtainingtheAcceptanceofData.
expected KeratinoSens™ prediction for the 10 Proficiency Substances recommended inTable 1 and by obtaining the EC1.5 and IC50 values that fall within the respective reference
Backgroundand8principles
test methods
includedin theKey Eventbasedrange for at leastout of the of
10theproficiency
substances.
These
Proficiency
Substances
Test
were Guidelines
selected to represent the range of responses for skin sensitisation hazards. Other
selection
criteria
were ofcommercial
availability,
availabilityof highquality
in vivo
5.
The
assessment
skin sensitisation
has typicallyinvolved
the useof laboratoryreference,
andavailabilityofhighquality
in
vitro
data
from
the
KeratinoSens™
test
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
method. of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess(GPMT)

both the induction and elicitation phases of skin sensitisation. The murine tests, theTable 1.
Recommended
demonstrating
technical
proficiency
with theLLNA(OECD
TG 429) substances(12) and itsfor
three
non-radioactive
modifications,
LLNA:
DA
TM
KeratinoSens
test
method(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
442B) (14), all assess
the induction
response exclusively,
and have gained
acceptanceProficiency
Physical
LLNA
Human
KeratinoSe
EC1.5 (µM )IC50 (µM )CASRN
TMsince they provide anForm
advantagePrediction
over the guinea
pig(2)tests innsterms of Reference
animal welfare
Reference
Substances
category
Prediction
together with an objective measurementof skin sensitisation.
(1) of the induction phase
Range (4)Range (5)(3)

6.
Mechanistically-based in chemico and in vitro test methods addressing the firstevents of theSolidskin sensitisation
been adopted
for contributing
Salicylicthree key
Non- AOP have
69-72-7
Cat. 6
Negative> 1000 to the> 1000
evaluation of the skin sensitisation
hazard potential of chemicals: the OECD TG 442C
acidsensitiser
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; theNonLactic acidCat. 6 addressing
Negativethe second> 1000present 50-21-5
Test GuidelineLiquidassesses keratinocyte
activation
key event> 1000
sensitiser
and the OECD TG 442E addresses the activation of dendritic cells, the third key event of
the skin sensitisation AOP (16). NonFinally, the fourth key event representing T-cell
Glycerol
56-81-5
LiquidCat. 6
Negative> 1000> 1000proliferation is indirectly assessed sensitiser
in the murine Local Lymph Node Assay (LLNA) (12).
7.
As keratinocyte activation represents only one key event of the skin sensitisation
LiquidCat. 5
Negative> 1000
AOP (2)67-63-0(17), information
generatedNonwith test methods
developed
to address
this specific> 1000
sensitiser
key event may not be sufficient to conclude on the presence or absence of skinof chemicals.
Therefore Cat.
data4 generated
with the 5test
Ethylenesensitisation
Sensitiser
97-90-5potentialLiquid
Positive- 125methods> 500
proposed to support the discrimination between skinglycol described in this Test Guideline are(weak)sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
dimethacrylateApproaches to Testing and Assessment (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key events
Cinnamylof the skinSensitiser
104-54-1
Cat. methods,3
Positive
25 - 175sensitisationSolid
AOP as well
as non-testing
including read-acrossfrom> 1000
alcohol(weak)
chemical analogues (17). Examples on the use of data generated with these methods
within Defined
Approaches,
i.e. Sensitiser
approaches standardised
bothin relation25to- 250
the set of> 500
2149-30-4
Solid
Cat. 3
Positive
information
sources
used
and
in
the
procedureappliedtoderivepredictions
have
been
Mercaptobe(moderate)
published
(17)
and
can
be
employedasusefulelements
withinIATA.
nzothiazole
Isopropanol

8.

The test methods described in this Test Guideline cannot be used on their own,

4Sensitiser
55-55-0
SolidCat. 3
Positive< 12.520 - 200
neither to sub-categorise skin sensitisers
Methylamin(strong)into subcategories 1A and 1B as defined by UN
ophenol GHS (1), for authorities implementing these two optional subcategories, nor to predict
sulfate potency for safety assessment decisions. However, depending on the regulatory

framework, positive results generated with these methods may be used on their own to

Methyldibro
Sensitiser 1.
classify35691-65-7
a chemical intoSolid
UN GHS categorymoCat. 2

Positive< 20

20 - 100© OECD 201850

OECD/OCDE

glutaronitril
which it was also(strong)ein relation to the
2,4-Dinitrochlorobenz
but can be usedene
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

442D │ 27

97-00-7

Solid

Sensitiser(extreme)

Cat. 1

Positive< 12.55 - 20Notes: (1) The in vivo hazard (and potency) predictions are based on LLNA data (7). The in vivo potency isderived using the criteria proposed by ECETOC (15); (2) According to Basketter and co-workers (32). Cat. 1
represents clear evidence of contact allergy, Cat. 2 a frequent cause of contact allergy, Cat. 3 a common causeof contact allergy, Cat. 4 an infrequent cause of contact allergy, Cat. 5 a rare cause of contact allergy, and Cat.
6 essentially absent evidence of contact allergy (32). (3) A KeratinoSensTM prediction should be consideredin the framework of a Defined Approach or of an IATA and in accordance with the provisions of paragraphs
7 and 8 of the general introduction. (4) Based on the historical observed values (6).

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
201851

228││

442D
442D

OECD/OCDE

underwent
PerformanceStandard-based validation
based on whichTM
it TEST
was also
APPENDIX
IA -aANNEX
2: ADAPTATION
OF THEstudy
KERATINOSENS
reviewed
and received
positive
opinion byTO
ESAC
(7) (8) (9)XENO-FREE(10).
METHODUSINGHUMANREAGENTSACHIEVECELL
CULTURE4.
The test methods included in this Test Guideline may differ in relation to theprocedure used to generate the data and the readouts measured but can be usedThe
following adaptation
the KeratinoSens™
may onbe theperformed
using
indiscriminately
to addresstocountries’
requirementstest
formethod
test results
keratinocytes
humanreagents(humanserum
andrecombinanthumantrypsin)toachievexeno-freecell
activation Key Event of the AOP for skin sensitisation while benefiting from the Mutual
culture,subject
todemonstrationoftechnical
proficiency(asdescribed
in
Annex
1)
using
Acceptance of Data.
the adapted method (33).

Background and principles of the test methods included in the Key Event based
Table 2. Summary of adaptations
Test Guidelines
5. AspectThe
assessment ofValidated
skin sensitisation
has typically involved
the use of laboratoryof theReference Method
Xeno-Free Adaptation (thisanimals.
The classical methods
that use(Appendix
guinea-pigs,
Maximisation Test
Method(KeratinoSens™)
1A) the Guinea Pig Annex)(GPMT)
ofMagnusson
andKligmanandtheBuehler
Test
(OECD
TG
406) (11), assess
Serum1both
the induction Statesand elicitation
phasesof skinmurineserum
tests, the“serum” (DB-ALM
protocol155 sensitisation.
SpecifiesThe
10% human(paragraph
13)
LLNA (OECD TG states429) Foetal(12) Calfand Serum)
its three
non-radioactive
modifications, LLNA: DA(OECD
TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
Cytotoxicity
MTT:the4hrs
incubation;response
solubilise in
10%
MTT
3hrs incubation;
442B)
(14),
induction
exclusively,
and(1mg/ml):
have gained
acceptancemeasurement2all assess
SDS overnight; read at 600nm
solubilise in isopropanol; read atsince they provide (paragraphan advantage
over the guinea pig tests
in terms of animal welfare
25)
570nm
together with anobjective
measurementoftheinduction
phaseof skin sensitisation.
2
Positive control

Cinnamic aldehyde
4-64µMand(paragraph18)testCinnamic
aldehyde8-128µM.
6.
Mechanistically-basedin chemico
in vitro
methods
addressing
the first
1
three
key events of the skin sensitisation AOP have been adopted for contributing to theTrypsinNot specified (DB-ALM protocol 155 states
Non-animal recombinant trypsin
evaluation of the skinsensitisation hazard potential of chemicals:
the OECD TG 442C
Trypsin EDTA)(TrypZean, Sigma-Aldrich T3499)
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; the2present
Test Guideline
keratinocyte
Note: 1adaptations
to achieve assesses
xeno-free conditions;otheractivation
adaptationsaddressing
to the methodthe(33).second key eventand the OECD TG 442E addresses the activation of dendritic cells, the third key event ofthesensitisation(16). theFinally,
the fourth cell
key line
eventshould
representing
T-cellPriorskin
to usefor testingAOP
purposes,
KeratinoSens™
be adaptedtoproliferation
is indirectly
the murine
Lymph
Assay
(LLNA)(12).
routine cultureusing 10%assessed
human inserum.
HumanLocal
serum(fromNodepooleddonors)shouldbeobtaineda reliableactivationcommercial
source,only
withconsent
and QC
7.
Asfromkeratinocyte
represents
oneappropriate
key event donorof the skinsensitisation
testingforcell
cultureapplications.
As
with
any
type
of
serum,
when
a
new
batch
is used,AOP (2) (17), information generated with test methods developed to address this specificanof thebatch including
cell onmorphology,
growthand ofImax
keyinternal
event validation
may not be
sufficient
to conclude
the presence
or rates
absenceskin/ECvalues
with
at
least
the
positive
control,andpreferably
representativereference
1.5
sensitisation potential of chemicals. Therefore data generated with the test methods
chemicals
leastsensitiser
one nonsensitiser)should be conducted,
described in(atthis
Testone
Guideline
areandproposed
to support
the discrimination
between withskin
subsequent
reservationofsuccessfully
performing
batches
for
long
term
use.
the cells
sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within IfIntegrated
have
previously
culturedfoetal calf serum,
theytogether
should bewith
weanedintorelevant
humanApproaches
to beenTesting
and inAssessment(IATA),
other
serum
over
at
least
3
passages.
Provided
that
the
cells
are
showing
healthymorphology
complementary information, e.g. derived from in vitro assays addressing other key events
andcomparable
growth AOP
rates withthose
in foetal calf
serum, including
a cell bankread-acrossshould thenbeof theskin sensitisation
as wellas non-testing
methods,
fromcreatedfuture use.
It should
be noted
that use
the KeratinoSens™
cellwithline, thesewhen methods
culturedchemicalforanalogues(17).
Exampleson theof data generatedinhuman
serum,
shouldbeculturedup
toamaximum
passagenumberof
22
forwithin Defined Approaches, i.e. approaches standardised both in relation to theoptimal
set ofperformance,
includingtheandnumbertaken
to adaptappliedthem totohumanTo achieve
fully
information sources
usedin theprocedure
deriveserum.
predictions
have been
xeno-freecell
culture,anon-animal
sourceofrecombinanttrypsin(forexample,published (17) and can be employed as useful elements within IATA.
TrypzeanTM) should be used to harvest the cells during sub-culture (33). In all other8.
Guideline
cannot be inused
their own,
respects,The
thetestcellsmethods
should describedbe culturedin inthis
theTestsame
way as describedthisonAppendixtoneithertosub-categoriseskinsensitisers
into
subcategories
1A
and
1B
as
definedbyUN
Test Guideline 442D and the DB-ALM protocol (9) for the reference KeratinoSens™ cell
GHS
line (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
With reference
to paragraph
18, the withxeno-freeof be
the used
KeratinoSens™
framework,
positive
results generated
these adaptation
methods mayon their owntest
tomethodusing
humanreagents
has
been
optimisedusing
cinnamic
aldehyde(CAS
No.
classify a chemical into UN GHS category 1.
14371-10-9, >98% purity) as a positive control, at a final concentration range from 8 to

© OECD 201852

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

442D │ 29

128µM. Other positive controls, preferentially providing EC1.5 values in the mid-range,
may be used if historical data are available to derive comparable run acceptance criteria(33).
With reference to paragraph 25, the xeno-free adaptation of the KeratinoSens™ test
method using human reagents has been optimised using the following method forcytotoxicity assessment. Medium is replaced after the 48 hour exposure time with fresh
medium containing MTT (3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide; CAS No. 298-93-1) at a concentration of 1mg/ml, and cells incubated for 3
hours at 37 ± 1°C in the presence of 5% CO2. The MTT medium is then removed andcells are solubilised by the addition of isopropanol. After shaking for 30 minutes, theabsorption is measured at 570 nm with a spectrophotometer.
All other aspects the xeno-free adaptation of the KeratinoSens™ test method using
human reagents should be conducted in the same way as described for the standard
method described in this Appendix to Test Guideline 442D and the DB-ALM protocol (9)for the reference KeratinoSens™ cell line.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
2018

53

230││

442D
442D

OECD/OCDE

underwent
PerformanceStandard-based
validationOF
study
based on which it was also
APPENDIX
IA -aANNEX
3: QUALITYCONTROL
LUMINESCENCE
reviewed and received positive opinion by ESAC (7) (8) (9) (10).
MEASUREMENTS
4.
The test methods included in this Test Guideline may differ in relation to the
Basicused
experiment
for ensuring
luminescencemeasurementsin theprocedure
to generate
the dataoptimaland thereadouts measured
but canbe usedKeratinoSensTM
test
methodindiscriminately to address countries’ requirements for test results on the keratinocytes
activation
Key three
Eventparametersof the AOPareforcriticalskin sensitisation
while benefiting
thewithMutualThefollowing
to ensure obtaining
reliable fromresults
theAcceptance of Data.
luminometer: having anda sufficientsensitivity
a stable included
backgroundcontrol
wells; based
Backgroundprinciplesof the giving
test methods
inintheKey Event
Test Guidelines
having no gradient over the plate due to long reading times; and

A
B
C
D
E
F
G
H

5.  having
The assessmentof skin sensitisation
has typicallyinvolved
use wells.
of laboratory
no light contaminationin adjacentwells fromstronglythe
activeanimals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
Prior
to oftesting
it is andrecommended
appropriate(GPMT)
Magnusson
Kligman and to
the ensure
Buehlerhaving
Test (OECDTG 406)luminescence(11), assessmeasurements,
bytestingacontrol
plate
set-upasdescribed
below(triplicate
analysis).
both the induction and elicitation phases of skin sensitisation. The murine
tests, the
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
Plateassetup
of first
training experiment(OECD TG 442A) Table(13) as1. well
LLNA:
BrdU-ELISA
and BrdU-FCM (OECD TG
442B) (14), all assess the induction response exclusively, and have gained acceptance
1 since they2
3
4
5 over the
6 guinea7 pig tests
8 in terms
9 of animal
10
11
providean advantage
welfaretogether with an objective measurement of the induction phase of skin sensitisation.

12

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

DMSO

6.
Mechanistically-based in chemico and in vitro test methods addressing the first
three DMSO
key eventsof theDMSO
skin sensitisation
AOP have
adoptedDMSO
for contributing
to the DMSO
DMSO
DMSO
DMSO
DMSO
DMSObeenDMSO
DMSO
DMSO
evaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes
Direct Peptide
Reactivity
Assay DMSO
(15) addressing
thekey event;the DMSODMSODMSO the DMSO
DMSO
DMSO
DMSO
DMSO
DMSOfirst DMSO
DMSO
present Test Guideline assesses keratinocyte activation addressing the second key event
EGDMA
EGDMA
EGDMA
EGDMA
EGDMAthe EGDMAEGDMAcells,
EGDMA
EGDMA
EGDMAof EGDMA
and theOECD
TG 442E
addresses
activationEGDMAof dendritic
the third
key event
0.98the skin1.95 sensitisation
3.9
7.8
15.6 Finally,
31.25 the 62.5
125 event
250 representing
500
1000
2000
AOP (16).
fourth key
T-cellproliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
7.
As keratinocyte activation represents only one key event of the skin sensitisation
AOP(2)
information
test methods
to address
this specificDMSO
DMSO(17),DMSO
DMSOgenerated
DMSO withDMSODMSO developed
DMSO
DMSO
DMSO
DMSO
key event may not be sufficient to conclude on the presence or absence of skin
DMSO
DMSO
DMSO
DMSO
DMSO Therefore
DMSO
DMSO
DMSO
DMSO
DMSO
sensitisation
potentialof chemicals.
data generated
with theDMSO
test methods
described in this Test Guideline are proposed to support the discrimination between skinDMSODMSO (i.e.
DMSO
DMSOCategory
DMSO 1) andDMSOnon-sensitisers
CA 4
CA when8CA
16 within
CA 32 Integrated
CA 64
sensitisers
UN GHS
used
Approaches to Testing and Assessment (IATA), together with other relevantNotes:EGDMA = Ethylene
glycol dimethacrylate
97-90-5)
a strongly
inducingothercompound.
complementary
information,e.g. derived (CAS
fromNo.:in vitro
assaysaddressing
key events
C = Cinnamic aldehyde, positive reference (CAS No.: 104-55-2). Concentrations are given in µMof the skin sensitisation AOP as well as non-testing methods, including read-across fromchemical analogues (17). Examples on the use of data generated with these methods
The quality control analysis should demonstrate:
within Defined Approaches, i.e. approaches standardised both in relation to the set of
information
sources usedincrease
and in theprocedureinduction
applied in
to rowderivepredictions
 a dose-dependent
in luciferase
D, withthe Imaxhave> 20 beenfoldpublished(17)backgroundand can be employed
as useful
within IATA.
above(in most cases
Imaxelements
values between
100 and 300 are reached);

DMSO
DMSO
Blank

8.  aThetest methods increase
describedin inthis Testinduction
Guideline
betoused
theirown,
dose-dependent
luciferase
in cannot
wells H7
H11,onwith
a foldneither to
sub-categorise
sensitisers
inductionof 2 to 8skinin well
H11; into subcategories 1A and 1B as defined by UN
GHS (1), for authorities implementing these two optional subcategories, nor to predict
 nofordose-dependent
increasedecisions.
in luciferase
induction dependingin row C andonE (nopotencysafety assessment
However,the induction
regulatory
value
equal
or
above
1.5
(ideally
not
above
1.3)
due
to
possiblelightframework, positive results generated with these methods may be used on their own toto strongly
active wells in the EGDMA row;classify contaminationa chemical intoespecially
UN GHSnext
category
1.

© OECD 2018

54

OECD/OCDE

442D │ 31

which it was alsono statistically significant difference between the rows A, B, C, E, F and G. (i.e.
no gradient over plate); andin relation to thebut can be used
the keratinocytes
from the Mutualvariability in any of the rows A, B, C, E, F and G and in the DMSO wells in row
H should be below 20% (i.e. stable background).

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
2018

55

232││

442D
442D

OECD/OCDE

underwent
PerformanceStandard-based
validation
studyLuciferase
based on which
it wasAppendix
IB: In aVitro
Skin Sensitisation:
The
ARE-Nrf2
LuSensTestalso
Methodreviewed and received positive opinion by ESAC (7) (8) (9) (10).
4.

The test methods included in this Test Guideline may differ in relation to the

INITIALCONSIDERATIONS,AND LIMITATIONS
procedure
used to generate APPLICABILITY
the data and the readouts
measured but can be used

indiscriminately to address countries’ requirements for test results on the keratinocytes
1.
TheKeytestEventmethodin thisto Test
Guideline
442D
addresses
theactivationof described
the AOP for
skinAppendix
sensitisation
while
benefitingfrom
the Mutual
second
key
event
of
the
skin
sensitisation
AOP(1),
namelykeratinocytes
activation,byAcceptance of Data.
assessing with the help of luciferase, the Nrf2-mediated activation of antioxidant response
element(ARE)-dependent
genes.
sensitisers
havebeen reported
inducegenes
that
Backgroundand principles
of Skinthe test
methods
includedin the to
Key
Event
based
are
regulated
by
the
ARE
(2)
(3).
Small
electrophilic
substances
such
as
skin
sensitisers
Test Guidelines
can act on the sensor protein Keap1 (Kelch-like ECH-associated protein 1), by e.g.
5.
assessment ofof skinsensitisation
has typicallythe use of laboratory
covalentThe
modification
its cysteine
residue,resulting involved
in its dissociationfrom theanimals.
The factor
classical
methods
thatfactor-erythroid
use guinea-pigs,2-related
the Guinea
Pig2).
Maximisation
Test
transcription
Nrf2(nuclearfactor
The dissociated(GPMT)
Magnusson
Kligman and theBuehler
TG 406)for(11),assessNrf2 canofthen
activateand
ARE-dependent
genes
suchTestas (OECD
those coding
phase
II
both
the induction
elicitation
phases of skin sensitisation. The murine tests, thedetoxifying
enzymesand(2) (4)(5).
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
2.
vitro ARE-Nrf2
luciferase
LuSenstest methodand(hereafter
called(OECDthe LuSens(OECD The
TG in
442A)
(13) as wellas LLNA:
BrdU-ELISA
BrdU-FCMTG
test
method)underwentaPerformanceStandard-based
validation
study
based
on the442B) (14), allassesstheinduction
response
exclusively,
andhave
gainedacceptanceTM
KeratinoSens
Validated
Reference(VRM)(6) (7)(8) (9),of followed
by ansince they provide
an advantage
overMethod
the guinea
pig tests
in terms
animal welfare
independent
peer
review
conducted
bytheEuropean
Union
Reference
Laboratory
together with an objective measurement of the induction phase of skin sensitisation. for
Alternatives to Animal Testing (EURL ECVAM) (10). The LuSens test method was
6.
Mechanistically-based
chemicoand inaddressing
the first
consideredscientifically validin to
be usedas vitro
part testof methods
an IATA,to support
the
three
key eventsbetweenof the skin
AOPnon-sensitisers
have been adopted
for purpose
contributing
to thediscriminationskinsensitisation
sensitisers andfor theof hazard
evaluationof (10).
the skin sensitisation hazard potential of chemicals: the OECD TG 442C
identification
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; the3.
Thetestassesses
method keratinocyte
proved to beactivationtransferable
to laboratories
experienced
inpresent
Test LuSens
Guideline
addressing
the second
key eventcell
culturetechniques
and
met
the
reproducibility
performancestandards
requiredbothand the OECD TG 442E addresses the activation of dendritic cells, the third key event ofwithinandsensitisation
between laboratories(10).Finally,
Additional
in-house T-cell
study
the
skin
AOP (16).
the information
fourth key fromeventearlier
representing
on
72
test
chemicals
showedsimilarpredictive
capacity
as
the
VRM
(74%
accuracy,
74%
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
sensitivity, and 74% specificity) for discriminating skin sensitisers (i.e. UN GHS Cat. 1)
7.
As keratinocyte
activation
represents
only
one key
eventindicatingof the skinthesensitisationfrom non-sensitiserswhencomparedto LLNA
results(7) (10),
usefulness
AOP
(2)
(17),
information
generatedwithtest
methods
developed
to sensitisation
address this specificof the LuSens test method to contribute to the identification of skinhazard.
key
event themay
not bevalues
sufficientconcludeon theor aabsenceof skin
However,
accuracy
giventohere
for LuSens
testpresence
method as
stand-alone
test
sensitisation
potentialof chemicals.
generatedwith thein test
methods
method, are only
indicative
since the Therefore
test methoddata
shouldbe considered
combination
described
this Test
Guideline are
to of
supportthe discrimination
betweenwith otherinsourcesof informationin proposed
the context
a Defined
Approach or an
IATA skinandsensitisers(i.e.withUN theGHS
Categoryof1)paragraphs
and non-sensitisers
used within
Integrated
in accordance
provisions
7 and 8 inwhen
the General
IntroductionofApproaches
toTesting
andAssessment(IATA),
togetherwithotherrelevant
this Test Guideline. Furthermore when evaluating non-animal methods for skincomplementary
information,e.g.inderived
fromvitro assays
otheranimal
key events
sensitisation, it should
be kept
mind thattheinLLNA
test asaddressing
well as othertests
ofthenotskinsensitisation
as wellas non-testing methods, including read-across from
may
fully
reflect the AOP
situationin humans.
chemical analogues (17). Examples on the use of data generated with these methods
4.
On the basisof the current
data available,
the LuSens
showntowithinDefinedApproaches,
i.e. approaches
standardised
bothtest
in method
relationwas
to theset ofbeapplicabletotest
chemicals
coveringavarietyoforganicfunctionalgroups,reaction
information sources used and in the procedure applied to derive predictions have beenmechanisms,
potency(as determined
inIATA.
vivo studies) and physicopublished(17)skinandsensitisation
can be employed
as usefulelements withwithinchemical properties (7) (8). The test method is applicable to test chemicals soluble or that
8.
The test
methodsindescribedin thisTest Guidelinecannotbe used onintheirform a stabledispersion
the exposure
medium(i.e. a colloid
or suspension
whichown,theneitherto sub-categoriseskin sensitisers
subcategories
anddifferent
1B as definedby TestUN
test chemical
does not settleor separateinto
fromthe solvent1A
intophases).
GHS(1), forimplementing
these two atoptional
subcategories,
nor to predict
chemicals
thatauthorities
do not fulfil
these conditions
the highest
final required
testing
potencyforsafetyassessment
decisions.
However,dependingontheregulatory
concentration (i.e. 2000 µM or 2000 µg/mL if no molecular weight is available) may still
framework,
results generatedInwith
methods
mayfulfillingbe used on
ownfortobe tested atpositive
lower concentrations.
suchthesea case,results
thetheircriteriaclassify
categorythe
1. identification of the test chemical as a skinpositivitya chemical
could stillinto
be UN
usedGHSto support
sensitiser. In cases where a negative result is obtained in a test with a maximal© OECD 201856

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

442D │ 33

concentrations < 2000 µM (or < 2000 µg/mL if no molecular weight is available) and nocytotoxicity is observed, the result should be considered as inconclusive (see prediction
model in paragraph 32). If cytotoxicity (<70% viability) is reached at a test concentration< 2000 µM (or < 2000 µg/mL if no molecular weight is available), criteria for negativity
can still be applied. In general mono constituent substances with a LogP above 7 may beinsoluble in the exposure medium, however, if solubility or stable dispersion can beobtained and documented, testing may still be conducted.
5.
Negative results should be interpreted with caution as substances with an
exclusive reactivity towards lysine-residues can be detected as negative by the test
method as the key mechanism leading to the activation of the Keap1-Nrf2-ARE pathway
appears to be the electrophilic reaction of stressors with nucleophilic thiols (cysteine
sulfhydryl groups) of Keap-1. Complementary information from peptide reactivity assays
may help addressing this uncertainty, in particular assays able to distinguish between
cysteine and lysine reactivity. Furthermore, because of the limited metabolic capability of
the cell line used (12) and because of the experimental conditions, pro-haptens (i.e.
chemicals requiring enzymatic activation for example via P450 enzymes) and pre-haptens(i.e. chemicals activated by auto-oxidation) in particular with a slow oxidation rate may
also provide negative results. However, it has been shown that the majority of pre-haptens(i.e. chemicals activated by auto-oxidation) and pro-haptens (i.e. chemicals requiring
enzymatic activation for example via P450 enzymes) are sufficiently well identified by a
combination of test methods covering key events 1, 2 and 3 on the AOP so that negative
results can in general be used to support classification (13) (14) (15). On the other hand,test chemicals that do not act as a sensitiser but are nevertheless chemical stressors maylead to false positive results as shown with the VRM (11). Finally, test chemicals thatinterfere with the luciferase enzyme can confound the activity of luciferase in cell-based
assays causing either apparent inhibition or increased luminescence (16). For example,
phytoestrogen concentrations higher than 1 µM were reported to interfere with theluminescence signals in other luciferase-based reporter gene assays due to over-activationof the luciferase reporter gene (17). As a consequence, luciferase expression obtained athigh concentrations of phytoestrogens or similar compounds suspected of producing
phytoestrogen-like over-activation of the luciferase reporter gene needs to be examined
carefully (17). In cases where evidence can be demonstrated on the non-applicability of
the LuSens test method to other specific categories of test chemicals, the test methodshould not be used for those specific categories.
6.
In addition to supporting discrimination between skin sensitisers (i.e. UN GHS
Category 1) and non-sensitisers, the LuSens test method also provides information (e.g.
dose- response) that may potentially contribute to the assessment of sensitising potency
when used in integrated approaches such as IATA such as described for the VRM (13).
However, further work, preferably based on human data, is required to determine how the
LuSens test method results can contribute to potency assessment, especially in the context
of an IATA (18). Examples on how to use the ARE-Nrf2 luciferase test methods in
combination with other information are reported in literature (15) (18).

sed on their own,
7.
Definitions are provided in Annex 1 of the General Introduction.
as defined by UN
es, nor to predict
PRINCIPLE OF THE TESTn the regulatory
d on their own to
8.
The LuSens test method makes use of an immortalised adherent cell line derived
from human keratinocytes stably harbouring a luciferase reporter gene under the control©©OECD
OECD2018
201857

234││

442D
442D

OECD/OCDE

underwenta Performance
validation
study(20).
basedon which
it wasonalsoof the antioxidant
responseStandard-based
element of the rat
NQO1 geneGenesdependent
thereviewed
positiveby ESAC (7)by(8)contact(9) (10).sensitisers (21) (22). The
ARE suchandas received
NQO1 areknownopinion
to be up-regulated
cell line contains the luciferase gene under the transcriptional control of a promoter fused
4.
The test methods included in this Test Guideline may differ in relation to thewith the ARE element (7). The luciferase signal reflects the activation by sensitisers ofprocedure used to generate the data and the readouts measured but can be usedendogenous Nrf2 dependent genes, and the dependence of the luciferase signal in theindiscriminately to address countries’ requirements for test results on the keratinocytes
recombinant cell line on Nrf2 has been directly demonstrated for the VMR (23), andactivation Key Event of the AOP for skin sensitisation while benefiting from the Mutual
indirectly demonstrated for the LuSens (7). This allows quantitative measurement (byAcceptance of Data.
luminescence detection) of luciferase gene induction, using well established light
producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription
Background and principles of the test methods included in the Key Event based
factor in cells following exposure to electrophilic test substances.

Test Guidelines

9.
Test chemicals are considered positive in the LuSens test method if they induce a
5.
The significant
assessment inductionof skin sensitisation
has typicallyusethresholdof laboratory
statisticallyof the luciferase
activityinvolved
above a the
given
(i.e.
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
 1.5 fold, or 50% increase) in at least two consecutive concentrations which do not(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess
significantly affect cell viability (i.e. at which the cellular viability is above 70%) (7) (8).
both the induction and elicitation phases of skin sensitisation. The murine tests, theFor this purpose, induction of the luciferase activity over solvent/vehicle control is
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
determined. Furthermore, parallel cytotoxicity measurements should be conducted to(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
assess whether luciferase activity induction levels occur at sub-cytotoxic concentrations.
442B) (14), all assess the induction response exclusively, and have gained acceptance10.
Priorprovide
to routine
use of the LuSens
methodadheresto thisGuideline,
since theyan advantage
over thetest
guineapigthattests
in termsof Testanimalwelfarelaboratories
demonstratetechnical
proficiency,
using
thesensitisation.
ten Proficiency
togetherwith should
an objective
measurementof the induction
phaseof skin
Substances listed in Annex 1 of this Appendix.
6.
Mechanistically-based in chemico and in vitro test methods addressing the first
three key events of the skin sensitisation AOP have been adopted for contributing to the
PROCEDURE
evaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; the11.
A DB-ALM protocol for the LuSens test method is available and should bepresent Test Guideline assesses keratinocyte activation addressing the second key event
employed when implementing and using the test method in the laboratory (24). A
and the OECD TG 442E addresses the activation of dendritic cells, the third key event ofsummary of the main protocol steps of the LuSens test method as compared to the VRM
the skin sensitisation AOP (16). Finally, the fourth key event representing T-cellis given in Annex 2 of this Appendix. Laboratories implementing this Test Guideline canproliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
obtain the recombinant cell line used in the test method by requests to the testdevelopers. The luciferase
reporterrepresents
gene assayonly
is subject
to eventa Promega
use licence7.
As 3keratinocyte
activation
one keyof thelimited
skin sensitisation
requiringi) theinformation
use of luminescentreagents
purchased
fromPromega;ii) toAOP(2) (17),
generated assay
with test
methods
developed
to address
thisorspecific
contact
Promega
to obtaina free license
for commercial
The following
paragraphs
key
event
may not
be sufficient
to conclude
on the use.
presenceor absenceof skinprovide with potential
a descriptionof the main
components
procedures
thetestLuSenstest
sensitisationof chemicals.
Therefore
dataandgeneratedwithofthe
methods
method. in this Test Guideline are proposed to support the discrimination between skindescribed
sensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to
Testingand Assessment
Preparationof thekeratinocyte
cultures (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key events
12.
The LuSens transgenic cell line having a stable insertion of the luciferase reporterof the skin sensitisation AOP as well as non-testing methods, including read-across fromgene under the control of the ARE-element should be used. Upon receipt, cells are
chemical analogues (17). Examples on the use of data generated with these methods
propagated as defined by the test method protocol (e.g. 1 to 3 passages) and stored frozen
within Defined Approaches, i.e. approaches standardised both in relation to the set ofas a homogeneous stock. Cells from this original stock can be propagated up to a
information sources used and in the procedure applied to derive predictions have beenmaximum of 20 passage number and are employed for routine testing using the
published (17) and can be employed as useful elements within IATA.
appropriate maintenance/growth medium (e.g. Dulbecco's Modified Eagle's medium
8.
Thecontaining
test methods
described
in this Testsuch
Guideline
cannot beinused
their own,
(DMEM)
serum
and antibiotics
as puromycin
the onmaintenanceneither
sub-categorise
into subcategories
and 1Bcontamination))as defined by UN
mediumto (forselection) skinand sensitisers
penicillin/streptomycin(to 1A
prevent
asGHS(1), within
for authorities
implementing
these
twoNo
optional
subcategories,
nor to predict
described
the test method’s
protocol(24).
antibiotics
are added however
to thepotency
for safetymedium during
testing.assessment decisions. However, depending on the regulatory
framework, positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.
3

BASF SE, 67056 Ludwigshafen, Germany.

© OECD 201858

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to442D │ 35

13.
For testing, cells should be 80-90% confluent, and care should be taken to ensurethat cells are never grown to full confluence. One day prior to testing cells are harvested,and distributed into 96-well plates at the appropriate cell density (i.e. 10 000 cells/well).
Attention should be paid to avoid sedimentation of the cells during seeding to ensure
homogeneous cell number distribution across wells. If this is not the case, this step may
result in high well-to-well variability. For each repetition of the main luciferase test foreach test chemical concentration, three replicates are used for the luciferase activity
measurements, and three replicates used for the cell viability assay.

Preparation of the test chemical and control substances
14.
The test chemical and control substances are prepared (or thawed in case of stablefrozen solutions) on the day of testing. Test chemicals are dissolved in a suitable solvent,e.g. dimethyl sulfoxide (DMSO, CAS No. 67-68-5,  99% purity) to the final
concentration that allows reaching the maximum concentration tested (e.g. 200 mM). The
DMSO solutions can be considered self-sterilising, so that no sterile filtration is needed.
Test chemicals not soluble in DMSO are dissolved in sterile water or culture medium, inwhich the appropriate measures should be taken to ensure that the final solutions aresterile. For a test chemical which has no defined molecular weight (MW), a stock solution
is prepared to a default concentration of 200 mg/mL or 20% (w/v). In case solvents otherthan DMSO, water or the culture medium are used, appropriate scientific rationale should
be provided.
15.
Based on the stock solutions of the test chemical, serial dilutions are made using
DMSO or for test chemicals not soluble in DMSO, using sterile water or culture medium,to obtain master concentrations of the chemical to be tested (e.g. 12 concentrations
ranging from 0.098 to 200 mM). Independent of the solvent used, the masterconcentrations, are then further diluted 25 fold into culture medium containing serum,
and finally used for treatment with a further 4 fold dilution factor so that the final
concentrations of the tested chemical are reached (e.g. ranging from 0.98 to 2000 µM
based on a dilution factor of 2). For a test chemical which has no defined MW, serial
dilutions are made using DMSO or a suitable solvent to obtain the desired final
concentrations of the test chemical (e.g. from 0.98 to 2000 µg/ml).
16.
A cytotoxicity pre-range dose finding test is first performed, based e.g. on theabove concentrations, to determine the concentration at which cell viability is reduced to75% (CV75). The CV75 is then used as a basis for determining the concentrations to betested in the main luciferase test and the parallel cytotoxicity test (e.g., one concentration
above CV75, the CV75 and four concentrations below CV75 using a serial dilution factor of
1.2 resulting in the concentrations CV75/2.07, CV75/1.73, CV75/1.44, CV75/1.2, CV75 andCV75x1.2 μM). Alternative concentrations may be used upon justification (e.g. in case of
too low or too high cytotoxicity or poor solubility) (24).
17.
A concurrent solvent/vehicle control should be tested within each repetition (e.g.
DMSO), for which a sufficient number of wells should be prepared per plate (e.g. 12 forthe cytotoxicity pre-range dose finding test and 24 for the main luciferase test as
described in the protocol (24)). The solvent/vehicle control undergoes the same dilutions
as described for the master concentrations in paragraph 15, so that the final
solvent/vehicle control concentration should correspond to the same concentration as in
the tested chemicals and in the positive control (i.e. 1%), and should not significantly
affect cell viability. For a test chemical not soluble in the used solvent (e.g. DMSO), for©©OECD
OECD2018
2018

59

236││

442D
442D

OECD/OCDE

underwenta PerformanceStandard-based
validation
based
on which
it final
was also
which the dilutions
were madein water, thesolvent study
level inall wellsof thetest
reviewedandreceived
positivemust
opinionby ESACto(7)be(8)equal(9) (10).
solution of
thistest chemicalbe adjusted
to the solvent concentration
used for the other test chemicals and control substances (i.e. 1%).
4.
The test methods included in this Test Guideline may differ in relation to theprocedure
used to generate
data should
and thebut repetition,
can be used18.
A concurrent
negative thecontrolalsoreadouts
be testedmeasured
within eachforindiscriminately
to address
for test resultson thekeratinocytes
which a sufficientnumbercountries’of wells requirements
should be preparedper plate(e.g.
3 for theactivation
Key
Event ofdose
the finding
AOP fortest
skin
while
benefiting
the Mutual
cytotoxicity
pre-rangeandsensitisation
6 for the main
luciferase
testfrom
as describedinAcceptance
Data.In the LuSens test method, the concurrent negative control tested isthe protocolof(24)).
5 000 μM (or 450 μg/mL) DL-Lactic acid (CAS No. 50-21-5, ≥ 99% purity), known to bea non-sensitiserand
andprinciples
to result inofa thenegative
predictionincluded
with the LuSens
test Eventmethod.based
Other
Backgroundtest methods
in the Key
suitablenegative
controls
may
be
used
if
historicaldata
areavailable
toderiveTest Guidelines
comparable run acceptance criteria. Furthermore, in the LuSens test method a sufficient5.
assessment
typically
involveduseandof 12laboratory
number The
of wells(e.g. 6offorskin
thesensitisation
cytotoxicity has
pre-rangedosefindingthetest
for theanimals.
The classicaluseprotocol
guinea-pigs,
Guinea blank
Pig Maximisation
Test
main luciferase
test as methods
describedthat
in the(24)) thecontaining
medium controls(GPMT)
ofMagnusson
andKligmanandtheBuehler
Test
(OECD
TG
406)
(11),
assessare prepared consisting of untreated cells and culture medium only.
both the induction and elicitation phases of skin sensitisation. The murine tests, the19.
A concurrent
positive
be tested in amodifications,sufficient number
of wells
LLNA(OECD
TG 429)(12)control
and itsshould
three also
non-radioactive
LLNA:
DA
within eachto asdemonstrate
appropriate
response andof thetest system(OECD(e.g. 2 TG
for
(OECD
TG repetition
442A) (13)well as LLNA:
BrdU-ELISA
BrdU-FCM
the cytotoxicity
findingresponse
test and exclusively,
5 for the main
luciferase
test asacceptancedescribed
442B)(14), all pre-range
assess thedose
inductionandhave gainedin thethey
protocolFor theover
LuSenstest method,µM ofEthylene
Glycolsince
provide(24)).
an advantage
the guinea
pig tests 120
in terms
animal welfare
Dimethacrylate(EGMDA,CAS
No.
97-90-5,≥99%
purity)
is
used.
The
positive
control
together with an objective measurement of the induction phase of skin sensitisation.
is prepared using the same dilution steps as described for the master concentrations in
6.
Mechanistically-based
and in vitro
test methods
firstparagraph
14 and as described inin chemico
the test method’s
protocols(24). If addressing
the positivethe
controlthree
key eventsof thesensitisation
AOP
been adopted
to theconcentrationof 120μMskinis tootoxic or not
ablehaveto induce
luciferasefor≥ contributing
2.5 (see paragraphevaluation
of
the
skin
sensitisation
hazardpotentialofchemicals:
the
OECD
TG
442C
31) due to e.g. a new laboratory facility or a new batch of EGMDA, the performing
describes
theDirectPeptideReactivity
Assay
(15)
addressing
the
first
key
event;
thelaboratory may run a range finder experiment with EGDMA (confirmed in at least twopresent
Test
Guideline
assesseskeratinocyte
activation
addressing
thesecondkey
event
more runs) in order to set the concentration at which luciferase induction is ≥ 2.5 folds
andthe OECD
TG 442E addresses
activationof celldendritic
cells,
thirdFinally,
key eventofcompared
to solvent/vehicle
control,theandfor whichviability
is the≥ 70%.
other
the
skin
sensitisation
AOP
(16).
Finally,thefourthkey
event
representing
T-cellsuitable positive controls, preferentially providing EC1.5 values in the mid-range, may be
proliferation
is indirectly
murineLocal Lymph
Node Assay(LLNA) (12).
used if historical
data are assessedavailablein
tothederivecomparable
run acceptancecriteria.
7.
As keratinocyte activation represents only one key event of the skin sensitisation
Application
the test chemical
substances
AOP (2) (17),ofinformation
generatedandwithcontrol
test methods
developed to address this specific
key event may not be sufficient to conclude on the presence or absence of skin20.
For each test chemical and positive control substance, one experiment is needed
sensitisation potential of chemicals. Therefore data generated with the test methods
to derive a prediction (positive or negative), consisting of at least two independent
described in this Test Guideline are proposed to support the discrimination between skinrepetitions containing each three replicates (i.e. n=6). In case of discordant resultssensitisersthe(i.e.
GHS Category
1) and anon-sensitisers
used within
betweentwoUN
independent
repetitions,third repetitionwhen
containing
three Integrated
replicates
Approaches to Testing and Assessment (IATA), together with other relevant
should be performed (i.e. n=9). Each independent repetition is performed on a different
complementary information, e.g. derived from in vitro assays addressing other key events
day with fresh stock solution of test chemicals and independently harvested cells. Cellsof the skin sensitisation AOP as well as non-testing methods, including read-across frommay come from the same passage however.
chemical analogues (17). Examples on the use of data generated with these methods
21.
After seeding
as describedin paragraph
13, cells arefor 24 tohours
in thewithin Defined
Approaches,
i.e. approaches
standardised
bothgrown
in relation
the setof
96-wells
microtiter
Theinmedium
is then removed
replacedwith fresh
cultureinformation
sourcesplates.
used andthe procedure
applied toand
derivepredictions
have
been
mediumDMEM
containing
serumelements
but without
antibiotics
published(i.e.
(17)150
andµlcanbe employed
as useful
withinIATA. as described within
the method’s protocol (24)) to which 50 µl of the 25 fold diluted test chemical and control
8.
The test methods described in this Test Guideline cannot be used on their own,
substances are added. At least one well per plate should be left empty (no cells and noneither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
treatment) to assess background values.
GHS (1), for authorities implementing these two optional subcategories, nor to predict22.
are then incubatedabout 48 hours
at 37±1°C
in theregulatory
presence
potencyThefortreatedsafetyplates
assessment
decisions. forHowever,dependingon theof
5% CO2.positive
Care shouldtaken to withavoidtheseevaporation
volatile
testonchemicals
framework,
resultsbegenerated
methods ofmay
be usedtheir ownand
tocross-contamination
between
wells
by
test
chemicals
bye.g.
covering
theplateswithaclassify a chemical into UN GHS category 1.
foil during incubation with the test chemicals.
© OECD 201860

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

Luciferase activity measurements23.

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

442D │ 37

The following factors are critical to ensure appropriate luminescence readings:


the choice of a sensitive luminometer,the use of a plate format with sufficient height to avoid light-cross-contamination,the use of a luciferase substrate with sufficient light output to ensure sufficientsensitivity and low variability; andan appropriate and stable background level.

Prior to testing, a control experiment setup as described in Annex 3 of this Appendix
should be carried out to ensure that these three points are met.
24.
After the 48 hour exposure time with the test chemical and control substances,cells are washed with a phosphate buffered saline, and the appropriate lysis buffer for
luminescence readings added to each well for a sufficient time (e.g. 5-10 min in dark).
25.
Plates with the cell lysate are then placed in the luminometer for reading using thespecific program prescribed within the test method’s protocol (24). In case alternative
settings are used, e.g. depending on the model of luminometer used, these should bejustified. Furthermore, a glow substrate may also be used provided that the quality control
experiment of Annex 3 of this Appendix is successfully conducted.

Cytotoxicity Assessment
26.
For the LuSens cell viability assay, medium is replaced after the 48 hour exposure
time with fresh medium containing 0.5 mg/ml MTT (3-(4,5-dimethylthiazol2-yl) -2,5 diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS
No. 298-93-1) and cells are incubated for 2 hours at 37±1°C in the presence of 5% CO2.
The MTT medium is then removed and cells are lysed by using an appropriate lysing
agent for a sufficient amount of time (e.g. 10 % (w/v) SDS and 0.4% (v/v) acetic acidsolution in DMSO for 5 min). After shaking, the absorption is then measured using theparameters described in the test method protocol (24).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated DATA AND REPORTING
other relevant
g other key events
Data evaluation
read-across from
27.
The following parameters are calculated in the LuSens test method (see Annex 4th these methodsof
thisAppendix
for the detailed equations):tion to the set of
ictions have been
 Fold luciferase activity induction at all concentrations of the tested chemical,
positive control and negative control.
sed on their own,
 Cellular viability (CV) at all concentrations of the tested chemical and for all
as defined by UN
controls to determine (by interpolation) the concentration value at which 75% ofes, nor to predictcell viability occurs (CV75).
n the regulatory
28.
For each concentration showing a luciferase activity induction equal or higher ()d on their own tothan 1.5, statistical significance is determined (e.g. using a two-tailed Student’s t-test) bycomparing the luminescence values of the three replicate samples with the luminescence©©OECD
OECD2018
2018

61

238││

442D
442D

OECD/OCDE

underwenta PerformanceStandard-based
validation
study
basedtheon luciferase
which it was
alsovalues in thesolvent/vehicle
control wells
to assesswhether
activity
reviewed
received positive
opinionby ESAC(7) (8) (9) (10).
induction andis statisticallysignificant(p <0.05).
Furthermore,
it should be checked that no
significant cytotoxic effects occur at these concentrations (i.e. that the cell viability is
4.
The test methods included in this Test Guideline may differ in relation to the
70% at theused
concentrations
to  1.5
procedure
to generateleading
the data
andfold
theluciferase
readouts induction).
measured but can be used

indiscriminately
to address thatcountries’
for test
results
on the29.
It is recommended
data arerequirements
visually checkedwiththe helpof keratinocytes
graphs. If noactivation
Key Eventcurveof theis AOP
for skin
while benefitingfrom theMutualclear
dose-response
observed,
or ifsensitisation
the dose-response
curve obtained
is biphasic
Acceptance
Data.
(i.e.
crossingofthethreshold of 1.5 twice), the experiment should be repeated to verify
whether this is specific to the test chemical or due to an experimental artefact. In case the
Backgroundandisprinciplesof themethods includedin thethelower
Key Event
based
biphasic
response
reproducible
in antest
independent
experiment,
concentration,i.e.,
the threshold of 1.5 is crossed the first time should be reported. However, aTestwhen
Guidelines
concentration delivering an EC1.5 is not a requirement.
5.
The assessment of skin sensitisation has typically involved the use of laboratory
animals.Finally,
The classicalthat test
use method
guinea-pigs,
thefold
GuineaPig Maximisation
Test
30.
when inmethods
the LuSensa  1.5
luciferase
activity induction(GPMT)of Magnusson
and Kligman
and the Buehler
Test406) (11),assessre-testingshouldbeisobserved
only at the lowest
tested concentration(e.g.
CV(OECD
75/2.07),TG
both the induction
and oneelicitation
phasesskin sensitisation. The murine tests, theconductedusing at least
additional
lowerofconcentration.
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA(OECD TG 442A)(13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
Acceptancecriteria
442B) (14), all assess the induction response exclusively, and have gained acceptance31.
The following acceptance criteria should be met when using the LuSens test
since they provide an advantage over the guinea pig tests in terms of animal welfare
method. If any of the criteria listed below is not met, the data should be discarded and atogether with an objective measurement of the induction phase of skin sensitisation.
new repetition should be performed.
6.
Mechanistically-based in chemico and in vitro test methods addressing the firstTheaverageluciferase
activity induction
obtained
the positive
control, to thethree key eventsof theskin sensitisation
AOP havebeen withadoptedfor contributing
120
µM
EGDMA
(or
comparable
concentration–seeparagraph
19)
should≥evaluation of the skin sensitisation hazard potential of chemicals: the OECD
TGbe
442C
2.5,theandDirect
the positive
shouldAssay
have a(15)relativecell viability≥ 70%
describes
Peptidecontrol
Reactivity
addressing
the first
key asevent; thecomparedtothesolvent/vehicle
control.
present Test Guideline assesses keratinocyte activation addressing the second key eventand the The
OECD
TG 442E
addresses
the induction
activationobtainedof dendritic
the thirdcontrol,
key eventaverageluciferase
activity
withcells,
the negative
i.e.,ofthe skin5000
sensitisation
AOP
(16).
Finally,thefourthkey
event
representing
T-cellμM DL-Lactic acid, as well as the basal expression of untreated cells should
proliferation
is indirectly
assessed toin thethe averagemurine Local
Lymph Node
Assay (LLNA) (12).
be < 1.5
fold as comparedsolvent/vehicle
control.
7.  TheAs keratinocyte
activation
representsofonly
one key eventreadingof the forskinthesensitisation
average coefficientof variation
the luminescence
AOP (2)solvent/vehicle(17), information
generatedwithtest
methods
developedtoaddress
this specificcontrols (e.g. DMSO) should be below 20% in each repetition.
key event may not be sufficient to conclude on the presence or absence of skin
 At least
three test
should haveviability withof at least
70%methods
relative
sensitisation
potential
ofconcentrations
chemicals. Therefore
datacell
generated
the testtothesolvent/vehicle
controls.
Moreover,incasearesultistobeconsidereddescribed in this Test Guideline are proposed to support the discrimination between skinnegative,at least
concentration
be cytotoxic,
i.e.used
havewithina cell viability
sensitisers(i.e. UN
GHSoneCategory
1) andshould
non-sensitisers
when
Integrated
<
70%,
or
the
maximum
concentration
of
2000
μM
(or
2000
µg/
mL
for relevant
Approaches to Testing and Assessment (IATA), together with othersubstanceswith no definedMW) should
tested.
complementary
information,e.g. derived
from inhave
vitrobeen
assaysaddressing other key eventsof the skinsensitisation
AOPchemicals
as well asmay
non-testing
including32.
In somecases, test
induce methods,
no cytotoxicity,
in read-acrosswhich casesfrom
thechemical analogues(17).tested
ExamplesuseμM
of datageneratedwithmethods
maximum
concentration
shouldonbethe
2000(or 2000µg/ mLfor thesetest chemicals
within Defined
standardised
both in relation
to the set i.e.
ofhavingundefinedApproaches,
MW). If ini.e.
the approaches
main luciferase
test no concentration
is cytotoxic,
information
sources
used
and
in
the
procedureappliedtoderivepredictions
have
been
has a cell viability < 70%, and no luciferase induction is observed, then a second
published should(17) andbecan
be employedusefulelements
within IATA.
repetition
performed
usingase.g.
a 1.44serial dilution
factor based on the CV75
(i.e.
starting
with
1.44
x
CV
)
insteadofthe
1.2
serialdilutionusedonintheir
the main
75
8.
The test methods described in this Test Guideline cannotfactor
be usedown,
luciferase
test.
If
in
the
secondrepetition
cytotoxicity
andluciferase
inductionarenotneither to sub-categorise skin sensitisers into subcategories 1A and 1B as definedstillby UN
observed,athirdrepetition
should
be
run
with
the
maximum
concentration
of
2000
μM
GHS (1), for authorities implementing these two optional subcategories, nor to predict(or2000 forµg/ safety
mL for assessment
substances withundefined
MW). depending
This repetition
then bepotency
decisions.
However,on should
the regulatory
confirmed
performing
a fourth
repetition.
framework,bypositive
results
generatedwith these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 2018

62

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the MutualInterpretation of results and prediction model
33.
A LuSens prediction is considered positive if the following conditions are met in
2 of 2 or in the same 2 of 3 repetitions, otherwise the LuSens prediction is considerednegative (Figure 1):

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

442D │ 39

the luciferase induction is above or equal to (≥) 1.5 fold and is statisticallysignificant compared to the solvent control in at least 2 consecutive non-cytotoxic
tested concentrations (i.e. cellular viability is equal or higher than (≥) 70%),
whereby at least three tested concentrations should be non-cytotoxic (cellularviability equal or higher than (≥) 70%).

34.
In addition, a negative result obtained with test chemicals that do not form astable dispersion and were not tested up to 2000 µM (or 2000 µg/mL for test chemicals
with no defined MW) and for which no cytotoxicity is observed in any of the tested
concentration (see paragraph 31) should also be considered as inconclusive (seeparagraph 4).
Figure 2. Overview of the criteria leading to a prediction in the LuSens test method.
A LuSens prediction should be considered in the framework of a Defined Approach or of an IATA and inaccordance with the provision paragraph 4 and paragraphs 7 and 8 of the general introduction.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

Induction ≥1.5 fold?
AND statistically significantly
higher than solvent control?
In at least 2 consecutive
concentrations for which cell
viability  70%?

No

At least one
concentration with cell
viability < 70% ?
OR no cytotoxicity
observed up to 2000 M(or 2000 g/mL if nodefined MW)

YESAre at least 3/6 tested
concentrations noncytotoxic?

No

No

Retest as specified byprotocol, e.g. withnew concentration
range

YES
Positive
Procedure for one repetition

Negative

YES
For a LuSens prediction perform
at least two independent
repetitions- If the two repetitions are positive,
final outcome is POSITIVE and nofurther testing is required.
- If the two repetitions are negative,
final outcome is: NEGATIVE andno further testing is required.
- If discordant results, a third
independent repetition needs to be
conducted to complete theexperiment.
- The sensitization potential of a test
substance is determined by the
results of the majority of repetitionsof an experiment.

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
2018

63

240││

442D
442D

OECD/OCDE

underwent
Test reporta Performance Standard-based validation study based on which it was also
reviewed and received positive opinion by ESAC (7) (8) (9) (10).
35.
The test report should include the following information:
4.
The test methods included in this Test Guideline may differ in relation to theprocedure
used to generate the data and the readouts measured but can be used
Test chemical
indiscriminately to address countries’ requirements for test results on the keratinocytes
 Mono-constituent
substance
activation
Key Event of the
AOP for skin sensitisation while benefiting from the Mutual
Acceptanceof Data. identification, such as IUPAC or CAS name(s), CAS number(s),o Chemical
SMILES or InChI code, structural formula, and/or other identifiers like batch/
Backgroundprinciplesof thelot andnumberand expiry
date;test methods included in the Key Event based

Test Guidelines

o Physical appearance, water solubility, DMSO solubility, molecular weight,
5.
Theandassessment
ofrelevant
skin sensitisation
has typicallyinvolved
use available;of laboratory
additional
physicochemical
properties,
to thetheextentanimals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Testo Statement on (in)solubility or stable dispersion in exposure media(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess
Purity, chemical
identity phasesof impurities
as appropriate
practicallyboth theo inductionand elicitationof skinsensitisation.andThemurine feasible,
tests, theetc; TG 429) (12) and its three non-radioactive modifications, LLNA: DA
LLNA (OECD(OECD oTGTreatment
442A) (13)as towellas LLNA:
BrdU-ELISA
and BrdU-FCM
prior
testing,
if applicable(e.g. warming,
grinding); (OECD TG
442B) (14), all assess the induction response exclusively, and have gained acceptanceo provide
Concentration(s)tested;over the guinea pig tests in terms of animal welfare
since theyan advantage
togetherowith
an objective
measurementof theinductionphase of skin sensitisation.
Storageconditions
and stability
to theextent available.

6.
Mechanistically-based in chemico and in vitro test methods addressing the first Multi-constituent substance, UVCB and mixture:
three key events of the skin sensitisation AOP have been adopted for contributing to theo ofCharacterisation
as far as hazard
possiblepotential
by e.g. chemical
identity
above),
evaluation
the skin sensitisationof chemicals:the(seeOECD
TG 442C
purity,quantitative
occurrenceAssayand relevant
physicochemical(seethedescribes the
DirectPeptide Reactivity(15) addressing
the firstproperties
key event;of theassesses
constituents,
to the extent
available;
present Testabove)
Guideline
keratinocyte
activation
addressing the second key eventand the oOECD
TG 442E
addresses
the solubility,
activation DMSO
of dendritic
cells, andthe additional
third key event of
Physicalappearance,water
solubility
the skin sensitisation
AOP
(16).
Finally,thefourthkey
event
representing
T-cellrelevant physicochemical properties, to the extent available;
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
o Statement of (in)solubility or stable dispersion in exposure media
7.
As keratinocyte activation represents only one key event of the skin sensitisation
Molecular
weightor apparent
weightin case toof address
mixtures/polymers
AOP (2)o (17),information
generatedwithmolecular
test methods
developed
this specificofknown
compositionsor
otherinformation
relevant
fortheconduct
key event may not be sufficient to conclude on the presence or absenceofoftheskinsensitisationstudy;
potential of chemicals. Therefore data generated with the test methods
described
inthisTest prior
Guideline
are proposed
to support
the discriminationo Treatment
to testing,
if applicable(e.g. warming,
grinding); between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integratedo Concentration(s)tested;Approaches
to Testing andAssessment (IATA), together with other relevant
complementary
information,
e.g.
from
in vitro
addressing other key eventso Storage conditions andderived
stability
to theextentassays
available.
of the skin sensitisation AOP as well as non-testing methods, including read-across fromchemicalControls analogues (17). Examples on the use of data generated with these methods
within Defined Approaches, i.e. approaches standardised both in relation to the set of Positive
control
information
sourcesused and in the procedure applied to derive predictions have been
published
(17)
and
can
be employed suchas usefulelements
withinIATA. CAS number(s),o Chemical identification,
as IUPAC
or CAS
name(s),or InChI
code, structural
formula,and/orotherbeidentifiers;
8.
TheSMILES
test methods
described
in this Test
Guideline
cannotused on their own,neither to
sub-categoriseskinsensitisers
into
subcategories
1A
and
1B
as definedby UNo Physical appearance, water solubility, DMSO solubility, molecularweight,GHS (1), forauthorities
implementing
these
two
optional
subcategories,
nor
topredict
and additional relevant physicochemical properties, to the extent available andpotency forwhere
safetyassessment decisions. However, depending on the regulatory
applicable;
framework, positive results generated with these methods may be used on their own toPurity, chemical
identityof impurities
as appropriate and practically feasible,
classify oa chemical
into UN GHS
category
1.
etc;© OECD 201864

OECD/OCDE

442D │ 41

o Treatment prior to testing, if applicable (e.g. warming, grinding);

which it was alsoo Concentration(s) tested;in relation to thebut can be used
the keratinocytes
from the Mutualo Storage conditions and stability to the extent available;o Reference to historical positive control results demonstrating suitable run
acceptance criteria, if applicable.


Solvent/vehicle control

ey Event basedo Chemical identification, such as IUPAC or CAS name(s), CAS number(s),and/or other identifiers;

use of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

o Purity, chemical identity of impurities as appropriate and practically feasible,etc;
o Physical appearance, molecular weight, and additional relevant
physicochemical properties in the case other solvents/vehicles than thosementioned in this Appendix are used and to the extent available;o Storage conditions and stability to the extent available;o Justification for choice of solvent/vehicle for each test chemical.
ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

Negative control
o Chemical identification, such as IUPAC or CAS name(s), CAS number(s),and/or other identifiers;
o Purity, chemical identity of impurities as appropriate and practically feasible,etc;
o Physical appearance, molecular weight, and additional relevant
physicochemical properties in the case other negative controls than thosementioned in this Appendix are used and to the extent available;o Storage conditions and stability to the extent available;o Justification for choice of the negative control in the case other negative
controls than those mentioned in the Test Guideline are used.

Test method conditions

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own toName and address of the sponsor, test facility and study director;

Description of test method used;

Cell line used, its storage conditions and source (e.g. the facility from which they
were obtained);

Passage number and level of confluence of cells used for testing;

Cell counting method used for seeding prior to testing and measures taken to
ensure homogeneous cell number distribution (cf. paragraph 13);

Luminometer used (e.g. model), including instrument settings, luciferase substrateused, and demonstration of appropriate luminescence measurements based on thecontrol test described in Annex 3 of this Appendix;©©OECD
OECD2018
2018

65

242││

442D
442D

OECD/OCDE

underwent
Performance
validation study
on which
it was also
 Theaprocedure
usedStandard-based
to demonstrate proficiencyof the basedlaboratory
in performing
reviewed
byproficiency
ESAC (7) (8)(9) (10). or to demonstrate
theandtestreceived
method positive(e.g. by opinion
testing of
substances)
reproducible
performance
of
the
test
method
over
time.
4.
The test methods included in this Test Guideline
may differ in relation to theprocedure used to generate the data and the readouts measured but can be used
Test
procedure to address countries’ requirements for test results on the keratinocytes
indiscriminately
activation
Key Eventof the AOP
skin sensitisation
while benefiting from the Mutual
 Numberof repetitions
and for
replicatesused;
Acceptance of Data.
 Test chemical concentrations, application procedure and exposure time used (ifdifferent
thanthe one recommended)
Backgroundandprinciplesof the test methods included in the Key Event based

Test Guidelines
Description of evaluation and decision criteria used;

5.  Description
The assessmentof skinsensitisation
hasused;typically involved the use of laboratoryof study
acceptancecriteria
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test Descriptionof anytheBuehler
test procedure.
(GPMT)of Magnusson
andmodifications
Kligman andofthe
Test (OECD TG 406) (11), assess
both the induction and elicitation phases of skin sensitisation. The murine tests, theResults
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA(OECD
TG 442A) of(13)well as induction
LLNA: BrdU-ELISA
and BrdU-FCM(OECDforTG Tabulation
foldasluciferase
activity and viability
values (i.e.CV75
442B) (14),allassesstheinduction
response
exclusively,
andhave
gainedacceptancethe LuSens test method) obtained for the test chemical and for the positive control
since they
providean advantage over the guinea pig tests in terms of animal welfare
for each
repetition;
together with an objective measurement of the induction phase of skin sensitisation.
 The mean values (i.e. arithmetic means of cell viability and luciferase activity
6.
Mechanistically-basedin chemicoand invitro
methods
addressing the firstinduction)and SD calculatedusing datafrom
all test
individual
repetitions;
three key events of the skin sensitisation AOP have been adopted for contributing to the An ofindication
the rating ofhazard
the testpotential
chemicalof
accordingto themodel;
evaluation
the skinofsensitisation
chemicals:
theprediction
OECD TG
442C
describes
theDirectPeptideReactivity
Assay
(15)
addressing
the
first
key
event;the Coefficient of variation obtained with the luminescence readings for thepresent solvent/vehicle
Test Guideline control
assessesforkeratinocyte
activation
addressing
thesecondkey
event
each experiment;
and the OECD TG 442E addresses the activation of dendritic cells, the third key event of
depictingdose-response
curvesinductionluciferase
activity andthe skinA graphsensitisation
AOP(16). Finally,
theforfourth
keyofevent
representing
T-cellviability;
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
7.  Description
As keratinocyte
activation
represents
only one if
key
event of the skin sensitisationof anyother relevant
observations,applicable.
AOP (2) (17), information generated with test methods developed to address this specific
key event may
be sufficient to conclude on the presence or absence of skin
Discussionof thenot
results
sensitisation potential of chemicals. Therefore data generated with the test methods
 Discussionof theresults obtainedwith tothesupport
LuSensthe
testdiscrimination
method;described
in this Test
Guideline
are proposed
between skinsensitisers(i.e. UN GHS
Category
1) andnon-sensitiserswhen ofusedwithin ifIntegrated
 Considerationof thetest method
results
within the context
an IATA,other
Approaches
toTesting
andAssessment(IATA),
togetherwithotherrelevant
relevant information is available.
complementary information, e.g. derived from in vitro assays addressing other key eventsof the skin sensitisation AOP as well as non-testing methods, including read-across from
Conclusion
chemical analogues (17). Examples on the use of data generated with these methods
within Defined Approaches, i.e. approaches standardised both in relation to the set of
information sources used and in the procedure applied to derive predictions have been
published (17) and can be employed as useful elements within IATA.
8.
The test methods described in this Test Guideline cannot be used on their own,neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
GHS (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
framework, positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 2018

66

OECD/OCDE

442D │ 43

which it was also
Literaturein relation to the 1)
but can be used
the keratinocytes
from the Mutual

ey Event based

OECD (2012). Series on Testing and Assessment No. 168. The Adverse Outcome Pathway for
Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence.
Organisation for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/PART1&docLanguage=En

2) Natsch A. (2010). The Nrf2-Keap1-ARE Toxicity Pathway as a Cellular Sensor for Skin
Sensitizers-Functional Relevance and Hypothesis on Innate Reactions to Skin Sensitizers.
Toxicological Sciences 113, 284-292.

use of laboratory 3)
Maximisation Test
406) (11), assess 4)
murine tests, theions, LLNA: DA
FCM (OECD TG 5)
ained acceptanceof animal welfare 6)
ensitisation.

ddressing the first
ontributing to theOECD TG 442C 7)
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12). 8)

Emter R., Ellis G., Natsch A.(2010). Performance of a novel keratinocyte-based reporter cell line
to screen skin sensitizers in vitro. Toxicology and Applied Pharmacology 245, 281-290.
Dinkova-Kostova A.T., Holtzclaw W.D., Kensler T.W. (2005). The role of Keap1 in cellular
protective responses. Chem. Res. Toxicol. 18, 1779-1791.
Kansanen E., Kuosmanen S.M., Leinonen H., Levonen A.L. (2013). The Keap1-Nrf2 pathway:
Mechanisms of activation and dysregulation in cancer. Redox Biol. 1, 45-49.
OECD (2015). Guidance Document No 213. Performance Standards for assessment of proposed
similar or modified in vitro skin sensitisation ARE-NrF2 luciferase test methods. Series on
Testing and Assessment. Available at: http://www.oecd.org/env/ehs/testing/series-testingassessment-publications-number.htm
Ramirez T., Mehling A., Kolle S.N., Wruck C.J., Teubner W., Eltze T., Aumann A., Urbisch D.,
van Ravenzwaay B., Landsiedel R. (2014). LuSens: a keratinocyte based ARE reporter geneassay for use in integrated testing strategies for skin sensitization hazard identification. Toxicol.
In Vitro 28, 1482-1497.
Ramirez T., Stein N., Aumann A., Remus T., Edwards A., Norman K.G., Ryan C., Bader J.E.,
Fehr M., Burleson F., Foertsch L., Wang X., Gerberick F., Beilstein P., Hoffmann S.,
Mehling A., van Ravenzwaay B., Landsiedel R. (2016). Intra- and inter-laboratory
reproducibility and accuracy of the LuSens assay: A reporter gene-cell line to detect keratinocyte
activation by skin sensitizers. Toxicol. In Vitro 32, 278-286.

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skin 9) EURL ECVAM (2018). The LuSens test method Validation Study Report. Accessible at:https://tsar.jrc.ec.europa.eu/test-method/tm2011-10
within Integrated
other relevant 10) ESAC (2016). ESAC opinion on the BASF-coordinated Performance Standards-based validationg other key eventsof the LuSens test method for skin sensitisation testing. Available at:read-across fromhttp://publications.jrc.ec.europa.eu/repository/bitstream/JRC103706/esac_opinion_2016th these methods
04_lusens_final.pdf.
tion to the set of
ictions have been 11) EURL-ECVAM (2014). Recommendation on the KeratinoSensTM assay for skin sensitisation
testing, 42 pp. Available at: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvamrecommendations/recommendation-keratinosens-skin-sensitisation.
sed on their own,
as defined by UN 12) Fabian E., Vogel D., Blatz V., Ramirez T., Kolle S., Eltze T., van Ravenzwaay B., Oesch F.,
Landsiedel R. (2013). Xenobiotic metabolizin enzyme activities in cells used for testing skines, nor to predict
sensitization in vitro. Arch Toxicol 87, 1683-1969.
n the regulatory
d on their own to 13) Jaworska J., Dancik Y., Kern P., Gerberick F., Natsch A. (2013). Bayesian integrated testing
strategy to assess skin sensitization potency: from theory to practice. J. Appl. Toxicol. 33,
1353-1364.
©©OECD
OECD2018
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442D
442D

OECD/OCDEa Performance
validation
basedS.,onDumontwhich itC.,wasalso A.T.,
14) underwent
Casati S., Aschberger
K., Standard-based
Asturiol D., Basketter
D.,study
Dimitrov
Karlberg
reviewed
andJ.P.,
received
positive
ESAC(7) (8)(10). Ability of non-animal methods
Lepoittevin
PatlewiczG., opinion
Robertsby
D.W.,
Worth
A.(9)
(2016).
for skin sensitisation to detect pre- and pro-haptens: Report and recommendations of an EURL
4.
The test methods included in this Test Guideline may differ in relation to the
ECVAM expert meeting. EUR 27752 EN. Available at : https://eurl-ecvam.jrc.ec.europa.eu/eurlprocedure used to generate the data and the readouts measured but can be used
ecvam-status-reports/pre-prohapten-workshop-report.
indiscriminately to address countries’ requirements for test results on the keratinocytes
Eventof the
AOP forT.,skinsensitisation
while
MutualW., van15) activation
Bauch C.,Key
Kolle
S.N.,
Ramirez
EltzeT., FabianE.,benefiting
Mehling fromA., theTeubner
Acceptance
Data.
RavenzwaayofB.,
Landsiedel R. (2012). Putting the parts together: combining in vitro methods totest for skin sensitizing potentials. Regul. Toxicol. Pharmacol. 63, 489-504.

Background and principles of the test methods included in the Key Event based16) Thorne N., Inglese J., Auld D.S. (2010). Illuminating Insights into Firefly Luciferase and Other
Test
GuidelinesReporters Used in Chemical Biology. Chemistry and Biology 17, 646-657.
Bioluminescentassessment
skin sensitisation
has typically involved
the uselaboratoryEstrogen17) 5.
OECD The(2012).
BG1Lucof
Estrogen
Receptor Transactivation
Test Method
for of
Identifying
animals.
classicalandmethods
that use guinea-pigs,
the Guinea
Maximisation
TestNo. 457.
ReceptorThe
Agonists
Antagonists.
OECD Guidelines
for Pig
ChemicalTesting(GPMT)
ofMagnusson
andKligmanandtheBuehler
Test
(OECD
TG
406)
(11),
assessOrganisation for Economic Cooperation and Development, Paris. Available at:both
the induction and elicitation phases of skin sensitisation. The murine tests, the[http://www.oecd.org/env/testguidelines].
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA
18) (OECDECETOC(2003).
Classificationaccordingto potency. European
Centre forTG442A)Contact(13) assensitization:
well as LLNA:
BrdU-ELISA
and BrdU-FCM(OECD TG
Ecotoxicology
Toxicology
of Chemicals(Technical
ReportNo.have87). gained acceptance
442B)(14), all&assess
the induction
response
exclusively,
andtheyD.,provide
advantage
overN.,theKolle
guineapig Mehlingtests in termsof animal
19) since
UrbischBeckeranM.,
HonarvarS.N.,
A., Teubner
W.,welfare
Wareing B.,
togetherwithanobjective
measurementoftheinduction
phase
of
skin
sensitisation.
Landsiedel R. (2016). Assessment of Pre- and Pro-haptens Using Non-animal Test Methods for
Skin Sensitization.
Chem. Res.inToxicol.
29,and
901-13.
6.
Mechanistically-based
chemicoin vitro test methods addressing the first
key eventsof thesensitisation
AOP have beenanalysis
adoptedoffor
contributing
the with20) three
Wasserman
W.W.,
FahlskinW.E.
(1997). Comprehensive
proteins
which tointeract
evaluation
of
the
skin
sensitisation
hazardpotentialofchemicals:
the
OECD
TG
442C
the antioxidant responsive element: correlation of ARE-BP-1 with the chemoprotective induction
describes
DirectPeptideReactivity
Assay (15) addressing the first key event; theresponse. theArch.
Biochem.
Biophys.
344, 387-396.
present Test Guideline assesses keratinocyte activation addressing the second key event21) andAdetheN,OECD
Leon F,
M, Peiffer
Kerdine-RomerS, cells,
Tissier
PA,ofFabre I,TGPallardy
442E addresses
the JL,
activationof dendritic
theMH,
thirdBonnet
key eventOurlinHMOX1
NQO1
genes theare upregulated
response
to contactT-cellsensitizers intheskinJC.(2009);
sensitisation
AOPand(16).
Finally,
fourth key inevent
representing
dendritic cells
and THP-1
cellin theline:role Localof theKeap1/Nrf2
pathway.
Sci.
proliferation
is indirectly
assessedmurineLymph
Node Assay(LLNA) Toxicol(12).
2009 Feb;107(2):451-60.
7.
As keratinocyte activation represents only one key event of the skin sensitisation
22) AOPRob J.
Jeroen generated
L. A. Pennings,
KirstenA. developed
Baken, Tessa
E. Pronk,
Boorsma,(2)Vandebriel,(17), information
with test
methods
to address
thisAndre
specific
Ralph
Gottschalk,
Henk
Van
Loveren;Keratinocyte
Gene
Expression
ProfilesDiscriminate
key event may not be sufficient to conclude on the presence or absence of skinSensitizing and
Irritating
Compounds,
Toxicological
Sciences,
Issue 1, 1
sensitisation
potentialof chemicals.
Therefore
data generated
withVolume
the test117,
methods
September
2010,
Pages
81–89,
https://doi.org/10.1093/toxsci/kfq182
described in this Test Guideline are proposed to support the discrimination between skin(i.e.derUNVeenGHSJ.W.,
Category
1) andnon-sensitisers
usedH.,within23) sensitisers
Emter R., vanAdamsonG., EzendamJ., vanwhen
LoverenNatschIntegrated
A. (2013). Gene
Approaches
toTesting
andAssessment(IATA),
togetherwithotherexpression changes induced by skin sensitizers in the KeratinoSens™ cellline:relevant
Discriminatingcomplementarye.g. derived
from Toxicol.
in vitro assays
addressing
other key events
Nrf2-dependentinformation,
and Nrf2-independent
events.
In Vitro
27, 2225-2232.
of the skin sensitisation AOP as well as non-testing methods, including read-across from24) chemical
DB-ALManalogues
Protocol(17).
No.Examples
184 (2017).
LuSensAvailable
at methods
https://ecvamon the useof dataAssay.
generatedwith thesedbalm.jrc.ec.europa.eu
within Defined Approaches, i.e. approaches standardised both in relation to the set ofsources
usedFoertschand in theprocedureapplied toJ.,derivepredictions
have P.
been
25) information
Natsch A., Ryan
C.A.,
L., Emter
R., Jaworska
Gerberick
G.F., Kern
(2013). A
published
(17)
and
can
be
employedasusefulelements
withinIATA.
dataset on 145 chemicals tested in alternative assays for skin sensitization undergoingprevalidation.
of Applied
Toxicology
33,Guideline
1337-1352.
8.
The testJournal
methods
described
in this Test
cannot be used on their own,neithertosub-categoriseskinsensitisers
into
subcategories
1A andN.,
1B Goebel
as definedUN26) Basketter D.A., Alépée N., Ashikaga T., Barroso J., Gilmour
C.,byHibatallah
J.,
GHS
(1),
for
authorities
implementing
these
two
optional
subcategories,
nor
topredict
Hoffmann S., Kern P., Martinozzi-Teissier S., Maxwell G., Reisinger K., Sakaguchi H., Schepky
potency
for safetyassessment
decisions.
However, ofdepending
the regulatory
A., Tailhardat
M., TemplierM. (2014).
Categorization
chemicals on
accordingto their relative
framework,
positive
results
generated
with
these
methods
may
be
used
on
their own tohuman skin sensitizing potency. Dermatitis 25, 11-21.
classify a chemical into UN GHS category 1.

© OECD 201868OECD/OCDE

442D │ 45

which it was also
APPENDIX IB - ANNEX 1: PROFICIENCY SUBSTANCES

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event based

In Vitro Skin Sensitisation: The ARE-Nrf2 Luciferase LuSens Test Method
Prior to routine use of a test method that adheres to this Appendix to Test Guideline
442D, laboratories should demonstrate technical proficiency by correctly obtaining the
expected prediction for the 10 Proficiency Substances recommended in Table 1 and byobtaining the raw values that fall within the respective reference range for at least eightout of the ten proficiency substances. These Proficiency Substances were selected torepresent the range of responses for skin sensitisation hazards. Other selection criteria
were commercial availability, availability of high quality in vivo reference, andavailability of high quality in vitro data from the LuSens test method.

use of laboratory
Maximisation Test
Table 1: Recommended substances for demonstrating technical proficiency with the LuSens
406) (11), assess
test method.
murine tests, theions, LLNA: DA
Proficiency Substances
CASRN
Physical
LLNA
Human
LuSensFCM (OECD TG
Form
Prediction
category (2)In Vitro
EC1.5 (µM)
CV75 (µM )(1)ained acceptancePrediction(3)
Reference
Referenceof animal welfare
Range
(4)
Range (4)ensitisation.

Salicylic acidddressing the first
ontributing to theOECD TG 442C
Glycerolst key event; thesecond key event
third key event Isopropanolofpresenting T-cellay (LLNA) (12).

Sulfanilamideskin sensitisation
dress this specificabsence of skin
Eugenol
the test methods
tion between skinwithin Integrated
Cinnamyl alcohol
other relevant
g other key events
read-across from
2th these methods
Mercaptobenzothiazoletion to the set of
4-Methylaminophenol
ictions have been
sulfateMethyldibromo
sed on their own,
glutaronitrile
as defined by UN
es, nor to predict
2,4-Dinitron the regulatory
chlorobenzene
d on their own to

69-72-7

Solid

Nonsensitiser

Cat. 6

Negative> 1000> 200056-81-5

LiquidNonsensitiser

Cat. 6

Negative> 1000> 200067-63-0LiquidNonsensitiser

Cat. 5

Negative> 1000> 200063-74-1

Solid

Nonsensitiser

Negative(Basketter et al.
1994)

Negative> 1000> 200097-53-0LiquidSensitiser(weak)

Cat. 3

Positive< 500< 1000104-54-1

Solid

Sensitiser(weak)

Cat. 3

Positive

< 170

> 420

149-30-4

Solid

Sensitiser(moderate)

Cat. 3

Positive< 800< 200055-55-0

Solid

Sensitiser(strong)Cat. 3

Positive

< 30

< 50

35691-65-7

SolidSensitiser(strong)Cat. 2

Positive

< 25

< 50

97-00-7

Solid

Sensitiser(extreme)

Cat. 1

Positive<5< 10©©OECD
OECD2018
201869

246││

442D
442D

OECD/OCDE

Notes:(1) TheainPerformancevivo hazard (andStandard-based
potency) predictions
are based on
LLNAbaseddata (25).
in vivoisunderwentvalidation
study
on Thewhich
it potency
was also
derivedusing
thecriteria
proposedby
ECETOC
(18).
reviewed and received positive opinion by ESAC (7) (8) (9) (10).

(2) According to Basketter and co-workers (26). Cat. 1 represents clear evidence of contact allergy, Cat. 2 afrequent cause
contact
allergy,included
Cat. 3 a commonof contact
allergy,Cat.differ
4 an infrequentcause toof the4.
The oftest
methods
in thiscause
Test
Guideline
may
in relation
contact
allergy,Cat. 5toa rarecause of contact
allergy,Cat. readouts
6 essentiallymeasured
absent evidence
contact
procedure
usedgeneratethe data
andandthe
but ofcan
be usedallergy.
indiscriminately
to address
countries’
requirements
for testin resultson the(3) An ARE-Nrf2 luciferase
test method
prediction
should be considered
the frameworkof akeratinocytes
Definedactivation
Eventandof intheAOP forskin
while benefitingfrom
the MutualApproach or Keyof an IATA
accordancewiththe sensitisation
provisions of paragraphs
7 and 8 of thegeneralintroduction.
Acceptance of Data.
(4) Based on the historical observed values (7) (8). Although the EC 1.5 is not part of the LuSens prediction
model, it can be calculated from the obtained data, and used to determine the ranges of LuSens response for
Backgroundand principles
ofvalues
the test
includedin the Key
Event based
the Proficiency Substances.
The EC 1.5
were methods
calculated according
to Appendix
IA (paragraph
26).

Test Guidelines

5.
The assessment of skin sensitisation has typically involved the use of laboratory
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess
both the induction and elicitation phases of skin sensitisation. The murine tests, the
LLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA: DA(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG
442B) (14), all assess the induction response exclusively, and have gained acceptancesince they provide an advantage over the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
6.
Mechanistically-based in chemico and in vitro test methods addressing the first
three key events of the skin sensitisation AOP have been adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
describes the Direct Peptide Reactivity Assay (15) addressing the first key event; thepresent Test Guideline assesses keratinocyte activation addressing the second key eventand the OECD TG 442E addresses the activation of dendritic cells, the third key event of
the skin sensitisation AOP (16). Finally, the fourth key event representing T-cellproliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).
7.
As keratinocyte activation represents only one key event of the skin sensitisation
AOP (2) (17), information generated with test methods developed to address this specific
key event may not be sufficient to conclude on the presence or absence of skinsensitisation potential of chemicals. Therefore data generated with the test methods
described in this Test Guideline are proposed to support the discrimination between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key eventsof the skin sensitisation AOP as well as non-testing methods, including read-across fromchemical analogues (17). Examples on the use of data generated with these methods
within Defined Approaches, i.e. approaches standardised both in relation to the set of
information sources used and in the procedure applied to derive predictions have been
published (17) and can be employed as useful elements within IATA.
8.
The test methods described in this Test Guideline cannot be used on their own,neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN
GHS (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
framework, positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 201870

OECD/OCDE

442D │ 47

which it was also
APPENDIX IB - ANNEX 2: Comparison of the main protocol steps of the LuSens

and the VRM KeratinoSensTM test methods

in relation to thebut can be used
the keratinocytes
from the Mutual

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to

VRM (KeratinoSensTM)

LuSens

Preparation of the keratinocyte cultures
Propagation

2 to 4 passages

1 to 3 passages

Cryopreserved storage

2 to 4 passages

3 passages

Cell passages before main
experiment

At least 2

At least 5

Maximal passage number
propagation from frozen
stocks25 passages

20 passages for cytotoxicity range findingtest
15 passages for main luciferase test

DMEM containing serum andGeneticin

DMEM containing serum,
penicillin/streptomycin and puromycin

Propagation medium
Cell confluence for testing

80-90%

Harvest of cells prior to
testing

1 day

Plate format used fortesting

96 well-plates

Cell number seeded fortesting

10 000 cells/well, except in the well that is used for measurement ofbackgroundNumber of replicates foreach test chemical
concentration (in each
repetition)

3 wells (on independent plates)
for luciferase measurement
1 well for cytotoxicity assessment

3 wells (in the same plate) for all tests
i.e. the cytotoxicity range finder test andthe main luciferase test (including 3 wells
for luciferase measurement, and 3 wells
for parallel cytotoxicity assessment)

Preparation of the test chemical and control substances
Preparation
Solvent

Same day of testing
DMSO, sterile water or media forthose test items not soluble in
DMSO

Stock concentration

DMSO or media for those test items not
soluble in DMSO
200 mM

Test items with no definedmolecular weight

Stock solution prepared to adefault concentrations (40 mg/mL
or 4% (w/v))

Stock solution prepared to a default
concentrations (200 mg /mL or 20% (w/v))

Final tested concentration
range in 96 well-plate

12 concentrations (2 fold dilution)
ranging from 0.98 to 2000 µM

Cytotoxicity range finder test:
12 concentrations (2 fold dilution) ranging
from 0.98 to 2000 µM
Main luciferase test:
6 concentrations (1.2 fold dilution) ranging
from CV75/2.074 to CV75x1.2 µM

Solvent control

1% DMSO(18 replicates per repetition)

1% DMSO(12 replicates per repetition forcyototoxicity range finder test, and24 replicates per repetition for main
luciferase test)

Negative control

See solvent control

5000 M DL-Lactic acid
(3 replicates per repetition for cyototoxicity
range finder test, and 6 replicates per
repetition for main luciferase test)

Positive control

Cinnamic aldehyde
Four concentrations (2 folddilution) ranging from 4 to 64 µM(3 replicates per repetition)

120 M EGDMA
or alternative concentration that induced
luciferase =2.5 folds, and for which cell
viability is  70%©©OECD
OECD2018
2018

71

248││

442D
442D

OECD/OCDE(2 replicates
cyototoxicity
underwent a Performance Standard-based validation study
based peronrepetition
which itforwas
also
range finder test, and 5 replicates per
reviewed and received positive opinion by ESAC (7) (8) (9) repetition(10). for main luciferase test)
MediumNot inapplicable
6 replicatesper repetition
for cyototoxicity
4. control
The test methods included
this Test Guideline
may differ
in relation
to therange finder test, and 12 replicates perprocedure used to generate the data and the readouts
measured
but
can
be
used
repetition for main luciferase test

indiscriminately to address countries’ requirements for test results on the keratinocytes
3 replicates per repetition
1 replicates per repetition
activation Key Event of the AOP for skin sensitisation while benefiting from the MutualApplication of the test chemical and control substances & endpoints assessed
Acceptance of Data.
Blank control (no cells)

At least two independent repetitions containing each three replicates (n='6),'
Number of repetitions forand in case of discordant results, a third repetition should be performed (n='9).'each test chemical
Background and principles
the testis methods
included
the
Event
based
concentration
Eachofrepetition
conducted ona differentin
day
withKey
freshly
preparedtest
chemicals
andindependently
harvested
cells
(but
eventually
havingthe same
Test Guidelines
passage number)

5.treatment
Themediumassessment of150
skinsensitisation
has typicallyuse of(i.e.
laboratory
Cellµl culture
DMEM containing
seruminvolved
but withoutthe
antibiotics
Geneticin,
penicillin/streptomycin
andpuromycin)
animals. The classical methods that use guinea-pigs, the Guinea Pig Maximisation Test
to which 50 µl of the 25 fold test chemical and control substances are added(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess

Exposuretime
48 hours
37±1°Cin the presenceof 5%
CO2 tests, theboth theinduction and elicitation phasesof atskinsensitisation.
ThemurinePlates are covered with a foil to avoid evaporation of volatile test chemicals
LLNA (OECD TG 429) (12) and its three
non-radioactive modifications,and cross-contamination
between wells LLNA: DA(OECD TG 442A) (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG

After exposure, the relevant lysis buffer for
Luminescence activity
After exposure, cells are washed
442B) (14), all assess thewith
induction
exclusively,
and havereadings
gained added
acceptancemeasurement
luminescence
to eachphosphateresponse
buffered saline,
well
for
5-10
min,
under
agitationin theandtherelevant
lysis
bufferforsince they provide an advantage over the guinea pig tests in terms of animal welfare
dark.
luminescence readings added to
together with an objective measurement
thephase of skin sensitisation.
each well for 20ofmin
at induction
room
Luminescence is measured for 2 seconds
temperature.
using a luminometer.
6.
Mechanistically-basedin chemico and in vitro test methods
addressing the first
Plates with the cell lysate are
Other conditions may apply depending on
three key events of the skinsensitisation
AOP havecontributing
placedin the luminometerfor been adopted
the forluminometer
used. to thereading whichhazard
is programmed
to: of chemicals: the OECD TG 442C
evaluation of the skin sensitisation
potentiali) add the luciferase substrate todescribes the Direct Peptide
Assay(15) addressing the first key event; theeach Reactivity
well, ii) wait for
1 second,
and iii) integrate
the luciferase
present Test Guideline assesses
keratinocyte
activation addressing the second key event
activity for 2 seconds.

and the OECD TG 442E addresses the activation of dendritic cells, the third key event of
After(16).
exposure,
5mg/mlthe
MTTfourth After
200 µL of MTT T-cellworking
AOP
Finally,
key exposure,
event representing
solution is added and cells are
solution (0.5mg/ml) are added and cellsooproliferation is indirectly assessedmurineLymph
Assay
incubatedin
4hthe
at 37±1
areNodeincubated
2h at(LLNA)
37±1 C in(12).
C inLocal
thethe

Cytotoxicity
the skin assessment
sensitisation

presence of 5% CO2

presence of 5% CO2

7.
As keratinocyte activation
represents
only one key
eventof theCells are thenlysed overnightCells
are lysed
for skin
5 minsensitisation(with 10% (w/v)(with
10% SDSwith
solution),
SDS and to
0.4%(v/v) acetic
in a
AOP (2) (17), information
generated
test agitatedmethods developed
address
this acidspecific
andabsorption
measured
at
600
DMSO
solution),andabsorption
key event may not be sufficient tonmconclude on the presenceor absence of skinmeasured at 570 and 690 nm
sensitisation potential of chemicals. Therefore data generated with the test methods
Endpoints evaluated
Imax: maximal average fold
Fold luciferase activity induction as an
described in this Test Guideline
are observed
proposedat todiscrimination
betweenskin
averageof each tested
concentrationinductionanysupport theconcentration
sensitisers (i.e. UN GHS Category
1) andtested
non-sensitisers
whenused aswithinIntegrated
Cellularviability
an averageof eachconcentration
1.5: interpolated
Approaches to TestingECand
Assessment(IATA), togethertested
withconcentration
other relevant
for which there is a 1.5 fold
CV75: interpolated concentration at which
complementary information,e.g.
derived
from
in
vitro
assaysaddressingotherkey events
induction of luciferase activity
75% cell viability occursof the skin sensitisation AOPICas50 well
non-testing methods, including read-across from/ IC30:as
interpolated
chemical analogues (17).concentration
Examples at
onwhichthe 50%use andof data generated with these methods
30% reduction of cell viability
within Defined Approaches, i.e.
approaches
standardised both in relation to the set ofoccursrespectively
information
sources used and in the procedure applied to derive predictions have been
Acceptancecriteria
published(17)
and can be >1.5
employed
as useful elements within
IATA.
Positive control luciferase
fold statistically significant= 2.5 fold induction with the positive
activity

induction in at least one of thecontrol (e.g. 120 M EGDMA) relative to
8.
The test methods describedin this Test Guidelinecannotbe used on their own,tested concentrations of thesolvent control at a non-cytotoxic
neither to sub-categorise skinsensitisers
into
subcategories
1A and 1Bi.e.,
as celldefinedby= UN
positive
control (4to 64µM
concentration,
viability70%
cinnamic aldehyde).
relative to solvent
GHS (1), for authorities implementing
these two optional subcategories,
norcontrol
to predict
EC1.5 valueof positive control
potency for safety assessment
decisions.
However, depending on the regulatory
should be between 2SDs offramework, positive results
generated
with
these
may be used on their own tohistorical mean (e.g. 2 to30 Mmethods
invalidation
dataset)classify a chemical into UN GHS category 1.
Average induction of 64 µM
cinnamic aldehyde should be© OECD 201872

OECD/OCDE

which it was also

in relation to thebut can be used
the keratinocytes
from the Mutual

between 2 and 8.
Negative control
luciferase activity

Not applicable< 1.5 fold induction with the negative
control (5000 µM DL-Lactic acid) relative
to solvent control

Coefficient of variation = 20%(18 replicates)

Coefficient of variation = 20%(of at least 21 replicates)

Not applicable

Mean basal expression of medium control(cells with medium only) should have <1.5 fold luciferase activity induction
relative to solvent control
At least three test concentrations (of 6 in
the main luciferase test) should be noncytotoxic (cell viability = 70%). In addition,in case of a negative result, at least one
tested concentration (of 6 in the main
luciferase test) should be cytotoxic (cellviability < 70%)

A prediction is considered
positive whenthe
following conditions are
met in 2 of 2 or in 2 of 3
repetitions, otherwise theprediction is considerednegative

1. Imax equal or higher than ()1.5 fold and statisticallysignificantly different to thesolvent control (two-tailed,unpaired Student’s T-test)
2.The cellular viability is higher
than (>) 70% at the lowest
concentration with induction of
luciferase activity equal or above
1.5 fold (i.e. at the EC1.5
determining concentration)
3.The EC1.5 value is less than (<)
1000 µM (or < 200 µg/mL for test
chemicals with no defined MW)
4. There is an apparent overall
dose-dependent increase inluciferase induction

1. A luciferase induction above or equal to() 1.5 fold as compared to the solvent
control is observed in at least
2 consecutive non-cytotoxic tested
concentrations (i.e. cellular viability isequal or higher than () 70%)
2. At least three tested concentrations
should be non-cytotoxic (cellular viability
equal or higher than () 70%).

Chemicals that do notform a stable dispersion

Negative result obtained with test
chemicals that do not form astable dispersion < 1000 µM (or <200 µg/mL for test chemicals withno defined MW), should beconsidered inconclusive

Negative result obtained with test
chemicals that do not form a stabledispersion and were not tested up to 2000
µM (or 2000 µg/mL for test chemicals withno defined MW) should be considered
inconclusive

Solvent control variability
Othersey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

442D │ 49

Prediction model

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to©©OECD
OECD2018
201873

250││

442D
442D

OECD/OCDE

underwent
PerformanceStandard-based
validationOF
study
based on which it was also
APPENDIX
IB -aANNEX
3: QUALITYCONTROL
LUMINESCENCE
reviewed and received positive opinion by ESAC (7) (8) (9) (10).
MEASUREMENTS
4.

The test methods included in this Test Guideline may differ in relation to theBasicexperimentensuring
in theprocedure
used to forgenerate
the optimaldata andluminescence
the readouts measurements
measured but canbe usedLuSenstest
methodindiscriminately to address countries’ requirements for test results on the keratinocytes

activation
Eventof the luminescence
AOP for skin measurements,
sensitisation while
benefitingfrom
MutualIn order toKey
ensureoptimalwhen
performing
thetheassay
forAcceptanceofData.
the first time, it is recommended to perform one or two runs of the LuSens test method
using increasing concentrations of EGDMA as a test substance and using the plate layout

Backgroundand principlesof the thesetest methods
includedin the Key
Event
basedbeas described below.
By performing
repetitions,the following
aspects
shouldTest
Guidelines
considered:
5.  luciferase
The assessmentof skinsensitisation
hasintypicallyinvolved theuse ofinduction
shouldbe increased
a dose-dependent
fashion(inlaboratory
wells
animals.A-C:1-6)
The classicalmethods thatguinea-pigs,
the Guinea
Pig Maximisation Test
after treatment
with use
increasing
concentrationsof EDGMA;(GPMT) of Magnusson and Kligman and the Buehler Test (OECD TG 406) (11), assess
dose-dependent
increase inluciferase
induction
should beobserved
wells theboth thenoinductionand elicitation
phasesof skinsensitisation.
Themurineintests,
D:1-6,
andA-D:
7(emptywells)
incomparison
toluminescencevaluesinwellsDALLNA (OECD TG 429) (12) and its three non-radioactive modifications, LLNA:
8-12; (13) as well as LLNA: BrdU-ELISA and BrdU-FCM (OECD TG(OECD A-D:
TG 442A)
442B)
(14),
all
assessthe induction
response
exclusively,
and havegained the average
percentage
Standard
Deviationof the variability
in atleast acceptancesince they
provide an advantage
over (F-G:
the guinea
testsbeinterms20%of andanimalwelfare21 solvent/vehicle
control wells
1-12) pigshouldbelow
shouldnot
togethershow
with any
an objective
measurementoftheinduction
phase
of
skin
sensitisation.
“gradient-like” pattern.

A

6.
Mechanistically-based in chemico and in vitro test methods addressing the first
Table
Plate setup offirsthavetraining
three key events of theskin1:sensitisation
AOP
beenexperiment
adopted for contributing to theevaluation of the skin sensitisation hazard potential of chemicals: the OECD TG 442C
Direct 3Peptide Reactivity
thethe
1describes the245Assay (15)
6 addressing
7
8 first9 key event;10
11
present Test Guideline assesses keratinocyte activation addressing the second key eventand the EGDMAOECD TGEGDMA
442E addresses
activationEGDMAof dendritic cells, the third key event of
EGDMA
EGDMA the EGDMA
theskin
sensitisation
AOP
(16).
Finally,thefourth key event representing T-cell
CV75/2.07
CV75/1.73
CV75/1.44
CV75/1.2
CV75
CV75x1.2
proliferation is indirectly assessed in the murine Local Lymph Node Assay (LLNA) (12).

B

EGDMA
EGDMA
EGDMAactivation
EGDMArepresents
EGDMA only
EGDMA
7.
As
keratinocyte
one key event of the skin sensitisation
CV75/2.07
CV75/1.73
CV75/1.44 generated
CV75/1.2 with
CV75
CV75x1.2 developed to address this specific
AOP (2)(17), information
test methods

C

EGDMA
EGDMApotential
EGDMA
EGDMA
EGDMA
EGDMA
sensitisationof chemicals.
Therefore
data generated with the test methods
CV75/2.07
CV75/1.73
CV75/1.44
CV75/1.2
CV75
CV75x1.2
described in this Test Guideline are proposed to support the discrimination between skin12

key event may not be sufficient to conclude on the presence or absence of skinGsensitisers (i.e. UN GHS Category 1) and non-sensitisers when used within Integrated
Approaches to Testing and Assessment (IATA), together with other relevant
complementary information, e.g. derived from in vitro assays addressing other key eventsof the skinsensitisation
as well asMedium
non-testing
methods,
read-acrossMediumMediumMediumAOP Medium
MediumMediumincluding
Medium Medium
Medium from
Medium Medium
chemical analogues (17). Examples on the use of data generated with these methods
within Defined
Approaches,i.e.
both DMSO
in relation
the setDMSOof DMSO
DMSO
DMSO
DMSO
DMSOapproaches
DMSO standardised
DMSO
DMSO
DMSO toDMSO
information sources used and in the procedure applied to derive predictions have been
published(17) and canbe employed
as usefulelements
within
IATA.
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO DMSO DMSO DMSO DMSO DMSO

H

Blank

D
E
F

8.
The test methods described in this Test Guideline cannot be used on their own,neither to sub-categoriseskinsensitisers
into subcategories 1A and 1B
as definedby UN
DL-Lactic acid
5000
M
EGDMA
120 M
GHS (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory
framework, positive results generated with these methods may be used on their own toclassify a chemical into UN GHS category 1.

© OECD 201874

OECD/OCDE

442D │ 51

which it was also
APPENDIX IB - ANNEX 4: CALCULATIONS USED IN THE LUSENS TESTMETHODin relation to thebut can be used
the keratinocytes
from the Mutual

1. The fold induction of luciferase activity (Imax) is calculated in the LuSens test methodby Equation 1, and the overall maximal fold induction (Imax) is calculated as the averageof the individual repetitions.
Equation 1:

ey Event baseduse of laboratory
Maximisation Test
406) (11), assess
murine tests, theions, LLNA: DA
FCM (OECD TG
ained acceptanceof animal welfare
ensitisation.

ddressing the first
ontributing to theOECD TG 442C
st key event; thesecond key event
third key event ofpresenting T-cellay (LLNA) (12).

skin sensitisation
dress this specificabsence of skin
the test methods
tion between skinwithin Integrated
other relevant
g other key events
read-across fromth these methods
tion to the set of
ictions have been

sed on their own,
as defined by UN
es, nor to predictn the regulatory
d on their own to

where

(𝐿𝐿

𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹𝐹 𝐹 𝐹 (𝐿𝐿𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠−𝐿𝐿𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 )𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 −𝐿𝐿𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 )Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and notreatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent
control
2. Viability in the LuSens test method is calculated by Equation 2:
Equation 2:
where

(𝑉𝑉

𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉𝑉 𝑉𝑉 (𝑉𝑉𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠−𝑉𝑉𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 )𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 −𝑉𝑉𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 )× 100

Vsample is the MTT-absorbance reading in the test chemical wellVblank is the MTT-absorbance reading in the blank well containing no cells and notreatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent
control
3. The concentration at which cell viability is reduced to 75% (CV75) is thencalculated in the LuSens test method by linear interpolation according to Equation 3, andthe overall the CV75 is calculated as the geometric mean of the individual replicates.
Equation 3:
where

75− 𝑉𝑉𝑏𝑏) + 𝐶𝐶𝑏𝑏
𝑉𝑉𝑏𝑏 − 𝑉𝑉𝑎𝑎

𝐶𝐶𝐶𝐶75 =  (𝐶𝐶𝑏𝑏 − 𝐶𝐶𝑎𝑎 ) × (Ca

is the tested concentration in µM with cell viability just above 75%

Cb

is the tested concentration in µM with cell viability just below 75%

Va

is the % viability obtained with Ca

Vb

is the % viability obtained with Cb©©OECD
OECD2018201875