新規試験法提案書

皮膚腐食性試験代替法

LabCyte EPI-MODEL24 SCT

令和 3 年12月

国立医薬品食品衛生研究所

新規試験法提案書
令和 3 年 12 月 7 日
No.

2021-02

皮膚腐食性試験代替法LabCyte EPI-MODEL24 SCTにに関す る提案
令和3年12月7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：本試験法において陽性の結果が得られた場合、被験物質を腐食性物質（国際連合化学品の分類および表示に関する世界調和システム（UN GHS）分類における区分 1）と判定することは可能である。UN GHS 細区分を考慮した評価においても、LabCyte EPI-MODEL24
SCT は、EpiSkin™および EpiDerm™ SCT と同様に評価できる試験法である。なお、本試
験法の利用にあたっては、適用範囲を十分に配慮した上で使用されるべきである。
この提案書は、皮膚腐食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 腐食性試験代替法 LabCyte EPI-MODEL24 SCT の使用を提案するものである。

西川秋佳
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
西 川 秋 佳 （国立医薬品食品衛生研究所 安全性生物試験研究センター/済生会宇都宮病院）：座長
板垣

宏 （ITACS コンサルティング）

中村るりこ （独立行政法人 製品評価技術基盤機構）
西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
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）
松 本 一 彦 （名古屋市立大学大学院）
任期：令和 2 年 4 月 1 日～令和 4 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
石 井 孝 司 （国立感染症研究所）
大久保貴之 （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）
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）
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）
北嶋聡 （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）

合 田 幸 広 （国立医薬品食品衛生研究所）
杉 山 圭 一 （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）
髙 橋 祐 次 （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）
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）

横 田 雅 彦 （独立行政法人 医薬品医療機器総合機構）
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
In Vitro Skin Corrosion: Reconstructed Human Epidermis (RhE) Test Method,
LabCyte EPI-MODEL24 SCT
At a meeting held on 7 December 2021 at the National Institute of Health Sciences (NIHS) in
Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: Although a positive result in an in vitro skin corrosion test using human skinmodels such as LabCyte EPI-MODEL24 SCT is generally considered sufficientfor predicting a test chemical to cause skin corrosion under the United Nations
Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS)

Category 1, this test as well as EpiSkin™ and EpiDerm™ SCT are consideredsufficient for predicting a test chemical to cause skin corrosion under the UN GHS
subcategories. Furthermore, thorough consideration must be given to the applicability
domain when using this test.
This statement was prepared following a review of the Organisation for Economic
Co-operation and Development (OECD) Test Guideline (TG) 431 In Vitro Skin Corrosion:
Reconstructed Human Epidermis (RhE) Test Method together with other materials preparedby the Skin Corrosion Testing JaCVAM Editorial Committee to acknowledge that the resultsof a review and study by the JaCVAM Regulatory Acceptance Board have confirmed theusefulness of this assay.
Based on the above, we propose the In Vitro Skin Corrosion: RhE Test Method, LabCyte
EPI-MODEL24 SCT as a useful means for assessing skin corrosion potential during safetyassessments by regulatory agencies.

Akiyoshi Nishikawa
Chairperson
JaCVAM Regulatory Acceptance Board

Yoko Hirabayashi
Chairperson
JaCVAM Steering Committee

December 7, 2021v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Mr. Akiyoshi Nishikawa (Center for Biological Safety and Research: CBSR, National
Institute of Health Sciences: NIHS / Saiseikai Utsunomiya Hospital) : Chairperson
Ms. Yoko Hirabayashi (CBSR, NIHS)
Mr. Hiroshi Itagaki (ITACS Consulting)
Mr. Kazuhiko Matsumoto (Nagoya City University)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Jihei Nishimura (Pharmaceuticals and Medical Devices Agency)
Term: From 1st April 2020 to 31st March 2022vi

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

Ms. Yoko Hirabayashi (CBSR, NIHS): Chairperson
Mr. Osamu Fueki (Pharmaceuticals and Medical Devices Agency)
Mr. Yukihiro Goda (NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, CBSR, NIHS)
Mr. Koji Ishii (National Institute of Infectious Diseases)
Mr. Yasunari Kanda (Division of Pharmacology, CBSR, NIHS)
Mr. Satoshi Kitajima (Division of Toxicology, CBSR, NIHS)
Ms. Kumiko Ogawa (Division of Pathology, CBSR, NIHS)
Mr. Takayuki Okubo (Ministry of Health, Labour and Welfare)
Mr. Keiichi Sugiyama (Division of Genetics and Mutagenesis, CBSR, NIHS)
Mr. Masahiro Takahata (Ministry of Health, Labour and Welfare)
Mr. Yuhji Taquahashi (Animal Management Section of the Division of Toxicology, CBSR, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Masahiko Yokota (Pharmaceuticals and Medical Devices Agency)
Mr. Takao Ashikaga (Division of Risk Assessment, CBSR, NIHS): Secretary
Mr. Hajime Kojima (Division of Risk Assessment, CBSR, NIHS): Secretaryvii

皮膚腐食性試験代替法LabCyte EPI-MODEL24 SCTにに関す る提案目次

評 価 会 議 報 告 書 -- ---------- ------- --------- ------- --------- ------- --------- ------- --------- ------- - 1
評 価 報 告 書 --- ------- --------- ------- --------- ------- --------- ------- --------- ------- --------- ------- 7
OECD GUIDELINE FOR TESTING OF CHEMICALS 431

IN VITRO SKIN CORROSION: RECONSTRUCTED HUMAN EPIDERMIS (RhE)TEST METHOD-------------------------------------------------------------------------------------31

評価会議報告書

ヒト表皮モデルを用いた皮膚腐食性試験代替法

JaCVAM 評価会議

令和 3 年（2021 年）12 月 7 日

1

JaCVAM 評価会議
西 川 秋 佳 （国立医薬品食品衛生研究所 安全性生物試験研究センター/済生会宇都宮病院）：座長
板垣

宏 （ITACS コンサルティング）

中村るりこ （独立行政法人 製品評価技術基盤機構）＊
西 村 次 平 （独立行政法人 医薬品医療機器総合機構）
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）
松 本 一 彦 （名古屋市立大学大学院）*：資料編纂委員会の委員でもある

2

任期：令和 2 年 4 月 1 日～令和 4 年 3 月 31 日

略語
EURL ECVAM：European Union Reference Laboratory for alternatives to animal testing(欧州代替法評価センター)
GHS：

Globally Harmonized System of Classification and Labelling of Chemicals (化学品の分類および表示に関する世界調和システム)

JaCVAM：

Japanese Center for the Validation of Alternative Methods(日本動物実験代替法評価センター)

MTT：

3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium BromideOECD：

Organisation for Economic Co-operation and Development (経済協力開発機構)

SCT：

Skin Corrosive Test (皮膚腐食性試験)

TG：

Test Guideline (試験法ガイドライン)

UN：

United Nations (国際連合)

3

JaCVAM 評価会議は、腐食性試験資料編纂委員会により作成された「ヒト表皮モデルを用いた皮膚腐食性試験代替法評価報告書」1)をもとに本試験法の科学的妥当性、社会的および行政的な受け入れについて検討した。
1. 試験法の定義
名称：ヒト表皮モデルを用いた皮膚腐食性試験代替法
代替する対象毒性試験：ウサギを用いる皮膚腐食性試験 2)
試験法の概略：
本試験法では、ウサギ皮膚の代わりに角質層を有する 3 次元再構築ヒト表皮モデル（以下、表皮モデル）を用い、被験物質が角質層下の表皮細胞層に対し細胞毒性を示す能力を評価する。表皮モデル表面に被験物質を一定時間適用した後洗浄し、表皮細胞の生存率を MTT の還元量から求め、皮膚腐食性を判定する。
試験法の科学的妥当性：
本試験法は、日本で開発された表皮モデルを用いた LabCyte EPI-MODEL24 SCT であり、JaCVAM によるバリデーション研究とそれに続く第三者評価により、実験動物を用いた皮膚腐食性試験の代替法として科学的に妥当であると報告されている 3,4)。EURL ECVAM でバリデートされた EpiSkin™および EpiDerm™ SCT と同様 5,6)、OECD TG431 にて承認されている 7)。JaCVAM 皮膚腐食性試験資料編纂委員会では、現在まで公開されている情報 8-11)
および先に評価された表皮モデルを用いた皮膚腐食性試験代替法と同様に、LabCyte EPIMODEL24 SCT の科学的妥当性について評価した。その結果、本試験法は、「腐食性物質が角質層の傷害または角質層への吸収を経て下層の表皮細胞に対し毒性を示す」という皮膚腐食性発現機序に基づき、細胞への毒性を指標にしたものであり、原理的にも妥当であると判断された。
2. 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、通常の培養技術を習熟した施設であれば実施できる試験法であり、OECD
TG431で承認された市販されている表皮モデルは他にもあるが、本モデルは国産であり、入手が容易であるとともに、コストも安い。また、生きた動物を用いないという点で、3Rs の精神に合致しており、社会的受け入れ性は高い。
行政上の利用性：
本試験法において陽性の結果が得られた場合、被験物質を腐食性物質（国連 GHS 分類における区分 1）12)と判定することは可能である。国連 GHS 細区分を考慮した評価においても、LabCyte EPI-MODEL24 SCT は、EpiSkin™および EpiDerm™ SCT と同様に評価できる

4

試験法である。なお、本試験法の利用にあたっては、適用範囲を十分に配慮した上で使用されるべきである。
参考文献
1) JaCVAM 腐食性試験資料編纂委員会：評価報告書 ヒト表皮モデルを用いた皮膚腐食性試験代替法（2021 年 12 月 7 日）
2) OECD, Guideline for the testing of chemicals. 404, Acute Dermal Irritation/Corrosion 2015.
3) JaCVAM Validation Management Team, Validation Study for in vitro skin corrosion test method
using reconstructed human epidermal tissue LabCyte EPI-MODEL24. 2019.
4) JaCVAM, Peer Review Panel Evaluation of the Performance-based Validation Study on the
LabCyte EPI-Model24 in vitro skin corrosion test method as a me-too test method according toOECD GD 219 and falling within the OECD TG 431, 2018.
5) ECVAM, Statement on the scientific validity of the Episkin test (An in vitro test for skincorrosivity) 1998.
6) ECVAM, Statement on the Application of the Epidermtm Human Skin Model for Skin Corrosivity
Testing 2000.
7) OECD, Guideline for the testing of chemicals. 431, In Vitro Skin Corrosion: Human skin model
test. 2016.
8) ICCVAM, NIH Publication No.02-4502 ICCVAM Evaluation of Episkin™, EpiDerm™ (EPI200), and the Rat Skin Transcutaneous Electrical Resistance (TER) Assay: In Vitro Test Methods
for Assessing Dermal Corrosivity Potential of Chemicals 2002.
9) OECD, Series on Testing and Assessment No. 190 Summary Document on the Statistical
Performance of Methods in OECD Test Guideline 431 for Sub-Categorisation, in Series on
Testing and Assessment. 2013.
10) OECD, Series on Testing & Assessment No. 203, Guidance Document on an Integrated
Approach on Testing and Assessment (IATA) for Skin Corrosion and Irritation. 2014.
11) OECD, Series on Testing & Assessment No. 219 Performance Standards for the Assessment of
Proposed Similar or Modified In Vitro Reconstructed Human Epidermis (RhE) Test Methods for
Skin Corrosion Testing as Described in TG 431. 2015.
12) GHS 関係省庁連絡会議仮訳, 化学品の分類および表示に関する世界調和システム（GHS）改訂 8 版. 化学工業日報社, 東京, 2020.

5評価報告書

ヒト表皮モデルを用いた皮膚腐食性試験代替法

皮膚腐食性試験資料編纂委員会

令和 3 年（2021 年）12 月 7 日

17

皮膚腐食性試験資料編纂委員会

髙橋

祐次 （委員長：国立医薬品食品衛生研究所 毒性部）

中村るりこ （独立行政法人 製品評価技術基盤機構
山城
小島

朋子 （株式会社資生堂

品質・安全性評価室／日本化学工業協会）

肇 （国立医薬品食品衛生研究所 安全性予測評価部）

28

化学物質管理センター）

List of Abbreviations（（略語一覧）
ANOVA：

Analysis of Variance Software（分散分析）

ESAC:

ECVAM Scientific Advisory Committee（ECVAM 科学諮問会議）

EURL ECVAM: European Union Reference Laboratory for alternatives to animal testing（欧州代替法評価センター）
GHS：

Globally Harmonized System of Classification and Labelling of Chemicals（化学品の分類および表示に関する世界調和システム）

ICCVAM：

Interagency Coordinating Committee on the Validation of Alternative Methods（米国の代替法に関する省庁間連絡会議）

JaCVAM：

Japanese Center for the Validation of Alternative Methods（日本動物実験代替法評価センター）

MTT：

3- (4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium BromideOECD：

Organisation for Economic Co-operation and Development（経済協力開発機構）

OD：

Optical densities（吸光度）PBS：

Phosphate buffered saline（リン酸緩衝液）

SCT：

Skin Corrosive Test（皮膚腐食性試験）

SDS：

Sodium dodecylsulphate

TER：

Transcutaneous electrical resistance assay（経皮電気抵抗性試験）

TG：

Test Guideline（試験法ガイドライン）

UN：

United Nations（国際連合）

4
9

要旨
ウサギを用いる皮膚腐食性試験の動物実験代替法（以下、代替法と記す）として、経済協力開発機構（OECD: Organisation for Economic Co-operation and Development）にて試験法ガイドライン（TG: Test Guideline）431 として承認されたヒト表皮モデルを用いる試験法の有用性を評価した。当該 TG は 2019 年の改定において、LabCyte EPI-MODEL24 が追加されたことから、他のヒト表皮モデルと信頼性と妥当性という視点において、比較、評価した結果、
TG431 に掲載されているモデルの中で、EpiSkin™、EpiDerm™と同様に、LabCyte EPIMODEL24 が腐食性の有無を評価できるモデルとして推奨できると考えられた。また、国際連合化学品の分類および表示に関する世界調和システム（UN GHS: United Nations Globally
Harmonized System of Classification and Labelling of Chemicals）分類の細区分を考慮した評価においても、LabCyte EPI-MODEL24 SCT（Skin Corrosive Test）は、EpiSkin™、EpiDerm™ SCT と同様に評価できる試験法である。当該モデルは、日本国内で製造されていることから、海外生産のモデルに比較してコスト、入手が容易である点で有用であると考えられた。

510

1. 試験法の科学的および規制面からの妥当性
皮膚腐食性試験は皮膚刺激性試験の一環として行われ、種々のガイドラインでは Draize
らにより提唱されたウサギを用いる方法が推奨されてきた 1)。この方法は被験物質の刺激性や腐食性を検出する試験として長く使用されてきたものの、判定を肉眼で行うため客観性に乏しく実験間や施設間での再現性が乏しい。更に動物に激しい痛みとストレスを与えることが社会的に問題となり、以前より動物を使用しない動物実験代替法（以下、代替法と記す）の開発が切望されていた。
この代替法として、経済協力開発機構（OECD: Organisation for Economic Co-operation andDevelopment）で試験法ガイドライン（TG: Test Guideline）431 には、皮膚腐食性試験として角質層を有し 3 次元的に再構築されたヒト表皮モデルを使用した評価方法が記載されている 2)。
この試験法は、腐食性物質が角質層の傷害または角質層に吸収された後拡散することにより、下層の細胞に到達して細胞毒性を示すという仮説に基づき、被験物質曝露後の細胞生存率を指標に皮膚腐食性を評価している。EpiSkin™や EpiDerm™等のヒト表皮モデルは欧米では既にバリデーション研究が実施され、欧州では化学物質の皮膚腐食性評価を目的として承認され、化学物質のリスク表示識別等に利用されていることや、昨今、国際連合 「化学品の分類および表示に関する世界調和システム（UN GHS: United Nations Globally Harmonized
System of Classification and Labelling of Chemicals）」 分類に従って評価されるケースが増えていることから、我が国においても腐食性試験の代替法として TG431 の 2016 年改訂版 3)について、JaCVAM 皮膚腐食性試験資料編纂委員会が信頼性と妥当性という視点から評価を行った。その結果、EpiSkin™、EpiDerm™、SkinEthics™および epiCS®が被験物質の皮膚腐食性を評価する試験法として推奨できるモデルであり、UN GHS 分類の細区分を考慮する場合
は EpiSkin™がもっとも有用であるとした評価報告書を作成した 4)。これはその後、JaCVAM
評価会議においても評価され、適用範囲を十分に配慮した上で使用されるべきであるとされた 4)。
さらに、UN GHS の最新の改訂版である 8 版（2019 年発行）5) においては、皮膚腐食性／刺激性の評価に対し、TG430、431 または 435 に従って実施された試験について一定の判定基準が設けられ、これらが腐食性区分 1（場合によっては細区分にも）に適用可能であることが示されたことから、代替法の行政利用の加速化が期待されているところである。
本評価書では、OECD TG431 の 2019 年改訂版 6) で新たに追加掲載されたヒト表皮モデル
LabCyte EPI-MODEL24 SCT（Skin Corrosive Test）についての腐食性試験法としての有用性を評価した。なお、本評価方法を用いる際の利便性を考慮して、既に評価済みである EpiSkin ™
および EpiDerm™ SCT を用いる腐食性試験法の情報についても併記した。

611

2. 試験プロトコル構成の妥当性
被験物質が角質層を通過して表皮細胞に曝露され、MTT〔3- (4,5-Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide〕の還元量から求めた細胞生存率の割合から皮膚腐食性を判定する。それらの概要を表１にまとめた。被験物質としては、液体、粘性物、半固体、固体、ロ
ウ様物、粘着性物質を対象にしている。実験操作上の違いは、前培養法および染色液の用量、組織からの抽出法等であり、基本的な曝露時間と判定基準については表 2 を参照されたい。
試験プロトコルとして、EpiSkin™、EpiDerm™ SCT および LabCyte EPI-MODEL24 SCT を例に説明する 6)。

1）EpiSkin™
12well プレートの各 well に培養液 2 mL を加えた後にヒト表皮モデルを置き、被験物質が液状の場合はピペッターで 50 µL 適用、粉末など固形の場合は 20mg を 100µL の NaCl 溶液（9 g/L）と合わせて適用する。被験物質を 3 分間、60 分間、および 240 分間曝露後、25 mLの PBS で被験物質を洗い流す。洗浄後、ペーパータオル等の上にて水分を切るか、表皮の破損に注意しながらピペッターを用い表面に残った PBS を吸い取った後、MTT 色素を含む培養液（0.3 mg/mL）を表皮モデルの下方に 2 mL 添加する。37℃、CO2 インキュベータ中に
180 分間培養した後、以下の手順にて抽出を行う。MTT 色素を含む培養液を除去後、パン
チ生検（biopsy punch）を用いて表皮モデルをくり抜き、酸性化イソプロパノール 0.5 mL 添加
したガラスチューブに浸漬し、一晩室温放置する。96 well プレートに抽出液を 200 µL ずつ
移し、マイクロプレートリーダーを用いて 545 nm-594 nm の領域での吸光度を測定する。酸性化イソプロパノールのみを加えた well をブランクとし、実測値とブランク値の差を求める。陰性対照である 240 分間曝露の NaCl 溶液（9 g/L）の吸光度を 100％とし、各検体の 3 分間、60 分間および 240 分間曝露時の吸光度を％として算出し細胞の生存率とする。
3 分間曝露したときの生存率が 35％未満を示す物質は“腐食性”と判定する。一方、240 分
間曝露したときに 35％以上の結果を示す物質は“非腐食性”と判定する。さらに、腐食性の区分として、3 分間処理したときの生存率が 35%未満の場合は区分１A とし、3 分間曝露したときの生存率が 35％以上で、かつ 60 分間曝露したときの生存率が 35％未満の結果を示
す物質、あるいは 60 分間曝露したときの生存率が 35％以上で、かつ 240 分間処理したときの生存率が 35％未満の結果を示す物質は、区分 1B/C とする。
2）EpiDerm™ SCT
6 well プレートの各 well に培養液 1 mL を加えた後にヒト表皮モデルを置き、被験物質が液状の場合はピペッターで 50 µL、粉末など固形の場合は 25 mg を 25 µL の水と合わせ、モデル上層に適用する 。被験物質を 3 分間および 60 分間曝露後、プレートから被験物質をデカンテーションにより除去した後、一定の弱流の PBS を用いて 20 回程度洗浄を行う。洗7
12

浄後、ペーパータオル等で水分を切り、破損に注意しながら新たな 24 well プレートに表皮モデルを移動する。MTT 色素を含む培養液（1.0 mg/mL）をヒト表皮モデルの下方に 0.3 mL添加する。37℃、CO2 インキュベータ中に 180 分間培養した後、MTT 色素を含む培養液を吸引し PBS を添加、吸引を 2 回程度繰り返すことで、余剰な色素を取り除く。インサートにイソプロパノールを 2 mL 添加し、一晩室温放置後、96 well プレートに抽出液を 200 µL
ずつ移し、マイクロプレートリーダーを用いて 540 nm あるいは 570 nm の領域での吸光度を測定する。イソプロパノールのみを加えた well をブランクとし、実測値とブランク値の差を求める。陰性対照である水の吸光度を 100％とし、各検体の 3 分間または 60 分間曝露
時の吸光度を％として算出し細胞の生存率とする。
3 分間曝露したときの生存率が 50％未満、あるいは 3 分間では生存率が 50％以上である
が 60 分間曝露したときに 15％未満の結果を示す物質を“腐食性”と判定する。一方、3 分間曝露したときに生存率が 50％以上で、かつ 60 分間曝露したときに 15％以上の物質は“非腐食性”と判定する。さらに、腐食性の区分として、3 分間曝露したときの生存率が 25%未満の場合は、区分１A、25%以上の場合は区分１B/C とする。

3）LabCyte EPI-MODEL24 SCT
24well プレートの各 well に培養液 0.5 mL を加えヒト表皮モデルを置き、被験物質が液状の場合はピペッターで 50 µL、粉末など固形の場合は 50 mg を 50 µL の水と合わせ、モデル上層に適用する 。被験物質を 3 分間および 60 分間曝露後、培養カップをピンセットで取り出し、タッピングすることで被験物質を除去した後、一定の強流の PBS を用いて 10 回程
度洗浄を行う。洗浄後、滅菌綿棒を用いて水分を取り除き、MTT 色素を含む培養液（0.5 mg/ml）
が 0.5 mL 入った新たなプレートにインサートを移動する。37℃、CO2 インキュベータ中に
180 分間培養した後、以下の手順にて抽出を行う。培養表皮をピンセットで取り出し、イソ
プロパノール 300 µL の入ったマイクロチューブに浸漬し、一晩冷暗所に放置後、96 well プレートに抽出液を 200 µL ずつ移す。マイクロプレートリーダーを用いて 570 nm および 650 nmの領域での吸光度を測定し、吸光度（570 nm）から吸光度（650 nm）を差し引いた値を測定値とする。イソプロパノールのみを加えた well をブランクとし、実測値とブランク値の差を求める。溶媒対照の水の吸光度を 100％とし各検体の 3 分間または 60 分間曝露時の吸光度を％として算出し細胞の生存率とする。
3 分間曝露したときの生存率が 50％未満、あるいは 3 分間では生存率が 50％以上である
が 60 分間曝露したときに 15％未満の結果を示す物質を“腐食性”と判定する。一方、3 分間処理したときに生存率が 50％以上、かつ 60 分間曝露したときに 15％以上の物質は“非腐食性”と判定する。さらに、腐食性の区分として、3 分間曝露したときの生存率が 15%未満の場合は、区分１A、15%以上の場合は区分１B/C とする。

8
13

14

曝露時間毎に 2 つ以上

液体および粘性物：50±3 µL(131.6µL/cm 2)

組織数

使用量と適用曝露時間毎に 2～3 つ

0.63 cm

2

EpiDerm™SCTを上に乗せる。

9

約 8 mm のフラットなディスク様の片ロウ様物：15 µL の水で湿らせた直径

液体：ナイロンメッシュを用いる場
合も用いない場合でも、50 µL (79.4µL/cm2)2
固体：20±2 mg (52.6 mg/cm )+ N
被験物質とナイロンメッシュとの親
塩化ナトリウム溶液(9 g/L）100 ±5 和性は予試験で確認する。
µL
半固体：50 µL (79.4 µL/cm2)
ロウ様/粘着性物質：ナイロンメッ
シュを用いて 50±2 mg (131.6
固体：25 mg (39.7 mg/cm2)＋25 µL の2
mg/cm )
水(必要であればそれ以上)

0.38cm2

EpiSkin™

モデル表面積テスト方法 要素

で示した数字は、ばらつきの許容範囲を示す。
±で

一部改 6)）

体および粘性物として処理する。

ロウ様物：容積式ピペットを用い、液固体：50±2 mg (166.7 mg/cm2)+50µLの水

液体および粘性物：50 µL(166.7µL/cm 2)

曝露時間毎に 2 つ以上

0.3cm2

LabCyte EPI-MODEL24 SCT

表１．皮膚腐食性試験のために確認された RhE 試験方法からなる主な試験方法（OECD TG431 補遺 2

15

曝露時間毎に曝露時間毎に

50 µL の水
曝露時間毎にPBS の一定の強流で 10 回以上のみ検討

露時のみで検討10

曝露時のみで検討

50 µL の氷酢酸で 240 分間曝露時 8 N 水酸化カリウム 50 µL で 60 分間曝 8N 水酸化カリウム 50 µL で 60 分間

50 µL の水

50 µL の NaCl 溶液（9g/L)

陰性対照

陽性対照

PBS を一定した弱流で 20 回

PBS 25 mL(すすぎ一回毎に 2 mL)

室温（18-28℃）で 3 分間、60 分間 室温で 3 分間、および 37℃、CO2 濃度 室温で 3 分間、および 37℃、CO2 濃度(±5 分間)および 240 分間（±10 分
5％、湿度 95％で 60 分間
5％、湿度 95％で 60 分間間）

えない対照をとる。

PBS によるすすぎ

曝露時間と温度

着色障害の事前確認

10 µL(液体)もしくは 10 mg(固体)に 50 µL(液体)もしくは 25 mg(固体)に 300 50 µL(液体)もしくは 50 mg(固体)に90 µL の水を加えて室温で 15 分間 µL の水を加えて 37℃、CO2 濃度 5％、 500 µL の水を加えて 37°C、CO2 濃度
攪拌する。
湿度 95％で 60 分間
5%、湿度 95% で 60 分間攪拌する→溶液が着色した場合、MTT のみ 攪拌する。
→溶液が着色した場合、MTT のみを加を加えない対照をとる。
→溶液が着色した場合、MTT のみを加 えない対照をとる。

EpiSkin™
EpiDerm™SCT
LabCyte EPI-MODEL24 SCT
50µL(液体)もしくは
25mg(固体)に
150µL（液体）もしくは 50 mg（固体）+50µL(液体)もしくは
20mg(固体)に
直接 MTT 還元性の事前
0.3 mg/mL MTT 溶液 2 mL を加え mg/mL MTT 溶液 1 mL を加えて、 0.5 mg/mL の MTT 溶液 500µL を加え
確認て、37℃、CO2 濃度 5％、湿度 95％ 37℃、CO2 濃度 5％、湿度 95％で 60 て 37°C、CO2 濃度 5%、湿度 95%でで 180±5 分間培養
分間培養
60 分間培養→溶液の色が青/紫に変わった場合、→溶液の色が青/紫に変わった場 →溶液の色が青/紫に変わった場合、凍凍結処理でモデル構成細胞を死滅さ
合、水処理でモデル構成細胞を死 結処理でモデル構成細胞を死滅させせたものに被験物質を処置する対照
滅させたものに被験物質を処置 たものに被験物質を処置する対照も もとる。
する対照もとる。
とる。

テスト方法 要素16
570nm (もしくは 540nm)
1％Triton X-100 で処理
4.08 時間≦ET50≦8.7 時間

570nm (545-595nm)

SDS で 18 時間処理

1.0 mg/mL≦IC50≦3.0 mg/mL

組織の品質確認

標準フィルターなしで標準フィルターなしでOD 測定条件

120 分間11

温で振とうした状態で（約 120rpm）

遮光し、室温で一晩抽出時間および温度
室温で振とうせずに一晩、もしくは室（分離組織を完全に浸漬）(インサート全体から抽出）(分離した組織を十分に浸漬）

300 µL のイソプロパノール

2 mL のイソプロパノール

500 µL の酸性化イソプロパノー
ル(0.04 N 塩酸を含むイソプロパノ
ール)

抽出溶媒

および温度

37℃、CO2 濃度 5％、湿度 95％で 180
分間

37℃、CO2 濃度 5％、湿度 95％で180 分間(±15 分間)

MTT 溶液での培養時間

1.4 mg/mL ≤ IC50 ≤ 4.0 mg/mL

SDS で 18 時間処理：

650 nm の標準フィルターにより 570 nm

遮光し、冷暗所で一晩分間（±15 分間）

37°C、CO2 濃度 5%、湿度 95% で 180

500 µL (0.5 mg/mL)

300 µL (1 mg/mL)

2 mL (0.3 mg/mL)

LabCyte EPI-MODEL24 SCT

MTT 溶液EpiDerm™SCT

EpiSkin™

テスト方法 要素

17

は2回た時は 2 回

30％を超えないこと。

の複製組織間の生存率の差は

1. 陰性対照(塩化ナトリウム溶液)で処理された複製組織の OD 値の平均は、いずれの曝露時間についても 0.6 以上 1.5 以下であること。
2. 陽性対照(氷酢酸)に 240 分間曝露された複製組織の生存率の平均値を、陰性対照に対する割合（％）で表した場合、20％以下であること。

1. 陰性対照(水)で処理された複製組
織の OD 値の平均は、全ての曝露時間において 0.8 以上 2.8 以下であること。
2. 陽性対照（8N 水酸化カリウム）に
60 分間曝露された複製組織の生存率の平均値陰性対照に対する割合（％）で表した場合、15％以下であること。
3. 生存率が 20-100％の場合におい3. 生存率の幅が 20~100％の範囲、 て、複製組織間の変動係数(CV）は30％を超えないこと。
かつ OD が 0.3 以上の場合、2 つ

1 回、明確な結果が得られなかった時

1 回、明確な結果が得られなかっ試験回数

適合判定基準EpiDerm™SCT

EpiSkin™

テスト方法 要素1. 陰性対照（水）で処理した複製組織の OD 値の平均は、いずれの曝露時間についても 0.7 以上 2.5 以下であること。
2. 陽性対照（8N 水酸化カリウム）に
60 分間曝露された複製組織の生存率の平均値陰性対照に対する割合（％）で表した場合、15％以下であること。
3. 生存率が 20～100％の範囲、かつOD が 0.3 以上の場合、2 つの複製
組織間の生存率の差は 30％を超え
ないこと。

は2回

1 回、明確な結果が得られなかった時

LabCyte EPI-MODEL24 SCT

表 2-1. EpiSkin™ の予測モデル

3 分間、60 分間および 240 分間曝露後の生存率

予測性の評価腐食性・UN GHS 細区分

3 分間曝露後の生存率が 35％未満

1A＊

3 分間曝露後の生存率が 35％以上で、かつ 60 分間曝露後の生存率が 35％未満の場合、もしくは 60 分間曝露後の生存率

腐食性・UN GHS 細区分 1B あるいは 1Cが 35％以上で、かつ 240 分間曝露後の生存率が 35％未満
240 分間曝露後の生存率が 35％以上

非腐食性*) 腐食性の細区分における RhE 試験法の有用性を評価するために作成したデータによると、EpiSkinTM 試験法により区分 1Aに分類された物質／混合物の約 22%が、実際には区分 1B または 1C に属するものである可能性がある（すなわち、過大評価）。

表 2-2. EpiDerm™SCT の予測モデル

3 分間および 60 分間曝露後の生存率予測性の評価段階 1
3 分間曝露後の生存率が 50％未満

腐食性

3 分間曝露後の生存率が 50％以上で、かつ 60 分間曝露後の生存率が 15％未満

腐食性

3 分間曝露後の生存率が 50％以上で、かつ 60 分間曝露後の生存率が 15％以上

非腐食性

段階 2

段階 1 で腐食性と判断された物質／混合物

3 分間曝露後の生存率が 25％未満
3 分間曝露後の生存率が 25％以上

UN GHS 細区分

1A＊

UN GHS 細区分 1B あるいは 1C*) 腐食性の細区分における RhE 試験法の有用性を評価するために作成したデータによると、EpiDerm™SCT により区分 1Aに分類された物質／混合物の約 29％実際には区分 1B または 1C に属するものである可能性がある（すなわち、過大評
価）。

18

13

表 2-3. LabCyte EPI-MODEL24 SCT 予測モデル
3 分間および 60 分間曝露後の生存率予測性の評価段階 1
3 分間曝露後の生存率が 50％未満

腐食性

3 分間曝露後の生存率が 50％以上で、腐食性

かつ 60 分間曝露後の生存率が 15％未満
3 分間曝露後の生存率が 50％以上で、非腐食性

かつ 60 分間曝露後の生存率が 15％以上
段階 2

段階 1 で腐食性と判断された物質／混合物

3 分間曝露後の生存率が 15％未満
3 分間曝露後の生存率が 15％以上

UN GHS 細区分

1A＊

UN GHS 細区分 1B あるいは 1C*）細区分を裏付けるため、RhE 試験法の有用性評価の観点から生成されたデータによると、LabCyte EPI-MODEL24 SCT 試
験法での細区分 1A の結果 30%は、実際には細区分 1B または細区分 1C の物質／混合物（すなわち、過大分類）からなる可能性があると示された。

3．
．開発および評価に使われた物質の分類、選択理由の妥当性、in vitroおおよび参照データの有無
LabCyte EPI-MODEL24 SCT の再現性は、TG431 性能標準に定められた 30 被験物質により調べられている 7)。EpiSkin™との再現性は、60 の被験物質により調べられている 8)。EpiDerm™ SCT の再現性は、24 の被験物質により調べられている 9)。さらに、EpiSkin™と EpiDerm™ SCT の予測性
が 80 被験物質で調べられていて 10)、LabCyte EPI-MODEL24 SCT においては 79 被験物質で調べられている 11)。評価に使用された被験物質の多くは欧州代替法評価センター（EURL ECVAM：
European Union Reference Laboratory for alternatives to animal testing）主導の皮膚腐食性試験バリデー
ション研究で使用された物質である。

．試験法の正確性（再現性）
4．
LabCyte EPI-MODEL24 SCT においては、TG431 の性能標準に定められた 30 の被験物質を用いた 3 施設での 3 回の実験において、3 施設とも一致しなかった物質や偽陰性物質はなく、施設内再現性は 90％以上（表 3）、および施設間再現性 83.3％（表 4）と性能標準に定められた基準内（施設内一致率：80％、施設間一致率：70％）であった。
EpiSkin™においては、60 物質を用いた 3 施設での 2-way の ANOVA 解析を用い、施設内および施設間の変動については、それらの間に有意な差はないと判断された 8)。60 物質（27 腐食性物質
および 33 非腐食性物質）のうち、42 物質は 3 施設とも施設内および施設間再現性が良好であった。残る 18 物質ではいずれかの結果が異なっていたが、EURL ECVAM は本試験法の信頼性と再現性は高いと判断した 12) 。この結論は、 ECVAM 科学諮問会議（ESAC: ECVAM Scientific
Advisory Committee ） お よ び 米 国 の 代 替 法 に 関 す る 省 庁 間 連 絡 会 議 （ ICCVAM ： Interagency
14

19

Coordinating Committee on the Validation of Alternative Methods）13) での評価においても確認された。
EpiDerm™ SCT においては、24 物質を用いた 3 施設での 2 回の実験において、21 物質の腐食性を 3 施設すべてで正しく予測できた 9)。EURL ECVAM は本試験法の信頼性と再現性は高いと判断した。この結論は、ESAC の評価後 14)、ICCVAM においても確認された 13)。

表 3. LabCyte EPI-MODEL24 SCT の施設内再現性結果

2015

表 4. LabCyte EPI-MODEL24 SCT の施設間再現性結果

施設間再現性 83.3％（25/30）

1621

5. 試験法の信頼性 7, 10, 15)
LabCyte EPI-MODEL24 SCT の 3 施設における細区分の予測性を表 5 にまとめた。3 施設の予測性はほぼ同等であったが、1-B および 1-C を 1-A とする場合は多く、正確に評価できた値は表 5に示す LabCyte EPI-MODEL24 requirement の基準である 55%を 43.3％と下回った。一致しなかった物質は（表 3 または表 4 の No.9、12、16～20）であった。非腐食性物質を 1-A とする結果（表 3または表 4 の No.9）から、5%の上記基準を上回り、平均 6.7％であった。
なお、LabCyte EPI-MODEL24 SCT を含む既存のモデルとの比較として、細区分の予測性を表 6にまとめた。79 物質を 2、3 回実験して得られた値を見る限り、いずれの値も基準を満たしており、モデル間の予測性にほとんど差はなかった。
これらモデルは MTT 還元物質への対応方法も TG4316) に記載されている。適用できない物質
としてはガス、エアロゾールのみが記載されている（バリデーション未実施）。これらモデルは物
理状態（液体・固体等）および水溶性の有無にかかわらず適用可能であり、ガス、エアロゾールを除く混合物でも適用可能とされている。なお、特定の種類の物質や混合物においてこれらモデルの適用性を否定するような明確な根拠が得られた場合には、適用範囲から除外するべきであるとされている。

表 5. OECD で集計した全セットの化学物質を用いた予測性の計算結果（細区分：UN GHS 区分 1A、、1B/1C、、 非 腐 食性 ）Specificity (C vs NC)

2217

表 6. OECD で集計した全セットの 79 化学物質を用いた予測性の計算結果（細区分：UN GHS 区分 1A、、1B/1C、、非腐食性）

EpiSkin™

EpiDerm™

LabCyte EPI-

Criteria

Sensitivity (for predictions C

98.5%

100%

100%

≥ 95%

Correctly classified 1A

83.3%

83.3%

86.1%

≥ 80%

1A underclassified NC

0%

0%

0%

0%

1B-and-1C overclassified 1A

21.5%

29.0%

vs. NC)

1A underclassified 1B-and-1C
Correctly classified 1B-and-1C

16.7%

76.3%

SCT

16.7%

≥ 55%

0%

0%

≤ 5%

NC overclassified 1A

0%

2.7%

Accuracy (C vs. NC)

89.6%

87.9%

NC overclassified 1B-and-1C
Accuracy (1A vs. 1B-and-1Cvs. NC)

20.7%

78.8%

≤ 20%

70.0%

2.2%

79.3%

13.9%

requirements

71.0%

1B-and-1C underclassified NC
Specificity (C vs NC)

MODEL24 SCT

30.0%

73.9%

78.4%

23.4%

18.9%

74.2%

2.7%

89.9%

76.4%

≤ 45%
≥ 70%
≤ 5%

≤ 30%

≥ 87%

≥ 72%

6. 他の科学的な報告との比較の有無
OECD の腐食性試験代替法ガイドラインとして、TG431 の他に 「TG430 TER（Transcutaneous
Electrical Resistance）Test Methods: 経皮電気抵抗試験」16) および 「TG435 In Vitro Membrane Barrier
Test Method for Skin Corrosion: in vitro 膜バリア試験」17) が承認されている。これらはいずれも EURLECVAM にてバリデーション研究が実施され、ICCVAM はこれらの試験法（Rat Skin TER, EpiSkin™、
EpiDerm™ SCT および Corrositex®）の正確度、感度および特異度について比較している（表 7）10, 13)。
これらの比較においては同じ物質を用いて評価されておらず、試験物質の数量や選択物質の種類が異なっているため、結果の数値だけをもって単純にヒト表皮モデルの優越性を比較評価する
ことは困難であるが、いずれの試験法も同等の予測性を有すると思われる。 LabCyte EPIMODEL24 SCT は EpiSkin™並びに EpiDerm™ SCT と比較されており、ヒト皮膚三次元モデルとしては十分な予測性を有していると考えられる。

18

23

表 7．
．試験法の比較結果 18)
TER

EpiSkin™

EpiDerm™
SCT

LabCyte EPI-

Corrositex

物質数

122

79*

79*

MODEL24 SCT

79

163

正確度

81%

89.6%

87.9%

89.9%

79%

感度

94%

98.5%

100%

100%

85%

特異度

71%

79.3%

73.9%

78.4%

72%

*:OECD EWG によって選択された 80 化学物質から商業的に入手不可の 1 化合物を除外
7. 3Rs 原則との関係（動物福祉面からの妥当性）
LabCyte EPI-MODEL24 SCT は他の表皮モデルと同様に動物を使用しておらず、動物福祉面から代替法として妥当である。

8. 試験法の有用性と限界（コスト、時間からの妥当性など）
化学物質の皮膚腐食性は、その物理化学的性質に強く依存したものであり、強い酸性（pH 2.0 以下）またはアルカリ性（pH 11.5 以上）物質は、強い局所作用を有する可能性が高いことから、皮膚腐食性と判断しても良いことになっている 19)。しかしながら、これは腐食性についての情報が他にない場合に行われるワーストケースとしての判断であり、例えば酸や塩基の添加により pH が変わり易い物質や混合物の場合は偽陽性の判断となる可能性も考えられる 20)。このため、このよ
うな物質に対して動物を用いる必要のない TG431 で皮膚腐食性を評価することは有用である。
各試験実施施設で本モデルを導入する際には、正しく評価できることを予め確認する必要がある。TG431 には、習熟度確認物質を正しく分類できるか否か試験することにより、専門技術の習熟について確認することができるとの記載がある（表 8）。
LabCyte EPI-MODEL24 は、日本国内で製造されていることから海外で開発された製品に比較して入手が容易であり、試験計画への柔軟な対応が可能になると考えられる。例えば、EpiDerm™
が 2 回／月の発送であるのに対し LabCyte EPI-MODEL24 は 1 回／週、モデルの入手コストは
EpiDerm™の約半分である。

24

19

表 8．
．習熟度確認物質

化学物質 1

OECD Test Guideline No 431 Table 16) 一部改

CASRN

化学物質
分類

UN GHSVRM(in vivo 試験) (in vitro 試験) 物理的
による
による
状態
区分
区分

区分 1A の in vivo 腐食性物質
79-08-3

有機酸

1A

1A

固体

13319-75-0

無機酸

1A

1A

液体

フェノール

108-95-2フェノール類

1A

1A

固体

ジクロロアセチ
ルクロリド

79-36-7

求電子剤

1A

1A

液体

ブロモ酢酸
3 フッ化ボロン
二水和物

区分 1B/1C の in vivo 腐食性物質
グリオキシル酸一水和物

563-96-2

有機酸

1B/1C

1B/1C

固体

乳酸

598-82-3

有機酸

1B/1C

1B/1C

液体

エタノールアミン

141-43-5

有機塩基

1B

1B/1C

粘稠性

塩酸（14.4%）

7647-01-0

無機酸

1B/1C

1B/1C

液体

in vivo 非腐食性物質
臭化フェネチル
4-アミノ-1,2,4トリアゾール
4-（メチルチオ）ベンズアルデヒド
ラウリン酸

103-63-9

求電子剤

NC

NC

液体

584-13-4

有機塩基

NC

NC

固体

3446-89-7

求電子剤

NC

NC

液体

143-07-7

有機酸

NC

NC

固体

＜略語＞
CASRN：CAS 登録番号UN GHS：国際連合「化学品の分類および表示に関する世界調和システム」
VRM：バリデーション済み標準試験法
NC：非腐食性

2025

9. その他
前回の皮膚腐食性試験代替法の評価後 4)、現在までの間に、UN GHS 専門家小委員会において、
動物実験代替法の導入に関する議論が進み、UN GHS の最新の改訂版である 8 版（2019 年発行）5)では、第 3.2 章 皮膚腐食性／刺激性に 「3.2.2.3 in vitro（試験管内）/ex vivo（生体外）のデータに基づく分類」 という項目が新たに追記された（Annex 1 参照）。
UN 文書においては商標登録された情報を記述することができないため、具体的なヒト表皮モ
デル名はないものの、それらの区分／細区分への判定基準が明記された。
これらが掲載された背景には JaCVAM をはじめ、EURL ECVAM や ICCVAM といった組織による動物実験代替法の検証結果が貢献するところが大きいと考えられる。

10. 結論
LabCyte EPI-MODEL24 を信頼性と妥当性という視点において評価した結果、他のヒト表皮モデ
ルを用いた皮膚腐食性試験（EpiSkin™、EpiDerm™ SCT）と同様に、被験物質の皮膚腐食性を評価する試験法として推奨できるモデルであると結論された。UN GHS 細区分を考慮した評価においても、LabCyte EPI-MODEL24 SCT は、EpiSkin™、EpiDerm™ SCT と同様に皮膚腐食性を評価できる試験法である。

26

21

11．
．参考文献
1)

OECD, Guideline for the testing of chemicals. 404, Acute Dermal Irritation/Corrosion 2015.

2)

OECD, Guideline for the testing of chemicals. 431, In Vitro Skin Corrosion: Human skin model test.
2004.

3)

OECD, Guideline for the testing of chemicals. 431, In Vitro Skin Corrosion: Human skin model test.
2016.

4)

JaCVAM 資料編纂委員会, 皮膚腐食性試験代替法

5)

GHS 関係省庁連絡会議仮訳, 化学品の分類および表示に関する世界調和システム（GHS）改訂
8 版. 化学工業日報社, 東京, 2020.

6)

OECD, Guideline for the testing of chemicals. 431, In Vitro Skin Corrosion: Reconstructed Human
Epidermis (RhE) Test Method. 2019.

7)

OECD, Series on Testing & Assessment No. 219 Performance Standards for the Assessment of
Proposed Similar or Modified In Vitro Reconstructed Human Epidermis (RhE) Test Methods for Skin
Corrosion Testing as Described in TG 431. 2015.

8)

Fentem, J. H. et al.: Toxicology In Vitro, 12: 483-524, 1998.

9)

Liebsch, M. et al.: Alternatives to laboratory animals : ATLA, 28: 371-401, 2000.

ヒト表皮モデル. 2017.

10) OECD, Series on Testing and Assessment No. 190 Summary Document on the Statistical Performanceof Methods in OECD Test Guideline 431 for Sub-Categorisation, in Series on Testing and Assessment.
2013.
11) JaCVAM Validation Management Team, Validation Study for in vitro skin corrosion test method using
reconstructed human epidermal tissue LabCyte EPI-MODEL24. 2019.
12) ECVAM, Statement on the scientific validity of the Episkin test (An in vitro test for skin corrosivity)
1998.
13) ICCVAM, NIH Publication No.02-4502 ICCVAM Evaluation of Episkin™, EpiDerm™ (EPI-200),
and the Rat Skin Transcutaneous Electrical Resistance (TER) Assay: In Vitro Test Methods forAssessing Dermal Corrosivity Potential of Chemicals 2002.
14) ECVAM, Statement on the Application of the Epidermtm Human Skin Model for Skin Corrosivity
Testing 2000.
15) Desprez, B. et al.: Toxicology In Vitro, 29: 2055-2080, 2015.
16) OECD, Guideline for the testing of chemicals. 430, In Vitro Skin Corrosion: Transcutaneous Electrical
Resistance Test Method (TER). 2015.
17) OECD, Guideline for the testing of chemicals. 435, In Vitro Membrane Barrier Test Method for Skin
Corrosion. 2015.
18) JaCVAM, Peer Review Panel Evaluation of the Performance-based Validation Study on the LabCyte
EPI-Model24 in vitro skin corrosion test method as a me-too test method according to OECD GD 219
and falling within the OECD TG 431, 2018.
19) OECD, Series on Testing & Assessment No. 203, Guidance Document on an Integrated Approach on
Testing and Assessment (IATA) for Skin Corrosion and Irritation. 2014.
20) Scheel, J. et al.: Toxicology In Vitro, 25: 1435-1447, 2011.

22

27

ANNEX 1: 化学品の分類および表示に関する世界調和システム（GHS））改訂 8 版より
3.2.2.3

in vitro（（試験管内）/ex vivo（（生体外）のデータに基づく分類

3.2.2.3.1

現在入手できるそれぞれの in vitro/ex vivo 試験方法は皮膚刺激性または皮膚腐食性のどちらかを評価するが、一つの試験で両方の影響は評価しない。従って in vitro/ex vivo 試験結果のみに基づいた分類は二つ以上の方法で得られたデータを必要とするであろう。区分 3 を導入する
所管官庁は、現在入手可能な国際的に検証され受け入れられている in vitro/ex vivo 試験方法では、
区分 3 と分類される物質を識別することはできないことを認識することが重要である。
3.2.2.3.2

可能な限り分類は国際的に検証され受け入れられている in vitro/ex vivo 試験方法を用いて取られたデータに基づくべきであり、これらの試験方法に記載されている分類判定基準が適用される必要がある。試験物質が用いられた試験方法の適用範囲内にある場合にのみ、in vitro/ex
vivo のデータは分類に使用することができる。公表された文献に記載されている追加的な制限も考慮されるべきである。

3.2.2.3.3

皮膚腐食性

3.2.2.3.3.1 OECD TG430、431 または 435 に従って試験が実施された場合、表 3.2.6 の判定基準に基づいて、物質は皮膚腐食性区分 1（また可能であり要求されていれば細区分 1A、1B または 1C）に分類される。
3.2.2.3.3.2 いくつかの in vitro/ex vivo 試験方法では細区分 1B および 1C（表 3.2.6 参照）を区別することはできない。細区分が所管官庁によって要求されており、既存の in vitro/ex vivo データが細区分を区別することができない場合には、これら二つの細区分を区別するための追加的な情報を考慮しなければならない。追加的な情報が無いまたは不十分である場合には、区分 1 が適用される。
3.2.2.3.3.3
3.2.2.3.4

腐食性とは同定されない物質は皮膚刺激性としての分類が検討されるべきである。

皮膚刺激性

3.2.2.3.4.1 腐食性に関する分類が除外され、しかも OECD TG439 に従った試験が実施された
場合には、物質は表 3.2.7 の判定基準に基づいて皮膚刺激性区分 2 としての分類が検討されるべきである。
3.2.2.3.4.2 所管官庁が区分 3 を採用している場合、現在入手可能な皮膚刺激性に関する in vitro/ex
vivo 試験方法（例えば OECD TG439）では、物質を区分 3 に分類することはできないことを認識することが重要である。このような場合、区分 1 または区分 2 に関するどちらの分類判定も実行されない場合、区分 3 または区分に該当しないを区別するために追加的な情報が必要とされる。
3.2.2.3.4.3 所管官庁が区分 3 を採用しない場合、皮膚刺激性に関して国際的に受け入れられ検証されている in vitro/ex vivo 試験、例えば OECD TG439 における陰性結果は皮膚刺激性の区分に28

23

該当しないと結論するために使用することができる。

表 3.2.6.

in vitro/ex vivo 方法に関する皮膚腐食性判定基準（TG431 部分のみ抜粋）
再構築ヒト表皮モデル試験： OECD TG431、区分

附属書 2、方法 1、2、3、4
試験化学品を、ヒトの皮膚の上層部に性質がよく似た 3 次元再構築ヒト表皮（RhE）に局所的に適用する 4 つの方法がある。試験方法は、腐食性化学品
は拡散または浸食により角質層を貫通し、かつ下層の細胞に対して毒性があるという前提に基づく。組織の生存率は、MTT 染料の酵素反応による青色ホ
ルマザン塩への変換を、青色ホルマザン塩を組織から抽出して定量することで評価する。腐食性化学品は、定められた閾値以下の組織の生存率を低下させる能力で決定される。
判定基準は、定義された曝露期間による組織の生存率に基づく。

1

方法 1

方法 2、3、4

3 分間、60 分間また 3 分間曝露後 50％未満；またはは 240 分間曝露後 3 分間曝露後 50％以上かつ 60 分間曝露後 15％未満
35％未満
1A

方法 1

方法 2

方法 3

方法 4

3 分間曝露後 35％未

3 分間曝露後

3 分間曝露後

3 分間曝露後 15％未

満

25％未満

18％未満

満

1B

3 分間曝露後 35％以

3 分間曝露後

3 分間曝露後 3 分間曝露後後 15％

1C

上かつ 60 分間曝露後

25％以上かつ区 18％以上かつ区 以上かつ区分 1 の判

35％未満または 60 分

分 1 の判定基準 分 1 の判定基準 定基準を満足

間曝露後 35％以上か

を満足

を満足

つ 240 分 間 曝 露 後
35％未満
皮膚腐食性と240 分間曝露後 35％は分類されない以上

3 分間曝露後 50％以上、かつ 60 分間曝露後 15％以上

24

29

OECD/OCDE

431

Adopted:14 June 2019

OECD GUIDELINE FOR TESTING OF CHEMICALSIn Vitro Skin Corrosion:
Reconstructed Human Epidermis (RhE) Test Method
INTRODUCTION
1.
Skin corrosion refers to the production of irreversible damage to the skinmanifested as visible necrosis through the epidermis and into the dermis, following theapplication of a test chemical [as defined by the United Nations (UN) Globally Harmonized
System of Classification and Labelling of Chemicals (GHS)] (1). This updated Test
Guideline 431 provides an in vitro procedure allowing the identification of non-corrosiveand corrosive substances and mixtures in accordance with UN GHS (1). It also allows apartial sub-categorisation of corrosives.
2.
The assessment of skin corrosion potential of chemicals has typically involved theuse of laboratory animals (OECD Test Guideline 404 (TG 404); originally adopted in 1981
and revised in 1992, 2002 and 2015) (2). In addition to the present TG 431, two other invitro test methods for testing corrosion potential of chemicals have been validated andadopted as OECD Test Guidelines 430 (3) and 435 (4). Furthermore the in vitro OECD TG
439 (5) has been adopted for testing skin irritation potential. A document on Integrated
Approaches to Testing and Assessment (IATA) for Skin Corrosion and Irritation describes
several modules which group information sources and analysis tools, and provides
guidance on (i) how to integrate and use existing testing and non-testing data for theassessment of skin irritation and skin corrosion potentials of chemicals and (ii) proposes anapproach when further testing is needed (6).
3.
This Test Guideline addresses the human health endpoint skin corrosion. It makes
use of reconstructed human epidermis (RhE) (obtained from human derived nontransformed epidermal keratinocytes) which closely mimics the histological,
morphological, biochemical and physiological properties of the upper parts of the humanskin, i.e. the epidermis. This Test Guideline was originally adopted in 2004 and updated in2013, 2016 and 2019 to include additional test methods using the RhE models. The Test
Guideline was also updated in 2015 to introduce the possibility to use the methods tosupport the sub-categorisation of corrosive chemicals, and to refer to the IATA guidance
document, and introduce the use of an alternative procedure to measure viability.

© OECD, (2019)You are free to use this material subject to the terms and conditions available at http://www.oecd.org/termsandconditions/.

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4.
Five validated test methods using commercially available RhE models are includedin this Test Guideline, as described below. Prevalidation studies (7), followed by a formal
validation study for assessing skin corrosion (8) (9) (10) have been conducted (11) (12) for
two of these commercially available test methods, EpiSkin™ Standard Model (SM), andEpiDerm™ Skin Corrosivity Test (SCT) (EPI-200) (referred to in the following text as the
Validated Reference Methods – VRMs, EpiSkinTM=VRM1, EpiDermTM= VRM2). The
outcome of these studies led to the recommendation that the two VRMs mentioned above
could be used for regulatory purposes for distinguishing corrosive (C) from non-corrosive(NC) substances, and that the EpiSkin™ could moreover be used to support subcategorisation of corrosive substances (13) (14) (15). Two other commercially available invitro skin corrosion RhE test methods have subsequently shown similar results to the
EpiDerm™ SCT according to PS-based Validation (16) (17) (18). These are the
SkinEthicTM RHE1 and epiCS® (previously named EST-1000) that can also be used forregulatory purposes for distinguishing corrosive from non-corrosive substances (19) (20).
Post validation studies performed by the RhE model producers in the years 2012 to 2014
with a refined protocol correcting interferences of unspecific MTT reduction by the test
chemicals improved the performance of both discrimination of C/NC as well as supporting
sub-categorization of corrosives (21) (22). Further statistical analyses of the post-validation
data generated with Epiderm™ SCT, SkinEthic™ RHE and epiCS® have been performed
to identify alternative predictions models that improved the predictive capacity for subcategorisation (23). Finally, the LabCyte EPI-MODEL24 is another commerciallyavailable in vitro skin corrosion RhE test that was shown to be scientific similar to the
VRMs and can therefore be used for regulatory purposes to distinguish corrosive from noncorrosive substances as well as support sub-categorization of corrosives (40) (41) (42)(43).
5.
Before a proposed similar or modified in vitro RhE test method for skin corrosion
other than the VRMs can be used for regulatory purposes, its reliability, relevance(accuracy), and limitations for its proposed use should be determined to ensure its similarity
to the VRMs, in accordance with the requirements of the Performance Standards (PS) (24)set out in accordance with the principles of Guidance Document No.34 (25). The Mutual
Acceptance of Data will only be guaranteed after any proposed new or updated test method
following the PS have been reviewed and included in this Test Guideline. The test methods
included in this Test Guideline can be used to address countries’ requirements for test
results on in vitro test method for skin corrosion, while benefiting from the Mutual
Acceptance of Data.

DEFINITIONS
6.

Definitions used are provided in Annex I.

INITIAL CONSIDERATIONS
7.
This Test Guideline allows the identification of non-corrosive and corrosive
substances and mixtures in accordance with the UN GHS (1). This Test Guideline further
supports the sub-categorisation of corrosive substances and mixtures into optional Subcategory 1A, in accordance with the UN GHS (1), as well as a combination of Subcategories 1B and 1C (21) (22) (23). A limitation of this Test Guideline is that it does notallow discriminating between skin corrosive Sub-category 1B and Sub-category 1C inaccordance with the UN GHS (1) due to the limited set of well-known in vivo corrosive
Sub-category 1C chemicals. The five test methods under this test guideline are able todiscriminate sub-categories 1A versus 1B-and-1C versus NC.
©OECD 201932

OECD/OCDE

431 │ 38.
A wide range of chemicals representing mainly individual substances has beentested in the validation studies supporting the test methods included in this Test Guideline.
The original database of the validation study conducted for identification of non-corrosives
versus corrosives amounted to 60 chemicals covering a wide range of chemical classes (8)(9) (10). Testing to demonstrate sensitivity, specificity, accuracy and within-laboratoryreproducibility of the assay for sub-categorisation was further performed by the test method
developers using 79 to 80 chemicals also covering a wide range of chemical classes, andresults were reviewed by the OECD (21) (22) (23). On the basis of the overall data
available, the Test Guideline is applicable to a wide range of chemical classes and physicalstates including liquids, semi-solids, solids and waxes. The liquids may be aqueous or nonaqueous; solids may be soluble or insoluble in water. Whenever possible, solids should beground to a fine powder before application; no other prior treatment of the sample isrequired. In cases where evidence can be demonstrated on the non-applicability of test
methods included in the Test Guideline to a specific category of test chemicals, these test
methods should not be used for that specific category of test chemicals. In addition, thisTest Guideline is assumed to be applicable to mixtures as an extension of its applicability
to substances. However, due to the fact that mixtures cover a wide spectrum of categories
and composition, and that only limited information is currently available on the testing of
mixtures, in cases where evidence can be demonstrated on the non-applicability of the Test
Guideline to a specific category of mixtures (e.g. following a strategy as proposed in (26)),
the Test Guideline should not be used for that specific category of mixtures. Whenconsidering 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 be given to whether the results of such testing will yield results thatare meaningful scientifically. Such considerations are not needed, when there is aregulatory requirement for testing of the mixture. Gases and aerosols have not beenassessed yet in validation studies (8) (9) (10). While it is conceivable that these can betested using RhE technology, the current Test Guideline does not allow testing of gases andaerosols.
9.
Test chemicals absorbing light in the same range as MTT formazan and test
chemicals able to directly reduce the vital dye MTT (to MTT formazan) may interfere withthe tissue viability measurements and need the use of adapted controls for corrections. The
type of adapted controls that may be required will vary depending on the type ofinterference produced by the test chemical and the procedure used to measure MTT
formazan (see paragraphs 25-31).
10.
While this Test Guideline does not provide adequate information on skin irritation,
it should be noted that OECD TG 439 specifically addresses the health effect skin irritationin vitro and is based on the same RhE test system, though using another protocol (5). For afull evaluation of local skin effects after a single dermal exposure, the Guidance Document
No. 203 on Integrated Approaches for Testing Assessment should be consulted (6). This
IATA approach includes the conduct of in vitro tests for skin corrosion (such as describedin this Test Guideline) and skin irritation before considering testing in living animals. It is
recognized that the use of human skin is subject to national and international ethicalconsiderations and conditions.

PRINCIPLE OF THE TEST11.
The test chemical is applied topically to a three-dimensional RhE model, comprisedof non-transformed, human-derived epidermal keratinocytes, which have been cultured to©OECD 2019

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OECD/OCDE

form a multi-layered, highly differentiated model of the human epidermis. It consists oforganized basal, spinous and granular layers, and a multi-layered stratum corneum
containing intercellular lamellar lipid layers representing main lipid classes analogous tothose found in vivo.
12.
The RhE test method is based on the premise that corrosive chemicals are able topenetrate the stratum corneum by diffusion or erosion, and are cytotoxic to the cells in theunderlying layers. Cell viability is measured by enzymatic conversion of the vital dye MTT[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide,Thiazolyl
blue
tetrazolium bromide; CAS number 298-93-1], into a blue formazan salt that isquantitatively measured after extraction from tissues (27). Corrosive chemicals are
identified by their ability to decrease cell viability below defined threshold levels (seeparagraphs 35 and 36). The RhE-based skin corrosion test methods have shown to bepredictive of in vivo skin corrosion effects assessed in rabbits according to the OECD
guideline 404 (2).

DEMONSTRATION OF PROFICIENCY
13.
Prior to routine use of any of the five validated RhE test methods that adhere to thisTest Guideline, laboratories should demonstrate technical proficiency by correctlyclassifying the twelve Proficiency Substances listed in Table 1. In case of the use of amethod for sub-classification, also the correct sub-categorisation should be demonstrated.
In situations where a listed substance is unavailable or where justifiable, another substancefor which adequate in vivo and in vitro reference data are available may be used (e.g. fromthe list of reference chemicals (24)) provided that the same selection criteria as describedin Table 1 are applied.
Table 1. List of Proficiency Substances1

Substance

CASRN

ChemicalClass2

UN
GHS
Cat.
Based
on In
Vivo
results 3

Cat. Basedon
In Vitro
results4

Mean cell viability for
VRMs
VRM1

VRM2

PhysicalState

3 min 60 min. 3 min. 60 min

Sub-category 1A In Vivo Corrosives
Bromoacetic
acid
Boron
trifluoride
dihydrate
Phenol79-08-3

Organic acid1A

(3) 1A

3

2.8

3.2

2.8

S

13319-7501

Inorganic acid1A

(3) 1A

2.4

4.2

4.4

10.1

L

108-95-2

Phenol1A(3) 1A

29.8

21.8

22.6

13.5

S

Dichloroacetyl
chloride79-36-7

Electrophile1A(3) 1A

5.6

6.3

1.3

1.4

L

(3) 1B-and-1C

110.4

22.5

90.4

3.1

S

(3) 1B-and-1C(3) 1B-and-1C(3) 1B-and-1C

80.2
66.2
69.3

9.4
40.3
5.7

90
69.7
80.8

3.5
9.3
9

L
Viscous
L

Combination of sub-categories 1B-and-1C In Vivo Corrosives
Glyoxylic acid563-96-2
Organic acid
1B-and-1C
monohydrate
Lactic acid598-82-3
Organic acid
1B-and-1C
Ethanolamine
141-43-5
Organic base
1B
Hydrochloric
7647-01-0
Inorganic acid
1B-and-1Cacid(14.4%)©OECD 201934

OECD/OCDE

431 │ 5In Vivo Non Corrosives
Phenethyl
bromide4-Amino1,2,4triazole
4-(methylthio)benzaldehyde
Lauric acid103-63-9

ElectrophileNC(3) NC

141

117.2

112.5

71.2

N

584-13-4

Organic baseNC(3) NC

116.8

120.6

105.7

88.2

N

3446-89-7

ElectrophileNC(3) NC

136.7

150.4

85.4

81.6

N

143-07-7

Organic acidNC(3) NC

102

117.4

90.7

64.4

N

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; UN GHS = United Nations Globally
Harmonized System (1); VRM = Validated Reference Method, EpiSkinTM=VRM1, EpiDermTM= VRM2;
NC = Not Corrosive
1The proficiency substances, sorted first by corrosives versus non-corrosives, then by corrosive sub-category
and then by chemical class, were selected from the substances used in the ECVAM validation studies EpiSkin™
and EpiDerm™ (8) (9) (10) and from post-validation studies based on data provided by EpiSkin™ (22),
EpiDerm™, SkinEthic™ and epiCS® developers (23). Unless otherwise indicated, the substances were tested
at the purity level obtained when purchased from a commercial source (8) (10). The selection includes, to theextent possible, substances that: (i) are representative of the range of corrosivity responses (e.g. non-corrosives;
weak to strong corrosives) that the VRMs are capable of measuring or predicting; (ii) are representative of thechemical classes used in the validation studies; (iii) have chemical structures that are well-defined; (iv) induce
reproducible results in the VRM; (v) induce definitive results in the in vivo reference test method; (vi) arecommercially available; and (vii) are not associated with prohibitive disposal costs.
2Chemical class assigned by Barratt et al. (8).
3The corresponding UN Packing groups are I, II and III, respectively, for the UN GHS 1A, 1B and 1C.
4The in vitro predictions reported in this table were obtained with all five test methods covered in TG 431; forphenol though the LabCyte EPI-MODEL24 had slightly discordant results across runs, i.e. 1A-1BC-1BC; othermethods achieved these classifications in validation or post-validation testing performed by the test method
developers.
5The viability values obtained in the ECVAM Skin Corrosion Validation Studies were not corrected for direct
MTT reduction (killed controls were not performed in the validation studies). However, the post-validation data
generated by the test method developers that are presented in this table were acquired with adapted controls
(23).

14.
As part of the proficiency exercise, it is recommended that the user verifies thebarrier properties of the tissues after receipt as specified by the RhE model manufacturer.
This is particularly important if tissues are shipped over long distance/time periods. Oncea test method has been successfully established and proficiency in its use has been
demonstrated, such verification will not be necessary on a routine basis. However, whenusing a test method routinely, it is recommended to continue to assess the barrier properties
in regular intervals.

PROCEDURE
15.
The following is a generic description of the components and procedures of the
RhE test methods for skin corrosion assessment covered by this Test Guideline. The RhEmodels endorsed as scientifically valid for use within this Test Guideline, i.e. the EpiSkin™
(SM), EpiDerm™ (EPI-200), SkinEthic™ RHE, epiCS® and LabCyte EPI-MODEL24(16) (17) (19) (28) (29) (30) (31) (32) (33) (40) (41), can be obtained from commercial
sources. Standard Operating Procedures (SOPs) for these five RhE models are available(34) (35) (36) (37) (42), and their main test method components are summarised in Annex
2. It is recommended that the relevant SOP be consulted when implementing and using oneof these methods in the laboratory. Testing with the five RhE test methods covered by thisTest Guideline should comply with the following:©OECD 2019

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431

OECD/OCDERHE TEST METHOD COMPONENTS
General conditions
16.
Non-transformed human keratinocytes should be used to reconstruct theepithelium. Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum
granulosum) should be present under a functional stratum corneum. The stratum corneum
should be multi-layered containing the essential lipid profile to produce a functional barrier
with robustness to resist rapid penetration of cytotoxic benchmark chemicals, e.g. sodium
dodecyl sulphate (SDS) or Triton X-100. The barrier function should be demonstrated andmay be assessed either by determination of the concentration at which a benchmark
chemical reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, orby determination of the exposure time required to reduce cell viability by 50% (ET50) upon
application of the benchmark chemical at a specified, fixed concentration (see paragraph18). The containment properties of the RhE model should prevent the passage of material
around the stratum corneum to the viable tissue, which would lead to poor modelling ofskin exposure. The RhE model should be free of contamination by bacteria, viruses,
mycoplasma, or fungi.

Functional conditions
Viability
17.
The assay used for quantifying tissue viability is the MTT-assay (27). The viable
cells of the RhE tissue construct reduce the vital dye MTT into a blue MTT formazan
precipitate, which is then extracted from the tissue using isopropanol (or a similar solvent).
The OD of the extraction solvent alone should be sufficiently small, i.e., OD < 0.1. The
extracted MTT formazan may be quantified using either a standard absorbance (OD)measurement or an HPLC/UPLC-spectrophotometry procedure (38). The RhE model users
should ensure that each batch of the RhE model used meets defined criteria for the negative
control. An acceptability range (upper and lower limit) for the negative control OD values
should be established by the RhE model developer/supplier. Acceptability ranges for thenegative control OD values for the five validated RhE test methods included in this Test
Guideline are given in Table 2. An HPLC/UPLC-Spectrophotometry user should use thenegative control OD ranges provided in Table 2 as the acceptance criterion for the negative
control. It should be documented that the tissues treated with negative control are stable inculture (provide similar OD measurements) for the duration of the exposure period.
Table 2. Acceptability ranges for negative control OD values to control batch quality
Lower acceptance limit

Upper acceptance limit

EpiSkin™ (SM)
EpiDerm™ SCT (EPI-200)

≥ 0.6

≤ 1.5

≥ 0.8

≤ 2.8

SkinEthic™ RHE

≥ 0.8

≤ 3.0

epiCS

≥ 0.8

≤ 2.8

LabCyte EPI-MODEL24 SCT

≥ 0.7

≤ 2.5

©OECD 201936

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431 │ 7

Barrier function
18.
The stratum corneum and its lipid composition should be sufficient to resist the
rapid penetration of certain cytotoxic benchmark chemicals (e.g. SDS or Triton X-100), as
estimated by IC50 or ET50 (Table 3). The barrier function of each batch of the RhE model
used should be demonstrated by the RhE model developer/vendor upon supply of thetissues to the end user (see paragraph 21).

Morphology
19.
Histological examination of the RhE model should be performed demonstrating
multi-layered human epidermis-like structure containing stratum basale, stratum spinosum,
stratum granulosum and stratum corneum and exhibits lipid profile similar to lipid profile
of human epidermis. Histological examination of each batch of the RhE model useddemonstrating appropriate morphology of the tissues should be provided by the RhE model
developer/vendor upon supply of the tissues to the end user (see paragraph 21).

Reproducibility
20.
Test method users should demonstrate reproducibility of the test methods over timewith the positive and negative controls. Furthermore, the test method should only be used
if the RhE model developer/supplier provides data demonstrating reproducibility over timewith corrosive and non-corrosive chemicals from e.g. the list of Proficiency Substances(Table 1). In case of the use of a test method for sub-categorisation, the reproducibility withrespect to sub-categorisation should also be demonstrated.

Quality control (QC)
21.
The RhE model should only be used if the developer/supplier demonstrates thateach batch of the RhE model used meets defined production release criteria, among whichthose for viability (paragraph 17), barrier function (paragraph 18) and morphology(paragraph 19) are the most relevant. These data are provided to the test method users, sothat they are able to include this information in the test report. Only results produced with
QC accepted tissue batches can be accepted for reliable prediction of corrosive
classification. An acceptability range (upper and lower limit) for the IC50 or the ET50 isestablished by the RhE model developer/supplier. The acceptability ranges for the fivevalidated test methods are given in Table 3.

©OECD 2019

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Table 3. QC batch release criterion
Lower acceptance limit

Upper acceptance limit

EpiSkin™ (SM)(18 hours treatment with SDS)(33)

IC50 = 1.0 mg/mL

IC50 = 3.0 mg/mL

EpiDerm™SCT (EPI-200)

ET50 = 4.0 hours

ET50 = 8.7 hours

ET50 = 4.0 hours

ET50 = 10.0 hours

epiCS (1% Triton X-100)(36)

ET50 = 2.0 hours

ET50 = 7.0 hours

LabCyte EPI-MODEL24 SCT

IC50 = 1.4 mg/mL

IC50 = 4.0 mg/mL(1% Triton X-100)(34)
SkinEthic™ RHE(1% Triton X-100)(35)(18 hours treatment with SDS) (42)

Application of the Test Chemical and Control Substances
22.
At least two tissue replicates should be used for each test chemical and controls foreach exposure time. For liquid as well as solid chemicals, sufficient amount of test chemical
should be applied to uniformly cover the epidermis surface while avoiding an infinite dose,
i.e. a minimum of 70 μL/cm2 or 30 mg/cm2 should be used. Depending on the methods,
the epidermis surface should be moistened with deionized or distilled water before
application of solid chemicals, to improve contact between the test chemical and theepidermis surface (34) (35) (36) (37) (42). Whenever possible, solids should be tested as afine powder. The application method should be appropriate for the test chemical (see e.g.
references (34-37). At the end of the exposure period, the test chemical should be carefully
washed from the epidermis with an aqueous buffer, or 0.9% NaCl. Depending on which of
the five validated RhE test methods is used, two or three exposure periods are used per test
chemical (for all five valid RhE models: 3 min and 1 hour; for EpiSkin™ an additional
exposure time of 4 hours). Depending on the RhE test method used and the exposure period
assessed, the incubation temperature during exposure may vary between room temperatureand 37ºC.
23.
Concurrent negative and positive controls (PC) should be used in each run todemonstrate that viability (with negative controls), barrier function and resulting tissue
sensitivity (with the PC) of the tissues are within a defined historical acceptance range. The
suggested PC chemicals are glacial acetic acid or 8N KOH depending upon the RhE model
used (see Annex 2 and relevant SOP for details). It should be noted that 8N KOH is a direct
MTT reducer that might require adapted controls as described in paragraphs 25 and 26. The
suggested negative controls are 0.9% (w/v) NaCl or water.

Cell Viability Measurements
24.
The MTT assay, which is a quantitative assay, should be used to measure cell
viability under this Test Guideline (27). The tissue sample is placed in MTT solution of
appropriate concentration (0.3, 0.5 or 1 mg/mL, see Annex 2 and relevant SOP for details)
for 3 hours. The precipitated blue formazan product is then extracted from the tissue using
a solvent (e.g. isopropanol, acidic isopropanol), and the concentration of formazan is©OECD 201938

OECD/OCDE

431 │ 9measured by determining the OD at 570 nm using a filter band pass of maximum ± 30 nm,
or by an HPLC/UPLC spectrophotometry procedure (see paragraphs 30 and 31) (38).
25.
Test chemicals may interfere with the MTT assay, either by direct reduction of the
MTT into blue formazan, and/or by colour interference if the test chemical absorbs,
naturally or due to treatment procedures, in the same OD range of formazan (570 ± 30 nm,
mainly blue and purple chemicals). Additional controls should be used to detect and correct
for a potential interference from these test chemicals such as the non-specific MTT
reduction (NSMTT) control and the non-specific colour (NSC) control (see paragraphs 26
to 30). This is especially important when a specific test chemical is not completely removed
from the tissue by rinsing or when it penetrates the epidermis, and is therefore present in
the tissues when the MTT viability test is performed. Detailed description of how to correct
direct MTT reduction and interferences by colouring agents is available in the SOPs for thetest methods (34) (35) (36) (37) (42).
26.
To identify direct MTT reducers, each test chemical should be added to freshly
prepared MTT medium (34) (35) (36) (37) (42). If the MTT mixture containing the test
chemical turns blue/purple, the test chemical is presumed to directly reduce the MTT, andfurther functional check on non-viable epidermis should be performed, independently ofusing the standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry
procedure. This additional functional check employs killed tissues that possess only
residual metabolic activity but absorb the test chemical in similar amount as viable tissues.
Each MTT reducing chemical is applied on at least two killed tissue replicates per exposure
time, which undergo the whole skin corrosion test. The true tissue viability is thencalculated as the percent tissue viability obtained with living tissues exposed to the MTT
reducer minus the percent non-specific MTT reduction obtained with the killed tissues
exposed to the same MTT reducer, calculated relative to the negative control run
concurrently to the test being corrected (%NSMTT).
27.
To identify potential interference by coloured test chemicals or test chemicals that
become coloured when in contact with water or isopropanol and decide on the need for
additional controls, spectral analysis of the test chemical in water (environment duringexposure) and/or isopropanol (extracting solution) should be performed. If the test chemical
in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, further colorant
controls should be performed or, alternatively, an HPLC/UPLC-spectrophotometry
procedure should be used in which case these controls are not required (see paragraphs 30
and 31). When performing the standard absorbance (OD) measurement, each interferingcoloured test chemical is applied on at least two viable tissue replicates per exposure time,
which undergo the entire skin corrosion test but are incubated with medium instead of MTT
solution during the MTT incubation step to generate a non-specific colour (NSCliving)
control. The NSCliving control needs to be performed concurrently per exposure time percoloured test chemical (in each run) due to the inherent biological variability of living
tissues. The true tissue viability is then calculated as the percent tissue viability obtainedwith living tissues exposed to the interfering test chemical and incubated with MTT
solution minus the percent non-specific colour obtained with living tissues exposed to theinterfering test chemical and incubated with medium without MTT, run concurrently to thetest being corrected (%NSCliving).
28.
Test chemicals that are identified as producing both direct MTT reduction (seeparagraph 26) and colour interference (see paragraph 27) will also require a third set ofcontrols, apart from the NSMTT and NSCliving controls described in the previous©OECD 2019

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431

OECD/OCDE

paragraphs, when performing the standard absorbance (OD) measurement. This is usually
the case with darkly coloured test chemicals interfering with the MTT assay (e.g., blue,
purple, black) because their intrinsic colour impedes the assessment of their capacity todirectly reduce MTT as described in paragraph 26. These test chemicals may bind to bothliving and killed tissues and therefore the NSMTT control may not only correct for potential
direct MTT reduction by the test chemical, but also for colour interference arising from thebinding of the test chemical to killed tissues. This could lead to a double correction forcolour interference since the NSCliving control already corrects for colour interference
arising from the binding of the test chemical to living tissues. To avoid a possible double
correction for colour interference, a third control for non-specific colour in killed tissues(NSCkilled) needs to be performed. In this additional control, the test chemical is applied
on at least two killed tissue replicates per exposure time, which undergo the entire testingprocedure but are incubated with medium instead of MTT solution during the MTT
incubation step. A single NSCkilled control is sufficient per test chemical regardless of thenumber of independent tests/runs performed, but should be performed concurrently to the
NSMTT control and, where possible, with the same tissue batch. The true tissue viability
is then calculated as the percent tissue viability obtained with living tissues exposed to thetest chemical minus %NSMTT minus %NSCliving plus the percent non-specific colour
obtained with killed tissues exposed to the interfering test chemical and incubated withmedium without MTT, calculated relative to the negative control run concurrently to thetest being corrected (%NSCkilled).
29.
It is important to note that non-specific MTT reduction and non-specific colour
interferences may increase the readouts of the tissue extract above the linearity range of thespectrophotometer. On this basis, each laboratory should determine the linearity range of
their spectrophotometer with MTT formazan (CAS # 57360-69-7) from a commercial
source before initiating the testing of test chemicals for regulatory purposes. In particular,
the standard absorbance (OD) measurement using a spectrophotometer is appropriate toassess direct MTT-reducers and colour interfering test chemicals when the ODs of thetissue extracts obtained with the test chemical without any correction for direct MTT
reduction and/or colour interference are within the linear range of the spectrophotometeror when the uncorrected percent viability obtained with the test chemical already defined
it as a corrosive (see paragraphs 35 and 36). Nevertheless, results for test chemicals
producing %NSMTT and/or %NSCliving ≥ 50% of the negative control should be takenwith caution.
30.
For coloured test chemicals which are not compatible with the standard absorbance(OD) measurement due to too strong interference with the MTT assay, the alternative
HPLC/UPLC-spectrophotometry procedure to measure MTT formazan may be employed(see paragraph 31) (37). The HPLC/UPLC-spectrophotometry system allows for the
separation of the MTT formazan from the test chemical before its quantification (38). For
this reason, NSCliving or NSCkilled controls are never required when using HPLC/UPLCspectrophotometry, independently of the chemical being tested. NSMTT controls should
nevertheless be used if the test chemical is suspected to directly reduce MTT or has a colourthat impedes the assessment of the capacity to directly reduce MTT (as described inparagraph 26). When using HPLC/UPLC-spectrophotometry to measure MTT formazan,
the percent tissue viability is calculated as percent MTT formazan peak area obtained withliving tissues exposed to the test chemical relative to the MTT formazan peak obtained withthe concurrent negative control. For test chemicals able to directly reduce MTT, true tissue
viability is calculated as the percent tissue viability obtained with living tissues exposed tothe test chemical minus %NSMTT. Finally, it should be noted that direct MTT-reducers©OECD 201940

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431 │ 11that may also be colour interfering, which are retained in the tissues after treatment andreduce MTT so strongly that they lead to ODs (using standard OD measurement) or peakareas (using UPLC/HPLC-spectrophotometry) of the tested tissue extracts that fall outsideof the linearity range of the spectrophotometer cannot be assessed, although these are
expected to occur in only very rare situations.
31.
HPLC/UPLC-spectrophotometry may be used also with all types of test chemicals(coloured, non-coloured, MTT-reducers and non-MTT reducers) for measurement of MTT
formazan (38). Due to the diversity of HPLC/UPLC-spectrophotometry systems,
qualification of the HPLC/UPLC-spectrophotometry system should be demonstrated
before its use to quantify MTT formazan from tissue extracts by meeting the acceptancecriteria for a set of standard qualification parameters based on those described in the U.S.
Food and Drug Administration guidance for industry on bio-analytical method validation(38) (39). These key parameters and their acceptance criteria are shown in Annex 4. Oncethe acceptance criteria defined in Annex 4 have been met, the HPLC/UPLCspectrophotometry system is considered qualified and ready to measure MTT formazanunder the experimental conditions described in this Test Guideline.

Acceptance Criteria
32.
For each test method using valid RhE models, tissues treated with the negative
control should exhibit OD reflecting the quality of the tissues as described in table 2 andshould not be below historically established boundaries. Tissues treated with the PC, i.e.
glacial acetic acid or 8N KOH, should reflect the ability of the tissues to respond to a
corrosive chemical under the conditions of the test method (see Annex 2 and relevant SOP
for details). The variability between tissue replicates of test chemical and/or control
substances should fall within the accepted limits for each valid RhE model requirements(see Annex 2 and relevant SOP for details) (e.g. the difference of viability between the twotissue replicates should not exceed 30%). If either the negative control or PC included in a
run fall out of the accepted ranges, the run is considered as not qualified and should be
repeated. If the variability of test chemicals falls outside of the defined range, its testingshould be repeated.

Interpretation of Results and Prediction Model
33.
The OD values obtained for each test chemical should be used to calculate
percentage of viability relative to the negative control, which is set at 100%. In case
HPLC/UPLC-spectrophotometry is used, the percent tissue viability is calculated aspercent MTT formazan peak area obtained with living tissues exposed to the test chemical
relative to the MTT formazan peak obtained with the concurrent negative control. The cutoff percentage cell viability values distinguishing corrosive from non-corrosive test
chemical (or discriminating between different corrosive sub-categories) are defined below
in paragraphs 35 and 36 for each of the test methods covered by this Test Guideline andshould be used for interpreting the results.
34.
A single testing run composed of at least two tissue replicates should be sufficient
for a test chemical when the resulting classification is unequivocal. However, in cases ofborderline results, such as non-concordant replicate measurements, a second run may beconsidered, as well as a third one in case of discordant results between the first two runs.
35.
The prediction model for the EpiSkin™ skin corrosion test method (9) (34) (22),associated with the UN GHS (1) classification system, is shown in Table 4:©OECD 2019

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Table 4. EpiSkin™ prediction model
Viability measured after exposure time
points (t='3,' 60 and 240 minutes)

Prediction to consider< 35% after 3 min exposure

Corrosive:
Optional Sub-category 1A *≥ 35% after 3 min exposure AND < 35% after
60 min exposure

Corrosive:OR

A combination of optional Subcategories 1B-and-1C≥ 35% after 60 min exposure AND < 35% after 240
min exposure≥ 35% after 240 min exposure

Non-corrosive*) According to the data generated in view of assessing the usefulness of the RhE test methods forsupporting sub-categorisation, it was shown that around 22 % of the Sub-category 1A results of the
EpiSkin™ test method may actually constitute Sub-category 1B or Sub-category 1C
substances/mixtures (i.e. over classifications) (see Annex 3).

36.
The prediction models for the EpiDerm™ SCT (10) (23) (35), the SkinEthic™
RHE (17) (18) (23) (36), the epiCS® (16) (23) (37) and LabCyte EPI-MODEL24 (41) (42)skin corrosion test methods, associated with the UN GHS (1) classification system, areshown in Table 5:©OECD 201942

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431 │ 13

Table 5. EpiDerm™ SCT, SkinEthic™ RHE epiCS® and LabCyte EPI-MODEL24

SCTViability measured after exposure time
points (t=3 and 60 minutes)

Prediction to be considered

STEP 1 for EpiDerm™ SCT, SkinEthic™ RHE, epiCS® and LabCyte EPI-MODEL24 SCT< 50% after 3 min exposure

Corrosive≥ 50% after 3 min exposure AND

Corrosive< 15% after 60 min exposure≥ 50% after 3 min exposure AND≥ 15% after 60 min exposure

Non-corrosive

STEP 2 for EpiDerm™ SCT - for substances/mixtures identified as Corrosive in step 1< 25% after 3 min exposure

Optional Sub-category 1A *≥ 25% after 3 min exposure

A combination of optional Sub-categories 1Band-1C

STEP 2 for SkinEthic™ RHE - for substances/mixtures identified as Corrosive in step 1< 18% after 3 min exposure

Optional Sub-category 1A *≥ 18% after 3 min exposure

A combination of optional Sub-categories 1Band-1C

STEP 2 for epiCS® - for substances/mixtures identified as Corrosive in step 1< 15% after 3 min exposure

Optional Sub-category 1A *≥ 15% after 3 min exposure

A combination of optional Sub-categories 1Band-1C

STEP 2 for LabCyte EPI-MODEL24 SCT - for substances/mixtures identified as Corrosive in step 1< 15% after 3 min exposure

Optional Sub-category 1A *≥ 15% after 3 min exposure

A combination of optional Sub-categories 1Band-1C* According to the data generated in view of assessing the usefulness of the RhE test methods forsupporting sub-categorisation, it was shown that around 29%, 31%, 33% and 30% of the Subcategory 1A results of the EpiDerm™ SCT, SkinEthic™ RHE epiCS® and LabCyte EPIMODEL24 SCT, respectively, may actually constitute Sub-category 1B or Sub-category 1C
substances/mixtures (i.e. over-classifications) (see Annex 3).

DATA AND REPORTING
Data
37.
For each test, data from individual tissue replicates (e.g. OD values and calculated
percentage cell viability for each test chemical, including classification) should be reported
in tabular form, including data from repeat experiments as appropriate. In addition, means
and ranges of viability and CVs between tissue replicates for each test should be reported.
Observed interactions with MTT reagent by direct MTT reducers or coloured test chemicals
should be reported for each tested chemical.

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Test Report
38.

The test report should include the following information:

Test Chemical and Control Substances:

Mono-constituent substance: chemical identification, such as IUPAC or CAS
name, CAS number, SMILES or InChI code, structural formula, purity, chemical
identity of impurities as appropriate and practically feasible, etc;

Multi-constituent substance, UVCB and mixture: characterised as far as possible
by chemical identity (see above), quantitative occurrence and relevant
physicochemical properties of the constituents;

Physical appearance, water solubility, and any additional relevant physicochemical
properties;

Source, lot number if available;Treatment of the test chemical/control substance prior to testing, if applicable (e.g.
warming, grinding);Stability of the test chemical, limit date for use, or date for re-analysis if known;

Storage conditions.

RhE model and protocol used and rationale for it (if applicable)
Test Conditions:

RhE model used (including batch number);

Calibration information for measuring device (e.g. spectrophotometer), wavelengthand band

pass (if applicable) used for quantifying MTT formazan, and linearity range ofmeasuring device;Description of the method used to quantify MTT formazan;

Description of the qualification of the HPLC/UPLC-spectrophotometry system, if
applicable;Complete supporting information for the specific RhE model used including its
performance. This should include, but is not limited to:o i) Viability;o ii) Barrier function;o iii) Morphology;o iv) Quality controls (QC) of the model;

Reference to historical data of the model. This should include, but is not limited toacceptability of the QC data with reference to historical batch data;

Demonstration of proficiency in performing the test method before routine use by
testing of the proficiency substances.

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Test Procedure:

Details of the test procedure used (including washing procedures used after
exposure period);

Doses of test chemical and control substances used;

Duration of exposure period(s) and temperature(s) of exposure;Indication of controls used for direct MTT-reducers and/or colouring test
chemicals, if applicable;

Number of tissue replicates used per test chemical and controls (PC, negative
control, and NSMTT, NSCliving and NSCkilled, if applicable), per exposure time;Description of decision criteria/prediction model applied based on the RhE model
used;



Description of any modifications of the test procedure (including washingprocedures).

Run and Test Acceptance Criteria:

Positive and negative control mean values and acceptance ranges based on
historical data;

Acceptable variability between tissue replicates for positive and negative controls;

Acceptable variability between tissue replicates for test chemical.

Results:


Tabulation of data for individual test chemicals and controls, for each exposure
period, each run and each replicate measurement including OD or MTT formazan
peak area, percent tissue viability, mean percent tissue viability, differences
between replicates, SDs and/or CVs if applicable;

If applicable, results of controls used for direct MTT-reducers and/or colouring test
chemicals including OD or MTT formazan peak area, %NSMTT, %NSCliving,%NSCkilled, differences between tissue replicates, SDs and/or CVs (if applicable),and final correct percent tissue viability;

Results obtained with the test chemical(s) and control substances in relation to thedefined run and test acceptance criteria;

Description of other effects observed;The derived classification with reference to the prediction model/decision criteria
used.

Discussion of the results:
Conclusions:©OECD 2019

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OECD/OCDELITERATURE
1. UN. (2013). United Nations Globally Harmonized System of Classification andLabelling of Chemicals (GHS), Fifth Revised Edition, UN New York and Geneva.
Availableat:[http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html]
2. OECD. (2015). Guideline for Testing of Chemicals. (No. 404.): Acute Dermal
Irritation, Corrosion, Organisation for Economic Cooperation and Development,
Paris.
3. OECD. (2015). Guideline for the Testing of Chemicals (No. 430.): In Vitro Skin
Corrosion: Transcutaneous Electrical Resistance (TER). Organisation for
Economic Cooperation and Development, Paris.
4. OECD. (2015). Guideline for the Testing of Chemicals (No. 435.): In Vitro
Membrane Barrier Test Method. Organisation for Economic Cooperation andDevelopment, Paris. (5) OECD. (2015). Guideline for the Testing of Chemicals(No. 439.): In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.
Organisation for Economic Cooperation and Development, Paris.
5. OECD. (2015). Guideline for the Testing of Chemicals (No. 439.): In Vitro Skin
Irritation: Reconstructed Human Epidermis Test Method. Organisation for
Economic Cooperation and Development, Paris.
6. OECD. (2014). Guidance Document on Integrated Approaches to Testing andAssessment of Skin Irritation/Corrosion. Environment, Health and Safety
Publications, Series on Testing and Assessment, (No. 203.) Organisation for
Economic Cooperation and Development, Paris.
7. Botham P.A., Chamberlain M., Barratt M.D., Curren R.D., Esdaile D.J., Gardner
J.R., Gordon V.C., Hildebrand B., Lewis R.W., Liebsch M., Logemann P.,
Osborne R., Ponec M., Regnier J.F., Steiling W., Walker A.P., and Balls M. (1995).
A Prevalidation Study on In Vitro Skin Corrosivity Testing. The report andRecommendations of ECVAM Workshop 6.ATLA 23, 219-255.
8. Barratt M.D., Brantom P.G., Fentem J.H., Gerner I., Walker A.P., and Worth A.P.
(1998). The ECVAM International Validation Study on In Vitro Tests for Skin
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12, 471-482.
9. Fentem J.H., Archer G.E.B., Balls M., Botham P.A., Curren R.D., Earl L.K.,
Esdaile D.J., Holzhutter H.- G., and Liebsch M. (1998). The ECVAM International
Validation Study on In Vitro Tests for Skin Corrosivity. 2. Results and Evaluationby the Management Team. Toxicol.in Vitro 12, 483-524.
10. Liebsch M., Traue D., Barrabas C., Spielmann H., Uphill, P., Wilkins S., WiemannC., Kaufmann T., Remmele M. and Holzhütter H. G. (2000). The ECVAM
Prevalidation Study on the Use of EpiDerm for Skin Corrosivity Testing, ATLA28, pp. 371-401.
11. Balls M., Blaauboer B.J., Fentem J.H., Bruner L., Combes R.D., Ekwall B., Fielder
R.J., Guillouzo A., Lewis R.W., Lovell D.P., Reinhardt C.A., Repetto G.,
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431 │ 17of Toxicity Test Procedures. The Report and Recommendations of ECVAM
Workshops, ATLA 23, 129-147.
12. ICCVAM (Interagency Coordinating Committee on the Validation of Alternative
Methods). (1997). Validation and Regulatory Acceptance of Toxicological Test
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13. ICCVAM (Interagency Coordinating Committee on the Validation of Alternative
Methods). (2002). ICCVAM evaluation of EpiDerm™ (EPI-200), EPISKIN™
(SM), and the Rat Skin Transcutaneous Electrical Resistance (TER) Assay: In Vitro
Test Methods for Assessing Dermal Corrosivity Potential of Chemicals. NIH
Publication No. 02-4502. National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods, National Institute ofEnvironmental Health Sciences, Research Triangle Park, NC, USA.Available
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14. EC-ECVAM. (1998). Statement on the Scientific Validity of the EpiSkin™ Test(an In Vitro Test for Skin Corrosivity), Issued by the ECVAM Scientific Advisory
Committee(ESAC10),
3
April
1998.
Availableat:[http://www.ecvam.jrc.ec.europa.eu.html].
15. EC-ECVAM. (2000). Statement on the Application of the EpiDerm™ Human Skin
Model for Skin Corrosivity Testing, Issued by the ECVAM Scientific Advisory
Committee(ESAC14),
21
March
2000.
Availableat:[http://ecvam.jrc.ec.europa.eu].
16. Hoffmann J., Heisler E., Karpinski S., Losse J., Thomas D., Siefken W., Ahr H.J.,
Vohr H.W. and Fuchs H.W. (2005). Epidermal-Skin-Test 1000 (EST-1000)-A New
Reconstructed Epidermis for In Vitro Skin Corrosivity Testing. Toxicol.In Vitro
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17. Kandárová H., Liebsch M., Spielmann,H., Genschow E., Schmidt E., Traue D.,
Guest R., Whittingham A., Warren N, Gamer A.O., Remmele M., Kaufmann T.,
Wittmer E., De Wever B., and Rosdy M. (2006). Assessment of the Human
Epidermis Model SkinEthic RHE for In Vitro Skin Corrosion Testing of Chemicals
According to New OECD TG 431. Toxicol.In Vitro 20, 547 559.
18. Tornier C., Roquet M. and Fraissinette A.B. (2010). Adaptation of the Validated
SkinEthic™ Reconstructed Human Epidermis (RHE) Skin Corrosion Test Method
to 0.5 cm2 Tissue Sample. Toxicol. In Vitro 24, 1379-1385.
19. EC-ECVAM. (2006). Statement on the Application of the SkinEthic™ Human Skin
Model for Skin Corrosivity Testing, Issued by the ECVAM Scientific Advisory
Committee(ESAC25),
17
November
2006.
Availableat:[http://www.ecvam.jrc.ec.europa.eu.html].
20. EC-ECVAM. (2009). ESAC Statement on the Scientific Validity of an In-Vitro
Test Method for Skin Corrosivity Testing: the EST-1000, Issued by the ECVAM
Scientific Advisory Committee (ESAC30), 12 June 2009. Available at:[http://www.ecvam.jrc.ec.europa.eu.html].
21. OECD. (2013). Summary Document on the Statistical Performance of Methods inOECD Test Guideline 431 for Sub-categorisation. Environment, Health, and Safety©OECD 2019

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Publications, Series on Testing and Assessment (No. 190.). Organisation for
Economic Cooperation and Development, Paris.

22. Alépée N., Grandidier M.H., and Cotovio J. (2014). Sub-Categorisation of Skin
Corrosive Chemicals by the EpiSkin™ Reconstructed Human Epidermis Skin
Corrosion Test Method According to UN GHS: Revision of OECD Test Guideline
431. Toxicol.In Vitro 28, 131-145.
23. Desprez B., Barroso J., Griesinger C., Kandárová H., Alépée N., and Fuchs, H.
(2015). Two Novel Prediction Models Improve Predictions of Skin Corrosive Subcategories by Test Methods of OECD Test Guideline No. 431. Toxicol. In Vitro
29, 2055 2080.
24. OECD. (2015). Performance Standards for the Assessment of Proposed Similar or
Modified In Vitro Reconstructed Human Epidermis (RHE) Test Methods For Skin
Corrosion in Relation to OECD TG 431. Environmental Health and Safety
Publications, Series on Testing and Assessment (No. 219). Organisation for
Economic Cooperation and Development, Paris
25. OECD. (2005). Guidance Document on the Validation and International
Acceptance of New or Updated Test Methods for Hazard Assessment.
.Environment, Health and Safety Publications, Series on Testing and Assessment(No. 34.), Organisation for Economic Cooperation and Development, Paris.
26. Eskes C. et al. (2012). Regulatory Assessment of In Vitro Skin Corrosion and
Irritation Data Within the European Framework: Workshop Recommendations.
Regul.Toxicol.Pharmacol. 62, 393-403.
27. Mosmann T. (1983). Rapid Colorimetric Assay for Cellular Growth and Survival:
Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 65, 5563.
28. Tinois E., et al. (1994). The Episkin Model: Successful Reconstruction of Human
Epidermis In Vitro. In: In Vitro Skin Toxicology. Rougier A.,. Goldberg A.M and
Maibach H.I. (Eds): 133-140.
29. Cannon C. L., Neal P.J., Southee J.A., Kubilus J. and Klausner M. (1994), New
Epidermal Model for Dermal Irritancy Testing. Toxicol.in Vitro 8, 889 - 891.
30. Ponec M., Boelsma E, Weerheim A, Mulder A, Bouwstra J and Mommaas M.
(2000). Lipid and Ultrastructural Characterization of Reconstructed Skin Models.
Inter. J. Pharmaceu. 203, 211 - 225.
31. Tinois E., Tillier, J., Gaucherand, M., Dumas, H., Tardy, M. and Thivolet J. (1991).
In Vitro and Post Transplantation Differentiation of Human Keratinocytes Grown
on the Human Type IV Collagen Film of a Bilayered Dermal Substitute. Exp. Cell
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32. Parenteau N.L., Bilbo P, Nolte CJ, Mason VS and Rosenberg M. (1992). The
Organotypic Culture of Human Skin Keratinocytes and Fibroblasts to Achieve
Form and Function. Cytotech. 9, 163-171.
33. Wilkins L.M., Watson SR, Prosky SJ, Meunier SF and Parenteau N.L. (1994).
Development of a Bilayered Living Skin Construct for Clinical Applications.
Biotech.Bioeng.43/8, 747-756.

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34. EpiSkin™ (December 2011)SOP,. INVITTOX Protocol (No. 118.). EpiSkin™
Skin Corrosivity Test.. Available at: [http://www.ecvam.jrc.ec.europa.eu.hm;].
35. EpiDremTM SOP. (February 2012). Version MK-24-007-0024 Protocol for : In
vitro EpiDerm™ Skin Corrosion Test (EPI-200-SCT), for Use with MatTek
Corporation's Reconstructed Human Epidermal Model EpiDerm. Available at:[http://www.ecvam.jrc.ec.europa.eu.html].
36. SkinEthic™ RHE SOP,INVITTOX Protocol (January 2012). SkinEthic™ Skin
Corrosivity Test. Available at: [http://www.ecvam.jrc.ec.europa.eu.html].
37. EpiCS® SOP, Version 4.1 (January 2012). In Vitro Skin Corrosion: Human Skin
Model Test Epidermal Skin Test 1000 (epiCS®) CellSystems. Available at:[http://www.ecvam.jrc.ec.europa.eu.html].
38. Alépée N., Barroso J., De Smedt A., De Wever B., Hibatallah J., Klaric M., Mewes
K.R., Millet M., Pfannenbecker U., Tailhardat M., Templier M., and McNamee P.
Use of HPLC/UPLCspectrophotometry for Detection of MTT Formazan in In Vitro
Reconstructed Human Tissue (RhT)- based Test Methods Employing the MTTAssay to Expand their Applicability to Strongly Coloured Test Chemicals.
Manuscript in Preparation.
39. US FDA. (2001). Guidance for Industry: Bioanalytical Method Validation. U.S.
Department of Health and Human Services, Food and Drug Administration. (May2001).
Availableat:[http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].
40. Katoh M., Hamajima F., Ogasawara T., and Ken-ichiro Hata. (2010). Assessmentof the human epidermal model LabCyte EPI-MODEL for in vitro skin corrosion
testing according to the OECD test Guideline 431. J Toxicol. Sci. 35, 411-417.
41. LabCyte Validation Management Team (2018). Validation Study for in vitro skincorrosion test method using reconstructed human epidermal tissue LabCyte EPIMODEL24. Available at: [http://www.jacvam.jp/files/doc/01_05/01_05_Z1.pdf].
42. LabCyte EPI-MODEL24 SCT SOP, Version 1.6. (May, 2017). Skin corrosion test
using the reconstructed human model “LabCyte EPI-MODEL24”. Available at:[http://www.jacvam.jp/files/doc/01_05/01_05_Z2.pdf ].
43. Report of the Peer-review of the validation study for LabCyte EPI-MODEL24 In
Vitro
Skin
Corrosion
Test
Method.
Availableat[http://www.jacvam.jp/files/doc/01_05/01_05_Z3.pdf].

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OECD/OCDEANNEX 1- DEFINITIONS
Accuracy: The closeness of agreement between test method results and accepted reference
values. It is a measure of test method performance and one aspect of relevance. The term
is often used interchangeably with “concordance” to mean the proportion of correct
outcomes of a test method (25).
Cell viability: Parameter measuring total activity of a cell population e.g. as ability ofcellular mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Thiazolyl blue), which
depending on the endpoint measured and the test design used, correlates with the total
number and/or vitality of living cells.
Chemical: means a substance or a mixture.
Concordance: This is a measure of test method performance for test methods that give acategorical result, and is one aspect of relevance. The term is sometimes usedinterchangeably with accuracy, and is defined as the proportion of all chemicals tested thatare correctly classified as positive or negative. Concordance is highly dependent on theprevalence of positives in the types of test chemical being examined (25).
ET50: Can be estimated by determination of the exposure time required to reduce cell
viability by 50% upon application of the benchmark chemical at a specified, fixed
concentration, see also IC50.
GHS (Globally Harmonized System of Classification and Labelling of Chemicals): A
system proposing the classification of chemicals (substances and mixtures) according tostandardized types and levels of physical, health and environmental hazards, andaddressing corresponding communication elements, such as pictograms, signal words,
hazard statements, precautionary statements and safety data sheets, so that to convey
information on their adverse effects with a view to protect people (including employers,
workers, transporters, consumers and emergency responders) and the environment (1).
HPLC: High Performance Liquid Chromatography.
IATA: Integrated Approach on Testing and Assessment.
IC50: Can be estimated by determination of the concentration at which a benchmark
chemical reduces the viability of the tissues by 50% (IC50) after a fixed exposure time, seealso ET50.
ET50. Infinite dose: Amount of test chemical applied to the epidermis exceeding theamount required to completely and uniformly cover the epidermis surface.
Mixture: means a mixture or solution composed of two or more substances in which they
do not react.
Mono-constituent substance: A substance, defined by its quantitative composition, in
which one main constituent is present to at least 80% (w/w).
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue
tetrazolium bromide.
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 <©OECD 201950

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431 │ 2180% (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.
NC: Non corrosive.
NSCkilled control: Non-Specific Colour control in killed tissues.
NSCliving control : Non-Specific Colour control in living tissues.
NSMTT: Non-Specific MTT reduction.
OD: Optical Density
PC: Positive Control, a replicate containing all components of a test system and treatedwith a substance known to induce a positive response. To ensure that variability in thepositive control response across time can be assessed, the magnitude of the positive
response should not be excessive.
Performance standards (PS): Standards, based on a validated test method, that provide abasis for evaluating the comparability of a proposed test method that is mechanistically andfunctionally similar. Included are; (i) essential test method components; (ii) a minimum listof Reference Chemicals selected from among the chemicals used to demonstrate theacceptable performance of the validated test method; and (iii) the similar levels of
reliability and accuracy, based on what was obtained for the validated test method, that the
proposed test method should demonstrate when evaluated using the minimum list ofReference Chemicals (25).
Relevance: Description of relationship of the test method to the effect of interest andwhether it is meaningful and useful for a particular purpose. It is the extent to which thetest method correctly measures or predicts the biological effect of interest. Relevance
incorporates consideration of the accuracy (concordance) of a test method (25).
Reliability: Measures of the extent that a test method can be performed reproducibly within
and between laboratories over time, when performed using the same protocol. It is assessedby calculating intra- and inter-laboratory reproducibility (25).
Run: A run consists of one or more test chemicals tested concurrently with a negative
control and with a PC.
Sensitivity: The proportion of all positive/active chemicals that are correctly classified by
the test method. It is a measure of accuracy for a test method that produces categoricalresults, and is an important consideration in assessing the relevance of a test method (25).
Skin corrosion in vivo: The production of irreversible damage of the skin; namely, visible
necrosis through the epidermis and into the dermis, following the application of a test
chemical for up to four hours. Corrosive reactions are typified by ulcers, bleeding, bloody
scabs, and, by the end of observation at 14 days, by discoloration due to blanching of theskin, complete areas of alopecia, and scars. Histopathology should be considered toevaluate questionable lesions.
Specificity: The proportion of all negative/inactive chemicals that are correctly classified
by the test method. It is a measure of accuracy for a test method that produces categoricalresults and is an important consideration in assessing the relevance of a test method (25).
Substance: means chemical elements and their compounds in the natural state or obtainedby any production process, including any additive necessary to preserve the stability of the©OECD 2019

51

22 │

431

OECD/OCDE

product and any impurities deriving from the process used, but excluding any solvent which
may be separated without affecting the stability of the substance or changing its
composition.
Test chemical: means what is being tested.
UPLC: Ultra-High Performance Liquid Chromatography.
UVCB: substances of unknown or variable composition, complex reaction products or
biological materials.

©OECD 2019

52

53

Treatment doses
and application

At least 2 per exposure time

Number oftissue replicates

mg/cm2) with a nylon meshg/L)
Waxy/sticky: 50 ± 2 mg (131.6

Liquids and viscous: 50 ± 3 μL
(131.6 μL/cm2)
Solids: 20± 2 mg (52.6 mg/cm2)+100 μL±5μL NaCl solution (90.38 cm2

EpiSkin™

1

Model surface

Test Method
Component

Nr.

At least 2 per exposure time

Liquids and viscous:40 ± 3 μL
(80μL/cm2) using nylonmesh
Pre-test compatibility of test
chemical with nylon mesh
Solids: 20 μL ± 2μl H2O + 20± 3
mg (40 mg/cm2)
Waxy/sticky: 20 ± 3 mg (40mg/cm2) with a nylon mesh

Liquids: 50 μL (79.4 μL/cm2)
with or without a nylon mesh
Pre-test compatibility of test
chemical with nylon meshSemi solids: 50 μL (79.4 μL/cm2)
Solids: 25 μL H2O (or necessary)+ 25 mg (39.7 mg/cm2)
Waxes: flat “disc like” piece of
ca. 8 mm diameter placed atop
the tissue wetted with 15μL H2O.

0.5 cm2

SkinEthic™ RHE

32-3 per exposure time

0.63 cm2

EpiDerm™ SCT

2

Liquids and viscous:50 μL(166.7μL/cm2)
Solids: 50± 2 mg (166.7 mg/cm2)+ 50 μL H2O
Waxy: Use a positive
displacement pipette and tip asliquid and viscous substance.

Liquids and viscous:50 μL(83.3μL/cm2) using nylonmesh
Pre-test compatibility of test
chemical with nylon meshSemi solids: 50 μL (83.3 μL/cm2)
Solids: 25 mg (41.7 mg/cm2) +25 μL H2O (or more if necessary)
Waxy/sticky: flat “cookie like”
piece of ca. 8 mm diameter
placed atop the tissue wettedwith 15μL H2O©OECD 2019

At least 2 per exposure time

0.3 cm2

LabCyte EPI-MODEL24 SCT

5431 │ 23

At least 2 per exposure time

0.6 cm2

epiCS®

4

ANNEX 2 - MAIN TEST METHOD COMPONENTS OF THE RhE TEST METHODS VALIDATED FOR SKIN
CORROSION TESTING

OECD/OCDE

54

3 min, 60 min (±5 min)and 240 min (±10 min)In ventilated cabinet
Room Temperature (RT, 1828oC)

Exposure time
and temperature

300 μL 1 mg/mL

50 μL 8N KOH
Tested with every exposure time
300 μL 1 mg/mL

50 μL Glacial acetic acid
Tested only for 4 hours

2 mL 0.3 mg/mL

180 min (±15 min) at 37oC, 5%

Positive control

MTT solution

MTT incubation

180 min at 37oC, 5% CO2, 95%

40 μL 8N KOH
Tested only for 1 hour

50 μL H2O
Tested with every exposure time

50 μL NaCl solution (9 g/L)
Tested with every exposure time

Negative control

180 min (±15 min) at 37oC, 5%40 μL H2O
Tested with every exposure time

20 times with a constant soft
stream of 1x PBS

20 times with a constant soft
stream of 1x PBS

3 min at RT,
and 60 min at 37oC, 5% CO2,
95% RH

40 μL (liquid) or 20 mg (solid)+ 300 μL H2O mixed for 60 min
at RT
 if solution becomes
coloured, living adapted
controls should be
performed

40 μL (liquid) or 20 mg (solid)+ 1 mL MTT 1 mg/mL solution
for 180±15 min at 37oC, 5%
CO2, 95% RH
 if solution turns
blue/purple, freeze-killed
adapted controls should be
performed

SkinEthic™ RHE

3

25 mL 1x PBS (2 mL/throwing)

Rinsing

3 min at RT,
and 60 min at 37oC, 5% CO2,
95% RH

50 μL (liquid) or 25 mg (solid)+ 300 μL H2O mixed for 60 min
at 37oC, 5% CO2, 95% RH
 if solution becomes
coloured, living adapted
controls should be
performed

10 μL (liquid) or 10 mg (solid)+ 90μL H2O mixed for 15 min atRT if solution becomes
coloured, living adapted
controls should be
performed

Pre-check forcolourinterference

Pre-check fordirect MTT
reduction

50 μL (liquid) or 25 mg (solid)+ 1 mL MTT 1 mg/mL solution
for 60 min at 37oC, 5% CO2,
95% RH if solution turns
blue/purple, freeze-killed
adapted controls should be
performed

2

50 μL (liquid) or 20 mg (solid)+ 2 mL MTT 0.3 mg/mL
solution for 180±5 min at 37oC,
5% CO2, 95% RH
 if solution turns
blue/purple, water-killed
adapted controls should be
performed

1

OECD/OCDE
EpiDerm™ SCT

Test Method
Component

Nr.

431

EpiSkin™

24 │180 min at 37oC, 5% CO2, 95%

300 μL 1 mg/mL

50 μL 8N KOH
Tested with every exposure time

50 μL H2O
Tested with every exposure time

20 times with a constant soft
stream of 1x PBS

3 min at RT,
and 60 min at 37oC, 5% CO2,
95% RH

50 μL (liquid) or 25 mg (solid)+ 300 μL H2O mixed for 60 min
at 37oC, 5% CO2, 95% RH
 if solution becomes
coloured, living adapted
controls should be
performed

50 μL (liquid) or 25 mg (solid)+ 1 mL MTT 1 mg/mL solution
for 60 min at 37oC, 5% CO2,
95% RH if solution turns
blue/purple, freeze-killed
adapted controls should be
performed

epiCS®

4©OECD 2019

180 min (±5 min) at 37oC, 5%

500 μL 0.5 mg/mL

50 μL 8N KOH
Tested only for 1 hour

50 μL H2O
Tested with every exposure time

10 times or more with a constantstrong stream of 1x PBS

3 min at RT,
and 60 min at 37oC, 5% CO2,
95% RH

50 μL (liquid) or 50 mg (solid)+ 500 μL H2O mixed for 60 min
at 37oC, 5% CO2, 95% RH
 if solution becomes
coloured, living adapted
controls should be
performed

50 μL (liquid) or 50 mg (solid)+ 500 μL MTT 0.5 mg/mL
solution for 60 min at 37oC, 5%
CO2, 95% RH
 if solution turns
blue/purple, freeze-killed
adapted controls should be
performed

LabCyte EPI-MODEL24 SCT

555

1.5 mL isopropanol(extraction from top and bottom
of insert)
Overnight without shaking at RT
or for 120 min with shaking(~120 rpm) at RT
570 nm (540 - 600 nm)
without reference filter
Treatment with 1% Triton X-100
4.0 hours ≤ ET50 ≤ 10.0 hours
1. Mean OD of the tissue
replicates treated with thenegative control (H2O) shouldbe ≥ 0.8 and ≤ 3.0 for every
exposure time
2. Mean viability of the tissue
replicates exposed for 1 hour(and 4 hours, if applicable)with the positive control (8NKOH), expressed as % of thenegative control, should be ≤15%
3. In the range 20-100% viability
and for ODs ≥ 0.3, differenceof viability between the two

2 mL isopropanol(extraction from top and bottom
of insert)
Overnight without shaking at RT
or for 120 min with shaking(~120 rpm) at RT
570 nm (or 540 nm)
without reference filter
Treatment with 1% Triton X-100
4.08 hours ≤ ET50 ≤ 8.7 hours
1. Mean OD of the tissue
replicates treated with thenegative control (H2O) shouldbe ≥ 0.8 and ≤ 2.8 for every
exposure time
2. Mean viability of the tissue
replicates exposed for 1 hourwith the positive control (8NKOH), expressed as % of thenegative control, should be ≤15%
3. In the range 20 - 100%
viability, the Coefficient of
Variation (CV) between tissue
replicates should be ≤ 30%

500 μL acidified isopropanol(0.04 N HCl in isopropanol)(isolated tissue fully immersed)

Overnight at RT, protected fromlight

570 nm (545 - 595 nm)
without reference filter

18 hours treatment with SDS
1.0mg/mL ≤ IC50 ≤ 3.0mg/mL

1. Mean OD of the tissue
replicates treated with thenegative control (NaCl) shouldbe ≥ 0.6 and ≤ 1.5 for every
exposure time
2. Mean viability of the tissue
replicates exposed for 4 hours
with the positive control(glacial acetic acid), expressed
as % of the negative control,
should be ≤ 20%
3. In the range 20-100% viability
and for ODs ≥ 0.3, differenceof viability between the twotissue replicates should not

Extraction time
And temperature

OD reading

Tissue Quality
Control

Acceptability
Criteria

SkinEthic™ RHE EIT

CO2, 95% RH

SkinEthic™ RHE

3

Extraction
solvent

RH

EpiDerm™ SCT

2

EpiDerm™ SCT

CO2, 95% RH

EpiSkin™

1

EpiSkin™ EIT

Test Method
Componenttime andtemperature

Test Method
Component

Nr.

OECD/OCDE

1. Mean OD of the tissue
replicates treated with thenegative control (H2O) shouldbe ≥ 0.8 and ≤ 2.8 for every
exposure time
2. Mean viability of the tissue
replicates exposed for 1 hourwith the positive control (8NKOH), expressed as % of thenegative control, should be ≤15%.
3. In the range 20-100% viability
and for ODs ≥ 0.3, differenceof viability between the twotissue replicates should not

Treatment with 1% Triton X-100
2.0 hours ≤ ET50 ≤ 7.0 hours

540 - 570 nm
without reference filter

Overnight without shaking at RT
or for 120 min with shaking(~120 rpm) at RT

2 mL isopropanol(extraction from top and bottom
of insert)

epiCS®

RH

epiCS®

4©OECD 2019

1. Mean OD of the tissue
replicates treated with thenegative control (H2O) shouldbe ≥ 0.7 and ≤ 2.5 for every
exposure time
2. Mean viability of the tissue
replicates exposed for 1 hourwith the positive control (8NKOH), expressed as % of thenegative control, should be ≤15%.
3. In the range 20-100% viability
and for ODs ≥ 0.3, differenceof viability between the twotissue replicates should not

18 hours treatment with SDS
1.4mg/mL ≤ IC50 ≤ 4.0 mg/mL

570 nm with reference filter 650
nm

Overnight at RT, protected fromlight

300 μL isopropanol(isolated tissue fully immersed)

LabCyte EPI-MODEL24 SCT

CO2, 95% RH

LabCyte EPI-MODEL24 SCT

5431 │ 25

56

26 │

Test Method
Component

Nr.

431

exceed 30%.

EpiSkin™

1
EpiDerm™ SCT

2tissue replicates should notexceed 30%.

SkinEthic™ RHE

3

OECD/OCDE
exceed 30%.

epiCS®

4©OECD 2019

exceed 30%.

LabCyte EPI-MODEL24 SCT

5OECD/OCDE

431 │ 27

ANNEX 3 - PERFORMANCE OF TEST METHODS FOR SUBCATEGORISATION
The table below provides the performances of the five test methods calculated based on aset of 79 or 80 chemicals tested by the five test developers. Calculations of four test
methods (EpiSkin™, EpiDerm™ SCT, SkinEthic™ RHE and epiCS®) were performed by
the OECD Secretariat, reviewed and agreed by an expert subgroup (21) (23). Calculationof LabCyte EPI-MODEL24 SCT was performed by the test developer, reviewed and agreed
by the validation management group and a peer review panel (41) (43).
STATISTICS ON PREDICTIONS OBTAINED ON THE ENTIRE SET OF CHEMICALS(n= 80 chemicals tested over 2 independent runs for epiCS® or 3 independent runs for
EpiDerm™ SCT, EpiSkin™ and SkinEthic™RHE i.e. respectively 159* or 240
classifications.
n= 79** chemicals tested over 3 independent runs for LabCyte EPI-MODEL24 SCT, i.e.
237 classification.)*one chemical was tested once in epiCS® because of no availability (23).
** one chemical was not tested in LabCyte EPI-MODEL24 SCT because of no availability.
EpiSkin EpiDerm SkinEthic epiCS
LabCyteEPIMODEL24
Overclassifications:
1B-and-1C overclassified 1A
21.5%
29.0%
31.2%
32.8%
30.0%
NC overclassified 1B-and-1C
20.7%
23.4%
27.0%
28.4%
18.9%
NC overclassified 1A
0.0%
2.7%
0.0%
0.0%
2.7%
Overclassified as Corrosive
20.7%
26.1%
27.0%
28.4%
21.6%
Global overclassification rate (all 17.9%
23.3%
24.5%
25.8%
21.5%
categories)
Underclassifications:
1A underclassified 1B-and-1C
16.7%
16.7%
16.7%
12.5%
13.9%
1A underclassified NC
0.0%
0.0%
0.0%
0.0%
0.0%
1B-and-1C underclassified NC
2.2%
0.0%
7.5%
6.6%
0.0%
Global underclassification rate
3.3%
2.5%
5.4%
4.4%
2.1%
(all categories)
Correct Classifications:
1A correctly classified
83.3%
83.3%
83.3%
87.5%
86.1%
1B-and-/1C correctly classified
76.3%
71.0%
61.3%
60.7%
70.0%
NC correctly classified
79.3%
73.9%
73.0%
71.62% 78.4%
78.8%
74.2%
70.0%
69.8%
76.4%
Overall Accuracy©OECD 2019

57

28 │

431

OECD/OCDEANNEX 4 - Key parameters and acceptance criteria for qualification of an
HPLC/UPLC-spectrophotometry system for measurement of MTT formazan
extracted from RhE tissues
Parameter
Selectivity

Accuracy

Protocol Derived from FDA Guidance (36)(38)
Analysis of isopropanol, living blank (isopropanol
extract from living RhE tissues without any
treatment),
dead blank (isopropanol extract from killed RhE
tissues without any treatment)
Quality Controls (i.e., MTT formazan at 1.6 g/mL, 16
g/mL and 160 g/mL ) in isopropanol (n=5)
Quality Controls in isopropanol (n=5)

Matrix EffectQuality Controls in living blank (n=5)

CarryoverAnalysis of isopropanol after an ULOQ2 standard

Reproducibility(intra-day)

3 independent calibration curves (based on 6
consecutive 1/3 dilutions of MTT formazan in
isopropanol starting at ULOQ, i.e., 200 g/mL);
Quality Controls in isopropanol (n=5)
Day 1: 1 calibration curve and Quality Controls in
isopropanol (n=3)
Day 2: 1 calibration curve and Quality Controls in
isopropanol (n=3)
Day 3: 1 calibration curve and Quality Controls in
isopropanol (n=3)
Quality Controls in living blank (n='3)' analysed the
day of the preparation and after 24 hours of storage
atroom temperature
Quality Controls in living blank (n='3)' analysed
theday of the preparation and after several days ofstorageat a specified temperature (e.g., 4ºC, -20ºC, 80ºC)

Precision

Reproducibility(inter-day)

Short Term
Stability of MTT
Formazan in RhE
Tissue Extract
Long Term
Stability of MTT
Formazan in RhE
Tissue Extract, if
required

Acceptance Criteria
Areainterference = 20%
of AreaLLOQ1

CV = 15% or = 20%
for the LLOQ
%Dev = 15% or =20% for LLOQ
85% = %Matrix
Effect= 115%
Areainterference = 20%
of AreaLLOQ
Calibration
Curves:%Dev = 15%
or = 20% for LLOQ
Quality Controls:%Dev= 15% and CV= 15%%Dev = 15%%Dev = 15%

Note:
1LLOQ: Lower Limit of Quantification, defined to cover 1-2% tissue viability, i.e., 0.8 μg/mL.
2ULOQ: Upper Limit of Quantification, defined to be at least two times higher than the highest expected MTT
formazan concentration in isopropanol extracts from negative controls i.e., 200 μg/mL.

©OECD 201958