᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 29 ᖺ 6 ᭶ 1 ᪥                                 No. 2017-01⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࣄࢺ⾲⓶ࣔࢹࣝἲ࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 29 ᖺ 5 ᭶ 11 ᪥࡟ᮾிࠊᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸JaCVAM ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢᥦ᱌ࡀ࡞ࡉࢀࡓࠋ  ᥦ᱌ෆᐜ㸸 ࣄࢺ⾲⓶ࣔࢹࣝ㸦EpiSkin™ࠊEpiDerm™ࠊSkinEthic™ࠊepiCS®㸧ࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂ἲ࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊ⿕㦂≀㉁ࢆ⭉㣗ᛶ≀㉁㸦ᅜ㐃 GHS
ศ㢮࡟࠾ࡅࡿ༊ศ 1㸧࡜ุᐃࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿࡀࠊUN GHS ศ㢮ࡢ⣽༊ศࡢࡓࡵ࡟ࡣ EpiSkinTM ࡢࡳ฼⏝ྍ⬟࡛࠶ࡿࠋ࡞࠾ࠊᮏヨ㦂ἲࡢ฼⏝࡟࠶ࡓࡗ࡚ࡣࠊ㐺⏝⠊ᅖࢆ༑ศ࡟㓄៖ࡋࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ
ࡇࡢᥦ᱌᭩ࡣࠊOrganisation for Economic Co-operation and Development (OECD) Test Guideline(TG) 431 In vitro skin corrosion: reconstructed human epidermis (RHE) test methodࢆࡶ࡜࡟ࠊ⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚ࠊJaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࣄࢺ⾲⓶ࣔࢹࣝἲࡢ౑
⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ ኱㔝Ὀ㞝                      すᕝ⛅ె
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗i

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧㔝 ཱྀ  ┿ ᕼ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥
                 
ii

JaCVAM 㐠Ⴀጤဨ఍す ᕝ ⛅ ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧㸸ጤဨ㛗ᕝ す  ᚭ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧኱ ཎ  ᣅ㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ㸧ᑠᕝஂ⨾Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ຍ ⸨  ⠜㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧ㅋ ⏣ Ὀ ᡂ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧᪥ୗ㒊ဴஓ㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧⠛ ⏣ ࿴ ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧㧗 ᮌ ⠜ ஓ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊 ື≀⟶⌮ᐊ㸧᮰ 㔝 ṇ ᫂㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧᪥ ⏣  ඘㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᖹ ᯘ ᐜ Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧ᗈ ℩ ᫂ ᙪ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶண ホ౯㒊㸧ᘅ ⏣ ග ᜨ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮏ 㛫 ṇ ඘ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧ᑠ ᓥ  ⫕ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊➨஧ᐊ㸧㸸஦ົᒁ

iii

iv

JaCVAM Statement on the
In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method
At a meeting held on 11 May 2017 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 EpiSkinTM, EpiDermTM, SkinEthicTM, or epiCS® is generally
considered sufficient for predicting a test chemical to cause skin corrosion under
UN GHS Category 1, only skin corrosion tests using EpiSkin are consideredsufficient for predicting a test chemical to cause skin corrosion under the UN GHS
subcategories. Furthermore, thorough consideration must be given to theapplicability 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: Reconstructed Human
Epidermis (RHE) Test Method as a useful means for assessing skin corrosion potential duringsafety assessments by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

June 1, 2017v

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. Yasuo Ohno (Kihara Memorial Yokohama Foundation for the Advancement ofLife Sciences) : Chairperson
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology)
Mr. Yuji Ishii (Biological Safety Research Center: BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Shunji Nakai (Japan Chemical Industry Association)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Ms. Maki Noguchi (Pharmaceuticals and Medical Devices Agency)
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Term: From 1st April 2016 to 31st March 2018vi

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

Mr. Akiyoshi Nishikawa (BSRC, NIHS): Chairperson
Mr. Toru Kawanishi (NIHS)
Mr. Mitsuru Hida (Ministry of Health, Labour and Welfare)
Ms. Yoko Hirabayashi (Division of Toxicology, BSRC, NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Ms. Mitsue Hirota (Pharmaceutical & Medical Devices Agency)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Yasunari Kanda (Division of Pharmacology, BSRC, NIHS)
Mr. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Tetsuya Kusakabe (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Taku Oohara (Ministry of Health, Labour and Welfare)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology, BSRC,
NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretary

vii

viii

⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࣄࢺ⾲⓶ࣔࢹࣝἲ┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------- 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------- 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 431,

In vitro skin corrosion: reconstructed human epidermis (RHE) test
method -----------------------------------------------------------------------------------------------35
PERFORMANCE STANDARDS FOR THE ASSESSMENT OF PROPOSEDSIMILAR OR MODIFIED IN VITRO RECONSTRUCTED HUMANEPIDERMIS (RHE) TEST METHODS FOR SKIN CORROSION TESTING
AS DESCRIBED IN TG 431 -----------------------------------------------------------------61ix

xホ౯఍㆟ሗ࿌᭩

⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࣄࢺ⾲⓶ࣔࢹࣝἲJaCVAM ホ౯఍㆟

ᖹᡂ 29 ᖺ㸦2017 ᖺ㸧5 ᭶ 11 ᪥

11

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ୖᬛᙲ㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒஭ᩍᏳ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰஭ಇྖ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧㔝 ཱྀ ┿ ᕼ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟⃝ ⪽㸦᪥ᮏẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥22ࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂ἲࡣࠊ࢘ࢧࢠࢆ⏝࠸ࡿ⓶⭵⭉㣗ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚㛤Ⓨࡉࢀࡓヨ㦂ἲ࡛࠶ࡿࠋᮏヨ㦂ἲ࡛ࡣࠊ⭉㣗ᛶ≀㉁ࡀゅ㉁ᒙࢆയᐖࡲࡓࡣゅ㉁ᒙ࡟྾཰ࡉࢀࡓᚋᣑᩓࡋࠊ⾲⓶⣽⬊࡟฿㐩ࡋ࡚⣽⬊ẘᛶࢆ♧ࡍ࡜࠸࠺௬ㄝ࡟ᇶ࡙ࡁࠊ⿕㦂≀㉁᭚㟢ᚋࡢ⣽⬊⏕Ꮡ⋡ࢆᣦᶆ࡟⓶⭵⭉㣗ᛶࢆホ౯ࡍࡿࠋ
ᮏヨ㦂ἲ࡟ࡘ࠸࡚ࡣࠊEpiSkin™ࠊEpiDerm™ࠊSkinEthic™ࠊepiCS®࡜࠸࠺㸲✀ࡢ⾲⓶ࣔࢹࣝࡀ࠶ࡆࡽ
ࢀ࡚࠸ࡿࠋ࠸ࡎࢀࡢࣔࢹࣝ࡟ࡼࡿヨ㦂ࡶࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࠊECVAM㸦European Centre for
the Validation of Alternative Methods㸸Ḣᕞ௦᭰ἲホ౯ࢭࣥࢱ࣮㸧࡟ࡼࡾࡑࡢಙ㢗ᛶ࡜෌⌧ᛶࡀ㧗࠸ࡇ࡜ࡀ☜ㄆࡉࢀࡓ 1~4)ࠋEpiSkin™ࠊEpiDerm™࡟࠾࠸࡚ࡣࠊESAC㸦ECVAM Scientific Advisory Committee㸸ECVAM⛉Ꮫㅎၥ఍㆟㸧࡛ࡢホ౯ᚋࠊࡑࡢ⤖ㄽࡣ ICCVAM㸦Interagency Coordinating Committee on the Validation of
Alternative Methods㸸⡿ᅜ௦᭰ἲ࡟㛵ࡍࡿ┬ᗇ㛫㐃⤡఍㆟㸧࡟࠾࠸࡚ࡶ☜ㄆࡉࢀࡓ 5,6) ࠋୖグ 4 ✀ࣔࢹࣝ࡟ࡘ࠸࡚ࡣ OECD㸦Organisation for Economic Co-operation and Development㸸⤒῭༠ຊ㛤Ⓨᶵᵓ㸧࡟࡚ࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ㸦TG㸧431 ࡜ࡋ࡚ᢎㄆࡉࢀࡓࠋࡇࡢ TG ࡣ᫖௒✀ࠎࡢⅬ࡛ᨵゞࡉࢀ࡚࠾ࡾࠊ⌧ᅾࡣ 2016ᖺ∧࡜࡞ࡗ࡚࠸ࡿ 7)ࠋ
JaCVAM ホ౯఍㆟ࡣࠊ⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࡢホ౯ሗ࿌᭩ࠖ8)ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 ࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲ௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 ࢘ࢧࢠࢆ⏝࠸ࡿ⓶⭵⭉㣗ᛶヨ㦂ヨ㦂ἲࡢᴫ␎㸸ᮏヨ㦂ἲ࡛ࡣࠊ࢘ࢧࢠ⓶⭵ࡢ௦ࢃࡾ࡟ゅ㉁ᒙࢆ᭷ࡍࡿ 3 ḟඖ෌ᵓ⠏ࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࠊ⿕㦂≀㉁ࡀゅ㉁ᒙୗࡢ⾲⓶⣽⬊ᒙ࡟ᑐࡋ⣽⬊ẘᛶࢆ♧ࡍ⬟ຊࢆホ౯ࡍࡿࠋ⾲⓶ࣔࢹࣝ⾲㠃࡟⿕㦂≀㉁ࢆ୍ᐃ᫬㛫㐺⏝ࡋࡓᚋὙίࡋࠊ⾲⓶⣽⬊ࡢ⏕Ꮡ⋡ࢆ MTTࠝ3 ( 4, 5 - Dimethylthiazol - 2 - yl ) - 2, 5-diphenyltetrazolium bromideࠞࡢ㑏ඖ㔞࠿ࡽồࡵࠊ⓶⭵⭉㣗ᛶࢆุᐃࡍࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱཬࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶᮏヨ㦂ἲࡣࠊ4 ✀ࡢ⾲⓶ࣔࢹࣝࡢ࠸ࡎࢀࡢࣔࢹࣝࡶࠊECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡜ࡑࢀ࡟⥆ࡃ ESAC ࡟ࡼࡿ➨୕⪅ホ౯࡟ࡼࡾࠊᐇ㦂ື≀ࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚⛉Ꮫⓗ࡟ጇᙜ࡛࠶
ࡿ࡜ሗ࿌ࡉࢀ࡚࠾ࡾ 1~4)ࠊEpiSkin™࠾ࡼࡧ EpiDerm™ࢆ⏝࠸ࡓヨ㦂ἲ࡟ࡘ࠸࡚ࡣ ICCVAM ࡟࠾࠸࡚ࡶྠ
ᵝ࡟☜ㄆࡉࢀࡓ 6)ࠋୖグ 4 ✀ࡢ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡿ᪉ἲ࡟ࡘ࠸࡚ࡣ⌧ᅾ OECD ࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ
࡚ᢎㄆࡉࢀ࡚࠸ࡿ 7)ࠋJaCVAM ⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡛ࡣࠊ⌧ᅾࡲ࡛බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗ 1~7,9~11)ࢆᇶ࡟ࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ホ౯ࡋࡓࠋࡑࡢ⤖ᯝࠊᮏヨ㦂ἲࡣࠊࠕ⭉㣗ᛶ≀㉁ࡀゅ㉁ᒙࡢയᐖࡲࡓࡣゅ㉁ᒙ࡬ࡢ྾཰ࢆ⤒࡚ୗᒙࡢ⾲⓶⣽⬊࡟ᑐࡋẘᛶࢆ♧ࡍࠖ࡜࠸࠺⓶⭵⭉㣗ᛶⓎ⌧ᶵᗎ࡟ᇶ࡙ࡁࠊ⣽⬊࡬ࡢẘᛶࢆᣦᶆ࡟ࡋࡓࡶࡢ࡛࠶ࡾࠊཎ⌮ⓗ࡟ࡶጇᙜ࡛࠶ࡿ࡜ุ᩿ࡉࢀࡓࠋ

3
3

㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
࠸ࡎࢀࡢ⾲⓶ࣔࢹࣝࡶື≀ࢆ౑⏝ࡋ࡚࠾ࡽࡎࠊື≀⚟♴㠃࠿ࡽ௦᭰ἲ࡜ࡋ࡚ጇᙜ࡛࠶ࡿࠋᙉ࠸㓟ᛶ㸦pH
2.0 ௨ୗ㸧ࡲࡓࡣ࢔ࣝ࢝ࣜᛶ㸦pH 11.5 ௨ୖ㸧≀㉁ࡣࠊᙉ࠸ᒁᡤയᐖࢆᘬࡁ㉳ࡇࡍྍ⬟ᛶࡀ࠶ࡿࡇ࡜࠿ࡽࠊ
⓶⭵⭉㣗ᛶ࡜ุ᩿ࡋ࡚ࡶⰋ࠸ࡇ࡜࡟࡞ࡗ࡚࠸ࡿ 9)ࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡇࢀࡣ⭉㣗ᛶ࡟ࡘ࠸࡚ࡢ᝟ሗࡀ௚࡟
࡞࠸ሙྜ࡟⾜ࢃࢀࡿ࣮࣡ࢫࢺࢣ࣮ࢫ࡜ࡋ࡚ࡢุ᩿࡛࠶ࡾࠊ౛࠼ࡤ⦆⾪స⏝ࡀᑠࡉ࠸≀㉁ࡸΰྜ≀ࡢሙྜࡣഇ㝧ᛶࡢุ᩿࡜࡞ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿ 10)ࠋࡇࡢࡓࡵࠊࡇࡢࡼ࠺࡞≀㉁࡟ᑐࡋ࡚ࡶື≀ࢆ⏝࠸ࡿᚲせࡢ࡞࠸ᮏヨ㦂ἲ࡛⓶⭵⭉㣗ᛶࢆホ౯ࡍࡿࡇ࡜ࡣ᭷⏝࡛࠶ࡿࠋ࠸ࡎࢀࡢࣔࢹࣝࡶ UN GHS (United Nations Globally Harmonized System of Classification and Labelling ofChemicals㸸ᅜ㐃 GHS) ศ㢮࡟࠾ࡅࡿ⭉㣗ᛶ㸦༊ศ 1㸧࡟ࡘ࠸࡚ࠊࡑࡢ᭷↓ࡢணᛶࡣ㧗ࡃࠊOECD ࡛㞟ィࡋࡓ໬Ꮫ≀㉁࡛ࡣࠊឤᗘࡣ 95㹼100㸣ࠊ≉␗ᗘࡣ 72㹼79㸣ࠊṇ☜ᗘࡣ 84㹼90%࡛࠶ࡾࠊഇ㝜ᛶ⋡ࡣ 0
㹼5%࡜ప࠸࡛ࣞ࣋ࣝ࠶ࡗࡓ㸦⾲ 1㸧ࠋྛࣔࢹࣝ࡟ࡼࡿᅜ㐃 GHS ศ㢮ࡢ⣽༊ศ㸦1A㹼1C㸧ࡢணᛶ࡟ࡘ࠸࡚ࡣࠊ80 ≀㉁ࢆ 2 ࡲࡓࡣ 3 ᅇᐇ㦂ࡋ࡚ᚓࡽࢀࡓ್ࢆぢࡿ㝈ࡾࠊEpiSkinTM ࡢࡳࡀ 1B/1C ࢆ 80㸣⛬ᗘ㆑ูྍ⬟࡛࠶ࡗࡓࠋࡑࡢ௚ࡢࣔࢹࣝࡣ⣽༊ศࡢホ౯࡟ࡣ฼⏝࡛ࡁ࡞࠸࡜ุ᩿ࡋࡓࠋ
MTT 㑏ඖ≀㉁࡟ᑐࡋ࡚ࡣࠊTG431 ࡟ᑐᛂ᪉ἲࡀグ㍕ࡉࢀ࡚࠾ࡾࠊࡇࢀ࡟‽ࡌࡿࡇ࡜࡛ホ౯ྍ⬟࡛࠶ࡿࠋ࢞ࢫࠊ࢚࢔ࣟࢰ࣮ࣝ࡟ࡘ࠸࡚ࡣࣂࣜࢹ࣮ࢩࣙࣥࡀᐇ᪋ࡉࢀ࡚࠾ࡽࡎࠊ㐺⏝ྍྰࡣุ᩿࡛ࡁ࡞࠸ࠋࡑ
ࢀࡽ௨እࡢ≀㉁࡟ࡘ࠸࡚ࡣࠊ≀⌮≧ែ㸦ᾮయ࣭ᅛయ➼㸧࠾ࡼࡧỈ⁐ᛶࡢ᭷↓࡟࠿࠿ࢃࡽࡎ㐺⏝ྍ⬟࡛࠶ࡾࠊΰྜ≀࡟ࡶ㐺⏝ྍ⬟࡞ሙྜࡀ࠶ࡿࠋ
⾲ 1㸬໬Ꮫ≀㉁ࡢ඲ࢭࢵࢺ*࡟࠾ࡅࡿண

ᛶ㸦⭉㣗ᛶ [ᅜ㐃 GHS ༊ศ㸯] ࡢ᭷↓㸧11)

EpiSkinTM

EpiDermTM

SkinEthicTM

epiCS®

ṇ☜ᗘ

89.6㸣

87.9%

84.6㸣

84.3㸣

ឤᗘ

98.5㸣

100%

94.6㸣

95.3㸣

≉␗ᗘ

79.3㸣

73.9%

73.0㸣

71.6㸣

ഇ㝧ᛶ⋡

20.7%

26.1%

27.0%

28.4%

ഇ㝜ᛶ⋡

1.5%

0%

5.4%

4.7%

*) OECD ࡢ㞟ィ࡟ࡼࡿࠋ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶཬࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣࠊ㏻ᖖࡢᇵ㣴ᢏ⾡ࢆ⩦⇍ࡋࡓ᪋タ࡛࠶ࢀࡤᐇ᪋࡛ࡁࡿヨ㦂ἲ࡛࠶ࡾࠊOECD TG431 ࡟ᢎㄆࡉࢀࡓ⾲⓶ࣔࢹࣝࡣᕷ㈍ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠾ࡾࠊ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲ࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊ⿕㦂≀㉁ࢆ⭉㣗ᛶ≀㉁㸦ᅜ㐃 GHS ศ㢮࡟࠾ࡅࡿ༊ศ
1㸧࡜ุᐃࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿࡀࠊUN GHS ศ㢮ࡢ⣽༊ศࡢࡓࡵ࡟ࡣ EpiSkinTM ࡢࡳ฼⏝ྍ⬟࡛࠶ࡿࠋ࡞࠾ࠊᮏヨ㦂ἲࡢ฼⏝࡟࠶ࡓࡗ࡚ࡣࠊ㐺⏝⠊ᅖࢆ༑ศ࡟㓄៖ࡋࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ

4
4

ཧ⪃ᩥ⊩
1)

ECVAM (1998) Statement on the scientific validity of the EpiSkin test (An in vitro test for skin corrosivity)

2)

ECVAM (2000) Statement on the application of the EpiDermTM human skin model for skin corrosivity testing

3)

ECVAM (2006) Statement on the application of the SkinEthicTM human skin model for skin corrosivity testing

4)

ECVAM (2009) ESAC Statement on the scientific validity of an in-vitro test for skin corrosivity testing

5)

ICCVAM (1999) NIH Publication No.99-4495. Corrositex: An in vitro test method for assessing dermal
corrosivity potential of chemicals.

6)

ICCVAM (2002) NIH Publication No.02-4502. ICCVAM Evaluation of EPISKINTM, and EpiDermTM (EPI200) and rat skin transcutaneous electrical resistance (TER) assay: in vitro test method for assessing dermal
corrosivity potential of chemicals.

7)

OECD (2016) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model test.

8)

JaCVAM ⓶⭵⭉㣗ᛶ㈨ᩱ⦅⧩ጤဨ఍㸸⓶⭵⭉㣗ᛶヨ㦂ホ౯ሗ࿌᭩ ࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࡢホ౯ሗ࿌᭩㸦2017 ᖺ 2 ᭶ 24 ᪥㸧㸬

9)OECD (2014) Series on Testing and Assessment No. 203, Guidance document on an Integrated Approach on
Testing and Assessment (IATA) for skin corrosion and irritation

10) Sheel J. et.al., Classification and labeling of industrial products with extreme pH by making use of in vitro
methods for the assessment of skin and eye irritation and corrosion in a weight of evidence approach,Toxicology in Vitro, 25, 1435-1447 (2011)11) OECD (2015) 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

5
5

6

ホ౯ሗ࿌᭩

⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࣄࢺ⾲⓶ࣔࢹࣝἲ

⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ ᖹᡂ 29 ᖺ㸦2017 ᖺ㸧2 ᭶ 24 ᪥

17⓶⭵⭉㣗ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㧘ᶫ ♸ḟ

ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ẘᛶ㒊୰ᮧࡿࡾࡇ⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ ໬Ꮫ≀㉁⟶⌮ࢭࣥࢱ࣮㡲᪉ ╩ኵ

ఫ཭໬Ꮫᰴᘧ఍♫ ࣞࢫ࣏ࣥࢩࣈࣝࢣ࢔㒊㸦໬ᏛရᏳ඲ࢢ࣮ࣝࣉ㸧㸭᪥ᮏ໬Ꮫᕤᴗ༠఍ᑠᓥ ⫕ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶண

2

8

ホ౯㒊

せ᪨
࢘ࢧࢠࢆ⏝࠸ࡿ⓶⭵⭉㣗ᛶヨ㦂ࡢື≀ᐇ㦂௦᭰ἲ㸦௦᭰ἲ㸧࡜ࡋ࡚⤒῭༠ຊ㛤Ⓨᶵᵓ(OECD:
Organisation for Economic Co-operation and Development㸧࡛ヨ㦂࢞࢖ࢻࣛ࢖ࣥ(TG: Test Guideline)
431 ࡜ࡋ࡚ᢎㄆࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡿヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡋࡓࠋಙ㢗ᛶ࡜ጇᙜᛶ࡜࠸
࠺どⅬ࡟࠾࠸࡚ࠊࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓヨ㦂ࢆホ౯ࡋࡓ⤖ᯝࠊTG431 ࡟ᥖ㍕ࡉࢀ࡚࠸ࡿࡍ࡭࡚
ࡢࣔࢹࣝ EpiSkin™ࠊEpiDerm™ࠊSkinEthic™ࠊepiCS®ࡀ⭉㣗ᛶࡢ᭷↓ࢆホ౯࡛ࡁࡿࣔࢹࣝ࡜ࡋ࡚
᥎ዡ࡛ࡁࡿ࡜⪃࠼ࡽࢀࡓࠋࡓࡔࡋࠊᅜ㐃໬Ꮫရࡢศ㢮࠾ࡼࡧ⾲♧࡟㛵ࡍࡿୡ⏺ㄪ࿴ࢩࢫࢸ࣒(UN
GHS: United Nations Globally Harmonized System of Classification and Labelling of Chemicals)ศ㢮ࡢ⣽༊ศࢆ⪃៖ࡍࡿሙྜࡣ EpiSkin™ࡀ⓶⭵⭉㣗ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚ࡶࡗ࡜ࡶ᭷⏝࡛࠶ࡿ࡜⤖ㄽ
ࡋࡓࠋ

3

9

1. ヨ㦂ἲࡢ⛉Ꮫⓗ࠾ࡼࡧつไ㠃࠿ࡽࡢጇᙜᛶ
⓶⭵⭉㣗ᛶヨ㦂ࡣ⓶⭵่⃭ᛶヨ㦂ࡢ୍⎔࡜ࡋ࡚⾜ࢃࢀࠊ✀ࠎࡢ࢞࢖ࢻࣛ࢖࡛ࣥࡣ Draize ࡽ࡟ࡼࡾᥦၐࡉࢀࡓ࢘ࢧࢠࢆ⏝࠸ࡿ᪉ἲࡀ᥎ዡࡉࢀ࡚ࡁࡓ 1)ࠋࡇࡢ᪉ἲࡣ⿕㦂≀㉁ࡢ่⃭ᛶࡸ⭉㣗ᛶࢆ᳨ฟࡍࡿヨ㦂࡜ࡋ࡚㛗ࡃ౑⏝ࡉࢀ࡚ࡁࡓࡶࡢࡢࠊุᐃࢆ⫗║࡛⾜࠺ࡓࡵᐈほᛶ࡟ஈࡋࡃᐇ㦂㛫ࡸ᪋タ㛫࡛ࡢ෌⌧ᛶࡀஈࡋ࠸ࠋ᭦࡟ື≀࡟⃭ࡋ࠸③ࡳ࡜ࢫࢺࣞࢫࢆ୚࠼ࡿࡇ࡜ࡀ♫఍ⓗ࡟ၥ㢟࡜࡞ࡾࠊ௨๓ࡼࡾື≀ࢆ౑⏝ࡋ࡞࠸ື≀ᐇ㦂௦᭰ἲ㸦௨ୗࠊ௦᭰ἲ࡜グࡍ㸧ࡢ㛤Ⓨࡀษᮃࡉࢀ࡚࠸ࡓࠋ
ࡇࡢ௦᭰ἲ࡜ࡋ࡚ࠊ⤒῭༠ຊ㛤Ⓨᶵᵓ(OECD: Organisation for Economic Co-operation andDevelopment㸧࡛ヨ㦂࢞࢖ࢻࣛ࢖ࣥ(TG: Test Guideline)431 ࡟ࡣࠊ⓶⭵⭉㣗ᛶヨ㦂࡜ࡋ࡚ゅ㉁ᒙࢆ
᭷ࡋ 3 ḟඖⓗ࡟෌ᵓ⠏ࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝࢆ౑⏝ࡋࡓホ౯᪉ἲࡀグ㍕ࡉࢀ࡚࠸ࡿ 2)ࠋࡇࡢヨ㦂ἲࡣࠊ⭉㣗ᛶ≀㉁ࡀゅ㉁ᒙࡢയᐖࡲࡓࡣゅ㉁ᒙ࡟྾཰ࡉࢀࡓᚋᣑᩓࡍࡿࡇ࡜࡟ࡼࡾࠊୗᒙࡢ⣽⬊࡟฿㐩ࡋ࡚⣽⬊ẘᛶࢆ♧ࡍ࡜࠸࠺௬ㄝ࡟ᇶ࡙ࡁࠊ⿕㦂≀㉁᭚㟢ᚋࡢ⣽⬊⏕Ꮡ⋡ࢆᣦᶆ࡟⓶⭵⭉㣗ᛶࢆホ౯ࡋ࡚࠸ࡿࠋEpiSkin™ࡸ EpiDerm™➼ࡢࣄࢺ⾲⓶ࣔࢹࣝࡣḢ⡿࡛ࡣ᪤࡟ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࠊḢᕞ࡛ࡣ໬Ꮫ≀㉁ࡢ⓶⭵⭉㣗ᛶホ౯ࢆ┠ⓗ࡜ࡋ࡚ᢎㄆࡉࢀࠊ໬Ꮫ≀㉁ࡢࣜࢫࢡ⾲♧㆑ู➼࡟฼⏝ࡉࢀ࡚࠸ࡿࠋ≉࡟᫖௒࡛ࡣᅜ㐃໬Ꮫရࡢศ㢮࠾ࡼࡧ⾲♧࡟㛵ࡍࡿୡ⏺ㄪ࿴ࢩࢫࢸ࣒(UN GHS: United Nations Globally Harmonized System of Classification and Labelling of Chemicals)ศ㢮࡟ᚑࡗ࡚ホ౯ࡉࢀࡿࢣ࣮ࢫࡀቑ࠼࡚࠸ࡿࠋࡇࡢ TG431 ࡣ᫖௒ࠊẖᖺ✀ࠎࡢⅬ࡛ᨵゞࡉࢀ࡚࠾ࡾࠊ⌧ᅾࡣ 2016 ᖺ∧࡜࡞ࡗ࡚࠸ࡿ 3)ࠋ
ᡃࡀᅜ࡛᪤Ꮡࡢ໬Ꮫ≀㉁ࢆホ౯ࡍࡿሙྜࠊOECD ࡛ᢎㄆࡉࢀࡓヨ㦂᪉ἲ࡟ࡼࡿ⤖ᯝࡣࠊ୍⯡ⓗ࡟⾜ᨻⓗ࡟ཷࡅධࢀࡽࢀࡿࡀࠊ⌧ᅾࡲ࡛௦᭰ἲ࡛ࡢ⤖ᯝࢆࡶ࡜࡟⾜ᨻⓗ࡟ホ౯ࡉࢀࡓ౛ࡣከࡃ࡞
࠸ࠋᏳ඲ᛶホ౯࡟࠾ࡅࡿ௦᭰ἲࡢᬑཬࡀษᮃࡉࢀ࡚࠸ࡿ⌧≧࡟࠾࠸࡚ࠊᡃࡀᅜ࡛ࡶ⛉Ꮫⓗ࡟ጇᙜ࡞ࡶࡢࡣ✚ᴟⓗ࡟ཷࡅධࢀࡿࡇ࡜ࡀᚲせ࡜࡞ࡗ࡚࠸ࡿࠋ࡞࠾ࠊᅜෆ௻ᴗ࠿ࡽࡶᅜ㝿ⓗ࡞ホ౯࡟⪏࠼࠺ࡿࣄࢺ⾲⓶ࣔࢹࣝࡀ㛤Ⓨࡉࢀ࡚࠸ࡿࠋᮏホ౯᭩࡛ࡣࠊOECD TG431 ࡟ᥖ㍕ࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝ EpiSkin ™ࠊEpiDerm™ࠊSkinEthic™
࠾ࡼࡧ epiCS®ࢆ⏝࠸ࡿ⭉㣗ᛶヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡋࡓ 3)ࠋ
2. ヨ㦂ࣉࣟࢺࢥࣝᵓᡂࡢጇᙜᛶ 3)⿕ 㦂 ≀ ㉁ ࡀ ゅ ㉁ ᒙ ࢆ ㏻ 㐣 ࡋ ࡚ ⾲ ⓶ ⣽ ⬊ ࡟ ᭚ 㟢 ࡉ ࢀ ࠊ MTT ࠝ 3(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromideࠞࡢ㑏ඖ㔞࠿ࡽồࡵࡓ⣽⬊⏕Ꮡ⋡ࡢ๭ྜ࠿ࡽ

⓶⭵⭉㣗ᛶࢆุᐃࡍࡿࠋࡑࢀࡽࡢᴫせࢆ⾲㸯࡟ࡲ࡜ࡵࡓࠋᐇ㦂᧯సୖࡢ㐪࠸ࡣࠊ๓ᇵ㣴ἲ࠾ࡼࡧᰁⰍᾮࡢ⏝㔞ࠊ⤌⧊࠿ࡽࡢᢳฟἲ➼࡛࠶ࡾࠊᇶᮏⓗ࡞᭚㟢᫬㛫࡜ุᐃᇶ‽࡟ࡘ࠸࡚ࡣ⾲ 2 ࢆཧ↷ࡉࢀࡓ࠸ࠋヨ㦂ࣉࣟࢺࢥࣝ࡜ࡋ࡚ࠊEpiDerm™ࢆ౛࡟ㄝ᫂ࡍࡿࠋ6 well ࣉ࣮ࣞࢺࡢྛ well ࡟ᇵ㣴ᾮ 1 mL ࢆຍ࠼ࡓᚋ࡟ࣄࢺ⾲⓶ࣔࢹࣝࢆ⨨ࡁࠊ⿕㦂≀㉁ࡀᾮ≧ࡢሙྜࡣࣆ࣌ࢵࢱ࣮࡛ 50 μLࠊ⢊ᮎ࡞࡝ᅛᙧ
ࡢሙྜࡣ 25 mg ࢆ 25 μL ࡢỈ࡜ྜࢃࡏࠊࣔࢹࣝୖᒙ࡟㐺⏝ࡍࡿ (n=2)ࠋ⿕㦂≀㉁ࢆ 3 ศࡲࡓࡣ 60ศฎ⌮ᚋࠊࣉ࣮ࣞࢺ࠿ࡽ⿕㦂≀㉁ࢆࢹ࢝ࣥࢸ࣮ࢩࣙࣥ࡟ࡼࡾ㝖ཤࡋࡓᚋࠊ10 mL ࡢ PBS ࡛ 2㹼3
ᅇ㍍ࡃὙίࡍࡿࠋὙίᚋࠊ࣮࣌ࣃ࣮ࢱ࢜ࣝ➼࡛Ỉศࢆษࡾࠊ◚ᦆ࡟ὀពࡋ࡞ࡀࡽูࡢ 24 well ࣉ࣮ࣞࢺ࡟ࣄࢺ⾲⓶ࣔࢹࣝࢆ⛣ືࡍࡿࠋMTT Ⰽ⣲ࢆྵࡴᇵ㣴ᾮࢆࣄࢺ⾲⓶ࣔࢹࣝࡢୗ᪉࡟ 0.3 mL ῧຍࡍࡿࠋ37ΥࠊCO2 ࢖࣮ࣥ࢟ࣗ࣋ࢱ୰࡟ 3 ᫬㛫㟼⨨ࡋࡓᚋࠊ௨ୗࡢᡭ㡰࡟࡚ᢳฟࢆ⾜࠺ࠋ࢖ࢯࣉ
ࣟࣃࣀ࣮ࣝࢆ 2 mL ῧຍࡋࠊ୍ᬌᐊ

ᨺ⨨ᚋࠊ96 well ࣉ࣮ࣞࢺ࡟ᢳฟᾮࢆ 200 μL ࡎࡘ⛣ࡋ㸦1 ≀
4

10

㉁࠶ࡓࡾ 2㹼3 well㸧ࠊ࣐࢖ࢡࣟࣉ࣮ࣞࢺ࣮ࣜࢲ࣮ࢆ⏝࠸࡚ 540 nm ࠶ࡿ࠸ࡣ 570 nm 㡿ᇦ࡛ࡢ྾ග
ᗘࢆᐃࡍࡿࠋ࢖ࢯࣉࣟࣃࣀ࣮ࣝࡢࡳࢆຍ࠼ࡓ well ࢆࣈࣛࣥࢡ࡜ࡋࠊᐇ

್࡜ࣈࣛࣥࢡ್ࡢᕪࢆ

ồࡵࡿࠋ⁐፹ᑐ↷ࡢ྾ගᗘࢆ 100㸣࡜ࡋྛ᳨యࡢ 3 ࡲࡓࡣ 60 ศ㛫ฎ⌮᫬ࡢ྾ගᗘࢆ㸣࡜ࡋ࡚⟬ฟࡋ⣽⬊ࡢ⏕Ꮡ⋡࡜ࡍࡿࠋ3 ศ㛫ฎ⌮ࡋࡓ࡜ࡁࡢ⏕Ꮡ⋡ࡀ 50㸣ᮍ‶ࠊ࠶ࡿ࠸ࡣ 3 ศ㛫࡛ࡣ⏕Ꮡ⋡ࡀ
50㸣௨ୖ࡛࠶ࡿࡀ 60 ศ㛫ฎ⌮ࡋࡓ࡜ࡁ࡟ 15㸣ᮍ‶ࡢ⤖ᯝࢆ♧ࡍ≀㉁ࢆ“⭉㣗ᛶ”࡜ุᐃࡍࡿࠋ୍
᪉ࠊ3 ศ㛫ฎ⌮ࡋࡓ࡜ࡁ࡟⏕Ꮡ⋡ࡀ 50㸣௨ୖࠊ60 ศ㛫ฎ⌮ࡋࡓ࡜ࡁ࡟ 15㸣௨ୖࡢ≀㉁ࡣ“㠀⭉㣗ᛶ”࡜ุᐃࡍࡿࠋヨ㦂ࡣ㸯ᅇ࡜ࡋࠊwell 㛫࡛␗࡞ࡿ⤖ᯝࡀᚓࡽࢀࡓሙྜࡢࡼ࠺࡟᫂☜࡞ホ౯ࡀ࡛ࡁ࡞࠿ࡗࡓሙྜࡣ㏣ຍヨ㦂ࢆᐇ᪋ࡋࠊ᭱⤊ホ౯࡜ࡍࡿࠋ

5

11

12

஦๓☜ㄆ

┤᥋ MTT 㑏ඖᛶࡢ

50 μL(ᾮయ)ࡶࡋࡃࡣ 20 mg(ᅛయ)࡟ 0.3 mg/mL MTT ⁐ᾮ 2 mL ࢆຍ࠼࡚ࠊ37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵
ᗘ 95㸣࡛ 180±5 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊỈฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋ

ࣟ࢘ᵝ/⢓╔ᛶ≀㉁㸸ࢼ࢖࣓ࣟࣥ
ࢵࢩࣗࢆ⏝࠸࡚ 50±2 mg (131.6
mg/cm2)

650 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 1 mg/mL MTT ⁐ᾮ 1 mL ࢆຍ࠼࡚ࠊ37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ95㸣࡛ 60 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊ෾⤖ฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆ
ฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋࣟ࢘ᵝ≀㸸15 μL ࡢỈ࡛‵ࡽࡏࡓ┤ᚄ⣙ 8 mm ࡢࣇࣛࢵࢺ࡞ࢹ࢕
ࢫࢡᵝࡢ∦ࢆୖ࡟஌ࡏࡿࠋ

ᅛయ㸸 25 mg (39.7 mg/cm )㸩25
μL ࡢỈ(ᚲせ࡛࠶ࢀࡤࡑࢀ௨ୖ)2

༙ᅛయ㸸50 μL (79.4 μL/cm )2ᾮయ㸸ࢼ࢖࣓ࣟࣥࢵࢩࣗࢆ⏝࠸ࡓ≧ែ࡛ࠊࡶࡋࡃࡣ⏝࠸࡞࠸ሙྜ࡛ࡶ 50 μL (79.4 μL/cm2)⿕㦂≀㉁࡜ࢼ࢖࣓ࣟࣥࢵࢩࣗ࡜ࡢぶ࿴ᛶࡣணヨ㦂࡛☜ㄆࡍࡿࠋᾮయ࠾ࡼࡧ⢓ᛶ≀㸸50±3 μL(131.6μL/cm 2)౑⏝㔞࡜㐺⏝

ᅛయ㸸20±2 mg (52.6 mg/cm2)+100±5 μL ࡢ NaCl ⁐ᾮ(9 g/L㸧᭚㟢᫬㛫ẖ࡟ 2-3᭚㟢᫬㛫ẖ࡟ᑡ࡞ࡃ࡜ࡶ 2

0.63 cm 2

0.38cm2ࣔࢹࣝ⾲㠃✚⤌⧊ᩘ

EpiDermTMSCT

EpiSkinTM

ࢸࢫࢺ᪉ἲ せ⣲±࡛♧ࡋࡓᩘᏐࡣࠊࡤࡽࡘࡁࡢチᐜ⠊ᅖࢆ♧ࡍࠋ⾲㸯㸬⓶⭵⭉㣗ᛶヨ㦂ࡢࡓࡵ࡟☜ㄆࡉࢀࡓ RhE ヨ㦂᪉ἲ࠿ࡽ࡞ࡿ୺࡞ヨ㦂᪉ἲ 3)

40 μL(ᾮయ)ࡶࡋࡃࡣ 20 mg(ᅛయ)࡟ 1 mg/mL MTT ⁐ᾮ 1 mL ࢆຍ࠼࡚ࠊ37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ95㸣࡛ 180±15 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊ෾⤖ฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆ
ฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋࣟ࢘ᵝ/⢓╔ᛶ≀㉁㸸ࢼ࢖࣓ࣟࣥ
ࢵࢩࣗࢆ⏝࠸ࡓ≧ែ࡛ 20±3 mg(40 mg/cm2)

ᅛయ㸸
20±3 mg (40 mg/cm2)㸩20±2 μL ࡢỈᾮయ࠾ࡼࡧ⢓ᛶ≀㸸ࢼ࢖࣓ࣟࣥ
ࢵࢩࣗࢆ౑⏝ࡋ࡚ 40±3 μL (80μL/cm2)⿕㦂≀㉁࡜ࢼ࢖࣓ࣟࣥࢵࢩࣗ࡜ࡢぶ࿴ᛶࡣணヨ㦂࡛☜ㄆࡍࡿࠋ᭚㟢᫬㛫ẖ࡟ᑡ࡞ࡃ࡜ࡶ 2

0.5 cm 2

SkinEthicTM RHE

50 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 1 mg/mL MTT ⁐ᾮ 1 mL ࢆຍ࠼࡚ 37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ95㸣࡛ 60 ศᇵ㣴ĺ⁐ᾮࡢⰍࡀ㟷/⣸࡟ኚࢃࡗࡓሙ
ྜࠊ෾⤖ฎ⌮࡛ࣔࢹࣝᵓᡂ⣽⬊ࢆṚ⁛ࡉࡏࡓࡶࡢ࡟⿕㦂≀㉁ࢆ
ฎ⨨ࡍࡿᑐ↷ࡶ࡜ࡿࠋࣟ࢘ᵝ≀㸸15 μL ࡢỈ࡛‵ࡽࡏࡓࣇࣛࢵࢺ࡞ࠊ┤ᚄ⣙ 8 mm ࡢࢡࢵ࣮࢟ᵝࡢ∦ࢆୖ࡟஌ࡏࡿࠋ

ᅛయ㸸25 mg (41.7 mg/cm2)+
25 μL ࡢỈ(ᚲせ࡛࠶ࢀࡤࡑࢀ௨
ୖ)༙ᅛయ㸸50 μL (83.3 μL/cm2)

ᾮయ㸸ࢼ࢖࣓ࣟࣥࢵࢩࣗࢆ⏝࠸ࡓ≧ែ࡛ 50 μL (83.3 μL/cm2)⿕㦂≀㉁࡜ࢼ࢖࣓ࣟࣥࢵࢩࣗ࡜ࡢぶ࿴ᛶࡣணヨ㦂࡛☜ㄆࡍࡿࠋ᭚㟢᫬㛫ẖ࡟ᑡ࡞ࡃ࡜ࡶ 2

0.6 cm 2

epiCS®

13

8 N Ỉ㓟໬࣒࢝ࣜ࢘ 40 μL ࡛ 1 ᫬㛫᭚㟢᫬ࡢࡳ

37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛
180 ศ(±15 ศ)
1.5 mL ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝ
(࢖ࣥࢧ࣮ࢺ඲య࠿ࡽᢳฟࡍࡿ㸧

8 N Ỉ㓟໬࣒࢝ࣜ࢘ 50 μL ࡛᭚㟢᫬㛫ẖ࡟⃰ᗘ 1 mg/mL 300 μL
37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛180 ศ
2 mL ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝ
(࢖ࣥࢧ࣮ࢺ඲య࠿ࡽᢳฟࡍࡿ㸧⃰ᗘ 0.3 mg/mL 2 mL

37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣
࡛ 180 ศ(±15 ศ)

500 μL ࡢ㓟ᛶ໬࢖ࢯࣉࣟࣃࣀ
࣮ࣝ
(0.04 N ሷ㓟ࢆྵࡴ࢖ࢯࣉࣟࣃ
ࣀ࣮ࣝ)(ศ㞳ࡋࡓ⤌⧊ࢆ༑ศ࡟ᾐₕࡉࡏࡿ㸧MTT ⁐ᾮ

MTT ⁐ᾮ࡛ࡢᇵ㣴

ᢳฟ⁐፹

᫬㛫࠾ࡼࡧ

㝧ᛶᑐ↷

ᗘ

7

40 μL ࡢỈ᭚㟢᫬㛫ẖ࡟

50 μL ࡢỈ᭚㟢᫬㛫ẖ࡟

㝜ᛶᑐ↷⃰ᗘ 1 mg/mL 300 μL

PBS ࢆ୍ᐃࡋࡓᙅὶ࡛ 20 ᅇ

PBS ࢆ୍ᐃࡋࡓᙅὶ࡛ 20 ᅇ

PBS 25 mL(ࡍࡍࡂ୍ᅇẖ࡟ 2
mL)
50 μL ࡢሷ໬ࢼࢺ࣒ࣜ࢘⁐ᾮ
㸦9g/L)
᭚㟢᫬㛫ẖ࡟
50 μL ࡢị㓑㓟࡛ 4 ᫬㛫᭚㟢᫬ࡢࡳ

PBS ࡟ࡼࡿࡍࡍࡂ

(࢖ࣥࢧ࣮ࢺ඲య࠿ࡽᢳฟࡍࡿ㸧

2 mL ࡢ࢖ࢯࣉࣟࣃࣀ࣮ࣝ

37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛180 ศ

⃰ᗘ 1 mg/mL 300 μL

8 N Ỉ㓟໬࣒࢝ࣜ࢘ 5 0μL ࡛᭚㟢᫬㛫ẖ࡟

50 μL ࡢỈ᭚㟢᫬㛫ẖ࡟

PBS ࢆ୍ᐃࡋࡓᙅὶ࡛ 20 ᅇ

ᐊ ࡛ 3 ศࠊ࠾ࡼࡧ
37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛60 ศ

ᐊ 㸦18-28Υ㸧࡛ 3 ศࠊ60 ศ ᐊ ࡛ 3 ศࠊ࠾ࡼࡧ 37ΥࠊCO2
㸦±5 ศ㸧࠾ࡼࡧ 240 ศ㸦±10 ศ㸧 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛ 60 ศ᥮Ẽࡉࢀࡓ࢟ࣕࣅࢿࢵࢺෆ

᭚㟢᫬㛫࡜

ᗘ

50 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 300 μL ࡢỈࢆຍ࠼࡚ 37Υࠊ
CO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛ 60 ศᨩᢾࡍࡿࠋĺ⁐ᾮࡢⰍࡀ╔Ⰽࡋ࡚࠸ࡿሙ
ྜࠊMTT ࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋᐊ ࡛ 3 ศࠊ࠾ࡼࡧ
37ΥࠊCO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛
60 ศ

40 μL(ᾮయ)ࡶࡋࡃࡣ 20 mg(ᅛయ)࡟ 300 μL ࡢỈࢆຍ࠼࡚ᐊ ࡛ 60ศᨩᢾࡍࡿࠋ
ĺ⿕㦂≀㉁ࡢⰍࡀ╔Ⰽࡋ࡚࠸ࡿ
ሙྜࠊMTT ࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋ

50 μL(ᾮయ)ࡶࡋࡃࡣ 25 mg(ᅛయ)࡟ 300 μL ࡢỈࢆຍ࠼࡚ 37Υࠊ
CO2 ⃰ᗘ 5㸣ࠊ‵ᗘ 95㸣࡛ 60 ศᨩᢾࡍࡿࠋĺ⁐ᾮࡀ╔ⰍࡋࡓሙྜࠊMTT ࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋ

10 μL(ᾮయ)ࡶࡋࡃࡣ 10 mg(ᅛయ)࡟ 90 μL ࡢỈࢆຍ࠼࡚ᐊ
࡛ 15 ศᨩᢾࡍࡿࠋ
ĺ⁐ᾮࡀ╔ⰍࡋࡓሙྜࠊMTTࡢࡳࢆຍ࠼࡞࠸ᑐ↷ࢆ࡜ࡿࠋ╔Ⰽ㞀ᐖࡢ஦๓☜ㄆ

epiCS®

SkinEthicTM RHE

EpiDermTMSCT

EpiSkinTM

ࢸࢫࢺ᪉ἲ せ⣲

14
1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿ࡗ
ࡓ᫬ࡣ 2 ᅇ1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿
ࡗࡓ᫬ࡣ 2 ᅇ

1.
1. 㝜ᛶᑐ↷(ሷ໬ࢼࢺ࣒ࣜ࢘⁐ᾮ)࡛ฎ⌮ࡉࢀࡓ⤌⧊ࡢ
OD ್ࡢᖹᆒࡣࠊ᭚㟢᫬㛫ẖ࡟ 0.6 ௨ୖ 1.5 ௨ୗࠋ
2. 㝧ᛶᑐ↷(ị㓑㓟)࡛ 4 ᫬㛫 2.
ฎ⨨ࡉࢀࡓ⤌⧊ࡢ⏕Ꮡ⋡ࡢ
ᖹᆒࡣ 20㸣௨ୗࠋ
3. ⏕Ꮡ⋡ࡢᖜࡀ 20-100㸣࠾ࡼࡧ OD ್ࡀ 0.3 ௨ୖࡢሙྜࠊ 3.
2 ࡘࡢ⤌⧊㛫ࡢ⏕Ꮡ⋡ࡢᕪࡣ 30㸣ࢆࡇ࠼࡞࠸ࠋヨ㦂ᅇᩘ㐺ྜุᐃᇶ‽

8㝜ᛶᑐ↷(Ỉ)࡛ฎ⨨ࡉࢀࡓ⤌⧊ࡢ OD ್ࡢᖹᆒࡣࠊ඲࡚ࡢ
᭚㟢᫬㛫࡟࠾࠸࡚ 0.8 ௨ୖ
2.8 ௨ୗࠋ
㝧ᛶᑐ↷㸦8N Ỉ㓟໬࣒࢝ࣜ࢘㸧࡟ 1 ᫬㛫᭚㟢ࡋࡓ⤌⧊」〇≀ࡢ ᐃ್ࡢ⏕Ꮡ⋡ࡣ
15㸣ᮍ‶ࠋ
⏕Ꮡ⋡ࡀ 20-100㸣ࡢሙྜ࡟࠾࠸࡚ࠊ⤌⧊㛫ࡢኚືಀᩘ(CV)ࡣ 30㸣௨ୗࠋ

4.08 ᫬㛫ӌET50ӌ8.7 ᫬㛫

1.0 mg/mLӌIC50ӌ3.0 mg/mL

1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿ࡗ
ࡓ᫬ࡣ 2 ᅇ1. 㝜ᛶᑐ↷(Ỉ)࡛ฎ⨨ࡋࡓ⤌⧊ࡢ OD ್ࡢᖹᆒࡣࠊྛ᭚㟢᫬㛫ẖ࡟ 0.8 ௨ୖ 2.8 ௨ୗࠋ
2. 1 ᫬㛫(ࡶࡋྍ⬟࡛࠶ࢀࡤ 4 ᫬㛫)㝧ᛶᑐ↷(8N Ỉ㓟໬࣒࢝ࣜ࢘)࡛ฎ⨨ࡉࢀࡓ⤌⧊」〇≀ࡢ ᐃ್ࡢ⏕Ꮡ⋡ࡣ 15㸣ᮍ‶ࠋ
3. 20-100㸣ࡢ⏕Ꮡ⋡࠾ࡼࡧ 0.3
௨ୖࡢ OD ್࡟࠾࠸࡚ࠊ2 ࡘࡢ⤌⧊㛫ࡢ⏕Ꮡ⋡ࡢᕪࡣ
30㸣ࢆ㉸࠼࡞࠸ࠋ

1. 㝜ᛶᑐ↷(Ỉ)࡛ฎ⨨ࡉࢀࡓ⤌⧊ࡢ OD ್ࡢᖹᆒࡣࠊ᭚㟢᫬㛫ẖ࡟ 0.8 ௨ୖ 3.0 ௨ୗࠋ
2. 1 ᫬㛫(ࡶࡋྍ⬟࡛࠶ࢀࡤ 4 ᫬㛫)㝧ᛶᑐ↷(8N Ỉ㓟໬࣒࢝ࣜ࢘)࡛ฎ⨨ࡉࢀࡓ⤌⧊」〇≀ࡢ ᐃ್ࡢ⏕Ꮡ⋡ࡣ 15㸣ᮍ‶ࠋ
3. 20-100㸣ࡢ⏕Ꮡ⋡࠾ࡼࡧ 0.3
௨ୖࡢ OD ್࡟࠾࠸࡚ࠊ2 ࡘࡢ⤌⧊㛫ࡢ⏕Ꮡ⋡ࡢᕪࡣ
30㸣ࢆ㉸࠼࡞࠸ࠋ

2.0 ᫬㛫ӌET50ӌ7.0 ᫬㛫1㸣Triton X-100 ࡛ฎ⌮

540-570nmཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

㸦㹼120rpm㸧120 ศࡃࡣᐊ ࡛᣺࡜࠺ࡋࡓ≧ែ࡛ᐊ ࡛᣺࡜࠺ࡏࡎ࡟୍ᬌࠊࡶࡋ

epiCS®

1 ᅇࠊ᫂☜࡞⤖ᯝࡀᚓࡽࢀ࡞࠿ࡗ
ࡓ᫬ࡣ 2 ᅇ4.0 ᫬㛫ӌET50ӌ10.0 ᫬㛫1㸣Triton X-100 ࡛ฎ⌮

1㸣Triton X-100 ࡛ฎ⌮

SDS ࡛ 18 ᫬㛫ฎ⌮

⤌⧊ࡢရ㉁☜ㄆ

570nm (545-600nm)

570nm (ࡶࡋࡃࡣ 540nm)ཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

㸦㹼120rpm㸧120 ศ࡛᣺࡜࠺ࡋࡓ≧ែ࡛࡛᣺࡜࠺ࡏࡎ࡟୍ᬌࠊࡶࡋ

ࡃࡣᐊᐊ

570nm (545-595nm)

㸦㹼120rpm㸧120 ศ࡛᣺࡜࠺ࡋࡓ≧ែ࡛࡛᣺࡜࠺ࡏࡎ࡟୍ᬌࠊࡶࡋ

ࡃࡣᐊᐊ

SkinEthicTM RHE

ཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

୍࡛ᬌ

EpiDermTMSCTཧ↷ࣇ࢕ࣝࢱ࣮࡞ࡋ࡛

OD

ᐃ᮲௳

㐽ගࡋࠊᐊ

ᢳฟ᫬㛫࠾ࡼࡧ

ᗘ

EpiSkinTM

ࢸࢫࢺ᪉ἲ せ⣲⾲ 2-1 EpiSkinTM ࡢண ࣔࢹࣝ 3)
3 ศࠊ60 ศ࠾ࡼࡧ 240 ศ᭚㟢ᚋ
ࡢ⏕Ꮡ⋡

ண

ᛶࡢホ౯

3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣ᮍ‶⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1A㸨

3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣௨ୖ࡛ࠊ࠿
ࡘ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣ᮍ‶ࡢሙྜࠊࡶࡋࡃࡣ
60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣௨ୖ࡛ࠊ࠿
ࡘ 240 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣ᮍ‶
240 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 35㸣௨ୖ⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1B ࠶ࡿ࠸ࡣ
1C

㠀⭉㣗ᛶ

*) ⭉㣗ᛶࡢ⣽༊ศ࡟࠾ࡅࡿ RhE ヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡍࡿࡓࡵ࡟సᡂࡋࡓࢹ࣮ࢱ࡟ࡼࡿ࡜ࠊEpiSkinTM ヨ㦂ἲ࡟ࡼࡾ༊ศ 1A ࡟ศ㢮ࡉࢀࡓ≀㉁㸭ΰྜ≀ࡢ⣙ 22%ࡀࠊᐇ㝿࡟ࡣ༊ศ 1B ࡲࡓࡣ 1C ࡟ᒓࡍࡿࡶࡢ࡛࠶ࡿྍ⬟ᛶࡀ࠶ࡿ 㸦ࡍ࡞ࢃࡕࠊ㐣኱ホ౯㸧ࠋ

⾲ 2-2 EpiDermTMSCTࠊSkinEthicTMRHE ࠾ࡼࡧ epiCS®ࡢண ࣔࢹࣝ 3)
3 ศ࠾ࡼࡧ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ண ᛶࡢホ౯
3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 50㸣ᮍ‶
3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 50㸣௨ୖ࡛ࠊ࠿
ࡘ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 15㸣ᮍ‶
3 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 50㸣௨ୖ࡛ࠊ࠿
ࡘ 60 ศ᭚㟢ᚋࡢ⏕Ꮡ⋡ࡀ 15㸣௨ୖ⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1A㸨
⭉㣗ᛶ࣭ᅜ㐃 GHS ⣽༊ศ 1B ࠶ࡿ࠸ࡣ1C㠀⭉㣗ᛶ *) ⭉㣗ᛶࡢ⣽༊ศ࡟࠾ࡅࡿ RhE ヨ㦂ἲࡢ᭷⏝ᛶࢆホ౯ࡍࡿࡓࡵ࡟సᡂࡋࡓࢹ࣮ࢱ࡟ࡼࡿ࡜ࠊEpiSkinTM ヨ㦂ἲ࡟ࡼࡾ༊ศ 1A ࡟ศ㢮ࡉࢀࡓ≀㉁㸭ΰྜ≀ࡢ⣙ 22%ࡀࠊᐇ㝿࡟ࡣ༊ศ 1B ࡲࡓࡣ 1C ࡟ᒓࡍࡿࡶࡢ࡛࠶ࡿྍ⬟ᛶࡀ࠶ࡿ 㸦ࡍ࡞ࢃࡕࠊ㐣኱ホ౯㸧ࠋ

3. 㛤Ⓨ࠾ࡼࡧホ౯࡟౑ࢃࢀࡓ≀㉁ࡢศ㢮ࠊ㑅ᢥ⌮⏤ࡢጇᙜᛶࠊin vitro ࠾ࡼࡧཧ↷ࢹ࣮ࢱࡢ᭷↓EpiSkinTM ࡢ෌⌧ᛶࡣࠊ60 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ
⢋ࢆ ANNEX 1 ࡟♧ࡍ 4)ࠋEpiDerm™ࡢ෌⌧ᛶࡣࠊ24 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁
ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ⢋ࢆ ANNEX 2 ࡟♧ࡍ 5)ࠋSkinEthic™ࡢ෌⌧ᛶࡣࠊ12 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽ
ࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ⢋ࢆ ANNEX 3 ࡟♧ࡍ 6)ࠋEpiCS®ࡢ෌⌧ᛶࡣࠊ12 ࡢ⿕㦂≀㉁࡟ࡼࡾㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡢᢤ⢋ࢆ ANNEX 4 ࡟♧ࡍ 2㸧ࠋࡉࡽ࡟ࠊୖグ㸲ࡘࡢࣔࢹࣝࡢண

ᛶࡀ 80 ⿕㦂≀㉁࡛ㄪ࡭ࡽࢀ࡚࠸ࡿࠋࡑࢀࡽࡢ≀㉁ࡀᥖ㍕ࡉࢀࡓㄽᩥࡢᢤ⢋ࢆ ANNEX 5 ࡟♧

ࡍ 7㸧ࠋホ౯࡟౑⏝ࡉࢀࡓ⿕㦂≀㉁ࡢከࡃࡣḢᕞ௦᭰ἲホ౯ࢭࣥࢱ࣮㸦ECVAM㸸European Centre for the
Validation of Alternative Methods㸧୺ᑟࡢ⓶⭵⭉㣗ᛶヨ㦂ࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑⏝ࡉࢀࡓ≀㉁࡛࠶ࡿࠋࡇ
ࢀࡽ⿕㦂≀㉁ࡢホ౯⤖ᯝࡣୗグࡢ㈨ᩱ࡜ࡋ࡚ᥦฟࡉࢀ࡚࠸ࡿࠋ
Liebsh et al., ATLA 20005), Barratt et al., Toxicol. In Vitro 19988), Fentem et al, Toxicol. In Vitro 19984),
Worth et al., ATLA 19989), Botham et al., ATLA 199510) , ICCVAM(1999) NIH Publication No:99-449511),
ICCVAM (2002) NIH Publication No: 02-450212)

915

4. ヨ㦂ἲࡢṇ☜ᛶ㸦෌⌧ᛶ㸧
EpiSkinTM ࡟࠾࠸࡚ࡣࠊ60 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ 2-way ࡢ ANOVA ゎᯒࢆ⏝࠸ࠊ᪋タෆ࠾ࡼࡧ᪋タ㛫ࡢኚື࡟ࡘ࠸࡚ࡣࠊࡑࢀࡽࡢ㛫࡟᭷ព࡞ᕪࡣ࡞࠸࡜ุ᩿ࡉࢀࡓ(Fentem et al 1998)4)ࠋ60 ≀㉁㸦27⭉㣗ᛶ≀㉁࠾ࡼࡧ 33 㠀⭉㣗ᛶ≀㉁㸧ࡢ࠺ࡕࠊ42 ≀㉁ࡣ 3 ᪋タ࡜ࡶ᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀⰋዲ࡛࠶ࡗࡓࠋṧࡿ 18 ≀㉁࡛ࡣఱ➼࠿ࡢ⤖ᯝࡀ␗࡞ࡗ࡚࠸ࡓࡀࠊECVAM ࡣᮏヨ㦂ἲࡢಙ㢗ᛶ࡜෌⌧ᛶࡣ㧗࠸࡜ุ᩿ࡋࡓ 13 㸧ࠋࡇࡢ⤖ㄽࡣࠊ ECVAM ⛉Ꮫㅎၥ఍㆟㸦ESAC: ECVAM Scientific Advisory
Committee 13 㸧 ࠾ࡼࡧ⡿ᅜࡢ௦᭰ἲ࡟㛵ࡍࡿ┬ᗇ㛫㐃⤡఍㆟㸦ICCVAM㸸Interagency Coordinating
Committee on the Validation of Alternative Methods 12㸧࡛ࡢホ౯࡟࠾࠸࡚ࡶ☜ㄆࡉࢀࡓࠋ
EpiDerm™࡟࠾࠸࡚ࡣࠊ24 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ 2 ᅇࡢᐇ㦂࡟࠾࠸࡚ࠊ21 ≀㉁ࡢ⭉㣗ᛶࢆ 3 ᪋タࡍ࡭࡚࡛ṇࡋࡃண

࡛ࡁࡓ㸦Liebsch et al 2000㸧5)ࠋECVAM ࡣᮏヨ㦂ἲࡢಙ㢗ᛶ࡜෌⌧ᛶࡣ㧗࠸࡜ุ᩿ࡋࡓࠋࡇࡢ⤖ㄽࡣࠊESAC ࡢホ౯ᚋ 14㸧ࠊICCVAM ࡟࠾࠸࡚ࡶ☜ㄆࡉࢀࡓ 11,12㸧ࠋSkinEthic™࡟࠾࠸࡚ࡣࠊ12 ≀㉁ࢆ⏝࠸ࡓ 3 ᪋タ࡛ࡢ 3 ᅇࡢᐇ㦂࡟࠾࠸࡚ࠊ93.2㸣ࡢ⤖ᯝࡀ୍⮴ࡋ
Kandarova et al 2006 㸴㸧ࠊESAC ࡟࠾࠸࡚᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀ㧗࠸࡜ุ᩿ࡉࢀࡓ 15㸧ࠋ
EpiCS®࡟࠾࠸࡚ࡣࠊTG431㸦2004㸧ࡢཧ↷ 12 ≀㉁ࢆ⏝࠸ࡓ 4 ᪋タ࡛ࡢ 3 ᅇࡢᐇ㦂࡟࠾࠸࡚ 2㸧ࠊࢸࢺࣛࢡ࢚ࣟࣟࢳࣞࣥࢆ㝖ࡃ 11 ≀㉁ࡢ⤖ᯝࡀ୍⮴ࡋࠊESAC ࡟࠾࠸࡚᪋タෆ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀㄆࡵࡽ
ࢀࡓ 16㸧ࠋ
5. ヨ㦂ἲࡢಙ㢗ᛶ 7ࠊ17ࠊ18㸧 ࠸ࡎࢀࡢࣄࢺ⾲⓶ࣔࢹࣝࡶ⭉㣗ᛶࡢ᭷↓ࡢணᛶࡣ㧗ࡃࠊṇ☜ᗘ1ࡣ 84㹼90%ࠊឤᗘ2ࡣ 95㹼100㸣ࠊ≉␗ᗘ3ࡣ 72㹼79㸣࡛࠶ࡾࠊഇ㝜ᛶ⋡ࡣ 0㹼5%࡜ప࠸࡛ࣞ࣋ࣝ࠶ࡗࡓ㸦⾲ 3㸧ࠋࡇࢀࡽࡢ್࠿ࡽࠊOECDࡢᛶ⬟ᶆ‽࡛ࡣṇ☜ᗘࠊឤᗘ࠾ࡼࡧ≉␗ᗘࢆࣂࣜࢹ࣮ࢩࣙࣥ࡟ᚲせ࡞ᇶ‽࡜ࡋ࡚ 82.5ࠊ95 ࠾ࡼࡧ 70㸣࡜ᐃࡵ࡚࠸ࡿࠋ ࡉࡽ࡟ࠊྛࣔࢹࣝࡢ⣽༊ศࡢணᛶࢆ⾲ 4 ࡲ࡜ࡵࡓࠋ80 ≀㉁ࢆ 2 ࡲࡓࡣ 3 ᅇᐇ㦂ࡋ࡚ᚓࡽࢀࡓ್ࢆ

ぢࡿ㝈ࡾࠊ⣽༊ศ࡛࠶ࡿ㸯B/1C ࢆ 80㸣⛬ᗘ࡛ホ౯࡛ࡁ࡚࠸ࡿࣔࢹࣝࡣ EpiSkinTM ࡢࡳ࡛࠶ࡗࡓࠋࡑࡢ
௚ࡢࣔࢹࣝࡣᅜ㐃 GHS ⣽༊ศࡢホ౯࡟ࡣ฼⏝࡛ࡁ࡞࠸࡜ุ᩿ࡋࡓࠋ
ࡇࢀࡽࣔࢹࣝࡣ MTT 㑏ඖ≀㉁࡬ࡢᑐᛂ᪉ἲࡶ TG431 ࡟グ㍕ࡉࢀ࡚࠸ࡿࠋ㐺⏝࡛ࡁ࡞࠸≀㉁࡜ࡋ࡚
ࡣ࢞ࢫࠊ࢚࢔ࣟࢰ࣮ࣝࡢࡳࡀグ㍕ࡉࢀ࡚࠸ࡿ㸦ࣂࣜࢹ࣮ࢩࣙࣥᮍᐇ᪋㸧ࠋࡇࢀࡽࣔࢹࣝࡣ≀⌮≧ែ㸦ᾮయ࣭ᅛయ➼㸧࠾ࡼࡧỈ⁐ᛶࡢ᭷↓࡟࠿࠿ࢃࡽࡎ㐺⏝ྍ⬟࡛࠶ࡾࠊ࢞ࢫࠊ࢚࢔ࣟࢰ࣮ࣝࢆ㝖ࡃΰྜ≀࡛ࡶ㐺⏝ྍ⬟࡜ࡉࢀ࡚࠸ࡿࠋ࡞࠾ࠊ≉ᐃࡢ✀㢮ࡢ≀㉁ࡸΰྜ≀࡟࠾࠸࡚ࡇࢀࡽࣔࢹࣝࡢ㐺⏝ᛶࢆྰᐃࡍ
ࡿࡼ࠺࡞᫂☜࡞᰿ᣐࡀᚓࡽࢀࡓሙྜ࡟ࡣࠊ㐺⏝⠊ᅖ࠿ࡽ㝖እࡍࡿ࡭ࡁ࡛࠶ࡿ࡜ࡉࢀ࡚࠸ࡿࠋ

1ṇ☜ᗘ㸸⿕㦂≀㉁࡟ࡘ࠸࡚ヨ㦂ἲ࡛ᚓࡽࢀࡿ⤖ᯝࡀࠊ⿕㦂≀㉁ࡢ᪤ᐃࡢཧ↷್࡜㢮ఝࡋ࡚࠸ࡿ⛬ᗘࠋឤᗘ㸸ヨ㦂ࡉࢀࡓ㝧ᛶ≀㉁ࡢ୰࡛ࠊヨ㦂ἲ࡛ṇࡋࡃ㝧ᛶ࡜ุᐃࡉࢀࡓࡶࡢࡢ๭ྜࠋ
3 ≉␗ᗘ㸸ヨ㦂ࡉࢀࡓ㝜ᛶ≀㉁ࡢ୰࡛ࠊヨ㦂ἲ࡛ṇࡋࡃ㝜ᛶ࡜ุᐃࡉࢀࡓࡶࡢࡢ๭ྜࠋ
10
2

16

⾲ 3㸬OECD ࡛㞟ィࡋࡓ඲ࢭࢵࢺࡢ໬Ꮫ≀㉁ࢆ⏝࠸ࡓண ᛶࡢィ⟬⤖ᯝ㸦⭉㣗ᛶ [ᅜ㐃 GHS ༊ศ㸯] ࡢ᭷↓㸧17㸧
EpiDermTM SkinEthicTM
epiCS®
EpiSkinTM
87.9%
ṇ☜ᗘ
89.6㸣
84.6㸣
84.3㸣
100%
ឤᗘ
98.5㸣
94.6㸣
95.3㸣
73.9%
≉␗ᗘ
79.3㸣
73.0㸣
71.6㸣
20.7%
26.1%
27.0%
28.4%
ഇ㝧ᛶ⋡
1.5%
0%
5.4%
4.7%
ഇ㝜ᛶ⋡

⾲ 4. OECD ࡛㞟ィࡋࡓ඲ࢭࢵࢺࡢ໬Ꮫ≀㉁ࢆ⏝࠸ࡓண
㸯Aࠊ㸯B/1Cࠊ㠀⭉㣗ᛶ㸧3㸧ᛶࡢィ⟬⤖ᯝ㸦⣽༊ศ㸸ᅜ㐃 GHS ༊ศ໬Ꮫရ඲యࡢ⤫ィࢹ࣮ࢱ㸦㸣㸧㸦80 ✀㢮ࡢ໬Ꮫရࢆ 2 ࡲࡓࡣ 3 ᅇヨ㦂ࠊࡍ࡞ࢃࡕࠊ159㸨ࡲࡓࡣ 24 ᅇࡢศ㢮⤖ᯝ㸧㸨
1 ရ┠ࡣࠊධᡭ୙ྍ⬟࡛࠶ࡗࡓࡓࡵ 1 ᅇࡢࡳヨ㦂

EpiSkinTM

EpiDermTM

SkinEthicTM

EpiCSR

㐣኱ホ౯









༊ศ 1BC ࡢ໬Ꮫရࡢ㸯A ࡬ࡢ㐣኱ホ౯

21.5

29.0

31.2

32.8

༊ศ NC ࡢ໬Ꮫရࡢ㸯B/㸯C ࡬ࡢ㐣኱ホ౯

20.7

23.4

27.0

28.4

༊ศ NC ࡢ໬Ꮫရࡢ㸯A ࡬ࡢ㐣኱ホ౯

0.0

2.7

0.0

0.0

㐣኱ホ౯ࡉࢀࡓ༊ศ NC ໬Ꮫရ

20.7

26.1

27.0

28.4

඲༊ศ࡛ࡢ㐣኱ホ౯⋡

17.9

23.3

24.5

25.8









༊ศ㸯A ࡢ㸯B/1C ࡬ࡢ㐣ᑠホ౯

16.7

16.7

16.7

12.5

༊ศ㸯A ࡢ NC ࡬ࡢ㐣ᑠホ౯

0.0

0.0

0.0

0.0

༊ศ㸯B/1C ࡢ NC ࡬ࡢ㐣ᑠホ౯

2.2

0.0

7.5

6.6

඲༊ศ࡛ࡢ㐣ᑠホ౯⋡

3.3

2.5

5.4

4.4

ṇ☜࡞ศ㢮









ṇࡋࡃศ㢮ࡉࢀࡓ㸯A ໬Ꮫရ

83.3

83.3

83.3

87.5

ṇࡋࡃศ㢮ࡉࢀࡓ㸯B/1C ໬Ꮫရ

76.3

71.0

61.3

60.7

ṇࡋࡃศ㢮ࡉࢀࡓ NC ໬Ꮫရ

79.3

73.9

73.0

71.6

୍⮴ᗘ(ண

78.8

74.2

70.0

69.8

㐣ᑠホ౯

⬟㸧

NC:㠀⭉㣗ᛶ

6. ௚ࡢ⛉Ꮫⓗ࡞ሗ࿌࡜ࡢẚ㍑ࡢ᭷↓ OECD ࡢ⭉㣗ᛶヨ㦂௦᭰ἲ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ࠊTG431 ࡢ௚࡟ࠕTG430 TER㸦Transcutaneous Electrical
Resistance Test Method㸸⤒⓶㟁Ẽ᢬ᢠᛶヨ㦂 ࠖ19㸧࠾ࡼࡧࠕTG435

In Vitro Membrane Barrier Test Method

for Skin Corrosion : in vitro ⭷ࣂࣜ࢔ヨ㦂ࠖ20㸧ࡀᢎㄆࡉࢀ࡚࠸ࡿࠋࡇࢀࡽࡣ࠸ࡎࢀࡶ ECVAM ࡟࡚ࣂࣜ1117ࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࠊICCVAM ࡣࡇࢀࡽࡢヨ㦂ἲ Rat Skin TER, EpiSkin™, EpiDerm™࠾ࡼࡧ
Corrositex® ࡢṇ☜ᗘࠊឤᗘ࠾ࡼࡧ≉␗ᗘ࡟ࡘ࠸࡚ẚ㍑ࡋ࡚࠸ࡿ㸦⾲ 5㸧11,12㸧ࠋࡇࢀࡽࡢẚ㍑࡟࠾࠸࡚ࡣྠࡌ≀㉁ࢆ⏝࠸࡚ホ౯ࡉࢀ࡚࠾ࡽࡎࠊヨ㦂≀㉁ࡢᩘ㔞ࡸ㑅ᢥ≀㉁ࡢ✀㢮ࡀ␗࡞ࡗ࡚࠸ࡿࡓࡵ⤖ᯝࡢᩘ್ࡔࡅࢆࡶࡗ࡚ࠊ༢⣧࡟ࣄࢺ⾲⓶ࣔࢹࣝࡢඃ㉺ᛶࢆẚ㍑ホ౯ࡍࡿࡇ࡜ࡣᅔ
㞴࡛࠶ࡿࡀࠊ࠸ࡎࢀࡢヨ㦂ἲࡶྠ➼ࡢணᛶࢆ᭷ࡍࡿ࡜ᛮࢃࢀࡿࠋ

⾲ 5㸬ヨ㦂ἲࡢẚ㍑⤖ᯝ 11,12㸧

≀㉁ᩘ
ṇ☜ᗘ
ឤᗘ
≉␗ᗘ

TER
122
81% (99/122)
94%(51/54)
71%(48/68)

EpiSkin
60
83% (50/60)
82%(23/28)
84%(27/32)

EpiDerm
24
92% (22/24)
92%(11/12)
83%(10/12)

Corrositex
163
79% (128/163)
85%(76/89)
72%(52/74)

7. 3Rs ཎ๎࡜ࡢ㛵ಀ㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧 ࠸ࡎࢀࡢ⾲⓶ࣔࢹࣝࡶື≀ࢆ౑⏝ࡋ࡚࠾ࡽࡎࠊື≀⚟♴㠃࠿ࡽ௦᭰ἲ࡜ࡋ࡚ጇᙜ࡛࠶ࡿࠋ

8. ヨ㦂ἲࡢ᭷⏝ᛶ࡜㝈⏺㸦ࢥࢫࢺࠊ᫬㛫࠿ࡽࡢጇᙜᛶ࡞࡝㸧ᙉ࠸㓟ᛶ㸦pH 2.0 ௨ୗ㸧ࡲࡓࡣ࢔ࣝ࢝ࣜᛶ㸦pH 11.5 ௨ୖ㸧≀㉁ࡣࠊᙉ࠸ᒁᡤస⏝ࢆ᭷ࡍࡿྍ⬟ᛶࡀ㧗࠸ࡇ࡜࠿ࡽࠊ⓶⭵⭉㣗ᛶ࡜ุ᩿ࡋ࡚ࡶⰋ࠸ࡇ࡜࡟࡞ࡗ࡚࠸ࡿ 21㸧ࠋࡋ࠿ࡋ࡞ࡀࡽࠊࡇࢀࡣ⭉㣗ᛶ࡟ࡘ࠸࡚ࡢ᝟ሗࡀ௚࡟࡞࠸ሙྜ࡟⾜ࢃࢀࡿ࣮࣡ࢫࢺࢣ࣮ࢫ࡜ࡋ࡚ࡢุ᩿࡛࠶ࡾࠊ౛࠼ࡤ㓟ࡸሷᇶࡢῧຍ࡟
ࡼࡾ pH ࡀኚࢃࡾ᫆࠸≀㉁ࡸΰྜ≀ࡢሙྜࡣഇ㝧ᛶࡢุ᩿࡜࡞ࡿྍ⬟ᛶࡶ⪃࠼ࡽࢀࡿ 22㸧ࠋࡇࡢࡓࡵࠊ
ࡇࡢࡼ࠺࡞≀㉁࡟ᑐࡋ࡚ື≀ࢆ⏝࠸ࡿᚲせࡢ࡞࠸ TG431 ࡛⓶⭵⭉㣗ᛶࢆホ౯ࡍࡿࡇ࡜ࡣ᭷⏝࡛࠶ࡿࠋ⾲ 6 ࡟♧ࡍ⩦⇍ᗘ☜ㄆ≀㉁ࢆṇࡋࡃศ㢮࡛ࡁࡿ࠿ྰ࠿ヨ㦂ࡍࡿࡇ࡜࡟ࡼࡾࠊᑓ㛛ᢏ⾡ࡢ⩦⇍࡟ࡘ࠸࡚☜ㄆࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ࡢグ㍕ࡀ TG431 ࡟࠶ࡿࠋ9. ࡑࡢ௚
 OECD ࡟ᢎㄆࡉࢀࡓࣄࢺ⾲⓶ࣔࢹࣝࡣᾏእ࡛㛤Ⓨࡉࢀࡓ〇ရ࡛࠶ࡾࠊࢥࢫࢺ㠃࡛ࡸࡸ๭㧗࡛࠶ࡿࠋ᪥ᮏ〇ࡢࣄࢺ⾲⓶ࣔࢹࣝ࡜ࡋ࡚ࠊࡇࢀࡲ࡛࡟ LabCyte EPI-Model24㸧ࡸ Vitrolife-Skin ࡀሗ࿌ࡉࢀ࡚࠸ࡿ
25㸧

ࠋ≉࡟ࠊVitrolife-Skin ࡣཌ⏕ປാ⛉Ꮫ◊✲⿵ຓ㔠஦ᴗ࡛ࣂࣜࢹ࣮ࢺࡉࢀ 26㸧ࠊJaCVAM ࡟࠾࠸࡚ࡶホ౯ࡀ࡞ࡉࢀ࡚࠸ࡿ 27㸧ࠋࡓࡔࡋࠊOECD ࡢᛶ⬟ᶆ‽࡟‽ࡌࡓࣂࣜࢹ࣮ࢩࣙࣥࡣᐇ᪋ࡉࢀ࡚࠾ࡽࡎࠊOECD
࡟᪂ࡓ࡞ヨ㦂ἲ࡜ࡋ࡚᥎ዡ࡛ࡁࡿࣔࢹ࡛ࣝࡣ࡞࠸ࠋࡼࡗ࡚ࠊࡇࢀࡽࡢࣔࢹࣝࡢホ౯ࡣᮏホ౯᭩࡛ࡣ⾜ࢃ࡞࠿ࡗࡓࠋ ≉チ࡟ࡘ࠸࡚ࡢ᝟ሗࡣ௒ᅇࡢ᳨ウ㈨ᩱ࡟♧ࡉࢀ࡚࠸࡞࠸ࠋ࡞࠾ࠊୖグࡋࡓࣄࢺ⾲⓶ࣔࢹࣝࡣ᪤࡟ᕷ㈍ࡉࢀ࡚࠾ࡾࠊ࠸ࡎࢀࡶ㉎ධྍ⬟࡛࠶ࡿࠋ

1218

10. ⤖ㄽ ಙ㢗ᛶ࡜ጇᙜᛶ࡜࠸࠺どⅬ࡟࠾࠸࡚ࠊࣄࢺ⾲⓶ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂ࢆホ౯ࡋࡓ⤖ᯝࠊ⿕㦂≀㉁ࡢ⓶⭵⭉㣗ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ EpiSkin™ࠊEpiDerm™ࠊSkinEthic™࠾ࡼࡧ epiCS®ࡀ᥎ዡ࡛ࡁࡿࣔࢹࣝ࡜ࡋ࡚ᣲࡆࡽࢀࡓࠋࡓࡔࡋࠊᅜ㐃 GHS ⣽༊ศࢆ⪃៖ࡍࡿሙྜࡣࠊEpiSkin™ࡀ⓶⭵⭉㣗ᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚ࡶࡗ࡜ࡶ᭷⏝࡛࠶ࡿ࡜⤖ㄽࡋࡓࠋ1319

⾲ 6㸬⩦⇍ᗘ☜ㄆ≀㉁ 3㸧໬Ꮫ≀㉁ 1

CASRN

໬Ꮫ≀㉁
ศ㢮 2

UN GHS
VRM㸦inMTT㸦in vivo ヨ㦂㸧 vitro ヨ㦂㸧࡟
5
㑏ඖ๣
3
࡟ࡼࡿ༊ศ
ࡼࡿ༊ศ 4≀⌮ⓗ≧ែ༊ศ 1A ࡢ in vivo ⭉㣗ᛶ≀㉁
79-08-3

᭷ᶵ㓟

1A(3) 1A--ᅛయ

3 ࣇࢵ໬࣎ࣟࣥ
஧Ỉ࿴≀13319-75-0

↓ᶵ㓟

1A

(3) 1A

--

ᾮయ

ࣇ࢙ࣀ࣮ࣝ

108-95-2

ࣇ࢙ࣀ࣮ࣝ㢮

1A(3) 1A--ᅛయࢪࢡࣟࣟ࢔ࢭࢳࣝ
ࢡࣟࣜࢻ

79-36-7

ồ㟁Ꮚ๣

1A(3) 1A

--

ᾮయ

ࣈࣟࣔ㓑㓟

༊ศ 1B/1C ࡢ in vivo ⭉㣗ᛶ≀ࢢࣜ࢜࢟ࢩࣝ㓟୍Ỉ࿴≀

563-96-2

᭷ᶵ㓟

1B/1C

(3) 1B/1C

--

ᅛయ

ங㓟

598-82-3

᭷ᶵ㓟

1B/1C

(3) 1B/1C--

ᾮయ࢚ࢱࣀ࣮ࣝ࢔࣑ࣥ

141-43-5᭷ᶵሷᇶ

1B(3) 1B/1C

ሷ㓟㸦14.4%㸧

7647-01-0

↓ᶵ㓟

1B/1C

(3) 1B/1C

⢓⛻ᛶ
--

ᾮయ

in vivo 㠀⭉㣗ᛶ≀㉁⮯໬ࣇ࢙ࢿࢳࣝ

103-63-9ồ㟁Ꮚ๣NC(3) NC

4-࢔࣑ࣀ-1,2,4ࢺࣜ࢔ࢰ࣮ࣝ

584-13-4

᭷ᶵሷᇶNC(3) NC

4-㸦࣓ࢳࣝࢳ࢜㸧࣋ࣥࢬ࢔ࣝࢹࣄࢻ

3446-89-7

ồ㟁Ꮚ๣

NC

(3) NCࣛ࢘ࣜࣥ㓟

143-07-7᭷ᶵ㓟NC(3) NC

ᾮయ
--

ᅛయ
ᾮయ

--

ᅛయ

␎ㄒ㸸CASRN㸻CAS Ⓩ㘓␒ྕ㸹UN GHS㸻ᅜ㐃່࿌ࠕ໬Ꮫရࡢศ㢮࠾ࡼࡧ⾲♧࡟㛵ࡍࡿୡ⏺ㄪ࿴ࢩࢫࢸ࣒ࠖ(1)㸹VRM
㸻ࣂࣜࢹ࣮ࢩࣙࣥ῭ࡳᶆ‽ヨ㦂ἲ㸹NC㸻㠀⭉㣗ᛶ
1ࡇࢀࡽࡢ໬Ꮫ≀㉁ࡣࠊࡲࡎ⭉㣗ᛶ࡛࠶ࡿ࠿㠀⭉㣗ᛶ࡛࠶ࡿ࠿࡟ࡼࡾศ㢮ࡋࠊࡉࡽ࡟⭉㣗ᛶࡢ⣽༊ศ࠾ࡼࡧ໬Ꮫ≀㉁ࡢ✀㢮࡟ࡼࡾ⣽ศ㢮ࡋࡓࠋ⾲࡟グ㍕ࡋࡓ໬Ꮫ≀㉁ࡣࠊECVAM ࡟ࡼࡿ EpiSkinTM ࠾ࡼࡧ EpiDermTM ࡢࣂࣜࢹ࣮ࢩࣙ
ࣥヨ㦂࡛౑⏝ࡉࢀࡓ≀㉁ 4,5,8)ࠊ࡞ࡽࡧ࡟ EpiSkinTM 23㸧ࠊEpiDermTMࠊSkinEthicTM ࠾ࡼࡧ epiCS® 7)ࡢ㛤Ⓨ⪅࡟ࡼࡾᥦ౪ࡉ
ࢀࡓࢹ࣮ࢱ࡟ᇶ࡙࠸ࡓࣂࣜࢹ࣮ࢩࣙࣥᚋヨ㦂ࡼࡾ㑅ᢥࡋࡓࡶࡢ࡛࠶ࡿࠋ≉࡟グ㍕ࡢ࡞࠸㝈ࡾࠊᕷ㈍ࡉࢀ࡚࠸ࡿ໬Ꮫ≀㉁ࡢ㉎ධ᫬ࡢ⣧ᗘ࡟࠾࠸࡚ヨ㦂ࢆ⾜ࡗࡓ 5,8)ࠋࡇࡢ㑅ᢥ࡟࠶ࡓࡗ࡚ࡣࠊྍ⬟࡞㝈ࡾ௨ୗࡢࡼ࠺࡞≀㉁ࢆྵࡵࡿࡇ࡜
࡜ࡋࡓ㸸(i) VRM ࡟ࡼࡾ ᐃࡲࡓࡣண ྍ⬟࡞⭉㣗ᛶ཯ᛂࡢ⠊ᅖ㸦౛࠼ࡤࠊ㠀⭉㣗ᛶࠊᙅ⭉㣗ᛶ࡞࠸ࡋᙉ⭉㣗ᛶ㸧ࢆ௦⾲ࡍࡿ໬Ꮫ≀㉁ࠊ(ii)ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ศ㢮ࢆ௦⾲ࡍࡿ≀㉁ࠊ
(iii)໬Ꮫᵓ㐀ࡀ᫂☜࡟ྠᐃࡉࢀ࡚࠸ࡿ≀㉁ࠊ(iv)VRM ࡟࠾࠸࡚෌⌧ᛶࡢ㧗࠸⤖ᯝࡀᚓࡽࢀࡿ≀㉁ࠊ(v)in vivo ࡛ࡢᶆ‽ヨ㦂ἲ࡟࠾࠸࡚☜ᐇ࡞⤖ᯝࡀᚓࡽࢀࡿ≀㉁ࠊ(vi)ᕷ㈍ࡉࢀ࡚࠸ࡿ≀㉁ࠊ࡞ࡽࡧ࡟(vii) 㠀ᖖ࡟㧗㢠࡞ᗫᲠࢥࢫࢺࡀ࠿࠿
ࡽ࡞࠸≀㉁ࠋ
2
Barratt ࡽ(1998)࡟ࡼࡿ໬Ꮫ≀㉁ศ㢮 8)ࠋ
3
UN GHS ࡢ⣽༊ศ 1Aࠊ1B ࠾ࡼࡧ 1C ࡟ࡣࠊᅜ㐃ໟ⿦➼⣭ IࠊII ࠾ࡼࡧ III ࡀࡑࢀࡒࢀᑐᛂࡍࡿࠋ
4⾲࡟グ㍕ࡋࡓ VRM ࡟ࡼࡿ in vitro ࡛ࡢศ㢮ண ࡣࠊヨ㦂ἲࡢ㛤Ⓨ⪅ࡀ⾜ࡗࡓࣂࣜࢹ࣮ࢩࣙࣥᚋヨ㦂࡟࠾࠸࡚
EpiSkinTM ࠾ࡼࡧ EpiDermTM ヨ㦂ἲ㸦VRM㸧࡟ࡼࡾᚓࡽࢀࡓࡶࡢ࡛࠶ࡿࠋ
5
ECVAM ࡟ࡼࡿ⓶⭵⭉㣗ᛶヨ㦂࡛ᚓࡽࢀࡓ⏕Ꮡ⋡ࡢ್ࡣࠊ┤᥋ⓗ࡞ MTT 㑏ඖࢆ⪃៖ࡋࡓ⿵ṇࢆ⾜ࡗ࡚࠸࡞࠸ࠋ
ࣂࣜࢹ࣮ࢩࣙࣥᚋヨ㦂ࡢࢹ࣮ࢱࡣࠊ㐺ษ࡞ᑐ↷ࢆ⏝࠸࡚ᚓࡓࡶࡢ࡛࠶ࡿࠋ

1420

11㸬ᩥ⊩
1) OECD (2015) Guideline for the testing of chemicals. 404, Acute Dermal Irritation/Corrosion
2) OECD (2004) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model
test.
3) OECD (2016) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model
test.
4) Fentem, J.H. et al. (1998) The ECVAM International Validation Study on in vitro tests for skincorrosivity. 2. Results and evaluation by the management team. Toxicology in Vitro, 12, 483-524.
5) Liebsch, M., et al. (2000) The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity
testing. ATLA, 28, 371-401.
6) Kandarova, H. et al. (2006) Assessment of the human epidermis model SkinEthic RHE for in vitro
skin corrosion testing of chemicals according to new OECD TG 431, Toxicology in Vitro, 20,
547-559.
7) Desprez, B. et al. (2015) Two novel prediction models improve prediction of skin corrosive
sub-category by test methods of OECD Test Guideline No. 431, Toxicol. In Vitro, 29,2055-2080.
8)

Barratt, M.D. et al. (1998) The ECVAM International Validation Study on in vitro tests for skincorrosivity. 1. Selection and Distribution of the test chemicals. Toxicology in Vitro, 12, 471-482.

9)

Worth, A.P. et al. (1998) An evaluation of the proposed OECD testing strategy for skin corrosion.
ATLA, 26, 709-720.

10)Botham, P.A. et al. (1995) A pre-validation study on in vitro skin corrosivity testing: The report andrecommendations of ECVAM workshop 6. ATLA, 23, 219-255.

11) ICCVAM (1999) NIH Publication No.99-4495. Corrositex: An in vitro test method for assessing
dermal corrosivity potential of chemicals.
12) ICCVAM (2002) NIH Publication No.02-4502. ICCVAM Evaluation of EPISKIN, and EpiDerm(EPI-200) and rat skin transcutaneous electrical resistance (TER) assay: in vitro test method forassessing dermal corrosivity potential of chemicals.
13) ECVAM (1998) Statement on the scientific validity of the Episkin test (An in vitro test for skincorrosivity)14) ECVAM (2000) Statement on the application of the EpiDermTM human skin model for skin corrosivity
testing
15)

ECVAM (2006) Statement on the application of the SkinEthicTM human skin model for skincorrosivity testing

16)

ECVAM (2009) ESAC Statement on the scientific validity of an in-vitro test for skin corrosivity
testing

17)

OECD (2015) Series on Testing & Assessment No. 219, Performance Standards for the assessmentof proposed similar or modified in vitro reconstructed human epidermis (RhE) test methods for skincorrosion testing as described in TG 431

18) OECD (2013) Series on Testing & Assessment No. 190, Summary document on the Statistical
Performance of Methods in OECD Test Guideline 431 for Sub-Categorisation
19) OECD (2015) Guideline for the testing of chemicals. 430, in vitro Skin Corrosion: Transcutaneouselectrical resistance test (TER).
15

2120) OECD (2015) Guideline for the testing of chemicals. 435, in vitro membrane barrier test method for
Skin Corrosion.
21) OECD (2014) Series on Testing and Assessment No. 203, Guidance document on an Integrated
Approach on Testing and Assessment (IATA) for skin corrosion and irritation
22) Sheel J. et.al. (2011) Classification and labeling of industrial products with extreme pH by making useof in vitro methods for the assessment of skin and eye irritation and corrosion in a weight of evidence
approach, Toxicology in Vitro, 25, 1435-1447
23) Alépée N., Grandidier M.H., and Cotovio J. (2014). Sub-Categorisation of Skin Corrosive

Chemicals by the EpiSkinTM Reconstructed Human Epidermis Skin Corrosion Test Method
according to UNGHS: Revision of OECD Test Guideline 431. Toxicol. In Vitro 28, 131-145.
24) Katoh, M., et al. (2010) Assessment of the human epidermal model LabCyte EPI-MODEL for In vitro
skin corrosion testing according to the OECD test guideline 431. J Toxicol Sci., 35(3):411-7.
25) Morikawa, N., et al. (2005) Experimental study on a novel chemical application procedure for invitro skin corrosivity testing using the Vitrolife-Skin TM human skin model, AATEX, 11, 1, 68-78.
26) Kojima H et al. (2008) Validation of human skin models for skin corrosivity tests in Japan, AATEX
13(1), 36-44.
27) JaCVAM (2008) ࣄࢺ⓶⭵ࣔࢹࣝࢆ⏝࠸ࡓ⓶⭵⭉㣗ᛶヨ㦂௦᭰ἲࡢ➨୕⪅ホ౯ሗ࿌᭩㸦Vitrolif-SkinTM㸧

16

22

ANNEX
1) Fentem J.H. et al. (1998), The ECVAM International Validation Study on in vitro tests for skincorrosivity. 2. Results and evaluation by the management team. Toxicology in Vitro, 12, 483-524 ᢤ⢋2) Liebsch, M. et al. (2000), The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity
testing. ATLA, 28, 371-401 ᢤ⢋
3) Kandarova, H. et al. (2006), Assessment of the human epidermis model SkinEthic RHE for in vitro skincorrosion testing of chemicals according to new OECD TG 431, Toxicology in Vitro, 20, 547-559ᢤ⢋4) OECD (2004) Guideline for the testing of chemicals. 431, in vitro Skin Corrosion: Human skin model
test. ᢤ⢋5) OECD (2013) GD190 Summary document on the Statistical Performance of Methods in OECD Test
Guideline 431 for Sub-Categorisation ᢤ⢋6) Desprez, B. et al. (2015), Two novel prediction models improve prediction of skin corrosive
sub-category by test methods of OECD Test Guideline No. 431, Toxicol. In Vitro, 29,2055-2080 ᢤ⢋

17

23

ANNEX㸯㸸Fentem et al (1998)4)ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑ࢃࢀࡓ 60 ≀㉁

18

24

ANNEX 2: Liebsch et al (2000)5)ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑ࢃࢀࡓ 24 ≀㉁

19

25

ANNEX 3: Kandarova et al (2006)6)ࡢࣂࣜࢹ࣮ࢩ࡛ࣙࣥ౑ࢃࢀࡓ 12 ≀㉁

ANNEX 4:OECDTG431 (2004)3)ࡢཧ↷≀㉁

Table 1: Reference Chemicals
1,2-Diaminopropane

CAS-No. 78-90-0

Severely Corrosive

Acrylic Acid
2-tert-Butylphenol

CAS-No. 79-10-7

Severely Corrosive

CAS-No. 88-18-6

Corrosive

Potassium hydroxide (10%)

CAS-No. 1310-58-3

Corrosive

Sulfuric acid (10%)

CAS-No. 7664-93-9

Corrosive

Octanoic acid (caprylic acid)

CAS-No. 124-07-02

Corrosive

4-Amino-1,2,4-triazole

CAS-No. 584-13-4

Not corrosive

Eugenol

CAS-No. 97-53-0

Not corrosive

Phenethyl bromide

CAS-No. 103-63-9

Not corrosive

Tetrachloroethylene

CAS-No. 127-18-4

Not corrosive

Isostearic acid

CAS-No. 30399-84-9

Not corrosive

4-(Methylthio)-benzaldehyde

CAS-No. 3446-89-7

Not corrosive

20

26

2

1

27

ANNEX 5: Desprez et al(2015)7) 80 ≀㉁ᢤ⢋

21

1

28
22

1

29

23

4

3

2

1

30
24

ANNEX 6: OECD (2013)18)ண

ᛶࢹ࣮ࢱᢤ⢋

⾲ A-1㸬EpiDermTM ࡢ⭉㣗ᛶ᭷↓ࡢண

ᛶ

25

31

⾲ A-2㸬EpiSkinTM ࡢ⭉㣗ᛶ᭷↓ࡢண

ᛶ

26

32

⾲ A-3㸬SkinEthicTM ࡢ⭉㣗ᛶ᭷↓ࡢண⾲ A-4㸬EpiCS®⭉㣗ᛶ᭷↓ࡢண

ᛶ

ᛶ

27

33

34

OECD/OCDE

431
Adopted:29 July 2016

OECD GUIDELINE FOR THE 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 skin manifested as visible
necrosis through the epidermis and into the dermis, following the application of a test chemical [as definedby 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-corrosive and 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 the use oflaboratory 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 in vitro test methods for testing corrosion
potential of chemicals have been validated and adopted 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 the assessment of skin irritation andskin corrosion potentials of chemicals and (ii) proposes an approach when further testing is needed (6).
3.
This Test Guideline addresses the human health endpoint skin corrosion. It makes use ofreconstructed human epidermis (RhE) (obtained from human derived non-transformed epidermal
keratinocytes) which closely mimics the histological, morphological, biochemical and physiologicalproperties of the upper parts of the human skin, i.e. the epidermis. This Test Guideline was originallyadopted in 2004 and updated in 2013 to include additional test methods using the RhE modelsand thepossibility to use the methods to support the sub-categorisation of corrosive chemicals, and updated in2015 to refer to the IATA guidance document and introduce the use of an alternative procedure to measure
viability.
4.
Four validated test methods using commercially available RhE models are included in this Test
Guideline. 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 c-200) (referred to in the following
text as the Validated Reference Methods
VRMs). The outcome of these studies led to therecommendation that the two VRMs mentioned above could be used for regulatory purposes for
distinguishing corrosive (C) from non1
© OECD, (2016)
You are free to use this material
http://www.oecd.org/termsandconditions/.

subject

to

the

terms

and

conditions

availableatThis Guideline was adopted by the OECD Council by written procedure on 29 July 2016 [C(2016)103].

35

431

OECD/OCDE

be used to support sub-categorisation of corrosive substances (13) (14) (15). Two other commerciallyavailable in vitro
1
according to PSand epiCS® (previously
named EST-1000) that can also be used for regulatory purposes for distinguishing corrosive from noncorrosive 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 subcategorisation of corrosives (21) (22). Further statistical analyses of the post-validation data generated with®
have been performed to identify alternative predictions
models that improved the predictive capacity for sub-categorisation (23).
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 therequirements of the Performance Standards (PS) (24) set out in accordance with the principles of GuidanceDocument 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 testin vitro test method for skin corrosion, while benefiting from the Mutual Acceptance of Data.
DEFINITIONS
6.

Definitions used are provided in Annex 1.

INITIAL CONSIDERATIONS
7.
This Test Guideline allows the identification of non-corrosive and corrosive substances andmixtures in accordance with the UN GHS (1). This Test Guideline further supports the sub-categorisationof corrosive substances and mixtures into optional Sub-category 1A, in accordance with the UN GHS (1),
as well as a combination of Sub-categories 1B and 1C (21) (22) (23). A limitation of this Test Guideline isthat it does not allow 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®
test methods are able tosub-categorise (i.e. 1A versus 1B-and-1C versus NC)
8.
A wide range of chemicals representing mainly individual substances has been tested in thevalidation supporting the test methods included in this Test Guideline when they are used for identificationof non-corrosives and corrosives; the empirical database of the validation study amounted to 60 chemicals
covering a wide range of chemical classes (8) (9) (10). Testing to demonstrate sensitivity, specificity,
accuracy and within-laboratory-reproducibility of the assay for sub-categorisation was performed by thetest method developers and results 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 physical states
including liquids, semi-solids, solids and waxes. The liquids may be aqueous or non-aqueous; solids may
be soluble or insoluble in water. Whenever possible, solids should be ground to a fine powder before
application; no other prior treatment of the sample is required. In cases where evidence can bedemonstrated on the non-applicability of test methods included in the Test Guideline to a specific categoryof test chemicals, these test methods should not be used for that specific category of test chemicals. In
1

The abbreviation RhE (=Reconstructed human Epidermis) is used for all models based on RhE technology. The
abbreviation RHE as used in conjunction with the SkinEthic TM model means the same, but, as part of the name ofthis specific test method as marketed, is spelled all in capitals.

2© OECD, (2016)36

OECD/OCDE

431

addition, this Test 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 specificcategory of mixtures. Before use of the test guideline on a mixture for generating data for an intended
regulatory purpose, it should be considered whether, and if so why, it may provide adequate results for that
purpose. Such considerations are not needed, when there is a regulatory requirement for testing of the
mixture. Gases and aerosols have not been assessed yet in validation studies (8) (9) (10). While it isconceivable that these can be tested using RhE technology, the current Test Guideline does not allow
testing of gases and aerosols.
9.
Test chemicals absorbing light in the same range as MTT formazan and test chemicals able todirectly reduce the vital dye MTT (to MTT formazan) may interfere with the tissue viability measurements
and need the use of adapted controls for corrections. The type of adapted controls that may be required willvary depending on the type of interference produced by the test chemical and the procedure used tomeasure 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 irritation in vitro and is based on thesame RhE test system, though using another protocol (5). For a full evaluation of local skin effects after asingle 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 skincorrosion (such as described in 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, comprised of nontransformed, human-derived epidermal keratinocytes, which have been cultured to form a multi-layered,highly differentiated model of the human epidermis. It consists of organized basal, spinous and granularlayers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo.
12.
The RhE test method is based on the premise that corrosive chemicals are able to penetrate thestratum corneum by diffusion or erosion, and are cytotoxic to the cells in the underlying layers. Cell
viability is measured by enzymatic conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS number 298-93-1], into a blue
formazan salt that is quantitatively measured after extraction from tissues (27). Corrosive chemicals are
identified by their ability to decrease cell viability below defined threshold levels (see paragraphs 35 and36). The RhE-based skin corrosion test methods have shown to be predictive of in vivo skin corrosion
effects assessed in rabbits according to the OECD guideline 404 (2).

3© OECD, (2016)

37

431

OECD/OCDE

DEMONSTRATION OF PROFICIENCY
13.
Prior to routine use of any of the four validated RhE test methods that adhere to this Test
Guideline, laboratories should demonstrate technical proficiency by correctly classifying the twelve
Proficiency Substances listed in Table 1. In case of the use of a method for sub-classification, also thecorrect sub-categorisation should be demonstrated. In situations where a listed substance is unavailable orwhere justifiable, another substance for which adequate in vivo and in vitro reference data are available
may be used (e.g. from the list of reference chemicals (24)) provided that the same selection criteria as
described in Table 1 is applied.

4© OECD, (2016)38

OECD/OCDE

431

Table 1: List of Proficiency Substances1

Substance

CASRN

Chemical Class

2UN GHS
Cat. Basedon In Vivo
results 3

VRM
Cat. Based on
In Vitro results4

MTT
Reducer5

PhysicalState

Sub-category 1A In Vivo Corrosives
Bromoacetic acid
Boron trifluoride
dihydrate
PhenolDichloroacetyl
chloride79-08-3

Organic acid1A

(3) 1A--S

13319-75-0

Inorganic acid1A

(3) 1A--L

108-95-2

Phenol1A(3) 1A--S

79-36-7

Electrophile1A(3) 1A

--

L

--

S

Combination of sub-categories 1B-and-1C In Vivo Corrosives
Glyoxylic acidmonohydrate
Lactic acid
Ethanolamine
Hydrochloric acid
(14.4%)

563-96-2

Organic acid

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

598-82-3

Organic acid

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

141-43-5

Organic base

1B

(3) 1B-and-1CY

Viscous7647-01-0

Inorganic acid

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

L

In Vivo Non Corrosives
Phenethyl bromide

103-63-9

ElectrophileNC(3) NCY

L

4-Amino-1,2,4triazole

584-13-4

Organic baseNC(3) NC--S

4-(methylthio)benzaldehyde

3446-89-7

ElectrophileNC(3) NCY

L

Lauric acid143-07-7

Organic acidNC(3) NC--S

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; UN GHS = United Nations Globally Harmonized System(1); VRM = Validated Reference Method; NC = Not Corrosive1
The proficiency substances, sorted first by corrosives versus non-corrosives, then by corrosive sub-category and then by chemicalTM(8) (9) (10) and fromclass, were selected from
TM
TM
TM
®
post-validation studies based on data provided by EpiSkin (22), EpiDerm , SkinEthic and epiCS developers (23). Unlessotherwise indicated, the substances were tested at the purity level obtained when purchased from a commercial source (8) (10).
The selection includes, to the extent possible, substances that: (i) are representative of the range of corrosivity responses (e.g. noncorrosives; weak to strong corrosives) that the VRMs are capable of measuring or predicting; (ii) are representative of the chemicalclasses 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) are commercially available; and (vii) are not associatedwith prohibitive disposal costs.
2
Chemical 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.
4
The VRM in vitro predictions reported in this table were obtained with the EpiSkin TM and the EpiDermTM test methods (VRMs)
during post-validation testing performed by the test method developers.
5
The 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 the barrier propertiesof the tissues after receipt as specified by the RhE model manufacturer. This is particularly important if
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and proficiency in its use has been demonstrated, such verification will not be necessary on a routine basis.
However, when using 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 RhE models endorsed as scientifically-200), SkinEthicTM RHE
valid for use within this Test Guideline, i.e. the EpiSkinTM®
and epiCS models (16) (17) (19) (28) (29) (30) (31) (32) (33), can be obtained from commercial sources.
Standard Operating Procedures (SOPs) for these four RhE models are available (34) (35) (36) (37), andtheir main test method components are summarised in Annex 2. It is recommended that the relevant SOP
be consulted when implementing and using one of these methods in the laboratory. Testing with the four
RhE test methods covered by this Test Guideline should comply with the following:

RHE TEST METHOD COMPONENTS
General Conditions
16.
Non-transformed human keratinocytes should be used to reconstruct the epithelium. 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 bedemonstrated and may 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, or by determinationof the exposure time required to reduce cell viability by 50% (ET50) upon application of the benchmark
chemical at a specified, fixed concentration (see paragraph 18). The containment properties of the RhEmodel should prevent the passage of material around the stratum corneum to the viable tissue, which
would lead to poor modelling of skin exposure. The RhE model should be free of contamination bybacteria, 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 the negative control OD values for the four
validated RhE test methods included in this Test Guideline are given in Table 2. An HPLC/UPLCSpectrophotometry user should use the negative control OD ranges provided in Table 2 as the acceptance6© OECD, (2016)40

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criterion for the negative control. It should be documented that the tissues treated with negative control arestable in culture (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-200)
SkinEthic

TM

RHE

®

epiCS

Barrier function
18.
The stratum corneum and its lipid composition should be sufficient to resist the rapid penetrationof certain cytotoxic benchmark chemicals (e.g. SDS or Triton X-100), as estimated by IC50 or ET50 (Table3). The barrier function of each batch of the RhE model used should be demonstrated by the RhE model
developer/vendor upon supply of the tissues 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 andstratum corneum and exhibits lipid profile similar to lipid profile of human epidermis. Histologicalexamination of each batch of the RhE model used demonstrating appropriate morphology of the tissues
should be provided by the RhE model developer/vendor upon supply of the tissues to the end user (seeparagraph 21).
Reproducibility
20.
Test method users should demonstrate reproducibility of the test methods over time with thepositive and negative controls. Furthermore, the test method should only be used if the RhE model
developer/supplier provides data demonstrating reproducibility over time with 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 subcategorisation, the reproducibility with respect to sub-categorisation should also be demonstrated.
Quality control (QC)
21.
The RhE model should only be used if the developer/supplier demonstrates that each batch of the
RhE model used meets defined production release criteria, among which those for viability (paragraph 17),barrier function (paragraph 18) and morphology (paragraph 19) are the most relevant. These data areprovided to the test method users, so that 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 is established by the
RhE model developer/supplier. The acceptability ranges for the four validated test methods are given in
Table 3.
Table 3: QC batch release criteria
TM

EpiSkin(SM)

Lower acceptance limit

Upper acceptance limit

IC50 = 1.0 mg/mL

IC50 = 3.0 mg/mL

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SDS)(33)-200)

ET50 = 4.0 hours

ET50 = 8.7 hours

ET50 = 4.0 hours

ET50 = 10.0 hours

ET50 = 2.0 hours

ET50 = 7.0 hours(1% Triton X-100)(34)
SkinEthicTM RHE(1% Triton X-100)(35)
epiCS®(1% Triton X-100)(36)

Application of the Test Chemical and Control Substances
22.
At least two tissue replicates should be used for each test chemical and controls for each exposure
time. For liquid as well as solid chemicals, sufficient amount of test chemical should be applied to2
uniformly cover the epidermis surface while avoiding an infinite dose, i.e. a minimum of 70
or 30
2
mg/cm 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 the epidermis surface (34) (35) (36) (37). Whenever possible, solids should be tested as a fine
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 anaqueous buffer, or 0.9% NaCl. Depending on which of the four validated RhE test methods is used, two or
three exposure periods are used per test chemical (for all four valid RhE models: 3 min and 1 hour; for
EpiSkinTM an additional exposure time of 4 hours). Depending on the RhE test method used and theexposure 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 to demonstrate that
viability (with negative controls), barrier function and resulting tissue sensitivity (with the PC) of thetissues are within a defined historical acceptance range. The suggested PC chemicals are glacial acetic acidor 8N KOH depending upon the RhE model used. 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 thisTest Guideline (27). The tissue sample is placed in MTT solution of appropriate concentration (0.3 or
1 mg/mL) for 3 hours. The precipitated blue formazan product is then extracted from the tissue using asolvent (e.g. isopropanol, acidic isopropanol), and the concentration of formazan is measured bydetermining the OD at 570 nm using a filter band pass of maximum ± 30 nm, or by an HPLC/UPLCspectrophotometry 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 notcompletely 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
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reduction and interferences by colouring agents is available in the SOPs for the test methods (34) (35) (36)
(37).
26.
To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT
medium (34) (35) (36) (37). If the MTT mixture containing the test chemical turns blue/purple, the test
chemical is presumed to directly reduce the MTT, and further functional check on non-viable epidermis
should be performed, independently of using the standard absorbance (OD) measurement or an
HPLC/UPLC-spectrophotometry procedure. This additional functional check employs killed tissues thatpossess 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 then calculated 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 thenegative 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 during exposure) 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/UPLCspectrophotometry 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 interfering coloured test
chemical is applied on at least two viable tissue replicates per exposure time, which undergo the entire skincorrosion test but are incubated with medium instead of MTT solution during the MTT incubation step togenerate a non-specific colour (NSCliving) control. The NSCliving control needs to be performed concurrently
per exposure time per coloured test chemical (in each run) due to the inherent biological variability ofliving tissues. The true tissue viability is then calculated as the percent tissue viability obtained with living
tissues exposed to the interfering test chemical and incubated with MTT solution minus the percent nonspecific colour obtained with living tissues exposed to the interfering test chemical and incubated withmedium without MTT, run concurrently to the test being corrected (%NSCliving).
28.
Test chemicals that are identified as producing both direct MTT reduction (see paragraph 26) andcolour interference (see paragraph 27) will also require a third set of controls, apart from the NSMTT and
NSCliving controls described in the previous 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 to directlyreduce MTT as described in paragraph 26. These test chemicals may bind to both living 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 the binding of the test chemical to killed tissues.
This could lead to a double correction for colour interference since the NSCliving control already corrects forcolour 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 testing procedure 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 the number 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 the test
chemical minus %NSMTT minus %NSCliving plus the percent non-specific colour obtained with killed
tissues exposed to the interfering test chemical and incubated with medium without MTT, calculated
relative to the negative control run concurrently to the test being corrected (%NSCkilled).
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It is important to note that non-specific MTT reduction and non-specific colour interferences may
increase the readoutsof the tissue extract above the linearity range of the spectrophotometer. 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 isappropriate to assess direct MTT-reducers and colour interfering test chemicals when the ODs of the tissue
extracts obtained with the test chemical without any correction for direct MTT reduction and/or colour
interference are within the linear range of the spectrophotometer or 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
taken with 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/UPLCspectrophotometry 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/UPLC-spectrophotometry, independently of the chemical being tested. NSMTT
controls should nevertheless be used if the test chemical is suspected to directly reduce MTT or has acolour that impedes the assessment of the capacity to directly reduce MTT (as described in paragraph 26).
When using HPLC/UPLC-spectrophotometry to measure MTT formazan, the percent tissue viability is
calculated as percent 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. For test chemicals able
to directly reduce MTT, true tissue viability is calculated as the percent tissue viability obtained with living
tissues exposed to the test chemical minus %NSMTT. Finally, it should be noted that direct MTT-reducers
that may also be colour interfering, which are retained in the tissues after treatment and reduce MTT sostrongly that they lead to ODs (using standard OD measurement) or peak areas (using UPLC/HPLCspectrophotometry) of the tested tissue extracts that fall outside of the linearity range of thespectrophotometer 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 thediversity of HPLC/UPLC-spectrophotometry systems,
qualification of the HPLC/UPLCspectrophotometry system should be demonstrated before its use to quantify MTT formazan from tissue
extracts by meeting the acceptance criteria for a set of standard qualification parameters based on thosedescribed 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. Once theacceptance criteria defined in Annex 4 have been met, the HPLC/UPLC-spectrophotometry system isconsidered qualified and ready to measure MTT formazan under the experimental conditions described inthis Test Guideline.
Acceptability 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 and should not be below historicallyestablished boundaries. Tissues treated with the PC, i.e. glacial acetic acid or 8N KOH, should reflect theability of the tissues to respond to a corrosive chemical under the conditions of the test method (see Annex2). The variability between tissue replicates of test chemical and/or control substances should fall within
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the accepted limits for each valid RhE model requirements (see Annex 2) (e.g. the difference of viability
between the two tissue 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 thevariability of test chemicals falls outside of the defined range, its testing should 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, thepercent tissue viability is calculated as percent 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 cut-off 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 and should be used for interpreting theresults.
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 of borderline results, such as
non-concordant replicate measurements, a second run may be considered, 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:

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Table 4:EpiSkinTM prediction model
Viability measured after exposure time
points (t=3, 60 and 240 minutes)

Prediction
to be consideredCorrosive:< 35% after 3 min exposure

Optional Sub-category 1A *AND
< 35% after 60 min exposure

Corrosive:OR

A combination of optional
Sub-categories 1B-and-1CAND< 35% after 240 min exposure

Non-corrosive*) According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subTM
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 SkinEthicTM RHE (17) (18)(23) (36), and the epiCS® (16) (23) (37) skin corrosion test methods, associated with the UN GHS (1)classification system, are shown in Table 5:
Table 5:EpiDermTM SCT, SkinEthicTM RHE and epiCS®
Viability measured after exposure time
points (t=3 and 60 minutes)

Prediction
to be considered®

< 50% after 3 min exposure

CorrosiveAND< 15% after 60 min exposure

CorrosiveAND

Non-corrosive

for substances/mixtures identified as Corrosive in step 1< 25% after 3 min exposure

Optional Sub-category 1A *A combination of optional Sub-categories 1B-and-1C

STEP 2 forfor substances/mixtures identified as Corrosive in step 1< 18 % after 3 min exposure

Optional Sub-category 1A *A combination of optional Sub-categories 1B-and-1C

STEP 2 for epiCS®

for substances/mixtures identified as Corrosive in step 1< 15 % after 3 min exposure

Optional Sub-category 1A *A combination of optional Sub-categories 1B-and-1C* According to the data generated in view of assessing the usefulness of the RhE test methods for supporting subcategorisation, it was shown that around 29%, 31% and 33% of the Sub-

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test methods, 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.
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 theconstituents;
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), wavelength and band
pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring
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:i)
Viability;ii)
Barrier function;iii)
Morphology;
iv)
Reproducibility and predictive capacity;
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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.

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 washing procedures).
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, eachrun 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 the defined
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

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LITERATURE(1)

UN. (2013). United Nations Globally Harmonized System of Classification and Labelling of Chemicals(GHS),
Fifth
RevisedEdition,
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 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 and Assessment 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 and Recommendations 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 ECVAMInternational Validation Study on In Vitro Tests for Skin Corrosivity. 1. Selection and distribution of the
Test Chemicals. Toxicol.In Vitro 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 Evaluation by the Management Team. Toxicol.in Vitro 12, 483-524.

(10) Liebsch M., Traue D., Barrabas C., Spielmann H., Uphill, P., Wilkins S., Wiemann C., Kaufmann T.,
Remmele M. and Holzhütter H. G. (2000). The ECVAM Prevalidation Study on the Use of EpiDerm for
Skin Corrosivity Testing, ATLA 28, 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., Sladowski D., Spielmann H. et Zucco F. (1995).
Practical Aspects of the Validation of Toxicity Test Procedures. The Report and Recommendations of
ECVAM Workshops, ATLA 23, 129-147.(12) ICCVAM (Interagency Coordinating Committee on the
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Validation of Alternative Methods). (1997). Validation and Regulatory Acceptance of Toxicological Test
Methods. NIH Publication No. 97-3981. National Institute of Environmental Health Sciences, Research
Triangle Park, NC, USA. Available at: [http://www.iccvam.niehs.nih.gov/docs/guidelines/validate.pdf].
(13) ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods). (2002).
ICCVAM evaluation of EpiDermTM (EPI-200), EPISKINTM (SM), and the Rat Skin Transcutaneous
Electrical Resistance (TER) Assay: In Vitro Test Methods for Assessing Dermal Corrosivity Potential ofChemicals. NIH Publication No. 02-4502. National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods, National Institute of Environmental Health Sciences,
Research
Triangle
Park,
NC,
USA.Available
at:[http://www.iccvam.niehs.nih.gov/methods/epiddocs/epis_brd.pdf](14) EC-ECVAM. (1998). Statement on the Scientific Validity of the EpiSkinTM Test (an In Vitro Test for
Skin Corrosivity), Issued by the ECVAM Scientific Advisory Committee (ESAC10), 3 April 1998.
Available at: [http://www.ecvam.jrc.ec.europa.eu.html].
(15) EC-ECVAM. (2000). Statement on the Application of the EpiDermTM Human Skin Model for Skin
Corrosivity Testing, Issued by the ECVAM Scientific Advisory Committee (ESAC14), 21 March 2000.
Available at: [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 19, 925-929.
(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 Testingof 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 SkinEthicTM
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 SkinEthicTM Human Skin Model for Skin
Corrosivity Testing, Issued by the ECVAM Scientific Advisory Committee (ESAC25), 17 November
2006. Available at: [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 in OECD Test Guideline
431 for Sub-categorisation. Environment, Health, and Safety Publications, Series on Testing andAssessment (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
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
16© OECD, (2016)

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OECD/OCDE

431

Prediction Models Improve Predictions of Skin Corrosive Sub-categories 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 Testingand 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 theEuropean 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, 55-63.
(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 andUltrastructural 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 ofa Bilayered Dermal Substitute. Exp. Cell Res. 193: 310-319.
(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.
(34)

INVITTOX Protocol (No. 118.). EpiSki
Available at: [http://www.ecvam.jrc.ec.europa.eu.hm;].

(35)-24-007-0024Protocol for: In Vitro
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)

INVITTOX Protocol (January 2012). SkinEthicTM 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].
17© OECD, (2016)

51

431

OECD/OCDE(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 MTT Assay 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 Healthand Human Services, Food and Drug Administration. (May 2001). Available at:[http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf].

18© OECD, (2016)52

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431

ANNEX 1DEFINITIONS
Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of relevance. The term is often used interchangeably
mes of a test method (25).
Cell viability: Parameter measuring total activity of a cell population e.g. as ability of cellular
mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium 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 a categorical result,and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and is definedas the proportion of all chemicals tested that are correctly classified as positive or negative. Concordance ishighly dependent on the prevalence 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 to standardized types andlevels of physical, health and environmental hazards, and addressing corresponding communication
elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data
sheets, so that to convey information on their adverse effects with a view to protect people (including
employers, workers, transporters, consumers and emergency responders) and the environment (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 theviability of the tissues by 50% (IC50) after a fixed exposure time, see also ET50.
Infinite dose: Amount of test chemical applied to the epidermis exceeding the amount required tocompletely 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.

19© OECD, (2016)

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431

OECD/OCDE

Multi-constituent substance: A substance, defined by its quantitative composition, in which more than-constituent
substance is the result of a manufacturing process. The difference between mixture and multi-constituent
substance is that a mixture is obtained by 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.
NSClivingcontrol : 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 treated with a substanceknown to induce a positive response. To ensure that variability in the positive control response across time
can be assessed, the magnitude of the positive response should not be excessive.
Performance standards (PS): Standards, based on a validated test method, that provide a basis forevaluating the comparability of a proposed test method that is mechanistically and functionally similar.
Included are; (i) essential test method components; (ii) a minimum list of Reference Chemicals selectedfrom among the chemicals used to demonstrate the acceptable 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 and whether it ismeaningful and useful for a particular purpose. It is the extent to which the test method correctly measuresor 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 assessed by calculating intra- andinter-laboratory reproducibility (25).
Run: A run consists of one or more test chemicals tested concurrently with a negative control and with aPC.
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 categorical results, and is an importantconsideration 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 the skin, complete areas of alopecia, and scars.
Histopathology should be considered to evaluate questionable lesions.

20© OECD, (2016)

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OECD/OCDE

431

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 categorical results and is an importantconsideration in assessing the relevance of a test method (25).
Substance: means chemical elements and their compounds in the natural state or obtained by any
production process, including any additive necessary to preserve the stability of the 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 biologicalmaterials.

21© OECD, (2016)

55

56

OECD/OCDEANNEX 2At least 2 per exposure time

Number oftissue replicates

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, waterkilled adapted controls should be
performed

Waxy/sticky: 20
using nylon mesh2
3 mg Semisolids: 50 μL (83.3 μL/cm )22

Liquids: 50 μL (83.3 μL/cm2) using
nylon mesh
Pre-test compatibility of test chemicalwith nylon mesh

At least 2 per exposure time

0.6 cm2

epiCS®

2

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 shouldbe performed

Waxy
mm diameter placed atop the tissue
wetted with 15 μL H2O

Solids: 25 mg (41.7 mg/cm2) + 25 μL
3 mg (40 mg/cm ) H2O (or more if necessary)

Solids: 20 μL ± 2μl H2O + 20
(40 mg/cm2)

Liquids and viscous: 40 μL ± 3μl (80μL/cm2) using nylon mesh
Pre-test compatibility of test chemicalwith nylon mesh

At least 2 per exposure time

0.5 cm2

SkinEthicTM RHE

50 μL (liquid) or 25 mg (solid)
40 μL (liquid) or 20 mg (solid)+ 1 mL MTT+ 1 mL MTT
1 mg/mL solution for 60 min
1 mg/mL solution for 180± 15 min at
at 37oC, 5% CO2, 95% RH
37°C, 5% CO2, 95% RH
if solution turns blue/purple, freezeif solution turns blue/purple,
killed adapted controls should befreeze-killed adapted controls should
performed
be performed

Waxes
mm diameter placed atop the tissue
wetted with 15 μL H2O.

Liquids and viscous: 50 μL ± 3 μL Liquids: 50 μL (79.4 μL/cm2) with or(131.6 μL/cm2)
without a nylon mesh
Pre-test compatibility of test chemical
Solids: 20 2 mg (52.6 mg/cm2) + 100 with nylon mesh
μL ± 5μL NaCl solution (9 g/L)
Semisolids: 50 μL (79.4 μL/cm2)
2 mg (131.6
Waxy/sticky: 50
Solids: 25 μL H2O (or more if
mg/cm2) with a nylon meshnecessary) + 25 mg (39.7 mg/cm2)© OECD, (2016)

Pre-check fordirect MTT
reduction

Treatment
doses andapplication

0.63 cm2

0.38 cm2

Model surface
2-3 per exposure time

EpiDermTM SCT

EpiSkinTM

Test Method
Components

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

431

57

if solution becomes coloured, living
adapted controls should be performedo

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

Tested with every exposure time

50 μL Glacial acetic acid
Tested only for 4 hours

2 mL 0.3 mg/mL

180 min ( 15 min) at 37oC, 5% CO2,
95% RH

Positive control

MTT solution

MTT
incubation
time andtemperature© OECD, (2016)

50 μL H2O
Tested with every exposure time

50 μL NaCl solution (9 g/L)

Negative
control

23

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

20 times with a constant soft streamof 1x PBS

at 37 C, 5% CO2, 95% RHo

25 mL 1x PBS (2 mL/throwing)

Room Temperature (RT, 18-28 C)

In ventilated cabinet

and 240 min ( 10 min)

3 min, 60 min ( 5 min)

180 min (± 15 min) at 37oC, 5% CO2,
95% RH

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

300 μL 1 mg/mL

50 μL 8N KOH
Tested with every exposure time

40 μL 8N KOH
Tested only for 1 hour
300 μL 1 mg/mL

50 μL H2O
Tested with every exposure time

20 times with a constant soft streamof 1x PBS

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

if solution becomes coloured,
living adapted controls should be
performed

for 60 min at 37oC, 5% CO2, 95% RH

50 μL (liquid) or 25 mg (solid) + 300
μL H2O

epiCS®

431

40 μL H2O
Tested with every exposure time

20 times with a constant soft streamof 1x PBS

at 37oC, 5% CO2, 95% RH

3 min at RT, and 60 min

if test chemical is coloured, living
adapted controls should be performedfor 60 min at 37oC, 5% CO2, 95% RH

3 min at RT, and 60 min

40 μL (liquid) or 20mg (solid) + 300
μL H2O mixed for 60
min at RT

50 μL (liquid) or 25 mg (solid) + 300
μL H2O

10 μL (liquid) or 10 mg (solid) + 90 μL
H2O mixed for 15 min at RT

if solution becomes coloured, living
adapted controls should be performed

SkinEthicTM RHE

EpiDermTM SCT

EpiSkinTM

Rinsing

Exposuretime andtemperature

Pre-checkfor colour
interference

Test Method
Components

OECD/OCDE

58
570 nm (or 540 nm)
without reference filter
Treatment with 1% Triton X-100
4.08 hours
50

570 nm (545 - 595 nm)
without reference filter

18 hours treatment with SDS
1.0 mg/mL
50

24

2. Mean viability of the tissue
replicates exposed for 1 hour withthe positive control (8N KOH),
expressed as % of the negative
control, should be 20%

2. Mean viability of the tissue 2. Mean viability of the tissue 2. Mean viability of the tissue
replicates exposed for 4 hours with replicates exposed for 1 hour with thereplicates exposed for 1 hour (and 4
the positive control (glacial acetic positive control (8N KOH), expressed
hours, if applicable) with theacid), expressed as % of the negative as % of the negative control, should
positive
control(8N KOH),
control, should be 20%
be < 15%
expressed as % of the negative
control, should be 15%
3. In the range 20-100% viability and 3. In the range 20 - 100% viability, thefor ODs 0.3, difference of viability Coefficient of Variation (CV) 3. In the range 20-100% viability, andbetween the two tissue replicates between tissue replicates should be
for ODs, difference of viability
should not exceed 30%.
between the two tissue replicates30%
should not exceed 30%

3. In the range 20-100% viability, andfor ODs, difference of viability
between the two tissue replicatesshould not exceed 30%

1. Mean OD of the tissue replicatestreated with the negative control(H2O) should be 0.8
2.8 forevery exposure time

Treatment with 1% Triton X-100
2.0 hours
50

540 - 570 nm
without reference filter

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

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

1. Mean OD of the tissue replicates 1. Mean OD of the tissue replicates 1. Mean OD of the tissue replicatestreated with the negative control treated with the negative control
treated with the negative control(NaCl) should be 0.6
1.5 for (H2O) should be
0.8
2.8 for (H2O) should be 0.8
3.0 forevery exposure time
every exposure time
every exposure time

Treatment with 1% Triton X-100
4.0 hours
50

570 nm (540 - 600 nm)
without reference filter

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

Overnight without shaking at RT or for
120 min with shaking (~120 rpm) at
RTOvernight at RT, protected from light(0.04 N HCl in isopropanol)(isolated tissue fully immersed)

1.5 mL isopropanol(extraction from top and bottom of
insert)2 mL isopropanol(extraction from top and bottom of
insert)500 μL acidified isopropanol

SkinEthicTM RHE

EpiDermTM SCT

OECD/OCDE

EpiSkinTM© OECD, (2016)

Acceptability
Criteria

Tissue Quality
Control

OD reading

Extraction timeandtemperature

Extraction
solvent

Test Method
Components

431

OECD/OCDE

431

ANNEX 3PERFORMANCE OF TEST METHODS FOR SUB-CATEGORISATION
The table below provides the performances of the four test methods calculated based on a set of 80
chemicals tested by the four test developers. Calculations were performed by the OECD Secretariat,
reviewed and agreed by an expert subgroup (21) (23).
EpiSkinTM, EpiDermTM , SkinEthicTM and epiCS® test methods are able to sub-categorise (i.e. 1A versus
1B-and-1C versus NC)
Performances, Overclassification rates, Underclassification rates, and Accuracy (Predictive capacity) of thefour test methods based on a set of 80 chemicals all tested over 2 or 3 runs in each test method:
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*one chemical was tested once in epiCS® because of no availability (23)

EpiSkinTM EpiDermTM
Overclassifications:
1B-and-1C overclassified 1A
NC overclassified 1B-and-1C
NC overclassified 1A
overclassified Corr.
Global overclassification rate (all categories)
Underclassifications:
1A underclassified 1B-and-1C
1A underclassified NC
1B-and-1C underclassified NC
Global underclassification rate (all categories)
Correct Classifications:
1A correctly classified
1B-and-/1C correctly classified
NC correctly classified
Overall Accuracy

SkinEthicTM

epiCS®

21.50%
20.7%
0.00%
20.7%
17.9%

29.0%
23.4%
2.7%
26.1%
23.3%

31.2%
27.0 %
0.0 %
27.0%
24.5%

32.8%
28.4 %
0.00%
28.4%
25.8%

16.7%
0.00%
2.2%
3.3%

16.7 %
0.00%
0.00%
2.5%

16.7%
0.00%
7.5%
5.4%

12.5 %
0.00%
6.6%
4.4%

83.3%

83.3%

83.3%

87.5%

76.3%

71.0%

61.3%

60.7%

79.3%

73.9%

73.0%

71.62%

78.8%

74.2%

70%

69.8%

NC: Non-corrosive

25© OECD, (2016)

59

431

OECD/OCDEANNEX 4

Key parameters and acceptance criteria for qualification of an HPLC/UPLC-spectrophotometry system formeasurement of MTT formazan extracted from RhE tissues

Parameter

Selectivity
Precision

Protocol Derived from FDA Guidance (37)(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 μg/mL, 16
μg/mL and 160 μg/mL ) in isopropanol (n=5)

Accuracy

Quality Controls in isopropanol (n=5)

Matrix EffectQuality Controls in living blank (n=5)

CarryoverAnalysis of isopropanol after an ULOQ2 standard

Reproducibility(intra-day)

Reproducibility(inter-day)
Short Term
Stability of MTT
Formazan in RhE
Tissue Extract
Long Term
Stability of MTT
Formazan in RhE
Tissue Extract, if
required1
2

3 independent calibration curves (based on 6
consecutive 1/3 dilutions of MTT formazan in
isopropanol starting at ULOQ, i.e., 200 μg/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)

Areainterference
AreaLLOQ1
for the LLOQ
20% for LLOQ
Areainterference
AreaLLOQ
Calibration Curves:
20% for LLOQ
Quality Controls:nd CV

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 the
day of the preparation and after several days of storageat a specified temperature (e.g., 4ºC, -20ºC, -80ºC)

LLOQ: Lower Limit of Quantification, defined to cover 1-2% tissue viability, i.e., 0.8 μg/mL.
ULOQ: 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.

26© OECD, (2016)

60

Acceptance Criteria

Unclassified

ENV/JM/MONO(2015)26

Organisation de Coopération et de Développement Économiques
Organisation for Economic Co-operation and Development

23-Jul-2015___________________________________________________________________________________________English - Or. English
ENVIRONMENT DIRECTORATEENV/JM/MONO(2015)26
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYPERFORMANCE STANDARDS FOR THE ASSESSMENT OF PROPOSED SIMILAR OR MODIFIEDIN VITRO RECONSTRUCTED HUMAN EPIDERMIS (RHE) TEST METHODS FOR SKIN
CORROSION TESTING AS DESCRIBED IN TG 431(Intended for the developers of new or modified similar test methods)
Series on Testing & Assessment
No. 219

English - Or. English

JT03380410
Complete document available on OLIS in its original format
This document and any map included herein are without prejudice to the status of or sovereignty over any territory, to the delimitation ofinternational frontiers and boundaries and to the name of any territory, city or area.

61

ENV/JM/MONO(2015)26

2

62

ENV/JM/MONO(2015)26
OECD Environment, Health and Safety Publications
Series on Testing and Assessment
No. 219

PERFORMANCE STANDARDS FOR THE ASSESSMENT OFPROPOSED SIMILAR OR MODIFIEDIN VITRO RECONSTRUCTED HUMAN EPIDERMIS (RHE) TEST METHODS FOR SKIN
CORROSION TESTING
AS DESCRIBED IN TG 4311
(Intended for the developers of new or modified similar test methods)

Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT
Paris 2015

1

Proposed new or modified test methods following the PS of this Test Guideline should be submitted to the OECD for
adoption and inclusion into the Test Guideline before being used for regulatory purposes.

363ENV/JM/MONO(2015)26
About the OECDThe Organisation for Economic Co-operation and Development (OECD) is an intergovernmentalorganisation in which representatives of 34 industrialised countries in North and South America, Europe
and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise
policies, discuss issues of mutual concern, and work together to respond to international problems. Most of
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Committees and working groups are served by the OECD Secretariat, located in Paris, France, which isorganised into directorates and divisions.
The Environment, Health and Safety Division publishes free-of-charge documents in eleven different
series: Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides;
Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety ofNovel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission
Scenario Documents; and Safety of Manufactured Nanomaterials. More information about theEnvironment, Health and Safety Programme and EHS publications is available on the OECD’s World
Wide Web site (http://www.oecd.org/chemicalsafety/).

This publication was developed in the IOMC context. The contents do not necessarily reflect the views orstated policies of individual IOMC Participating Organisations.
The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was established in
1995 following recommendations made by the 1992 UN Conference on Environment and Development tostrengthen co-operation and increase international co-ordination in the field of chemical safety. The
Participating Organisations are FAO, ILO, UNDP, UNEP, UNIDO, UNITAR, WHO, World Bank andOECD. The purpose of the IOMC is to promote co-ordination of the policies and activities pursued by theParticipating Organisations, jointly or separately, to achieve the sound management of chemicals in
relation to human health and the environment.

464

ENV/JM/MONO(2015)26
This publication is available electronically, at no charge.
Also published in the Series on Testing and Assessment link
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/chemicalsafety/)or contact:
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ENV/JM/MONO(2015)26
FOREWORD

This document contains the Performance Standards (PS) for the validation of similar or modified RhE
methods for skin corrosion testing as described in TG 431. In the past, PS were usually annexed to TGs.
However, in view of separating information on the use of a test method as contained in the TG frominformation needed to validate test methods as contained in the PS, TGs and PS will now both be standalone documents. This approach had been agreed by the Working Group of the National Coordinators of
the Test Guidelines Programme (WNT). In case of the current PS for skin in vitro corrosion methods
according to TG 431, the text was reviewed in regard to harmonising with other relevant documents
addressing skin irritation and skin corrosion. The PS were reviewed by the OECD Expert Group on Skin
Irritation/Corrosion in November 2014. The PS are intended for the developers of new or modified similar
test methods to the validated reference method. The present document was approved by the WNT in April
2015, declassified and published under the responsibility of the Joint Meeting of the Chemicals Committee
and the Working Party on Chemicals, Pesticides, and Biotechnology on 10 July 2015.

666

ENV/JM/MONO(2015)26

INTRODUCTION
1.
This document contains Performance Standards which allow, in accordance with the principles of
Guidance Document No. 34 (1), determining the validation status (reliability and relevance) of similar andmodified skin corrosion test methods that are structurally and mechanistically similar to the RhE test
method in OECD Test Guideline 431 (2).
2.
These PS include the following sets of information: (i) Essential Test Method Components thatserve to evaluate the structural, mechanistic and procedural similarity of a new similar or modified
proposed test method, (ii) a list of 30 Reference Chemicals to be used for validating new or modified test
methods and (iii) defined target values of reproducibility and predictive capacity that need to be met byproposed test methods in order to be considered similar to the validated reference methods.
3.
The purpose of Performance Standards (PS) is to provide the basis by which new similar or
modified test methods, both proprietary (i.e. copyrighted, trademarked, registered) and non-proprietary,
can be deemed to be structurally and mechanistically similar to a Validated Reference Method (VRM) anddemonstrate to have sufficient reliability and relevance for specific testing purposes (i.e., scientificallyvalid), in accordance with the principles of Guidance Document No. 34 (1). The PS, based on scientificallyvalid and accepted test method(s), can be used to evaluate the reliability and relevance of test methods thatare based on similar scientific principles and measure or predict the same biological or toxic effect (1).
Such methods are referred to as similar or “me-too” test methods. Moreover, the PS may be used toevaluate modified test methods, which may propose potential improvements in comparison to approved
earlier versions of a method. In such cases the PS can be used to determine the effect of the proposedchanges on the test method’s performance and the extent to which such changes may affect the information
available for other components of the validation process (e.g. relating to Essential Test Method
Components). However, depending on the number and nature of the proposed changes as well as the data
and documentation available in relation to these changes, modified test methods may: i) either be foundunsuitable for a PS-based validation (e.g. if the changes are so substantial that the method is not any longer
deemed sufficiently similar with regard to the PS), in which cases they should be subjected to the same
validation process as described for a new test method (1), or ii) suitable for a limited assessment of
reliability and relevance using the established PS (1). Similar or modified new test methods (i.e., “me-too”
tests) successfully validated according to Performance Standards can be added to TG 431. However,
Mutual Acceptance of Data (MAD) will only be guaranteed for those test methods reviewed and adopted
by the OECD. Proposed similar or modified test methods validated according to these PS should therefore
be submitted to the OECD for adoption and inclusion into TG 431 before being used for regulatory
purposes.
4.
These PS are based on the ICCVAM PS (3) for evaluating the validity of new or modified RhE
test methods. The PS consists of: (i) Essential Test Method Components; (ii)
Recommended Reference Chemicals, and; (iii) Defined Reliability and Predictive Capacity Values that the
proposed similar or modified test method should meet or exceed. The VRMs used as to develop the present
PS are the EpiSkinTM (SM) and EpiDermTM SCT (EPI-200) test methods as described in TG 431 (2).
Definitions are provided in Annex I.
5.
Similar (me-too) or modified test methods proposed for use under Test Guideline 431 (2) shouldbe evaluated to determine their reliability and predictive capacity using Reference Chemicals representing
the full range of the TG 404 in vivo corrosivity scores (Table 5) prior to their use for testing new test
chemicals, in order to ensure that these methods are able to identify correctly non-corrosive and corrosive
chemicals, and possibly also to discriminate UN GHS Sub-category 1A from a combination of Subcategories 1B and 1C corrosive chemicals (4) (5). The proposed similar or modified test methods should
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ENV/JM/MONO(2015)26
have reproducibility, sensitivity, specificity and accuracy values which are equal or better than thosederived from the two VRM and as described in paragraphs 29 to 32 of these PS (Tables 6 and 7) (6) (7) (8).
ESSENTIAL TEST METHOD COMPONENTS
6.
The Essential Test Method Components consist of essential structural, functional, and proceduralelements of scientifically valid test methods (the VMRs) that should be included in the protocol of a
proposed, mechanistically and functionally similar or modified test method. These components include
unique characteristics of the test method, critical procedural details, and quality control measures.
Adherence to essential test method components will help to assure that a similar or modified proposed test
method is based on the same concepts as the corresponding VRMs (1) (2). The essential test method
components to be considered for similar or modified test methods related to TG 431 are described in detailin the following paragraphs.
7.
For specific parameters (e.g., for Tables 1, 2, 3 and 4) or modified procedures, adequate values orprocedures should be provided for the proposed similar or modified test method, these specific values orprocedures may vary depending on the specific test method and/or its modification.

General Conditions
8.
Non-transformed human keratinocytes should be used to reconstruct the epithelium. The RhEmodel is prepared in inserts with a porous synthetic membrane through which nutrients can pass to the cells.
Multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum) should bepresent under a functional stratum corneum. The test chemical is applied topically to the three-dimensional
RhE model, which should have a surface in direct contact with air so as to allow for an exposure similar to thein vivo situation. The stratum corneum should be multi-layered containing the essential lipid profile toproduce 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 and may
be assessed either by determination of the concentration at which a benchmark chemical reduces theviability of the tissues by 50% (IC50) after a fixed exposure time, or by determination of the exposure timerequired to reduce cell viability by 50% (ET50) upon application of the benchmark chemical at a specified,
fixed concentration (see paragraph 8). The containment properties of the RhE model should prevent thepassage of test chemical around the stratum corneum to the viable tissue, which would lead to poor
modelling of skin exposure. The RhE model should be free of contamination by bacteria, viruses,
mycoplasma, and fungi.
Functional Conditions
Viability
9.
The assay used for quantifying tissue viability is the MTT-assay (9). The viable cells of the RhE
tissue construct can reduce the vital dye MTT into a blue MTT formazan precipitate, which is thenextracted from the tissue using isopropanol (or a similar solvent). The Optical Density (OD) of theextraction solvent alone should be sufficiently small, i.e., OD < 0.1. The extracted MTT formazan may bequantified using either a standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry
procedure (10). 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 the negative
control OD values for the RhE VRMs are given in Table 1. An HPLC/UPLC-Spectrophotometry user
should use the negative control OD ranges provided in Table 1 as the acceptance criterion for the negative

8

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ENV/JM/MONO(2015)26
control. It should be documented that the tissues treated with negative control are stable in culture (provide
similar OD measurements) for the duration of the exposure period.
Table 1: Acceptability ranges for negative control OD values to control batch quality of the VRMs
Lower acceptance limit

Upper acceptance limit

EpiSkin™ (SM)

≥ 0.6

≤ 1.5

EpiDerm™ SCT (EPI-200)

≥ 0.8

≤ 2.8

Barrier function
10.
The stratum corneum and its lipid composition should be sufficient to resist the rapid penetrationof certain cytotoxic benchmark chemicals (e.g. SDS or Triton X-100), as estimated by IC50 or ET50 (Table
2).
Morphology
11.
Histological examination of the RhE model should be performed demonstrating multi-layered
human epidermis-like structure containing stratum basale, stratum spinosum, stratum granulosum andstratum corneum and exhibits lipid profile similar to lipid profile of human epidermis.
Reproducibility
12.
Test results of the positive and negative controls of the test method should demonstrate
reproducibility of the test method over time. In case of the use of a test method for sub- categorization, thereproducibility with respect to sub-categorization should also be demonstrated.
Quality control (QC)
13.
The RhE model should only be used if the developer/supplier demonstrates that each batch of the
RhE model used meets defined production release criteria, among which those for viability (paragraph 7),
barrier function (paragraph 8) and morphology (paragraph 9) are the most relevant. An acceptability range(upper and lower limit) for the barrier function as measured by the IC50 or ET50 (see paragraphs 6 and 8) shouldbe established by the RhE model developer/supplier. The acceptability range of the VRMs are given in Table 2.
Adequate ranges should be provided for any new similar or modified test method. These may vary depending on
the specific test method. Data demonstrating compliance with all production release criteria should be provided
by the RhE model developer/supplier. Only results produced with tissues fulfilling all of these production
quality release criteria can be accepted for reliable prediction of corrosive classification.
Table 2: QC batch release criteria of the VRMs
TM

EpiSkin(SM)

Lower acceptance limit

Upper acceptance limit

IC50 = 1.0 mg/mL

IC50 = 3.0 mg/mL

ET50 = 4.0 hours

ET50 = 8.7 hours(18 hours treatment with SDS)
(11)
EpiDerm™ SCT (EPI-200)(1% Triton X-100) (12)969ENV/JM/MONO(2015)26
Procedural Conditions
Application of the Test Chemical and Control Substances
14.
At least two tissue replicates should be used for each test chemical and each control substance foreach exposure time in each run. 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 minimumof 70 μL/cm2 or 30 mg/cm2 should be used). Whenever possible, solids should be tested as a fine powder.
15.
Concurrent negative and positive controls (PC) should be used in each run to demonstrate that
viability (with negative controls), and sensitivity (with the PC) of the tissues are within a defined historicalacceptance range. The concurrent negative control also provides the baseline (100% tissue viability) to
calculate the relative percent viability of the tissues treated with the test chemical. The positive control
suggested for the VRMs are glacial acetic acid or 8N KOH depending upon the RhE model used. It should
be noted that 8N KOH is a direct MTT reducer that might require adapted controls as described inparagraphs 15 and 16. The suggested VRMs negative controls are 0.9% (w/v) NaCl or water.
Cell Viability Measurements
16.
The MTT assay, which is a quantitative assay, should be used to measure tissue viability (9). It is
compatible with use in a three-dimensional tissue construct. The tissue sample is placed in MTT solutionof an appropriate concentration (e.g. 0.3 or 1 mg/mL in the VRMs) for 3 hours. The vital dye MTT is
reduced into a blue formazan precipitate by the viable cells of the RhE model. The precipitated blue formazan
product is then extracted from the tissue using a solvent (e.g. isopropanol, acidic isopropanol), and theconcentration of formazan is quantified by determining the OD at 570 nm using a filter band pass of
maximum ± 30 nm, or by an HPLC/UPLC-spectrophotometry procedure (10). The same procedure shouldbe employed for the concurrently tested negative and positive controls.
17.
Optical properties of the test chemical or its chemical action on MTT may interfere with themeasurement of MTT formazan leading to a false estimate of tissue viability. Test chemicals may interferewith 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 offormazan (i.e. 570 ± 30 nm, mainly blue and purple chemicals). Pre-checks should be performed before
testing to allow identification of potential direct MTT reducers and/or colour interfering chemicals. The
corresponding procedures should be standardised and part of the SOP. Additional controls should be used
to 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 16 to 19). 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.
For coloured test chemicals or test chemicals that become coloured in contact with water or isopropanol,
which are not compatible with the standard absorbance (OD) measurement due to too strong interferencewith the MTT assay (i.e., strong absorption at 570 ± 30 nm), an HPLC/UPLC-spectrophotometry
procedure to measure MTT formazan may be employed (10). A detailed description of how to correct
direct MTT reduction and colour interferences by the test chemical should be available in the test method’s
SOP. A description of the control measures used in the VRMs are summarised in paragraphs 16 to 19
below.
18.
To identify direct MTT reducers, each test chemical should be added to freshly prepared MTT
medium. If the MTT mixture containing the test chemical turns blue/purple, the test chemical is presumed
to directly reduce the MTT, and further functional check on non-viable epidermis should be performed,
independently of using the standard absorbance (OD) measurement or an HPLC/UPLC-spectrophotometry

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ENV/JM/MONO(2015)26
procedure. This additional functional check employs killed tissues (by e.g., exposure to low temperature("freeze-killed" tissues) or by other means) that possess only residual metabolic activity but absorb andretain the test chemical in a similar way as viable tissues. Each MTT reducing chemical is applied on atleast two killed tissue replicates per exposure time, which undergo the entire testing procedure. The true
tissue viability is calculated 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 thesame MTT reducer, calculated relative to the negative control run concurrently to the test being corrected(%NSMTT).
19.
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 during exposure) 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/UPLCspectrophotometry procedure should be used in which case these controls are not required (see paragraph19). When performing the standard absorbance (OD) measurement, each interfering coloured test chemical
should be applied on at least two viable tissue replicates per exposure time, which undergo the entire
testing procedure but are incubated with medium instead of MTT solution during the MTT incubation stepto generate a non-specific colour (NSCliving) control. The NSCliving control needs to be performed
concurrently per exposure time to the testing of the coloured test chemical (in each run) due to the inherent
biological variability of living tissues. The true tissue viability is calculated as the percent tissue viability
obtained with 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 the interfering test chemical
and incubated with medium without MTT, run concurrently to the test being corrected (%NSCliving).
20.
Test chemicals that are identified as producing both direct MTT reduction (see paragraph 16) andcolour interference (see paragraph 17) should also require a third set of controls when performing thestandard absorbance (OD) measurement, apart from the NSMTT and NSCliving controls described in theprevious paragraphs. 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 16. These test chemicals may be retained in both living andkilled 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 the retention of the test chemical by killed
tissues. This could lead to a double correction for colour interference since the NSCliving control alreadycorrects for colour interference arising from the retention of the test chemical by living tissues. To avoid apossible 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 testing procedure but are incubatedwith medium instead of MTT solution during the MTT incubation step. A single NSCkilled control issufficient per test chemical regardless of the number 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 calculated as the percent tissue viability obtained with living tissues exposed to the test
chemical minus %NSMTT minus %NSCliving plus the percent non-specific colour obtained with killed
tissues exposed to the interfering test chemical and incubated with medium without MTT, calculated
relative to the negative control run concurrently to the test being corrected (%NSCkilled).
21.
NSCliving or NSCkilled controls are never required when using HPLC/UPLC-spectrophotometry,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 colour (intrinsic or when mixed with water) that
impedes the assessment of the capacity to directly reduce MTT as described in paragraph 16. When using
HPLC/UPLC-spectrophotometry to measure MTT formazan, the percent tissue viability is calculated as
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percent 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. For test chemicals able to directlyreduce MTT, true tissue viability is calculated as the percent tissue viability obtained with living tissues
exposed to the test chemical minus %NSMTT. Finally, it should be noted that in very rare cases, direct
MTT-reducers or MTT-reducers that are also colour interfering and are retained in the tissues after
treatment may not be assessable by the VRMs if they lead to ODs (using standard OD measurement) or
peak areas (using UPLC/HPLC-spectrophotometry) of the tested tissue extracts that fall outside of thelinearity range of the spectrophotometer.
Acceptability Criteria
22.
For each run, tissues treated with the negative control should exhibit OD reflecting the quality ofthe tissues that followed shipment, receipt steps and all protocol processes and should not be outside of thehistorically established boundaries (see paragraph 7 and table 1). Similarly, tissues treated with the positive
control, should show a mean tissue viability (relative to the negative control) within an historicallyestablished range, thus reflecting the ability of the tissues to respond to a corrosive chemical under theconditions of the test method. The variability between tissue replicates of test chemicals and/or control
substances should fall within the accepted limits also established from historical values (e.g. the differenceof viability between the two tissue replicates should not exceed 30%). If either the negative control or PC
included in a run fall outside of the accepted ranges, the run is considered non-qualified and should be
repeated. If the variability between tissue replicates of test chemicals falls outside of the accepted range,
the test chemical should be re-tested. Paragraph 33 provides more details on re-testing in case of nonqualified runs during validation studies. Importantly, an increased frequency of non-qualified runs may
indicate problems with either the test system (e.g. the intrinsic RhE tissue quality) or with the handling(e.g. shipment, SOP execution). Therefore, occurrence of non-qualified runs in validation studies should be
carefully monitored and all non-qualified runs need to be reported.
Interpretation of Results and Prediction Model
23.
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, thepercent tissue viability is calculated as percent 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 cut-off value of percentage cell viability distinguishing corrosive from non-corrosive test
chemical (and/or discriminating between different corrosive sub-categories), and the statisticalprocedure(s) used to evaluate the results should be clearly defined, documented, and proven to beappropriate. The cut-offs defined for the VRMs are defined below in paragraphs 23 and 24.
24.
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 of borderline results, such as
non-concordant replicate measurements, a second run may be considered, as well as a third one in case of
discordant results between the first two runs.
25.
The prediction model for the VRM EpiSkin skin corrosion test method (6) (8) (11), associatedwith the UN GHS (4) classification system, is shown in Table 3:
Table 3: Prediction model of the VRM EpiSkinTM
Viability measured after exposure time
points (t=3, 60 and 240 minutes)< 35% after 3 min exposure

Corrosive:

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Prediction
to be consideredENV/JM/MONO(2015)26
x Optional Sub-category 1A≥ 35% after 3 min exposure AND< 35% after 60 min exposure

Corrosive:x A combination of optional
Sub-categories 1B-and-1COR≥ 35% after 60 min exposure AND< 35% after 240 min exposure≥ 35% after 240 min exposure

Non-corrosive

26.
The prediction models for the VRM EpiDerm SCT (7) (12) (13) test method associated with
the UN GHS (4) classification system, are shown in Table 4:
Table 4: Prediction model of the VRM EpiDermTM SCT
Viability measured after exposure time
points (t=3 and 60 minutes)

Prediction
to be consideredCorrosive:< 50% after 3 min exposurex Optional Sub-category 1A≥ 50% after 3 min exposure AND< 15% after 60 min exposure≥ 50% after 3 min exposure AND≥ 15% after 60 min exposure

Corrosive:x A combination of optional
Sub-categories 1B-and-1C
Non-corrosive

MINIMUM LIST OF REFERENCE CHEMICALS
27.
Reference Chemicals are used to determine whether the reliability and predictive capacity of a
proposed similar or modified test method, proven to be structurally and functionally sufficiently similar tothe VRM, or representing a minor modification of the VRM, are equal or better than those derived fromthe VRMs (6) (7) (8). The 30 recommended Reference Chemicals listed in Table 5 include chemicals
representing different chemical classes (i.e. chemical categories based on functional groups), and are
representative of the full range of TG 404 in vivo skin corrosion scores. The chemicals included in this list
comprise representatives of the following UN GHS (Sub-)categories: 10 Sub-category 1A chemicals, 10
chemicals of sub-categories 1B and 1C (the in vivo data do not permit distinction between the twocategories) as well as 10 non-corrosive chemicals. The Reference Chemicals were selected from the test
chemicals used in the validation studies of the VRMs (6) (7) (8) (14) using the selection criteria as
described in Table 5 (foot-note 1), with due regard to e.g., chemical functionality and physical state.
28.
The 30 Reference Chemicals listed in Table 5 represent the minimum number of chemicals thatshould be used to evaluate the reliability and predictive capacity of a proposed similar or modified test
method able to discriminate between Subcategory 1A, a combination of Sub-categories 1B and 1C as well
as non-corrosive substances and mixtures in accordance with the UN GHS (4) (1A vs. 1B-and-1C vs. NC).
For similar or modified test methods able to discriminate corrosive from non-corrosive substances andmixtures but not able to support sub-categorisation of corrosive chemicals (C vs. NC), only 20 of the 30
chemicals listed in Table 5 (the ones not in italics) need to be evaluated: 5 Sub-category 1A chemicals, 5chemicals of the combined Sub-categories 1B and 1C as well as 10 non-corrosive chemicals. The exclusive
use of these Reference Chemicals for the development/optimization of new similar test methods should be
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ENV/JM/MONO(2015)26
avoided to the extent possible. In situations where a listed Reference Chemical is unavailable, or cannot be
used for other justified reasons, another chemical could be used provided it fulfils the selection criteria as
described in Table 5 (foot-note 1) and adequate in vivo reference data are available, e.g. preferentially fromthe test chemicals used during the validation studies of the VRMs (6) (7) (8) (14). To gain further
information on the predictive capacity of the proposed test method, additional chemicals representing otherchemical classes and for which adequate in vivo reference data are available may be tested in addition to
the minimum list of Reference Chemicals.

1474ENV/JM/MONO(2015)26

Table 5: Minimum list of Reference Chemicals for determination of Reproducibility and Predictive Capacityof similar or modified in vitro RhE-based skin corrosion test methods. The 20 chemicals NOT in italics shouldbe tested with similar or modified test methods proposed to discriminate Corrosive from Non-Corrosive
chemicals (without sub-categorization). Additional reference chemicals should be tested with similar or
modified test methods proposed to identify Sub-category 1A, a combination of Category 1B and 1C (referred
to as 1B/1C below) and non-corrosive test chemicals. These additional reference chemicals are indicated initalics.
Chemical1

CASRN

ChemicalClass2

Physical
EpiSkinTM 4 EpiDermTM 4
State

SkinEthicTM 4

epiCS® 4

Non-corrosive chemicals based on in vivo results3
Phenethyl
bromide*4-Amino-1,2,4triazole
4-(methylthio)benzaldehyde*

103-63-9

ElectrophileL(3) NC(3) NC(3) NC(2) NC

584-13-4

Organic baseS(3) NC(3) NC(3) NC(2) NC3446-89-7

ElectrophileL(3) NC(3) NC(3) NC(2) NC

Lauric acid143-07-7

Organic acidS(3) NC(3) NC(2) NC

1,9-Decadiene

1647-16-1

Neutralorganic

L(3) NC(3) NC(2) NC95-68-1

Organic baseL(2) NC(1) 1B/1C(1) NC(2) 1B/1C(2) 1B/1C(1) 1A(1) NC(1) 1B/1C

1119-62-6

Organic acidS(3) NC(3) NC(3) NC(2) NC

112-39-0

Neutralorganic

S(3) NC(3) NC(2) NC

594-61-6

Organic acidS(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1C

3398-33-2

Soap /
Surfactant

L(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1C

2,4Dimethylaniline
3,3Dithiopropionicacid
Methyl palmitate
2-Hydroxyisobutyric acid
Sodiumundecylenate(33%)(3) NC(3) NC(3) NC

Combination of UN GHS Sub-categories 1B and 1C based on in vivo results3
Glyoxylic acidmonohydrate

563-96-2

Organic acidS(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1C

Lactic acid598-82-3

Organic acidL(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1C

Sodiumbisulphatemonohydrate

10034-88-5 Inorganic salt

S(3) 1B/1C(3) 1B/1C(2) 1B/1C(1) NC(2) 1B/1C

Ethanolamine*141-43-5

Viscous(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1CL(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1CL(3) 1B/1C(3) 1B/1C(3) 1B/1C(2) 1B/1C(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A

Organic base60/40
Octanoic/decano 68937-75-7 Organic acidic acid
Hydrochloric
7647-01-0 Inorganic acidacid (14.4%)
Inorganic
Fluoroboric acid 16872-11-0
acid
Propionic acid79-09-4

Organic acid(3) 1AL(3) 1A

L

15

75

ENV/JM/MONO(2015)26

CASRN

ChemicalClass2

88-18-6

PhenolL(3) 1B/1C

108-91-8

Organic baseL(3) 1B/1C

Acrylic acid79-10-7

UN GHS Sub-category 1A based on in vivo results3(3) 1A
Organic acidL(3) 1A

Bromoacetic acid79-08-3

Organic acidS

Boron trifluoride
13319-75-0 Inorganic aciddehydrateL

Phenol108-95-2

PhenolS

Phosphorus
tribromide

7789-60-8

Inorganicacid

L

Silver nitrate

7761-88-8

Inorganic salt

S

Formic acid64-18-6

Organic acidL

79-36-7

ElectrophileL

7664-93-9

Inorganic
acid10563-29-8 Organic baseChemical1
2-tertButylphenol*
Cyclohexyl
amine*

Dichloroacetyl
chloride
Sulphuric acid(98%)
N,N-Dimethyl
dipropylene
triamine*

Physical
EpiSkinTM 4 EpiDermTM 4
State

SkinEthicTM 4

epiCS® 4(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(2) 1A(3) 1A(3) 1A(3) 1A(2) 1AL(3) 1A(3) 1AL(3) 1B/1C(3) 1B/1C(3) 1A(3) 1A(3) 1A(3) 1A(1) 1A(2) 1B/1C(3) 1A(3) 1A(3) 1B/1C(2) 1A(2) 1B/1C

Abbreviations: CASRN = Chemical Abstracts Service Registry Number; UN GHS = United Nations Globally
Harmonized System (4); NC = Not Corrosive1
The reference chemicals, sorted first by corrosives versus non-corrosives, then by corrosive sub-category, wereselected from the test chemicals used in the ECVAM validation studies of EpiSkin™ and EpiDermTM SCT (6) (7)(14) and from post-validation studies based on data generated by EpiSkinTM (8), EpiDermTM, SkinEthicTM and epiCS®
developers. Unless otherwise indicated, these chemicals were tested at the purity level obtained when purchased froma commercial source (6) (7). The selection includes, to the extent possible, chemicals that: (i) are representative of therange of corrosivity responses (e.g. non-corrosives; weak to strong corrosives) that the VRMs are capable ofmeasuring or predicting; (ii) are representative of the chemical classes used in the validation studies; (iii) reflect theperformance characteristics of the VRM; (iv) have chemical structures that are well-defined; (v) induce reproducible
results in the VRM; (vi) induce definitive results in the in vivo reference test method; (vii) are commerciallyavailable; and (viii) are not associated with prohibitive disposal costs. Chemicals marked with an * are potential direct
MTT reducers.
2

Chemical class assigned by Barratt et al. (14).

3

The corresponding UN Packing groups are I, II and III, respectively, for the UN GHS 1A, 1B and 1C.
The in vitro predictions reported in this table were obtained with the various test methods during post-validation
testing performed by the test method developers. The numbers in brackets indicate, for each chemical, the number ofthe corresponding type of in vitro predictions for the test method considered. These predictions were corrected for direct
4

MTT reduction using killed control tissues.

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DEFINED RELIABILITY AND PREDICTIVE CAPACITY VALUES
29.
For purposes of establishing the reliability (i.e., within- and between laboratory
reproducibility) and predictive capacity (i.e., sensitivity, specificity and accuracy) of proposed similar or
modified RhE test methods to be used by several independent laboratories, all 30 (or 24 for methods notable to sub-categorize corrosive chemicals) Reference Chemicals listed in Table 5 should be tested in atleast three laboratories. In each laboratory, all relevant Reference Chemicals should be tested for eachexposure time in three independent runs performed with different tissue batches and at sufficiently spaced
time points. Each run should consist of at least two concurrently tested tissue replicates per exposure timefor each test chemical, negative control, positive control and adapted controls for direct MTT reduction
and/or colour interference.
30.
The calculation of the within-laboratory reproducibility, between-laboratory reproducibility,
accuracy, sensitivity and specificity values of the proposed test method should be done according to therules described below to ensure that a predefined and consistent approach is used:1.

Within-laboratory reproducibility (WLR) should be calculated based on concordance ofclassifications using only qualified tests obtained with Reference Chemicals for which at least
two qualified tests are available. In addition, it should be reported the number and identity of
the Reference Chemicals which per laboratory have none or only one qualified test (notconsidered for WLR calculations), as well as how many and which Reference Chemicals perlaboratory have two or three qualified tests (used for WLR calculations).

2.

For the calculation of between-laboratory reproducibility (BLR) the final classification foreach Reference Chemical in each participating laboratory should be obtained by using thearithmetic mean value of viability over the different qualified tests performed. BLR should be
calculated based on concordance of classifications using only qualified tests from Reference
Chemicals for which at least one qualified test per laboratory is available. It should bereported how many and which Reference Chemicals do not have at least one qualified test perlaboratory (not considered for BLR calculations), as well as how many and which Reference
Chemicals have 3, 4, 5, 6, 7, 8 or 9 qualified tests that can be used to calculate BLR (with atleast one qualified test per laboratory).

3.

The calculation of predictive capacity (e.g. sensitivity, specificity and accuracy for corrosive
vs. non-corrosive) as well as, in case of subcategorisation, over- and under-prediction rates,
should be done using all qualified tests obtained for each Reference Chemical in eachlaboratory. The calculations should be based on the individual predictions of each qualified
test for each Reference Chemical in each laboratory and not on the arithmetic mean values of
viability over the different qualified tests performed (15).

In this context, a qualified test consists of a test that meets the criteria for an acceptable test, as defined in
the corresponding SOP, and is within a qualified run. Otherwise, the test is considered as non-qualified. A
qualified run consists of a run that meets the test acceptance criteria for the negative control and positive
control, as defined in the corresponding SOP. Otherwise, the run is considered as non-qualified.
Within-laboratory reproducibility
31.
An assessment of within-laboratory reproducibility for similar or modified test method
proposed to discriminate corrosive from non-corrosive chemicals (but not to sub-categorize corrosive
chemicals), should show in every laboratory, a concordance of predictions (corrosive or non-corrosive)
obtained in different, independent tests of the 24 relevant Reference Chemicals equal or higher (≥) than
90% (actual for EpiSkinTM: 100%, 100% and 96% in each laboratory, respectively).
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ENV/JM/MONO(2015)26

32.
An assessment of within-laboratory reproducibility for similar or modified test method
proposed to discriminate between Sub-category 1A, a combination of Sub-categories 1B and 1C as well as
non-corrosive chemicals should show in every laboratory, a concordance of predictions obtained indifferent, independent tests of the 30 Reference Chemicals equal or higher (≥) than 80% (actual for
EpiSkinTM: 96%, 96% and 88% in each laboratory, respectively).
Between-laboratory reproducibility
33.
For similar or modified test methods proposed to discriminate corrosive from non-corrosive
chemicals (but not to sub-categorize corrosive chemicals), the concordance of predictions (corrosive or
non-corrosive) between a minimum of three laboratories, obtained for the 24 relevant Reference
Chemicals, should be equal or higher (≥) than 80% (actual for EpiSkinTM: 88%). For similar or modified
test methods proposed to discriminate between Sub-category 1A, a combination of Sub-categories 1B and
1C as well as non-corrosive chemicals, the concordance of predictions between a minimum of threelaboratories, obtained for the 30 Reference Chemicals, should be equal or higher (≥) than 70% (actual for
EpiSkinTM: 80%).
Predictive capacity
34.
The predictive capacity of the proposed similar or modified RhE test method should be equal or
better than the target values derived from the VRMs. For similar or modified test methods proposed todiscriminate corrosive from non-corrosive chemicals but unable to support sub-categorisation of corrosive
chemicals, the sensitivity, specificity and accuracy obtained with the 20 relevant Reference Chemicals(Table 5) should be equal or higher (≥) than 95%, 70% and 82.5% respectively (Table 6). For similar or
modified test methods proposed to discriminate between Sub-category 1A, a combination of Subcategories 1B and 1C as well as non-corrosive chemicals, the minimum predictive capacity values thatshould be obtained with the 30 Reference Chemicals (Table 5) are indicated in Table 7. A distinction ismade between RhE-based test methods similar to EpiSkin™ on the one hand and similar to EpiDerm™ on
the other hand due to their differences in Sub-categorization predictive capacities.
Table 6: Required sensitivity, specificity and accuracy for similar or modified RhE test methods tobe considered valid to discriminate corrosive from non-corrosive chemicals (C vs. NC) but not able
to support sub-categorisation of corrosive chemicals.
Sensitivity

Specificity

Accuracy

≥ 95%

≥ 70%

≥ 82.5%(actual for EpiSkin™: 100%;
actual for EpiDerm™: 100%)1(actual for EpiSkin™: 76.7%;
actual for EpiDerm™: 73.3%)1(actual for EpiSkin™: 88.3%;
actual for EpiDerm™: 86.7%)1

1

Values are based on the results of the two VRMs (EpiSkin™ and EpiDerm™) for the 20 Reference Chemicals
not in italics from Table 5.

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ENV/JM/MONO(2015)26

Table 7: Required predictive capacity for similar or modified RhE test method to be consideredvalid to discriminate between Sub-category 1A, a combination of Sub-categories 1B and 1C (referred
to as 1B-and-1C below) and non-corrosive chemicals *.
VRM

EpiSkin™ 1

EpiDerm™ 1

Sensitivity(for predictions C vs NC)

≥ 95%
TM
(actual for EpiSkin : 100.0%)≥ 80%
TM(actual for EpiSkin : 83.3%)≤ 20%
TM(actual for EpiSkin : 16.7%,)0%TM(actual for EpiSkin : 0.0%)≥ 80%
TM(actual for EpiSkin : 80.0%)≤ 20%
TM(actual for EpiSkin : 20.0%)≤ 5%TM(actual for EpiSkin : 0.0%)≥ 70%
TM(actual for EpiSkin : 76.7%)≤ 5%
TM(actual for EpiSkin : 0.0%)≤ 30%
TM(actual for EpiSkin : 23.3%)≥ 87%
TM(actual for EpiSkin : 92.2%)

≥ 95%
TM
(actual for EpiDerm : 100.0%)≥ 90%TM(actual for EpiDerm : 90.0%)≤ 10%TM(actual for EpiDerm : 10.0%,)0%TM(actual for EpiDerm : 0.0%)≥ 55%
TM(actual for EpiDerm : 60.0%)≤ 45%
TM(actual for EpiDerm : 40.0%)≤ 5%TM(actual for EpiDerm : 0.0%)≥ 70%
TM(actual for EpiDerm : 73.3%)≤ 5%
TM(actual for EpiDerm : 0.0%)≤ 30%
TM(actual for EpiDerm : 26.7%)≥ 87%
TM(actual for EpiDerm : 91.1%)

≥ 78%
TM
(actual for EpiSkin : 80.0%)

≥ 72%
TM
(actual for EpiDerm : 74.4%)

Correctly classified 1A
1A underclassified 1Band-1C
1A underclassified NC
Correctly classified 1Band-1C
1B-and-1C overclassified
1A
1B-and-1C
underclassified NC
Specificity ( i.e., correct
NC predictions)
NC overclassified 1A
NC overclassified 1Band-1C
Accuracy(C vs. NC)
Accuracy(1A vs. 1B-and-1C vs.
NC)
1

Actual values are based on the results of the two VRMs (EpiSkin™ and EpiDerm™) for the 30 Reference
Chemicals (see table 5).

* Depending on the results obtained with a similar or modified RhE test method for the 30 Reference Chemicals, it
may be considered similar to EpiSkin™ or similar to EpiDerm™ for the purpose of this Test Guideline. The
TM
TMEpiSkin and EpiDermtest methods are able to sub-categorize (i.e. 1A versus 1B-and-1C versus NC) butTMdifferences are observed (SkinEthic and epiCS® are considered similar to EpiDerm™). For RhE test methods thatTM
demonstrate similarity to EpiSkin , results can be directly used based on the outcoming predictions. For RhE testTMmethods that demonstrate similarity to EpiDerm , chemicals that are classified as Sub-category 1B-and-1C can beconsidered as Sub-category 1B-and-1C, whereas chemicals for which cell viability at 3 minutes is below 50% should
be considered as Category 1, since the Sub-category 1A predictions of these three test methods contain a high rate ofover-predictions of chemicals of Sub-categories 1B-and-1C (see also paragraph 7 of the Test Guideline 431 (2)). The
regulatory framework in member countries will decide how this Test Guideline will be used, e.g. acknowledging thesignificant probability of overclassification, a Sub-category 1A classification may still be accepted or further testing
may be conducted to confirm the result.

Study Acceptance Criteria
35.

It is possible that one or several tests pertaining to one or more Reference Chemical does/do not

1979ENV/JM/MONO(2015)26
meet the test acceptance criteria (non-qualified tests) or is/are not acceptable for other reasons such astechnical reasons or because they were obtained in a non-qualified run due to failure of the concurrent
positive and/or negative control. To complement missing data, a maximum of two additional tests for each
Reference Chemical is admissible per laboratory ("re-testing"). More precisely, since in case of re-testingalso the positive and negative control substances have to be concurrently tested, a maximum number of
two additional runs may be conducted for each Reference Chemical in each laboratory. Non-qualified tests
should be documented and reported. Importantly, each laboratory should not produce more than three
qualified tests per Reference Chemical. Excess production of data and subsequent data selection are
regarded as inappropriate. All tested tissues should be reported. The extent of unacceptable tests/runsshould be documented and the basis for the likely cause of each should be provided.
36.
It is conceivable that even after re-testing, three qualified tests are not obtained for every
Reference Chemical in every participating laboratory, leading to an incomplete data matrix. In such cases
the following three criteria should all be met in order to consider the datasets acceptable for purposes ofPS-based validation studies:1.

All relevant Reference Chemicals (24 for Category 1 vs. Non Corrosive; 30 for Sub-cat. 1Avs. Sub-cat. 1B-and-1C vs. Non Corrosive) should have at least one complete test sequence in
one laboratory.

2.

Each of at least three participating laboratories should have a minimum of 85% complete test
sequences (for 24 Reference Chemicals: 3 incomplete test sequences are allowed perlaboratory; for 30 Reference Chemicals: 4 incomplete test sequences are allowed perlaboratory).

3.

At least 90% of all test sequences from at least three laboratories need to be complete (for 24
Reference Chemicals tested in 3 laboratories: a total of 7 incomplete test sequences areallowed; for 30 Reference Chemicals tested in 3 laboratories: a total of 9 incomplete test
sequences are allowed).

In this context, a test sequence consists of the total number of independent tests performed for a single
Reference Chemical in a single laboratory, including any re-testing (a total of 3 to 5 tests). A test sequence
may include both qualified and non-qualified tests. A complete test sequence consists of a test sequence
containing three qualified tests. A test sequence containing less than 3 qualified tests is considered asincomplete.

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LITERATURE(1)OECD (2005), OECD Series on Testing and Assessment No. 34. Guidance Document on the
Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment.
Available at:[http://www.oecd.org/document/30/0,3343,en_2649_34377_1916638_1_1_1_1,00.html].

(2)

OECD (2015), OECD Guideline for the Testing of Chemicals No. 431. Reconstructed humanepidermis (RhE) method for skin corrosion testing. OECD, Paris.

(3)

ICCVAM (2004), Recommended Performance Standards for In Vitro Test Methods for Skin
Corrosion. NIH Publication Number 04-4510. Research Triangle Park, NC: National Institute ofEnvironmental Health Sciences. Available at:[http://iccvam.niehs.nih.gov/docs/dermal_docs/ps/ps044510.pdf](4)

UN (2013), United Nations Globally Harmonized System of Classification and Labelling ofChemicals (GHS), Fifth revised edition, UN New York and Geneva. Available at:[http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html](5)OECD (2015), OECD Guideline for Testing of Chemicals. No. 404: Acute Dermal Irritation,
Corrosion. OECD, Paris.

(6)

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 evaluation by the Management Team. Toxicol.in Vitro 12,483-524.

(7)

Liebsch et al., (2000), The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity
testing, ATLA 28, pp. 371-401.

(8)

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.

(9) Mosmann, T. (1983), Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55-63.
(10) 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/UPLC-spectrophotometry for detection of MTT formazan in in vitro Reconstructed human
Tissue (RhT)-based test methods employing the MTT assay to expand their applicability to strongly
coloured test chemicals. Manuscript in preparation.
(11) EpiSkin™ SOP, INVITTOX Protocol No. 118 (December 2011), EpiSkin™ Skin Corrosivity Test.
Available at: [http://ecvam.jrc.ec.europa.eu].
(12) EpiDerm™ SOP, Version MK-24-007-0024 (February 2012), Protocol for: In vitro EpiDerm™ skincorrosion test (EPI-200-SCT), For use with MatTek Corporation's reconstructed human epidermal
model EpiDerm. Available at: [http://ecvam.jrc.ec.europa.eu].

2181ENV/JM/MONO(2015)26(13) EC-ECVAM (2000), Statement on the application of the EpiDermTM human skin model for skincorrosivity testing, issued by the ECVAM Scientific Advisory Committee (ESAC 14), 21 March
2000. Available at: [http://ecvam.jrc.ec.europa.eu].
(14) 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 corrosivity. 1. Selection anddistribution of the test chemicals. Toxicol.in Vitro 12, 471-482.
(15) OECD (2013), OECD Serires on Testing and Assessment No. 190. Summary Document on theStatistical Performance of Methods in OECD Test Guideline 431 for Sub-Categorisation

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ANNEX 1DEFINITIONS
Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of relevance. The term is often used interchangeably
with “concordance” to mean the proportion of correct outcomes of a test method (1).
Between-laboratory reproducibility: A measure of the extent to which different qualified laboratories,
using the same protocol and testing the same substances, can produce qualitatively and quantitatively
similar results. Between-laboratory reproducibility is determined during the prevalidation and validation
processes, and indicates the extent to which a test can be successfully transferred between laboratories, also
referred to as inter-laboratory reproducibility (1).
C: Corrosive.
Cell viability: Parameter measuring total activity of a cell population e.g. as ability of cellular
mitochondrial dehydrogenases to reduce the vital dye MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium 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.
Complete test sequence: A test sequence containing three qualified tests. A test sequence containing lessthan 3 qualified tests is considered as incomplete (see also definition of “test sequence” below).
Concordance: This is a measure of test method performance for test methods that give a categorical result,and is one aspect of relevance. The term is sometimes used interchangeably with accuracy, and is definedas the proportion of all chemicals tested that are correctly classified as positive or negative. Concordance ishighly dependent on the prevalence of positives in the types of test chemical being examined (1).
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 to standardized types andlevels of physical, health and environmental hazards, and addressing corresponding communication
elements, such as pictograms, signal words, hazard statements, precautionary statements and safety data
sheets, so that to convey information on their adverse effects with a view to protect people (including
employers, workers, transporters, consumers and emergency responders) and the environment (4).
HPLC: High Performance Liquid Chromatography.
IC50: Can be estimated by determination of the concentration at which a benchmark chemical reduces theviability of the tissues by 50% (IC50) after a fixed exposure time, see also ET50.
Infinite dose: Amount of test chemical applied to the epidermis exceeding the amount required tocompletely and uniformly cover the epidermis surface.

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Me-too test: A colloquial expression for a test method that is structurally and functionally similar to avalidated and accepted reference test method. Such a test method would be a candidate for catch-up
validation (1). The term is interchangeably used with similar test method.
Mixture: means a mixture or solution composed of two or more substances in which they do not react (4).
MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Thiazolyl blue tetrazolium bromide.
NC: Non corrosive.
NSCkilled: Non-Specific Colour in killed tissues.
NSC: Non-Specific Colour in living tissues.
NSMTT: Non-Specific MTT reduction.
OD: Optical Density
PC: Positive Control, a replicate containing all components of a test system and treated with a substanceknown to induce a positive response. To ensure that variability in the positive control response across time
can be assessed, the magnitude of the positive response should not be excessive.
Performance standards (PS): Standards, based on a validated test method, that provide a basis forevaluating the comparability of a proposed test method that is mechanistically and functionally similar.
Included are; (i) essential test method components; (ii) a minimum list of Reference Chemicals selectedfrom among the chemicals used to demonstrate the acceptable 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 (1).
Prediction Model: a formula or algorithm (e.g., formula, rule or set of rules) used to convert the results
generated by a test method into a prediction of the (toxic) effect of interest. Also referred to as decision
criteria. A prediction model contains four elements: (i) a definition of the specific purpose(s) for which thetest method is to be used; (ii) specifications of all possible results that may be obtained, (iii) an algorithm
that converts each study result into a prediction of the (toxic) effect of interest, and (iv) specifications as tothe accuracy of the prediction model (e.g., sensitivity, specificity, and false positive and false negative
rates). Prediction models are generally not used in in vivo ecotoxicological tests (1).
Predictive Capacity: The predictive capacity reflects the test method performance in terms of correct andincorrect predictions in comparison to reference data. It gives quantitative information (e.g. correct
prediction rate) on the relevance of the test method. It comprises, amongst others, the sensitivity andspecificity of the test method.
Qualified run: A run that meets the test acceptance criteria for the NC and PC, as defined in thecorresponding SOP. Otherwise, the run is considered as non-qualified.
Qualified test: A test that meets the criteria for an acceptable test, as defined in the corresponding SOP,
and is within a qualified run. Otherwise, the test is considered as non-qualified.
Reference Chemicals: Chemicals selected for use in the validation process, for which responses in the invitro or in vivo reference test system or the species of interest are already known. These chemicals should
be representative of the classes of chemicals for which the test method is expected to be used, and should24

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represent the full range of responses that may be expected from the chemicals for which it may be used,from strong, to weak, to negative. Different sets of reference chemicals may be required for the different
stages of the validation process, and for different test methods and test uses (1).
Relevance: Description of relationship of the test method to the effect of interest and whether it ismeaningful and useful for a particular purpose. It is the extent to which the test method correctly measuresor predicts the biological effect of interest. Relevance incorporates consideration of the accuracy(concordance) of a test method (1).
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility (1).
Reproducibility: The agreement among results obtained from testing the same substance using the sametest protocol (1).
Run: A run consists of one or more test chemicals tested concurrently, by one laboratory, with a negative
control and with a positive control.
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 categorical results, and is an importantconsideration in assessing the relevance of a test method (1).
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 the skin, complete areas of alopecia, and scars.
Histopathology should be considered to evaluate questionable lesions (5).
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 categorical results and is an importantconsideration in assessing the relevance of a test method (1).
Substance: means chemical elements and their compounds in the natural state or obtained by any
production process, including any additive necessary to preserve the stability of the 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 (4).
Test: A single test substance concurrently tested in a minimum of two tissue replicates as defined in thecorresponding SOP.
Test sequence: The total number of independent tests performed for a single test substance in a single
laboratory, including any re-testing. A test sequence may include both qualified and non-qualified tests.
Validated Reference Method(s) (VRM(s)): one (or more) test method(s) officially endorsed as scientificvalid that was(were) used to develop the related official Test Guidelines and Performance Standards (PS).
The VRM is considered the reference test method to compare new proposed similar or modified test
methods in the framework of a PS-based validation study.

25

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Within-laboratory reproducibility: determination of the extent that qualified people within the samelaboratory can successfully replicate results using a specific protocol at different times, also referred to as
intra-laboratory reproducibility (1).

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