ᣂⷙ⹜㛎ᴺឭ᩺ᦠ             ᐔᚑ 25 ᐕ 11 ᦬ 20 ᣣ
                                  No. 2013-01⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺ LabCyte EPI-MODEL24 ߦ㑐ߔࠆឭ᩺
 ᐔᚑ 25 ᐕ 4 ᦬ 19 ᣣߦ᧲੩‫ޔ‬࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲߦߡ㐿௅ߐࠇߚᣂⷙ⹜㛎ᴺ⹏ଔળ⼏㧔ㅢ
⒓㧦JaCVAM ⹏ଔળ⼏㧕ߦ߅޿ߡએਅߩឭ᩺߇ߥߐࠇߚ‫ޕ‬    ឭ᩺ౝኈ㧦⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺ LabCyte EPI-MODEL24 ߪ୶ℂ⊛ߦఝࠇߚ⹜㛎ᴺߢ޽ࠅ‫ޔ‬ㆡಾߥ೑↪᧦ઙਅߢㆡ↪ߔࠆߥࠄ߫‫ޔ‬ൻቇ‛⾰ߩ⊹⤏ߦኻߔࠆ 4 ᤨ㑆ㆡ↪ߩ৻ᰴೝỗᕈࠍ⹏ଔߔࠆߎߣ߇น⢻ߢ޽ࠆ‫ޕ‬ ߎߩឭ᩺ᦠߪ‫ޔ‬European Center for the Validation of Alternative Methods(ECVAM)ߦࠃࠆ╙ਃ⠪⹏ଔ⾗ᢱ߅ࠃ߮OECD Test Guideline㧔TG㧕439ߩᚑ┙ᤨߦࠃࠅḰ஻ߐࠇߚ⾗ᢱࠍ߽ߣߦ‫⤏⊹ޔ‬ೝỗᕈ⹜㛎ઍᦧᴺ⹏ଔᆔຬળߦࠃࠅ߹ߣ߼ࠄࠇߚᢥᦠࠍ↪޿ߡJaCVAM⹏ଔળ⼏߇⹏ଔ߅ࠃ߮ᬌ⸛ߒ
ߚ⚿ᨐ‫↪᦭ߩߘޔ‬ᕈ߇⏕⹺ߐࠇߚߎߣ߆ࠄ૞ᚑߐࠇߚ‫ޕ‬ એ਄ߩℂ↱ߦࠃࠅ‫ⴕޔ‬᡽ᒰዪߩ቟ోᕈ⹏ଔᣇᴺߣߒߡ‫⤏⊹ޟ‬ೝỗᕈ⹜㛎ઍᦧᴺLabCyte

EPI-MODEL24‫ࠍ↪૶ߩޠ‬ឭ᩺ߔࠆ߽ߩߢ޽ࠆ‫ޕ‬ ศ↰ᱞ⟤                        ⷏Ꮉ⑺૫

JaCVAM ⹏ଔળ⼏ ⼏㐳               JaCVAM ㆇ༡ᆔຬળ ᆔຬ㐳

i

,C%8#/ ⹏ଔળ⼏ศ↰ᱞ⟤㧔ᣣᧄᲥᕈቇળ㧕㧦ᐳ㐳

ᵻ㊁ື⑲

㧔ᣣᧄⅣႺᄌ⇣ේቇળ㧕੖ච፲⦟᣿ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ↢ᵴⴡ↢ൻቇㇱ㧕৻㝩 ീ㧔ᣣᧄൻቇᎿᬺදળ㧕*ᄢ㊁ᵏ㓶㧔ᐳ㐳ផ⮈㧕*ዊ㊁ኹඳᔒ 㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕㤥Ỉ ദ㧔ᣣᧄേ‛ታ㛎ઍᦧᴺቇળ㧕᧖ጊ⌀ℂሶ 㧔ᣣᧄൻ♆ຠᎿᬺㅪวળ㧕⼱↰ᥓሶ㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕*

⷏Ꮉ⑺૫

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕’ ᩕੑ㧔ᣣᧄ఺∉Ქᕈቇળ㧕Ⴧ↰శノ㧔ᐳ㐳ផ⮈㧕ጊ↰㓉ᔒ㧔⁛┙ⴕ᡽ᴺੱ ⵾ຠ⹏ଔᛛⴚၮ⋚ᯏ᭴㧕*ᮮ㑐ඳ㓶㧔ᣣᧄ⊹⤏ࠕ࡟࡞ࠡ࡯࡮ធ⸅⊹⤏Ἳቇળ㧕ศ↰ ✛

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ∛ℂㇱ㧕ศ᧛ ഞ㧔ᐳ㐳ផ⮈㧕

ᷰㇱ৻ੱ㧔ᣣᧄ⵾⮎Ꮏᬺදળ㧕                  છᦼ㧦ᐔᚑ 24 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣ*㧦ᐔᚑ 25 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣii

,C%8#/ ㆇ༡ᆔຬળ
⷏Ꮉ⑺૫

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕㧦ᆔຬ㐳Ꮉ⷏ ᔀ

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ㧕ዊᎹਭ⟤ሶ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ∛ℂㇱ㧕⩲㊁ ⚐

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ Ქᕈㇱ㧕ୖᜬᙗ〝㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ൻቇ‛⾰቟ోኻ╷ቶ㧕*ᢪ⮮๺ᐘ

㧔⁛┙ⴕ᡽ᴺੱ ක⮎ຠක≮ᯏེ✚วᯏ᭴㧕૒‫ᧁޘ‬ᱜᐢ 㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ൻቇ‛⾰቟ోኻ╷ቶ㧕㑐㊁␭ሶ㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ⮎ℂㇱ㧕㜞ᧁ◊਽㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ Ქᕈㇱ േ‛▤ℂቶ㧕ᐢἑ᣿ᒾ  㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ✚ว⹏ଔ⎇
ⓥቶ㧕
ᧄ㑆ᱜల

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ᄌ⇣ㆮવㇱ㧕శጟବᚑ㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ክᩏ▤ℂ⺖㧕ጊᧄ㗅ੑ㧔ෘ↢ഭ௛⋭ ක⮎㘩ຠዪ ൻቇ‛⾰቟ోኻ╷ቶ㧕*ዊፉ ⡸㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ⮎ℂㇱᣂⷙ⹜㛎⴫⹏ଔቶ㧕㧦੐ോዪ*㧦ᐔᚑ 25 ᐕ 8 ᦬ 1 ᣣࠃࠅ

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JaCVAM statement
on in vitro assays using the reconstructed human epidermis models, LabCyte EPIMODEL24
for skin irritation testing
At the meeting concerning the above method, held on 19 April 2013 at the National Instituteof Health Sciences (NIHS), Tokyo, Japan, the members of the Japanese Center for the
Validation of Alternative Methods (JaCVAM) Regulatory Acceptance Board unanimously
endorsed the following statement:
The in vitro assay using the reconstructed human epidermis models such as LabCyte
EPI-MODEL24 is considered to have sufficient accuracy and reliability for prediction ofskin irritating test substances applied for 4 hours under proper conditions.
Following the review of the results of the ECVAM(European Center for the Validation of
Alternative Methods) statements and OECD (Organisation for Economic Co-operation andDevelopment) Test Guideline revised No. 439, it is concluded that two in vitro assays using thereconstructed human epidermis models such as EpiDerm and SkinEthics for skin irritation
testing are clearly beneficial.
The JaCVAM Regulatory Acceptance Board has been regularly kept informed of the
progress of the study, and this endorsement is based on an assessment of various documents,
including, in particular, the evaluation report prepared by the JaCVAM ad hoc peer review
panel for skin irritation testing.

Takemi Yoshida
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

20 November, 2013v

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. Takemi Yoshida (Japanese Society of Toxicology): Chairperson
Mr. Norihide Asano (Japanese Environmental Mutagen Society)
Mr. Tsutomu Ichiki (Japan Chemical Industry Association)*
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Tsutomu Miki Kurosawa (Japanese Society for Animal Experimentation)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Mitsuteru Masuda (nominee by Chairperson)
Mr. Akiyoshi Nishikawa (NIHS)
Mr. Yasuo Ohno (nominee by Chairperson)*
Mr. Hiroshi Onodera (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Tomoko Tanita (Pharmaceuticals and Medical Devices Agency)*
Mr. Takashi Yamada (National Institute of Technology and Evaluation)*
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact
Dermatitis)
Ms. Midori Yoshida (NIHS)
Mr. Isao Yoshimura (nominee by Chairperson)
Mr. Kazuto Watanabe (Japan Pharmaceutical Manufacturers Association)
Term: From 1st April 2012 to 31st March 2014*: From 1st April 2013 to 31st March 2014vi

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. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Jun Kanno (Division of Cellular and Molecular Toxicology, BSRC, NIHS)
Mr. Toru Kawanishi (NIHS)
Mr. Kenji Kuramochi (Ministry of Health, Labour and Welfare)*
Mr. Toshinari Mitsuoka (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Kazuyuki Saito (Pharmaceutical & Medical Devices Agency)
Mr. Masahiro Sasaki (Ministry of Health, Labour and Welfare)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
Mr. Atsuya Takagi (Animal Management Section of the Division of Cellular andMolecular Toxicology, BSRC, NIHS)
Mr. Junji Yamamoto (Ministry of Health, Labour and Welfare)*Mr. Hajime Kojima (Section for the Evaluation of Novel Methods, Division of
Pharmacology, BSRC, NIHS): Secretary* Arrival at post day: 1st August 2013

vii

viii

⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺ LabCyte EPI-MODEL24⋡ ᰴ⹏ଔળ⼏ႎ๔ᦠ ----------------------------------------------------------------------------------- 1⹏ଔႎ๔ᦠ ----------------------------------------------------------------------------------------- 7ࡃ࡝࠺࡯࡚ࠪࡦႎ๔ᦠ ------------------------------------------------------------------------ 21
PEER REVIEW REPORT ------------------------------------------------------------------- 71

OECD TG439 -------------------------------------------------------------------------------- 207
ផᅑࡊࡠ࠻ࠦ࡞ ------------------------------------------------------------------------------- 229

ix


 



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⷏Ꮉ⑺૫㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕㐳⼱Ꮉ㓉৻㧔⁛┙ⴕ᡽ᴺੱ ⵾ຠ⹏ଔᛛⴚၮ⋚ᯏ᭴㧕’ ᩕੑ㧔ᣣᧄ఺∉Ქᕈቇળ㧕Ⴧ↰శノ㧔ᐳ㐳ផ⮈㧕ᮮ㑐ඳ㓶㧔ᣣᧄ⊹⤏ࠕ࡟࡞ࠡ࡯࡮ធ⸅⊹⤏Ἳቇળ㧕ศ↰ ✛㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯ ∛ℂㇱ㧕ศ᧛ ഞ㧔ᐳ㐳ផ⮈㧕ᷰㇱ৻ੱ㧔ᣣᧄ⵾⮎Ꮏᬺදળ㧕   છᦼ㧦ᐔᚑ  ᐕ  ᦬  ᣣ㨪ᐔᚑ  ᐕ  ᦬  ᣣ

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3
3

㧚ᒰ⹥⹜㛎ᴺߣߘߩ࠺࡯࠲ߪ‫ޔ‬ㅘ᣿ߢ⁛┙ߥ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆ߆‫ޕ‬ᣣᧄേ‛ታ㛎ઍᦧᴺቇળߦࠃࠅ‫ޔ‬1'%&6) ߦᕈ⢻ᮡḰ  ߣߒߡឝタߐࠇߚൻቇ‛⾰࡝ࠬ࠻ࠍ߽
ߣߦࡃ࡝࠺࡯࡚ࠪࡦ߇ⴕࠊࠇ‫ߦࠄߐޔ‬ᕈ⢻ᮡḰߩᄌᦝߦࠃࠅㅊടࡃ࡝࠺࡯࡚ࠪࡦ߇ታᣉߐࠇߚ  ‫ߎޕ‬ߩ⚿ᨐࠍ߹ߣ߼ߚႎ๔ᦠߪ‫ޔ‬+PFGRGPFGPV+PVGTPCVKQPCN5EKGPVKHKE2GGT4GXKGY2CPGN ߦࠃࠅ⹏ଔߐࠇߚ‫ޕ‬ߘߩᓟ‫ޔ‬2GGT4GXKGY2CPGN ߩᜰ៰  ߦኻߒ‫ޔ‬ᡷ⸓ࡊࡠ࠻ࠦ࡞  ࠍၮߦࡃ࡝࠺࡯࡚ࠪࡦ߇ታᣉߐ
ࠇ‫ޔ‬1'%& ⊹⤏ೝỗኾ㐷ኅࠣ࡞࡯ࡊߦࠃࠆ⏕⹺ࠍ⚻ߡ 1'%&6) ߦㅊ⸥ߐࠇߚ  ‫ޕ‬એ਄ߩ⚻✲ߩࠃ߁ߦ‫ޔ‬ㅘ᣿ߢ⁛┙ߥ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆ‫ޕ‬ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߩઍᦧᴺߣߒߡ‫⾰‛ߥ߁ࠃߩߤޔ‬෶ߪ⵾ຠࠍ⹏ଔߔࠆߎߣࠍ⋡⊛ߣߒߡ޿ࠆ߆‫ޕ‬ᒰ⹥⹜㛎ᴺߪ‫⥸৻ޔ‬ൻቇ‛⾰ߩ⊹⤏ೝỗᕈࠍ 1'%&6) ߩಽ㘃ߦߒߚ߇ߞߡ‫⹏ޔ‬ଔߔࠆߎߣࠍ⋡⊛ߣߒߡ޿ࠆ‫ޕ‬㧚ᒰ⹥⹜㛎ᴺߪ‫⹏࠼࡯ࠩࡂޔ‬ଔ޽ࠆ޿ߪ࡝ࠬࠢ⹏ଔߩߤߜࠄߦ᦭↪ߢ޽ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ൻቇ‛⾰ߩ⊹⤏ೝỗᕈߦߟ޿ߡ‫ޔ‬70)*5%.2 ⷙ೙ߩಽ㘃ߦߒߚ߇ߞߡ‫ޔ‬ೝỗᕈߣ㕖ೝỗᕈࠍ⼂೎ߢ߈ࠆ‫ޕ‬ߒߚ߇ߞߡ‫ޔ‬ᒰ⹥⹜㛎ᴺߪᦑ㔺ߐࠇߚൻቇ‛⾰ߩࡂࠩ࡯࠼ࠍ⹏ଔߔࠆߩߦ᦭↪ߢ޽ࠆ߇‫⹏ࠢࠬ࡝ޔ‬ଔ߳ߩ᦭↪ᕈߪ⏕⹺ߐࠇߡ޿ߥ޿‫ޕ‬㧚ᒰ⹥⹜㛎ᴺߪ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏ଔߢ߈ࠆ߆‫ߩߘޕ‬႐ว‫ޔ‬ᒰ⹥⹜㛎ᴺߩㆡ↪᧦ઙ߇᣿⏕ߦߥߞߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔ‬70)*5%.2 ⷙ೙ߦၮߠߊಽ㘃ࠍ⋡⊛ߣߒߡ޿ࠆߚ߼‫⹏ࠍ⾰‛ߚߒ␜ߢ ޔ‬ଔߔࠆߎߣߪน⢻ߢ޽ࠆ‫ޕ‬
ᒰ⹥⹜㛎ᴺߩㆡ↪᧦ઙߪ᣿⏕ߦߥߞߡ޿ࠆ‫ޕ‬/66 ㆶరᴺߦࠃࠅ⚦⢩↢ሽ₸ࠍ᳞߼ߡ⹏ଔߦ↪޿ࠆߎߣ߆ࠄ‫ࠆࠃߦ⦡⌕ޔ‬ᓇ㗀߿ㆶర‛⾰ߦࠃࠆᓇ㗀ࠍฃߌࠆ‫ޔߚ߹ޕ‬㐿᡼ㆡ↪ߦࠃࠅ⹏ଔࠍⴕ߁ߚ߼‫ޔ‬ࠛࠕࡠ࠱࡞߿ࠟࠬߪㆡ↪ᄖߣߥࠆ‫ޕ‬㧣㧚ᒰ⹥⹜㛎ᴺߪࡊࡠ࠻ࠦ࡞ߩᓸ⚦ߥᄌᦝߦኻߒߡචಽ㗎ஜߢ޽ࠆ߆‫ޕ‬ᡷ⸓ࡊࡠ࠻ࠦ࡞  ߦߒߚ߇ߞߡታᣉߐࠇࠆ㒢ࠅ‫ޔ‬㗎ஜߢ޽ࠆ‫ޕ‬㧚ᒰ⹥⹜㛎ᴺߩᛛⴚ⠌ᓧߪ‫ޔ‬ㆡಾߥ⸠✵ߣ⚻㛎ࠍ⚻ߡ޿ࠆᜂᒰ⠪ߦߣߞߡኈᤃߥ߽ߩߢ޽ࠆ߆‫⹜ޕ‬
㛎ᴺߩታᣉߦ․ᱶߥ⸳஻߇ᔅⷐ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߦᔅⷐߥ⸳஻ߪ‫ޔ‬ၭ㙃⸳஻߅ࠃ߮ࡈࠜ࡞ࡑࠩࡦߩ↢ᚑ㊂ࠍ᷹ቯߔࠆߚ߼ߩࡑࠗࠢࡠࡊ࡟
࡯࠻࡝࡯࠳࡯ߩߺߢ޽ࠅ‫․ޔ‬ᱶߥ⸳஻ߪਇⷐߢ޽ࠆ‫ޕ‬44

 .CD%[VG'2+/1&'. ߪ ,6% ߆ࠄ⾼౉น⢻ߢ޽ࠆߚ߼‫ޔ‬ၭ㙃ߦ㑐ߔࠆㆡಾߥ⸠✵ߣ⚻㛎ࠍ⚻ߡ޿ࠆᜂᒰ⠪ߢ޽ࠇ߫ᛛⴚ⠌ᓧ߇ኈᤃߢ޽ࠆ‫ޕ‬㧚ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߣᲧߴߡᤨ㑆⊛⚻⾌⊛ߦఝࠇߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߦ߆߆ࠆᤨ㑆ߪ‫ ޔ‬ᣣ㑆ߢ޽ࠆ‫⹜ޕ‬㛎ᦼ㑆ߪ 1'%&6) ߣห⒟ᐲߢ޽ࠆ߇‫ޔ‬㚔ൻᦼ㑆ߩᔅⷐߥേ‛ታ㛎ࠃࠅఝࠇߡ޿ࠆ‫ޕ‬
ᒰ⹥⹜㛎ᴺߢߪ‫ޔ‬േ‛ታ㛎ߦ઻߁⚻⾌߇೥ᷫߢ߈‫ߦ⊛⾌⚻ޔ‬ఝࠇߡ޿ࠆ  ࠠ࠶࠻YGNN㧦ਁ౞ ‫ޕ‬ ᒰ⹥⹜㛎ᴺߪ‫ޔ‬േ‛⑔␩ߩⷰὐ෸߮⑼ቇ⊛⷗࿾߆ࠄ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏ଔߔ
ࠆઍᦧᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪ߔࠆߎߣߪน⢻߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔ‬േ‛ࠍ↪޿ߕߦ⊹⤏ೝỗᕈࠍ⹏ଔߢ߈ࠆߩߢ‫ޔ‬േ‛⑔␩ߩⷰὐ߆ࠄ᦭↪ߢ޽ࠆ‫ޕ‬߹ߚ‫ޔ‬ᒰ⹥⹜㛎ᴺߪൻቇ‛⾰ߩ  ᤨ㑆ㆡ↪ߦࠃࠆ⊹⤏ೝỗᕈࠍ⹏ଔߔࠆᣇᴺߢ޽ࠆ‫▸ߩߘޕ‬࿐㧔⮎੐ᴺ‫ޔ‬ㄘ⮎ข✦ᴺ‫ޔ‬ഭ௛቟ోⴡ↢ᴺ㧕ߦ߅޿ߡⴕ᡽⊛ߥ೑↪ߪน⢻ߢ޽ࠆ߇‫ޔ‬ᚒ߇࿖ߩක⮎ㇱᄖຠ‫ޔ‬ൻ♆ຠߦᔅⷐߣߐࠇࠆ  ᤨ㑆ㆡ↪ߦࠃࠆ⊹⤏ೝỗᕈ߳ߩᔕ↪ߩน⢻ᕈߦߟ޿ߡߪ⹏ଔߐࠇߡ޿ߥ޿‫ޕ‬ ᤨ㑆ㆡ↪ߦߡ⹺߼ࠄࠇࠆ⊹⤏ೝỗᕈߪ‫ᤨ ޔ‬㑆ㆡ↪ߒߚ႐ว‫ޔ‬Ⴧᒝߔࠆߎߣ߇⍮ࠄࠇߡ޿ࠆ
10)‫ޔߡߞ߇ߚߒޕ‬ᒰ⹥⹜㛎ᴺߢೝỗᕈߥߒ㧔)*5 ೝỗᕈ඙ಽߦߡήೝỗᕈ㧕ߣ⹏ଔߐࠇߡ߽‫ޔ‬ᤨ㑆ㆡ↪ߢߪೝỗᕈ޽ࠅߣ⹏ଔߐࠇࠆ႐ว߇޽ࠆ‫ᤨ ޔࠄ߇ߥߒ߆ߒޕ‬㑆ㆡ↪ߢᬌ಴น⢻ߥᒝ޿⊹⤏ೝỗᕈ㧔)*5 ೝỗᕈ඙ಽߦߡೝỗᕈ㧕ࠍ⹏ଔน⢻ߢ޽ࠆߎߣ߆ࠄ‫ࠣࡦ࠾࡯࡝ࠢࠬޔ‬ᴺߣߒߡ೑↪ߢ߈ࠆน⢻ᕈ߇޽ࠆ‫ޕ‬ෳ⠨ᢥ₂
 '%'%8#/  㧘5VCVGOGPVQPVJGXCNKFKV[QHKPXKVTQVGUVUHQTUMKPKTTKVCVKQPKUUWGF
D[VJG'%8#/5EKGPVKHKE#FXKUQT[%QOOKVVGG '5#% #RTKN#XCKNCDNGCV=JVVRGEXCOLTEGEGWTQRCGW?
 ⿒᧻ᶈᒾ㧘ኙ᳓ቁม.CD%[VG'2+/1&'.⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺߩ╙ਃ⠪⹏ଔႎ๔ᦠߩ╙ਃ⠪⹏ଔႎ๔ᦠ 0QX  1'%&  &TCHV7RFCVGF6)+PXKVTQUMKPKTTKVCVKQP4GEQPUVTWEVGF*WOCP'RKFGTOKU6GUV/GVJQFVJ/GGVKPIQHVJG9QTMKPI)TQWRQH0CVKQPCN%QQTFKPCVQTUQH
VJG6GUV)WKFGNKPGU2TQITCOOG 906 2CTKU#XCKNCDNGCV=JVVRYYYQGEFQTIGPXVGUVIWKFGNKPGU?

55 '%'%8#/  2GTHQTOCPEG5VCPFCTFUHQTCRRN[KPIJWOCPUMKPOQFGNUVQKPXKVTQUMKPKTTKVCVKQPVGUVKPI#XCKNCDNGCV=JVVRGEXCOLTEGEGWTQRCGW?
 -QLKOC*#PFQ;+FGJCTC--CVQJ/-QUCMC6/K[CQMC'5JKPQFC5
5W\WMK6;COCIWEJK;;QUJKOWTC+;WCUC#9CVCPCDG;CPF1OQTK6  
8CNKFCVKQP5VWF[QHVJG+P8KVTQ5MKP+TTKVCVKQP6GUVYKVJVJG.CD%[VG'2+/1&'.
#NVGTP.CD#PKO
 1'%&  8CNKFCVKQPTGRQTVHQTVJGUMKPKTTKVCVKQPVGUVOGVJQFWUKPI.CD%[VG'2+/1&'.1'%&5GTKGUQP6GUVKPICPF#UUGUUOGPV0Q1'%&2CTKU#XCKNCDNGCV=JVVRYYYQGEFQTIGPXVGUVIWKFGNKPGU?
 1'%&  2GGTTGXKGYTGRQTVQHXCNKFCVKQPQHVJGUMKPKTTKVCVKQPVGUVWUKPI.CD%[VG'2+/1&'.1'%&5GTKGUQP6GUVKPICPF#UUGUUOGPV0Q1'%&2CTKU#XCKNCDNGCV=JVVRYYYQGEFQTIGPXVGUVIWKFGNKPGU?
 -CVQJ/CPF*CVC-  4GHKPGOGPVQH.CD%[VG'2+/1&'.UMKP+TTKVCVKQPVGUVOGVJQFHQTCFCRVCVKQPVQVJGTGSWKTGOGPVUQH1'%&VGUVIWKFGNKPG##6':

 -QLKOC*-CVQJ/5JKPQFC5*CIKYCTC55W\WMK6+\WOKK4;COCIWEJK;0CMCOWTC/-CUCJCTC65JKDCK#  /GVQQ8CNKFCVKQP5VWF[QHKPXKVTQ5MKP+TTKVCVKQP6GUVWUKPIVJGTGEQPUVTWEVGFJWOCPGRKFGTOKU.CD%[VG'2+/1&'.,#RRNKGF6QZKEQNQI[CEEGRVGF
 %TW\CP)&CNDG[9'&̉CNGQ%,CPF5WIGT',  #EQORQUKVGOQFGNHQTOWNVKRNGCUUC[UQHUMKPKTTKVCVKQP6QZKEN+PF*GCNVJ  

એ਄

6
6

LabCyte EPI-MODEL24 ⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺߩ⹏ଔႎ๔ᦠ

ᐔᚑ 24 ᐕ 10 ᦬ 31 ᣣ

17⊹⤏ೝỗᕈ⹜㛎⹏ଔᆔຬળ
ᆔຬฬ㧦⿒᧻ᶈᒾ㧔ᆔຬ㐳㧦⮮↰଻ஜⴡ↢ᄢቇකቇㇱ㧕ኙ᳓ቁม㧔੩ㇺᄢቇ㧕

8

2

↪⺆㓸
Accuracy㧦ᱜ⏕ᐲConcordance: ৻⥌₸European Centre for Validation of Alternative Methods㧔ECVAM㧕㧦᰷Ꮊઍᦧᴺᬌ⸽࠮ࡦ࠲࡯
False negative rate㧦ன㒶ᕈ₸
False positive rate㧦ன㓁ᕈ₸Globally Harmonized System of Classification and Labelling of Chemicals (GHS): ൻቇຠߩಽ㘃߅ࠃ߮⴫␜ߦ㑐ߔࠆ਎⇇⺞๺ࠪࠬ࠹ࡓ
Independent International Scientific Peer Review Panel㧦╙ਃ⠪ኾ㐷ᛛⴚ⹏ଔ࿖㓙ᆔຬળ
Inter-laboratory validation study:ᣉ⸳㑆ࡃ࡝࠺࡯࡚ࠪࡦ
Intra-laboratory validation study:ᣉ⸳ౝࡃ࡝࠺࡯࡚ࠪࡦ
Japanese Centre for Validation of Alternative Methods㧔JaCVAM㧕㧦ᣣᧄേ‛ታ㛎ઍᦧᴺ⹏ଔ࠮ࡦ࠲࡯
Performance standard㧦ᕈ⢻ᮡḰ
Reliability㧦ା㗬ᕈOrganisation for Economic Co-operation and Development㧔OECD㧕㧦⚻ᷣදജ㐿⊒ᯏ᭴
Test Guideline (TG):࠹ࠬ࠻ࠟࠗ࠼࡜ࠗࡦ
Validation study㧦ࡃ࡝࠺࡯࡚ࠪࡦ
Validation study management team㧦ࡃ࡝࠺࡯࡚ࠪࡦㆇ༡ᆔຬળ

39㧚ᧄ⹜㛎ᴺߩ⑼ቇ⊛ᅷᒰᕈߣⷙ೙⹜㛎ᴺߣߒߡߩᅷᒰᕈ
LabCyte EPI-MODEL24 ࠍ↪޿ߚ⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺߪ‫ޔ‬ᩣᑼળ␠ࠫࡖࡄࡦ࡮࠹ࠖࠪࡘ࡮
ࠛࡦࠫ࠾ࠕ࡝ࡦࠣ㧔J-TEC㧕ߦࠃߞߡ㐿⊒ߐࠇߚᣣᧄ⵾ߩၭ㙃⴫⊹ࡕ࠺࡞ߢ޽ࠆ‫ߣ࡞࠺ࡕᧄޕ‬㘃ૃߒߚၭ㙃⴫⊹ࡕ࠺࡞ߩࡃ࡝࠺࡯࡚ࠪࡦ߇ ECVAM㧔European Center for the Validation of
Alternative Methods㧕ߦࠃࠅࡃ࡝࠺࡯࠻ߐࠇ‫ޔ‬EU ߢ⹺⸽ߐࠇߚ 1㧕‫ࠍࠇߎޕ‬ฃߌ‫ޔ‬ᣣᧄߢ߽ᣣᧄ
േ‛ታ㛎ઍᦧᴺቇળߦߡࡊࡠ࠻ࠦ࡞⏕ቯߩߚ߼ߩࡊ࡟ࡃ࡝࠺࡯࡚ࠪࡦ(phaseΣ)ࠍ⚻ߡ‫ޔ‬
ECVAM ߢ૞ᚑߐࠇߚ GHS ಽ㘃 1㧕ߦࠃࠆ⊹⤏ೝỗᕈ⹜㛎ઍᦧᴺߣߒߡߩ performance standard㧔ᕈ⢻ᮡḰ㧕 2) ߦឝタߐࠇߚൻቇ‛⾰࡝ࠬ࠻ࠍ߽ߣߦ‫ޔ‬me-too㧔ዊⷙᮨ㧕ࡃ࡝࠺࡯࡚ࠪࡦ
(phaseΤ)߇ታᣉߐࠇߚ‫ߩࡦ࡚ࠪ࡯࠺࡝ࡃޕ‬ታᣉਛ‫ޔ‬ECVAM ᕈ⢻ᮡḰߩ࡝ࠬ࠻߇ᡷ⸓ߐࠇߚߎ
ߣ߽޽ࠅ‫ޔ‬ㅊടࡃ࡝࠺࡯࡚ࠪࡦ(phaseΥ)ࠍታᣉߒߚ 3㧕‫ߚ߼ߣ߹ࠍࠄࠇߎޕ‬ႎ๔ᦠ߇ Independent
International Scientific Peer Review Panel㧔Peer Review㧕ߦࠃࠅ⹏ଔߐࠇ‫⚿ߩߘޔ‬ᨐߣߒߡ‫ޔ‬1-bromohexane ߩன㒶ᕈࠍᡷༀߔࠆᜰ␜߇޽ߞߚ 4㧕‫ߩ࡞ࠦ࠻ࡠࡊޕ‬ᡷ⦟߇ J-TEC ߦࠃࠅታᣉߐ
ࠇ 5㧕‫ޔ‬ᡷ⸓ࡊࡠ࠻ࠦ࡞ࠍ↪޿‫ޔ‬ᣂߚߦ 3 ᣉ⸳ߩදജߦࠃࠆ JaCVAM㧔Japanese Centre for the
Validation of Alternative Methods㧕ߦࠃࠆࡃ࡝࠺࡯࡚ࠪࡦ(phaseΦ)߇ታᣉߐࠇߚ‫ޔߦࠄߐޕ‬phaseΦߢ᣿ࠄ߆ߦߥߞߚ߫ࠄߟ߈ߩᄢ߈ߐࠍᡷༀߔࠆߚ߼‫ࠍࠇߘޔࠇߐ৻⛔߇࡞ࠦ࠻ࡠࡊޔ‬↪޿ߚㅊടࡃ࡝࠺࡯࡚ࠪࡦ(phaseΧ)߇ታᣉߐࠇߡ޿ࠆ‫ޕ‬ ᧄ⹏ଔᦠߪ‫ޔ‬phaseΦ߆ࠄΧߦ޿ߚࠆࡃ࡝࠺࡯࡚ࠪࡦ⚿ᨐࠍ߽ߣߦ‫ޔ‬LabCyte EPI-MODEL24ࠍ↪޿ߚ⊹⤏ೝỗᕈ⹜㛎ߩ⹏ଔࠍⴕߞߚ߽ߩߢ޽ࠆ‫⚿ߩࠄࠇߎޔ߅ߥޕ‬ᨐߪ‫ޔ‬OECD ⊹⤏ೝỗᕈኾ㐷ኅࠣ࡞࡯ࡊߦ߅޿ߡ߽⏕⹺ߐࠇ‫ޔ‬OECD ၭ㙃⴫⊹ࡕ࠺࡞ࠍ↪޿ߚ in vitro ⊹⤏ೝỗ
ᕈ⹜㛎ᴺߣߒߡ‫ޔ‬LabCyte EPI-MODEL24 ߇ OECD test guideline 㧔TG439㧕ߦㅊ⸥ߐࠇࠆߎ
ߣߦߥߞߚ 6㧕‫ޕ‬࿑ 1 ߦ‫⎇ࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬ⓥㆊ⒟ࠍ␜ߔࡈࡠ࡯࠴ࡖ࡯࠻ࠍ␜ߔ‫ޕ‬

Phase Iࡊ࡟ࡃ࡝࠺࡯࡚ࠪࡦ

ࡊࡠ࠻ࠦ࡞⏕ቯ

ECVAM ᕈ⢻ᮡḰPhase II

Me too ࡃ࡝࠺࡯࡚ࠪࡦ

19 ‛⾰
ECVAM ᕈ⢻ᮡḰ࡝ࠬ࠻

ECVAM ᕈ⢻ᮡḰᡷ⸓
Phase IIIㅊടࡃ࡝࠺࡯࡚ࠪࡦ

6 ‛⾰
ᡷ⸓ ECVAM ᕈ⢻ᮡḰ࡝ࠬ࠻

Peer Review Panel ᡷༀᜰ␜ࡊࡠ࠻ࠦ࡞ᡷ⸓
Phase IV

Me too ࡃ࡝࠺࡯࡚ࠪࡦ

OECD TG439

20 ‛⾰OECD TG ᕈ⢻ᮡḰ࡝ࠬ࠻ࡃ࡜࠷ࠠߩ໧㗴ࡊࡠ࠻ࠦ࡞ᡷ⸓
Phase Vㅊട౒ห⎇ⓥ

6 ‛⾰



࿑1
10

LabCyte EPI-MODEL24 ⊹⤏ೝỗᕈ⹜㛎ᴺࡃ࡝࠺࡯࡚ࠪࡦ⎇ⓥߩ⚻ㆊ

4㧞㧚⹜㛎ᴺߩᅷᒰᕈ ⹜㛎ࡊࡠ࠻ࠦ࡞ߪ‫ޔ‬TG 439 ߦ␜ߐࠇࠆෳᾖ⹜㛎ᴺ㧔EpiSkinTM ⹜㛎ᴺ㧕ߣ߶߷ห᭽ߢ޽ࠆ‫ޕ‬⹜㛎ࡊࡠ࠻ࠦ࡞ߪ‫ࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬㧔phaseΥ㧕߆ࠄࡃ࡝࠺࡯࡚ࠪࡦ㧔phaseΦ㧕ߦ߅޿ߡ‫ޔ‬Peer
Review ߆ࠄߩᜰ␜ࠍฃߌߡ ver.8.2 ߣߒߡᡷ⦟ߐࠇߚ‫ࡦ࡚ࠪ࡯࠺࡝ࡃޔߚ߹ޕ‬㧔phaseΦ㧕ߢߪ‫ޔ‬ෳട 3 ᣉ⸳ߩ߁ߜ‫ޔ‬1 ᣉ⸳߇․⇣ᐲߦ߅޿ߡ TG439 ߦ␜ߔၮḰࠍḩߚߐߥ߆ߞߚ‫ߩߘޕ‬ේ࿃ߣߒߡ‫߇߈ߟࠄ߫ޔ‬ᄢ߈޿ߎߣ߇ේ࿃ߣ್ᢿߐࠇߚߚ߼‫ࠍ߈ߟࠄ߫ޔ‬ᷫࠄߔ⋡⊛ߢ‫ࡊޔ‬ࡠ࠻ࠦ࡞ࠍᄢ߈ߊᄌᦝߒߥ޿▸࿐ౝߢߩᵞᵺᣇᴺߩ⹦⚦ߥ⛔৻߇࿑ࠄࠇߚ㧔SOPver.8.3㧕7)‫ޕ‬ᄌᦝߐࠇߚὐࠍ⴫ 1 ߦ␜ߔ‫ࡦ࡚ࠪ࡯࠺࡝ࡃޕ‬㧔phase V㧕ߢߪ‫ޔ‬SOPver.8.3 ߦᓥߞߡታᣉߐࠇ
ߚ⚿ᨐ‫⹜ޔ‬㛎ߦ߅ߌࠆ߫ࠄߟ߈ߪᷫዋߒߚ‫ޕ‬
⴫1

SOPver.8.3 ߦ߅ߌࠆⵍ㛎‛⾰ᵞᵺᣇᴺߩ᣿⏕ൻᵞᵺᠲ૞
1.ᵞᵺ೨ߩⵍ㛎‛⾰ߩ㒰෰
2.ᵞᵺᶧ㊂3.ฦᵞᵺᠲ૞ᓟߩ PBS 㒰෰
4.ᦨ⚳ᵞᵺᓟߩ PBS 㒰෰

SOP ver.8.2
SOPver.8.3
᣿⸥ߐࠇߡ޿ߥ޿‫ ޕ‬ᵞᵺ⋥೨ߦࠗࡦࠨ࡯࠻ࠍ௑ߌߡᑄ᫈ߔࠆ‫ޕ‬᣿⸥ߐࠇߡ޿ߥ޿‫࠻࡯ࠨࡦࠗ ޕ‬ኈ㊂એ਄ߩᄢ㊂ߩ PBS ࠍ↪޿ߡᵞᵺߔࠆ‫ޕ‬᣿⸥ߐࠇߡ޿ߥ޿‫ ޕ‬ᑄᶧࡆࡦߩ਄ߢシߊߚߚ޿ߡᑄᶧߔࠆᠲ૞㧔࠲࠶ࡇࡦࠣ㧕ࠍ৻࿁ⴕ߁‫ޕ‬᣿⸥ߐࠇߡ޿ߥ޿‫ࠍ࠻࡯ࠨࡦࠗ ޕ‬௑ߌߡ㒰෰ߔࠆᠲ૞ߩߺߢ਄ㅀߩ࠲࠶ࡇ
ࡦࠣᠲ૞ߪⴕࠊߥ޿‫ޕ‬⹜㛎ᴺ㧔SOPver.8.3㧕ߩ᭎ⷐࠍએਅߦ␜ߔ‫ޕ‬24 ࠙ࠚ࡞ࡊ࡟࡯࠻ߩฦ࠙ࠚ࡞ߦ‫ߦ࠻࠶ࠠޔ‬஻߃ߟߌߩၭ㙃ᶧ 0.5mL ࠍട߃ߡ LabCyte ߩࠗࡦࠨ࡯࠻ࠍ⸳⟎ߒ‫᥅৻ޔ‬೨ၭ㙃ߒߡ‫⹜ޔ‬㛎ߦଏߒߚ‫ⵍޕ‬㛎‛⾰߇ᶧ૕ߩ႐วߪ 25ȝL ࠍ LabCyte ⴫㕙ߦㆡ↪ߒߚ‫ⵍޕ‬㛎‛⾰߇࿕૕ߩ႐วߦߪ
25mg ࠍࡑࠗࠢࡠ࠴ࡘ࡯ࡉߦ⒊㊂ߒߡ߅߈‫⇐⫳߼੍ޔ‬᳓ 25ȝL ࠍᷝടߒߡ߅޿ߚၭ㙃⴫⊹⴫㕙ߦㆡ↪ߒߚ‫ޕ‬1 ⵍ㛎‛⾰ߦߟ߈‫ޔ‬3 ࠗࡦࠨ࡯࠻ࠍ૶↪ߒߚ‫ޕ‬15 ಽ㑆ቶ᷷ߢ㕒⟎ߒߡⵍ㛎‛⾰ࠍ᥸㔺ߒߚ‫ޕ‬ㆡ↪ᤨ㑆⚳ੌᓟ‫ࠍ࠻࡯ࠨࡦࠗޔ‬௑ߌߡࠗࡦࠨ࡯࠻ౝߩⵍ㛎‛⾰ࠍ㒰෰ߒ‫࡝ࡐޔ‬ᵞᵺࡆࡦߦ޿ࠇߚ PBS㧔࡝ࡦ㉄✭ⴣᶧ㧕ࠍࠗࡦࠨ࡯࠻ౝߦ็߈ઃߌࠆߎߣߦࠃࠅᵞᵺߒ‫ࠗޔ‬ࡦࠨ࡯࠻ࠍ௑ߌߡ PBS ࠍ㒰෰ߒߚ‫ߩߎޕ‬ᠲ૞ࠍ 10 ࿁એ਄➅ࠅ㄰ߒ‫⴫❱⚵ޔ‬㕙ߩⵍ㛎‛⾰ࠍขࠅ㒰޿ߚ‫ߩߘޕ‬ᓟ‫ޔ‬ᣂߒ޿࠙ࠚ࡞ߦၭ࿾ 1mL ࠍട߃‫ࠍ࠻࡯ࠨࡦࠗޔ‬⒖േߒ‫ޔ‬42 ᤨ㑆ၭ㙃ࠍⴕߞߚ‫ߩߘޕ‬ᓟ‫ޔ‬MTT ࠍᷝടߒߚၭ࿾ 0.5mL ࠍട߃ߚᣂߒ޿࠙ࠚ࡞ߦࠗࡦࠨ࡯࠻ࠍ⒖േߒ‫ޔ‬ၭ㙃ߒߚ‫ޕ‬3 ᤨ㑆ᓟ‫ޔ‬ฦࠗࡦࠨ࡯࠻ߩၭ㙃⴫⊹⚵❱ࠍࡑࠗࠢࡠ࠴ࡘ࡯ࡉߦฦ‫ࡊ࠰ࠗޔࠇ౉ޘ‬ࡠࡄࡁ࡯࡞ࠍ 300ȝL ട߃ߡ৻᥅એ਄㕒⟎ߒߡ MTT ࡈࠜ࡞ࡑࠩࡦࠍ᛽಴ߒߚ‫ޕ‬96 ࠙ࠚ࡞ࡊ࡟
࡯࠻ߦ᛽಴ᶧࠍ 200ȝL ߕߟ⒖ߒ‫ ߡ޿↪ࠍ࡯࠳࡯࡝࠻࡯࡟ࡊࡠࠢࠗࡑޔ‬570nm‫ ߮ࠃ߅ޔ‬650nm
ߩๆశᐲࠍ᷹ቯߒߚ‫ޕ‬570nm ࠍ᷹ቯᵄ㐳ߣߒߚๆశᐲ߆ࠄ‫ޔ‬650nm ࠍෳᾖᵄ㐳ߣߒߚๆశᐲ
ࠍᏅߒᒁ޿ߚ୯ࠍ᷹ቯ୯ߣߒߚ‫ࠍߺߩ࡞࡯ࡁࡄࡠࡊ࠰ࠗޕ‬ട߃ߚ࠙ࠚ࡞ࠍࡉ࡜ࡦࠢߣߒߡ‫ޔ‬ታ᷹୯ߣࡉ࡜ࡦࠢ୯ߩᏅࠍ᳞߼ߚ‫ޕ‬㒶ᕈኻᾖߩๆశᐲࠍ 100%ߣߒߚᤨߩฦ‛⾰ߩ⚦⢩↢ሽ₸(%)ࠍ⸘▚ߒߚ‫ ߚߒ↪૶ޕ‬3 ⚵❱ߩᐔဋ⚦⢩↢ሽ₸߇ 50%એਅߩ⚿ᨐࠍ␜ߔ‛⾰ࠍ⊹⤏ೝỗᕈ
㧔GHS ಽ㘃 1‫ ߪ޿ࠆ޽ޔ‬2㧕ߣ್ቯߒߚ‫ޕ‬㒶ᕈኻᾖߪ⫳⇐᳓ࠍ↪޿ߚ‫ޕ‬㓁ᕈኻᾖߪ 5㧑 SDS
㧔࡜࠙࡝࡞⎫㉄࠽࠻࡝࠙ࡓ㧕᳓ṁᶧࠍ↪޿ߚ‫ߩࠄࠇߎޕ‬ታ㛎ࠍ‫ࠅ➅ޔ‬㄰ߒ 3 ࿁ታᣉߒ‫ߩߘޔ‬ᐔဋ୯߆ࠄ㓁ᕈ߆㒶ᕈ߆ࠍ್ቯߒߚ‫ޕ‬㧟㧚ࡃ࡝࠺࡯࡚ࠪࡦ⹜㛎ߦ↪޿ߚ‛⾰ߩಽ㘃ߣᅷᒰᕈ
 ࡃ࡝࠺࡯࡚ࠪࡦ⹜㛎ߢߪ‫ޔ‬phaseΦߢ⴫ 2-1 ߅ࠃ߮ phaseΧߢ⴫ 2-2 ߦ␜ߔߘࠇߙࠇ 20 ߅ࠃ
߮ 6 ‛⾰‫ޔ‬ว⸘ 21 ‛⾰߇ଏߐࠇߚ‫ ߪࠄࠇߎޕ‬TG 439 ߩᕈ⢻ᮡḰߦឝタߐࠇߚ࡝ࠬ࠻ਛߩ 20‛⾰ࠍߔߴߡ฽ࠎߢ޿ࠆ‫ ⴫ޕ‬2-1 ߩ‛⾰ No.20 1,1,1-trichloroethane ߪ OECD TG ᩺ߦߪឝタߐ ࠇ ߡ ޿ ߚ ߇ ‫ ࡦ ࡚ ࠪ ࡯ ࠺ ࡝ ࡃ ޔ‬phase Φ ࠍ ታ ᣉ ਛ ߦ ౏ ቯ ൻ ߐ ࠇ ߚ ᦨ ⚳ Ბ 㓏 ߢ ߪ
tetrachloroethylene㧔⴫ 2-2‫ ⾰‛ޔ‬No.21㧕ߦ⟎߈឵ࠊߞߚ‫ ⴫ޕ‬1-2 ߦ⸥タߐࠇߚᱷࠅߩ 5 ‛⾰ߪ‫ ⴫ޔ‬2-1 ߦ߽ឝタߐࠇߡ޿ࠆ‫ޕ‬ ㆬ߫ࠇߡ޿ࠆ 20 ‛⾰ߪ‫ޔ‬ೝỗᕈᒝᐲ㧔GHS ࡜ࡦࠢ㧕‫‛ޔ‬ᕈ‫ޔ‬࿖㓙Ꮢ႐ߢߩ౉ᚻߒ߿ߔߐߥ
ߤࠍ⠨ᘦߒߡㆬᛮߐࠇ‫ޔ‬ᕈ⢻ᮡḰߢߪ‫ޔ‬ዊⷙᮨࡃ࡝࠺࡯࡚ࠪࡦߦ߅޿ߡ‫↪ࠍ⾰‛ߩࠄࠇߎޔ‬޿ߡၮḰએ਄ߩ⚿ᨐ߇ᓧࠄࠇࠆߎߣ߇⸽᣿ߐࠇࠇ߫‫ޔ‬ᣢሽࡕ࠺࡞ߣห⒟ᐲߣᛒ߁ߣߐࠇߡ޿
ࠆ‫ޔ߅ߥޕ‬category 3 ߦዻߔࠆ‛⾰ No.8~10 ߪ TG439 ߦḰ᜚ߒߡήೝỗᕈߣಽ㘃ߐࠇߡ޿ࠆ‫ޕ‬511

⴫ 2-1. Minimum List of Reference Chemicals for Determination of Accuracy and Reliability
Values for Similar or Modified RhE Skin Irritation Test Methods and Codes
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Name
1-bromo-4-chlorobutane
diethyl phthalate
naphthalene acetic acidallyl phenoxy-acetate
Isopropanol
4-methylthio-benzaldehyde
methyl stearate
heptyl butyrate
hexyl salicylate
Cinnamaldehyde
1-decanol
cyclamen aldehyde
1-bromohexane
2-chloromethyl-3,5-dimethyl-4-methoxypyridine HCl
di-n-propyl disulphidepotassium hydroxide 5%
benzynethiol,5-(1,1-dimethylethyl)-2-methyl
1-methyl-3-phenyl-1-piperazine
Heptanal
1,1,1-trichloroethane

CAS number
6940-78-9
84-66-2
86-87-37493-74-5
67-63-03446-89-7
112-61-8
5870-93-9
6259-76-3
104-55-2
112-30-1
103-95-7
11-25-1
86604-75-3
629-19-6
1310-58-3
7340-90-1
5271-27-2
111-71-7
71-55-6

UN GHS in vivo Cat.
Supplier
No Cat.
WPCI
No Cat.
WPCI
No Cat.
WPCI
No Cat.
WPCI
No Cat.
WPCI
No Cat.
WPCI
No Cat.
KCC
No Cat. (Optional Cat. 3) Sigma-Aldrich
No Cat. (Optional Cat. 3) Sigma-Fluka
No Cat. (Optional Cat. 3) Sigma-Aldrich
Cat.2
WPCI
Cat.2
WPCI
Cat.2
WPCI
Cat.2
WPCI
Cat.2
WPCI
Cat.2
WPCI
Cat.2
TCI
Cat.2
TCI
Cat.2
KCC
Cat.2
WPCI

1) CAS No.: Chemical abstracts service registry number.
KCC = Kanto Chemical Co. Inc.; TCI = Tokyo Chemical Industry Co. Ltd; WPCI = Wako Pure Chemical
Industries Ltd;No cat. = ήೝỗᕈ Cat3 = ήೝỗᕈ, .Cat.2 = Category 2㧦ೝỗᕈ⴫ 2-2 The list of chemicals and code with the additional study
No.
Name
2
diethyl phthalate
4allyl phenoxy-acetate
5Isopropanol
13
1-bromohexane
15
di-n-propyl disulphide
21
Tetrachloroethylene
WPCI = Wako Pure Chemical Industries

CAS number
84-66-2
7493-74-5
67-63-0
11-25-1
629-19-6
3446-89-7

Supplier
WPCI
WPCI
WPCI
WPCI
WPCI
WPCI

㧠㧚⹜㛎ᴺߩᱜ⏕ᕈࠍ⹏ଔߔࠆߚ߼ߦ↪޿ࠄࠇߚෳᾖൻว‛ߩ KPXKXQ ෳᾖ࠺࡯࠲
in vivo ෳᾖ࠺࡯࠲ߪ‫ޔ‬GHS ೝỗᕈ඙ಽࠍ 2.3 ߣߒߡ‫ࠇߘޔ‬એ਄㧔Category2㧕ࠍ 10 ‛⾰‫ࠇߘޔ‬ᧂḩࠍ 10 ‛⾰ㆬቯߒߡ޿ࠆ‫ޕ‬Category3 ೝỗᕈ 1.7-2.3 ߪήೝỗᕈߦ඙ಽߐࠇߡ޿ࠆ 9㧕‫ߘޕ‬ߩᖱႎߪ‫ޔ‬TG439 ᕈ⢻ᮡḰߢ⏕⹺ߢ߈ࠆ‫ޕ‬㧡㧚⹜㛎ᴺߩ࠺࡯࠲ߣ⚿ᨐߩ೑↪ᕈ ࡃ࡝࠺࡯࡚ࠪࡦ PhaseIV ߩ⚿ᨐࠍ⴫ 3 ߦ‫ޔ‬phaseV ߩ⚿ᨐࠍ⴫ 4 ߦ␜ߔ‫ޕ‬ᮡḰ஍Ꮕ㧔SD㧕߇
18%ࠍ⿥߃ࠆ⚿ᨐߪ‫ޔ‬ណ↪ߐࠇߡ߅ࠄߕ‫ޔ‬ㅊടታ㛎⚿ᨐ߇ណ↪ߐࠇߚ‫ ⴫ޕ‬3 ߦ‛⾰ No.15 ߩᣉ⸳ b ߪ㧝㨪3 ࿁⋡߹ߢߩ⚿ᨐ߇ណ↪ߐࠇߥ߆ߞߚ‫ޕ‬OECD ᕈ⢻ᮡḰߦߪㅊടታ㛎ߪ 2 ࿁߹ߢߣߐࠇߡ޿ࠆߎߣ߆ࠄ 10)‫ޔ‬4 ߅ࠃ߮ 5 ࿁⋡ߩ⚿ᨐߩߺ߇ណ↪ߐࠇߚ‫⚿ߩߎޕ‬ᨐ߆ࠄ‫ޔ‬ᣉ⸳㨎ߦ߅ߌࠆ‛⾰ No.15 ߦߟ޿ߡߪ➅ࠅ㄰ߒᢙ߇ 3 ࿁ᧂḩߥߩߢ‫್ޔ‬ቯ⚿ᨐߪᰳ᷹ߦߥߞߚ‫ ⴫ޕ‬3ߦ␜ߔࠃ߁ߦ‫ࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬㧔phaseV㧕ߢߪ‫ޔ‬ᣉ⸳ b ߦ߅ߌࠆ‛⾰ No.15 ߪ‫ޔ‬2 ࿁⋡߆ࠄ 4࿁⋡ࠍ߽ߣߦ‫್ޔ‬ቯߐࠇߚ‫ޕ‬ߥ߅‫ ⴫ޔ‬3 ߦឝタߐࠇߚ‛⾰ No.20 ߩ⚿ᨐߪ‫ޔ‬3 ▵ߢㅀߴߚℂ↱߆ࠄ⸃ᨆߦߪ೑↪ߒߡ޿ߥ
޿‫ޕ‬

12

6

⴫ 3. Mean viability of chemicals at each laboratory (Phase V)
Chem. No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
*
#

1
12.4
91.7
108.0
19.1
89.6
16.2
110.0
109.0
105.0
15.7
14.2
8.9
*48
2.1
19.9
0.9
6.9
6.7
9.5
8.7

Lab a
2
3
11.3 19.0
81.5 69.6
113.0 105.0*43.4 65.1
77.0 67.6
15.9 17.0
110.0 104.0
122.0 111.0
111.0 102.0
20.3 16.0
16.5
9.4
15.9 10.0
16.2 16.1
4.3
4.1
95.9 83.5
1.7
1.6
46.6
1.0
4.5
3.6
10.3 10.4
12.0
7.8

4

59.3

15.5

Lab b
Lab c
1
2
3
4
5
1
2
3
16.5 10.7 10.6
9.0
9.8
9.8
60.9 57.5 *65.5 69.5
90.5 *77.4 102.0
96.5 96.7 90.2
89.4 *90.8 106.0
66.6 70.6 *48.1 66.2
90.1 93.0 93.2
75.9 *57.5 74.8 77.1
*68.5 86.6 *66.4
17.3 13.5 11.4
15.5 16.1 12.0
98.8 93.1 76.3
91.2 102.0 108.0
93.1 106.0 86.6
95.5 106.0 119.0
98.0 95.7 83.5
99.6 100.0 113.0
11.5 15.9 11.4
17.3 14.1 14.9
12.4 17.3 16.2
22.1 15.1 14.1
11.0
7.8
9.0
6.0
7.4
5.7
*39.5 6.6 *49.6 17.2 19.0 17.5 17.0 16.2
4.9
5.2
9.1
2.8
3.4
3.2
*39.1 *28 *52.7 17.5 18.5 81.1 83.2 86.3
4.6
2.0
3.3
0.9
3.1
11.6
10.6 21.0 11.6
6.3
5.0
6.6
9.8
10.9 11.0
1.3
1.8
2.2
9.5
7.0
9.5
11.9 10.2 10.9
9.1
7.9
37.6 #17.4
7.6
7.0
6.8

4

5

93.0
98.9

#66.7
#96

67.2

74.4

Lab c
2
77.8
84.3
86.2
11.5
78.6
7.1

3
92.5
79.2
82.1
7.6
70.7
7.0

SD >18%, Not accepted data.
Additional experiments,Not accepted data.
In vitro skin no irritantIn vitro skin irritant⴫ 4. Mean cell viability at each Lab in the additional validation study (Phase V)
No
2
4
5
13
15
21

Name
diethyl phthalate
allyl phenoxy-acetate
Isopropanol
1-bromohexane
di-n-propyl disulphide
Tetrachloroethylene
*

1
94.5
83.1
77.3
17.5
96.5
5.6

Lab a
2
90.7
79.9
79.2
17.1
84.5
5.0

3
92.5
67.3
80.4
18.0
86.4
5.3

1
*49.9
*64.7
79.0
14.3
*72.1
7.1

Lab b
2
3
48.2
53.6
62.4
57.8
78.9
78.2
25.0
20.3
31.1
29.6
9.9
13.0

4
67.3
64.0

43.8

1
94.4
91.0
93.7
11.7
36.9
8.7

SD >18%In vitro skin no irritantIn vitro skin irritant㧢㧚⹜㛎ᴺߩᱜ⏕ᕈ
⴫ 3 ߅ࠃ߮ 4 ߩ⚿ᨐࠍ߽ߣߦ‫ޔ‬ᐔဋ୯߆ࠄ᳞߼ࠄࠇߚ್ቯ⚿ᨐࠍ⴫ 5 ߅ࠃ߮ 6 ߦ␜ߔ‫⴫ޕ‬
4 ߦ␜ߔࠃ߁ߦ‫ ⾰‛ޔ‬No.4 ᣉ⸳ a ߩ್ቯ⚿ᨐߪ 3 ࿁ਛ 2 ࿁߇㒶ᕈߢ޽ߞߚ߇‫ޔ‬3 ࿁ߩᐔဋ୯
ࠍ↪޿ࠆߣ㓁ᕈߥࠅ‫⚿ޔ‬ᨐ߇㘩޿㆑ߞߚ‫ޕ‬ㅊടࡃ࡝࠺࡯࡚ࠪࡦߢߪ‫ߩ࡞ࠦ࠻ࡠࡊࠆߥࠄߐޔ‬ᡷ⦟ߢߔߴߡߩᣉ⸳ߩߔߴߡߩታ㛎ߢ㒶ᕈߣߥߞߚ‫ ⴫ޕ‬5 ߅ࠃ߮ 6 ߦ␜ߔࠃ߁ߦ‫ ⾰‛ޔ‬No.13
ߪߔߴߡߩᣉ⸳ߩߔߴߡߩታ㛎ߢ㓁ᕈߣߥࠅ‫ߩ࡞ࠦ࠻ࡠࡊޔ‬ᡷ⦟߇⏕⹺ߢ߈ߚ‫ޕ‬
 TG439 ᕈ⢻ᮡḰߦḰ᜚ߒ‫ޔ‬PhaseΦ߅ࠃ߮Χߩ⚿ᨐࠍ⚵ߺวࠊߖߡ‫ ⾰‛ޔ‬2‫ޔ‬4‫ޔ‬5‫ޔ‬13‫ޔ‬
15‫ޔ‬2㧝ߦߟ޿ߡߪ‫ޔ‬phaseV ߩ⚿ᨐࠍ‫ࠇߘޔ‬એᄖߩ‛⾰ߪ PhaseΦߩ⚿ᨐࠍᦨ⚳⚿ᨐߣߒߡ⴫
8 ߦ߹ߣ߼ࠆߣ‫ޔ‬ᗵᐲ߇ 90㧑㧔2 ᣉ⸳㧕‫ޔ‬100%㧔1 ᣉ⸳㧕‫⇣․ޔ‬ᐲ߇ 70㧑㧔3 ᣉ⸳㧕‫ޔ‬ᱜ⏕ᐲ 80%
㧔2 ᣉ⸳㧕‫ޔ‬85%㧔1 ᣉ⸳㧕ߣߥࠅ‫ޔ‬ᕈ⢻ᮡḰߩၮḰߢ޽ࠆᗵᐲ 90%‫⇣․ޔ‬ᐲ 70%‫ޔ‬ᱜ⏕ᐲ 80%ߦోߡㆡวߒߚ‫࡞ࠦ࠻ࡠࡊߥ⊛⚳ᦨޔࠅࠃߦࠇߎޕ‬㧔SOPver.8.3㧕ࠍ↪޿ࠇ߫‫ޔ‬LabCyte ࠍ↪
޿ߚ⊹⤏ೝỗᕈ⹜㛎ߩᱜ⏕ᕈߪ㜞޿ߣ್ᢿߒߚ‫ޔߒߛߚޕ‬14 ‛⾰ߦߟ޿ߡߪ‫ޔ‬ᣥࡊࡠ࠻ࠦ࡞
㧔SOPver.8.2㧕ߢታᣉߐࠇߚ⚿ᨐߢ޽ࠅ‫࡞ࠦ࠻ࡠࡊ⚳ᦨޔ‬㧔SOPver.8.3㧕ߦㆡวߔࠆ߆ߪਇ᣿
ߢ޽ࠆ‫ߢ࡞ࠦ࠻ࡠࡊ⚳ᦨޔߢߎߘޕ‬ታᣉߐࠇߚ 14 ‛⾰ࠍ฽ࠎߛࠃࠅᐢ▸ߥ‛⾰ߩ࠺࡯࠲㧔⴫
9‫ޔ‬࿑ 2,3㧕ࠍ J-TEC ࠃࠅឭଏࠍฃߌ‫ߩ࡞ࠦ࠻ࡠࡊޔ‬ᡷ⸓ߦ઻߁⚿ᨐ߳ߩᓇ㗀ࠍ⹏ଔߒߚ‫ޕ‬ᴪ713⚿ᨐ‫ޔ‬஍ࠅߪዊߐߊ‫♖ޔ‬ᐲߩะ਄ߦነਈߒߡ޿ࠆߣ್ᢿߒߚ‫ޕ‬
⴫ 5. Classification using three independent viabilities (Phase IV)
Lab a
Lab b
UN GHS
in vivo Cat
1
2
3
F
1
2
3
F
1
1
No Cat.
P
P
P
P
P
P
P
P
P
2
No Cat.
N
N
N
N
N
N
N
N
N
3
No Cat.
N
N
N
N
N
N
N
N
N
4
No Cat.
P
N
N
P
N
N
N
N
N
5
No Cat.
N
N
N
N
N
N
N
N
N
6
No Cat.
P
P
P
P
P
P
P
P
P
7
No Cat.
N
N
N
N
N
N
N
N
N
8
No Cat.
N
N
N
N
N
N
N
N
N
9
No Cat.
N
N
N
N
N
N
N
N
N
10
No Cat.
P
P
P
P
P
P
P
P
P
11
Cat.2
P
P
P
P
P
P
P
P
P
12
Cat.2
P
P
P
P
P
P
P
P
P
13
Cat.2
P
P
P
P
P
P
P
P
P
14
Cat.2
P
P
P
P
P
P
P
P
P
15
Cat.2
P
N
N
N
P
P
ND
ND
N
16
Cat.2
P
P
P
P
P
P
P
P
P
17
Cat.2
P
P
P
P
P
P
P
P
P
18
Cat.2
P
P
P
P
P
P
P
P
P
19
Cat.2
P
P
P
P
P
P
P
P
P
20
Cat.2
P
P
P
P
P
P
P
P
P
P: Positive, N: Negative, F: Final detemination by median, ND: Not detected for invalid

No.

⴫ 6.

Classification using three independent viabilities (Phase V)

No
2
4
5
13
15
21

Name
diethyl phthalate
allyl phenoxy-acetate
Isopropanol
1-bromohexane
di-n-propyl disulphide
Tetrachloroethylene

⴫7Classification using three independent viabilities (Phase IV and phase V)

No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

14

Lab c
2
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

UN GHS
in vivo Cat
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
Cat.2
Cat.2
Cat.2
Cat.2
Cat.2
Cat.2
Cat.2
Cat.2
Cat.2
Cat.2

Lab.a
Mean
Judge
92.5
NI
76.8
NI
79.0
NI
17.5
I
89.1
NI
5.3
I

Lab.b
Mean
Judge
56.4
NI
61.4
NI
78.7
NI
19.9
I
34.8
I
10.5
I

Lab a
1
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

2
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

F
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

1
P
P
N
N
N
P
N
N
N
P
P
P
P
P
P
P
P
P
P
P

2
P
N
N
N
N
P
N
N
N
P
P
P
P
P
P
P
P
P
P
P

8

F
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

3
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

F
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

Lab.c
Mean
Judge
88.3
NI
84.8
NI
87.3
NI
10.3
I
62.1
NI
7.6
I

Lab b
3
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

3
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

Lab c
3
P
N
N
N
N
P
N
N
N
P
P
P
P
P
P
P
P
P
P
P

F
P
N
N
N
N
P
N
N
N
P
P
P
P
P
P
P
P
P
P
P

1
P
P
N
N
N
P
N
N
N
P
P
P
P
P
P
P
P
P
P
P

2
P
N
N
N
N
P
N
N
N
P
P
P
P
P
N
P
P
P
P
P

⴫ 8. 2x2 tables merged with additional study
Lab a

In vitro prediction

Irritant
Non-irritantTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

Irritant
91
10

Irritant
Non-irritantTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

Irritant
100
10Sensitivity (%)
Specificity (%)
Accuracy (%)

In vivo classification
Non-Irritant
3
7
10

Total
13
7
20

In vivo classification
Non-Irritant
3
7
10

Total
12
8
20

100.0
70.0
85.0

Lab c

In vitro prediction

Total
12
8
20

90.0
70.0
80.0

Lab b

In vitro prediction

In vivo classification
Non-Irritant
3
7
10

Irritant
Non-irritant
Total

Irritant
9
1
10
90.0
70.0
80.0

9

15

⴫ 9. SOPver.8.2 ߣ SOPver.8.3 ࠍ↪޿ߚ LabCyte EPI-MODEL24 ⊹⤏ೝỗᕈ⹜㛎ChemicalName
1-bromo-4-chlorobutane
diethyl phthalate
di-propylene glycol
naphtalen acetic acid
3-ChloronitroBenzene
3,3-Dithiodipropionic Acid
4,4-Methylenebis(2,6-di-tert-buthylphenol)

4-Amino-1,2,4-Triazole
Benzyl Benzoate
Sodium Bicarbonate
Erucamide
1,5-hexadiene
Polyethlene glycol 400
Glycerol3,3-Dimethylpentane
allyl phenoxy-acetate
Isopropanol
Benzyl Salicylate
Lauric Acid
4-methyl-thio-benzaldehyde
Methyl Stearate
Benzyl Acetate
Hydroxycitronellal
Isopropyl Myristate
Isopropyl Palmitate
n-Buthyl Propionate
Sodium Bisulphite
Benzyl Alcohol
allyl heptanoate
heptyl butyrate
2-Ethoxy Ethyl Methacrylate
hexyl salicylate
Linalyl Acetate
terpinyl acetate
Linalol
Cinnamaldehyde
Eugenol
cyclamen aldehyde
1-decanol
1-bromohexane
alpha-Terpineol
1-BromoPentane
2-chloromethyl-3,5-dimethyl-4-methoxypyridine HC

butyl methacrylate
di-n-propyl disulphide
Potassium Hydroxide 5%
Heptanal
benzynethiol, 5-(1,1-dim ethylethyl) -2-methyl

1-methyl-3-phenyl-1-piperazine
SLS (20% aq)
1,1,1 Trichloroethane
Tetrachlotroethylene
Capric acid (decanoic acid)
SLS (5% aq)

16

LabCyte EPI-MODEL24 SIT

In vivo In vivo
score class
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0.3
0.3
0.3
0.3
1
1
1
1
1
1
1
1
1.3
1.7
1.7
1.7
2
2
2
2
2
2
2.3
2.3
2.7
2.7
2.7
2.7
3
3
3
3.3
3.3
3.3
4
4
4
4

NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

Ver.8.2
Viability (%)
19.8 ± 3.4
87.6 ± 27.9
101.0 ± 8.5
99.7 ± 7.6
99.2 ± 4.7
96.3 ± 5.5
101.3 ± 2.2
99.1 ± 1.8
101.9 ± 3.3
100.0 ± 0.3
95.0 ± 6.6
91.2 ± 5.1
102.7 ± 4.3
109.1 ± 14.7
79.8 ± 9.8
82.3 ± 22.9
84.6 ± 2.7
97.2 ± 3.3
104.4 ± 9.0
22.6 ± 1.8
104.4 ± 7.7
26.2 ± 13.4
24.2 ± 7.1
100.8 ± 6.2
107.5 ± 7.3
36.8 ± 12.1
49.0 ± 33.5
13.6 ± 7.0
103.0 ± 6.8
108.1 ± 2.0
49.0 ± 23.1
106.7 ± 16.3
93.5 ± 7.3
32.1 ± 5.3
16.6 ± 8.8
29.1 ± 7.2
27.0 ± 7.4
30.1 ± 5.9
32.3 ± 8.8
39.9 ± 3.3
21.4 ± 10.3
21.9 ± 3.6
12.7 ± 3.2
28.9 ± 4.6
71.7 ± 7.5
3.0 ± 0.5
17.3 ± 3.2
27.6 ± 8.4
15.2 ± 4.6
12.0 ± 2.3
11.8 ± 1.7
17.0 ± 5.7
11.2 ± 5.9
13.4 ± 1.7

10

In vitro Cla ss

I/NI
I
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
NI
I
I
NI
NI
I
NI
I
NI
NI
I
NI
NI
I
I
I
I
I
I
I
I
I
I
I
NI
I
I
I
I
I
I
I
I
I

Ver8.3Viability (%)
27.8 ± 0.8
90.8 ± 15.7
95.4 ± 3.8
94.6 ± 6.6
88.8 ± 7.1
99.0 ± 2.4
99.1 ± 5.6
94.5 ± 2.1
96.5 ± 2.8
101.0 ± 4.5
97.0 ± 3.5
64.9 ± 10.1
96.3 ± 2.4
96.6 ± 4.6
65.1 ± 7.8
81.4 ± 9.1
74.7 ± 7.4
98.5 ± 7.7
93.6 ± 4.1
33.1 ± 2.4
90.8 ± 5.1
25.6 ± 1.8
26.2 ± 1.9
96.0 ± 8.6
100.0 ± 8.7
33.8 ± 3.5
66.9 ± 1.8
12.6 ± 2.0
88.7 ± 9.9104.2 ± 5.9
31.5 ± 10.7
97.2 ± 4.9
95.0 ± 10.0
28.9 ± 8.8
26.6 ± 4.9
30.2 ± 5.9
35.4 ± 4.4
26.8 ± 1.9
27.9 ± 6.8
32.7 ± 4.0
25.1 ± 0.5
29.8 ± 3.5
13.1 ± 1.3
30.0 ± 6.6
69.8 ± 20.3
0.1 ± 0.2
21.4 ± 0.2
28.1 ± 4.9
5.3 ± 6.9
12.7 ± 1.6
20.9 ± 2.6
22.9 ± 1.4
17.0 ± 1.3
15.9 ± 0.2

In vitro Class

I/NI
I
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
NI
I
I
NI
NI
I
NI
I
NI
NI
I
NI
NI
I
I
I
I
I
I
I
I
I
I
I
NI
I
I
I
I
I
I
I
I
I

㪭㫀㪸㪹㫀㫃㫀㫋㫐㩷㫀㫅㩷㪪㪦㪧㩷㫍㪼㫉㪅㪏㪅㪊㩷㩿㩼㪀

㪈㪌㪇

㪈㪇㪇

㪌㪇

㫐㩷㪔㩷㪇㪅㪐㪋㪋㫏㪩 㪉 㩷㪔㩷㪇㪅㪐㪌㪏

㪇
㪇

㪌㪇

㪈㪇㪇

㪈㪌㪇

㪭㫀㪸㪹㫀㫃㫀㫋㫐㩷㫀㫅㩷㪪㪦㪧㩷㫍㪼㫉㪅㪏㪅㪉㩷㩿㩼㪀࿑2

SOPver.8.2 ߣ SOPver.8.3 ࠍ↪޿ߚ LabCyte EPI-MODEL24 ⊹⤏ೝỗᕈ⹜㛎ߦ߅ߌࠆ↢⚦⢩₸ߩ⋧㑐㪈 㪄 㪹㫉㫆 㫄 㫆 㪄 㪋 㪄 㪺 㪿 㫃㫆 㫉㫆 㪹㫌 㫋㪸㫅 㪼㪋㪇㪻㫀㪼 㫋㪿 㫐㫃㩷 㫇㪿 㫋㪿 㪸㫃㪸㫋㪼㪻㫀㪄 㫇㫉㫆 㫇㫐㫃㪼 㫅 㪼 㩷 㪾㫃㫐㪺 㫆 㫃㫅 㪸㫇㪿 㫋㪸㫃㪼 㫅 㩷 㪸㪺 㪼 㫋㫀㪺 㩷 㪸㪺 㫀㪻㪊 㪄 㪚㪿 㫃㫆 㫉㫆 㫅 㫀㫋㫉㫆 㪙㪼 㫅 㫑㪼 㫅 㪼㪊 㪃㪊 㪄 㪛㫀㫋㪿 㫀㫆 㪻㫀㫇㫉㫆 㫇㫀㫆 㫅 㫀㪺 㩷 㪘㪺 㫀㪻㪋 㪃㪋 㪄 㪤 㪼 㫋㪿 㫐㫃㪼 㫅 㪼 㪹㫀㫊㩿 㪉 㪃㪍 㪄 㪻㫀㪄 㫋㪼 㫉㫋㪄 㪹㫌 㫋㪿 㫐㫃㫇㪿 㪼 㫅 㫆 㫃㪀㪋 㪄 㪘㫄 㫀㫅 㫆 㪄 㪈 㪃㪉 㪃㪋 㪄 㪫㫉㫀㪸㫑㫆 㫃㪼㪙㪼 㫅 㫑㫐㫃㩷 㪙㪼 㫅 㫑㫆 㪸㫋㪼㪪㫆 㪻㫀㫌 㫄 㩷 㪙㫀㪺 㪸㫉㪹㫆 㫅 㪸㫋㪼㪜㫉㫌 㪺 㪸㫄 㫀㪻㪼㪈 㪃㪌 㪄 㪿 㪼 㫏㪸㪻㫀㪼 㫅 㪼㪧㫆 㫃㫐㪼 㫋㪿 㫃㪼 㫅 㪼 㩷 㪾㫃㫐㪺 㫆 㫃㩷 㪋 㪇 㪇㪞㫃㫐㪺 㪼 㫉㫆 㫃㪊㪇㪊 㪃㪊 㪄 㪛㫀㫄 㪼 㫋㪿 㫐㫃㫇㪼 㫅 㫋㪸㫅 㪼㪸㫃㫃㫐㫃㩷 㫇㪿 㪼 㫅 㫆 㫏㫐㪄 㪸㪺 㪼 㫋㪸㫋㪼㪠㫊㫆 㫇㫉㫆 㫇㪸㫅 㫆 㫃㪙㪼 㫅 㫑㫐㫃㩷 㪪㪸㫃㫀㪺 㫐㫃㪸㫋㪼㪣㪸㫌 㫉㫀㪺 㩷 㪘㪺 㫀㪻㪋 㪄 㫄 㪼 㫋㪿 㫐㫃㪄 㫋㪿 㫀㫆 㪄 㪹㪼 㫅 㫑㪸㫃㪻㪼 㪿 㫐㪻㪼㪤 㪼 㫋㪿 㫐㫃㩷 㪪㫋㪼 㪸㫉㪸㫋㪼㪙㪼 㫅 㫑㫐㫃㩷 㪘㪺 㪼 㫋㪸㫋㪼㪟㫐㪻㫉㫆 㫏㫐㪺 㫀㫋㫉㫆 㫅 㪼 㫃㫃㪸㫃㪪㪛㩷㩿㩼㪀㪠㫊㫆 㫇㫉㫆 㫇㫐㫃㩷 㪤 㫐㫉㫀㫊㫋㪸㫋㪼㪠㫊㫆 㫇㫉㫆 㫇㫐㫃㩷 㪧㪸㫃㫄 㫀㫋㪸㫋㪼㫅 㪄 㪙㫌 㫋㪿 㫐㫃㩷 㪧㫉㫆 㫇㫀㫆 㫅 㪸㫋㪼㪉㪇㪪㫆 㪻㫀㫌 㫄 㩷 㪙㫀㫊㫌 㫃㫇㪿 㫀㫋㪼㪙㪼 㫅 㫑㫐㫃㩷 㪘㫃㪺 㫆 㪿 㫆 㫃㪸㫃㫃㫐㫃㩷 㪿 㪼 㫇㫋㪸㫅 㫆 㪸㫋㪼㪿 㪼 㫇㫋㫐㫃㩷 㪹㫌 㫋㫐㫉㪸㫋㪼㪉 㪄 㪜㫋㪿 㫆 㫏㫐㩷 㪜㫋㪿 㫐㫃㩷 㪤 㪼 㫋㪿 㪸㪺 㫉㫐㫃㪸㫋㪼㪿 㪼 㫏㫐㫃㩷 㫊㪸㫃㫀㪺 㫐㫃㪸㫋㪼㪣㫀㫅 㪸㫃㫐㫃㩷 㩷 㪘㪺 㪼 㫋㪸㫋㪼㫋㪼 㫉㫇㫀㫅 㫐㫃㩷 㪸㪺 㪼 㫋㪸㫋㪼㪣㫀㫅 㪸㫃㫆 㫃㪚㫀㫅 㫅 㪸㫄 㪸㫃㪻㪼 㪿 㫐㪻㪼㪜㫌 㪾㪼 㫅 㫆 㫃㪺 㫐㪺 㫃㪸㫄 㪼 㫅 㩷 㪸㫃㪻㪼 㪿 㫐㪻㪼㪈㪇㪈 㪄 㪻㪼 㪺 㪸㫅 㫆 㫃㪈 㪄 㪹㫉㫆 㫄 㫆 㪿 㪼 㫏㪸㫅 㪼㪸㫃㫇㪿 㪸㪄 㪫㪼 㫉㫇㫀㫅 㪼 㫆 㫃㪈 㪄 㪙㫉㫆 㫄 㫆 㪧㪼 㫅 㫋㪸㫅 㪼㪉 㪄 㪺 㪿 㫃㫆 㫉㫆 㫄 㪼 㫋㪿 㫐㫃㪄 㪊 㪃㪌 㪄 㪻㫀㫄 㪼 㫋㪿 㫐㫃㪄 㪋 㪄 㫄 㪼 㫋㪿 㫆 㫏㫐㫇㫐㫉㫀㪻㫀㫅 㪼 㩷 㪟㪚㪹㫌 㫋㫐㫃㩷 㫄 㪼 㫋㪿 㪸㪺 㫉㫐㫃㪸㫋㪼㪻㫀㪄 㫅 㪄 㫇㫉㫆 㫇㫐㫃㩷 㪻㫀㫊㫌 㫃㫇㪿 㫀㪻㪼㪧㫆 㫋㪸㫊㫊㫀㫌 㫄 㩷 㪟㫐㪻㫉㫆 㫏㫀㪻㪼 㩷 㪌 㩼㪟㪼 㫇㫋㪸㫅 㪸㫃㪹㪼 㫅 㫑㫐㫅 㪼 㫋㪿 㫀㫆 㫃㪃㩷 㪌 㪄 㩿 㪈 㪃㪈 㪄 㪻㫀㫄 㪼 㫋㪿 㫐㫃㪼 㫋㪿 㫐㫃㪀 㪄 㪉 㪄 㫄 㪼 㫋㪿 㫐㫃㪈 㪄 㫄 㪼 㫋㪿 㫐㫃㪄 㪊 㪄 㫇㪿 㪼 㫅 㫐㫃㪄 㪈 㪄 㫇㫀㫇㪼 㫉㪸㫑㫀㫅 㪼㪪㪣 㪪㩷 㩿 㪉 㪇 㩼 㩷 㪸㫈㪀㪇㪈 㪃㪈 㪃㪈 㩷 㪫㫉㫀㪺 㪿 㫃㫆 㫉㫆 㪼 㫋㪿 㪸㫅 㪼㪫㪼 㫋㫉㪸㪺 㪿 㫃㫆 㫋㫉㫆 㪼 㫋㪿 㫐㫃㪼 㫅 㪼㪭㪼㫉㪅㪏㪅㪉

㪭㪼㫉㪏㪅㪊

㪚㪸㫇㫉㫀㪺 㩷 㪸㪺 㫀㪻㩷 㩿 㪻㪼 㪺 㪸㫅 㫆 㫀㪺 㩷 㪸㪺 㫀㪻㪀㪪㪣 㪪㩷 㩿 㪌 㩼 㩷 㪸㫈㪀

㪪㪦㪧

࿑3

SOPver.8.2 ߣ SOPver.8.3 ࠍ↪޿ߚ LabCyte EPI-MODEL24 ⊹⤏ೝỗᕈ⹜㛎ߦ߅ߌࠆ↢⚦⢩₸ SD ߩ⋧㑐1117㧣㧚⹜㛎ᣇᴺߩା㗬ᕈ ⴫ 10 ߦ␜ߔࠃ߁ߦ‫ޔ‬phaseΦߢߪ 5 ‛⾰ߦ߅޿ߡ‫ߩ߈ߟࠄ߫ޔ‬ᄢ߈ߥ⚿ᨐ߇ᓧࠄࠇߚ‫ޕ‬ฦ‛⾰ߢ SD ߇ 18%ࠍ⿧߃ࠆ࠺࡯࠲߇ 2㨪3 ߺࠄࠇ‫ޔ‬ว⸘ 13 ࠺࡯࠲ߣߥߞߚ‫ߒ߼ߒߪ࠲࡯࠺ޕ‬ߡ޿ߥ޿߇‫ߩ߈ߟࠄ߫ޔ‬ᄢ߈߆ߞߚ 5 ‛⾰ߩ SD ߪ phaseΤߣᲧߴ‫ޔ‬phaseΦߢߪᄢ߈ߊߥߞߡ
޿ࠆ‫ޔߜࠊߥߔޕ‬1-bromohexsane ߩன㒶ᕈࠍᡷༀߔࠆߚ߼ߦⴕߞߚⵍ㛎‛⾰ߩᵞ޿ᣇᡷߪㆡ
ಾߢߪߥ߆ߞߚߣ޿߃ࠆ‫ߩ࡞ࠦ࠻ࡠࡊࠆߥࠄߐޔߢߎߘޕ‬ᡷༀ߇ߥߐࠇߚ phaseΧߢߪ‫ޔ‬SD୯ߪዊߐߊߥࠅ‫ ⴫ޔ‬3 ߦ␜ߔࠃ߁ߦ‫ޔ‬SD ߇ 18%ࠍ⿧߃ࠆ୯ߪ 3 ࠺࡯࠲ߩߺߦᡷༀߐࠇߚ‫ޕ‬ ࡃ࡝࠺࡯࡚ࠪࡦ PhaseIV‫ ߮ࠃ߅ޔ‬V ࠍ⚵ߺวࠊߖߚᣉ⸳ౝߩౣ⃻ᕈߦߟ޿ߡߪ‫ ⴫ޔ‬8 ߦ␜ߔࠃ߁ߦ‫ޔ‬3 ࿁ߩ➅ࠅ㄰ߒ⹜㛎ߢ್ቯ⚿ᨐ߇ోߡหߓ⚿ᨐߣߥߞߚ‛⾰ߩഀวߪ‫ޔ‬95㧑㧔2 ᣉ⸳㧦ᣉ⸳ b, c 19/20 ‛⾰㧕‫ޔ‬100%㧔1 ᣉ⸳㧕ߣߥࠅ‫ޔ‬ᕈ⢻ᮡḰߩၮḰߢ޽ࠆ 90%એ਄ߦోᣉ⸳
߇ㆡวߒߚ‫ޕ‬ࡃ࡝࠺࡯࡚ࠪࡦ PhaseIV‫ ߮ࠃ߅ޔ‬V ࠍ⚵ߺวࠊߖߚᣉ⸳㑆ߩౣ⃻ᕈߦߟ޿ߡߪ‫␜ߦ ⴫ޔ‬ߔࠃ߁ߦ‫ޔ‬3 ᣉ⸳ߩ್ቯ⚿ᨐ߇ోߡหߓ⚿ᨐߣߥߞߚ‛⾰ߩഀวߪ‫ޔ‬95%㧔19/20 ‛⾰‫ޔ‬㒰ߊ‛⾰ No.15㧕ߣߥࠅ‫ޔ‬ᕈ⢻ᮡḰߩၮḰߢ޽ࠆ 80%એ਄ߦㆡวߒߚ‫ޕ‬એ਄ߩ⚿ᨐ߅ࠃ߮‫ ⴫ޔ‬5 ߅ࠃ߮ 7 ߦ␜ߔᣉ⸳ౝ‫ޔ‬ᣉ⸳㑆ߩ㜞޿ౣ⃻ᕈ⚿ᨐ߆ࠄᧄ⹜㛎ᴺߩ
ᡷ⸓ࡊࡠ࠻ࠦ࡞ߩା㗬ᕈߪ㜞޿ߣ್ᢿߒߚ‫ޕ‬
⴫ 10. Chemicals obtained large variations at me-too validation studyData number of SD > 18㧑
No. Name
Lab a
Lab b
Lab c
2
diethyl phthalate
0/3
1/4
1/4
4
allyl phenoxy-acetate
1/4
1/4
0/3
5
Isopropanol
0/3
1/4
2/5
13 1-bromohexane
1/4
2/5
0/3
15 di-n-propyl disulphide
0/3
3/5
0/3

Total
2/11
2/11
3/12
3/12
3/11

㧤㧚࠺࡯࠲ߩ⾰ ⹜㛎ߦෳടߒߚᣉ⸳ߪ GLP ߩ♖␹ߦೣࠅ‫⹜ޔ‬㛎ߦ㑐ߔࠆߔߴߡߩ⸥㍳ࠍᱷߒ‫⸥ޔ‬㍳↪⚕ࠍฦᣉ⸳ߩ⹜㛎⽿છ⠪߇⏕⹺ߒߚᓟ‫ࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬ታⴕᆔຬ㐳ߦㅍઃߒߚ‫ࡦ࡚ࠪ࡯࠺࡝ࡃޕ‬ታⴕᆔຬ㐳ߪߔߴߡߩ⸥㍳↪⚕ࠍ⏕⹺ߒ‫ޔ‬ਇ᣿ߥὐ߇޽ࠇ߫໧޿วࠊߖ‫⋧ޔ‬੕ߢ⏕ቯߒߚ࠺࡯࠲ߩߺࠍ⸃ᨆߦ↪޿ߡ޿ࠆ‫್ߣࠆ޿ߡࠇߐ଻⏕ߪ⾰ߩ࠲࡯࠺ޔߡߞࠃޕ‬ᢿߒߚ‫ޕ‬㧥㧚⹜㛎ᴺߩ᦭↪ᕈߣ㒢⇇
LabCyte EPI-MODEL24㧔24 ࠗࡦࠨ࡯࠻/ࠠ࠶࠻㧕ߪ‫ޔ‬1 ࠠ࠶࠻ 5 ਁ 4 ජ౞ 1 ࠠ࠶࠻ߢᧂ⍮ᬌ
૕ࠍ 6 ‛⾰㧔ઁߦ㒶ᕈኻᾖ‫ޔ‬㓁ᕈኻᾖ㧕⹏ଔน⢻ߢ޽ࠆ‫ޕ‬േ‛ታ㛎ߣᲧセߒߡ‫ޔ‬5 ᣣ㑆ߣ޿߁⍴ᤨ㑆ߢ⚿ᨐࠍᓧࠆߎߣ߇ߢ߈ࠆ‫৻ޕ‬ᣇ‫ޔ‬LabCyte
EPI-MODEL24 ౉ᚻᓟߪ 4 ᣣએౝߦ⹜㛎ࠍ㐿ᆎߔࠆᔅⷐ߇޽ࠅ‫⹜ޔ‬㛎ታᣉߦ 5 ᣣ㑆ࠍⷐߔࠆߚ
߼‫ޔ‬Ἣᦐᣣ೔⌕ߢߔߋߦ⹜㛎ࠍታᣉߒߥ޿႐ว‫ޔ‬࿯ᦐᣣ‫ޔ‬ᣣᦐᣣߢߩ૞ᬺ߇⊒↢ߔࠆ‫ޕ‬OECD TG404 ߦ␜ߐࠇࠆ࠼࡟ࠗ࠭⹜㛎ߪ‫ޔ‬3 ඘ߩ࠙ࠨࠡࠍ↪޿ߡ 4 ᤨ㑆㐽Ⴇ⾍ઃࠍⴕ޿‫ޔ‬㒰෰ᓟ‫ޔ‬1,24,48,72 ᤨ㑆ᓟߩ෻ᔕࠍ࠼࡟ࠗ࠭⹏ଔၮḰߢ⹏ଔߔࠆᣇᴺߢ޽ࠆ‫ޕ‬GHS ಽ㘃ߦ߅޿ߡߪ‫ޔ‬඙ಽ 2㧔ೝỗᕈ㧦ዋߥߊߣ߽ 2 ୘૕ߢ 2.3 એ਄㧕‫ޔ‬඙ಽ 1㧔ᒙೝỗᕈ㧦ዋߥߊߣ߽ 2
୘૕ߢ 1.7 એ਄ 2.3 ᧂḩ㧕‫ޔ‬඙ಽᄖ㧔㕖ೝỗᕈ㧦1.7 ᧂḩ㧕ߣߥߞߡ޿ࠆ‫ޔߜ߁ߩߎޕ‬TG439 ߢߪ඙ಽ 3 ࠍಽ㘃ߔࠆߎߣߪߢ߈ߥ޿ߣߐࠇߡ޿ࠆ‫ޕ‬ㆡ↪㒢⇇ߣߒߡ‫ޔ‬MTT ᐓᷤ૞↪ߩ޽ࠆ‛⾰ߣ⌕⦡ᕈ‛⾰߇᜼ߍࠄࠇࠆ‫ޔߪߦ࡞ࠦ࠻ࡠࡊޕ‬ߎࠇࠄߩ‛⾰ߩᬌ಴ᣇᴺߦߟ޿ߡߩ⸥タ߇޽ࠅ‫ߩߘޔ‬ᓇ㗀߇᣿ࠄ߆ߥ‛⾰ߦߟ޿ߡߪ‫ߩߘޔ‬⒟ᐲࠍቯ㊂ߔࠆᔅⷐ߇޽ࠆߣ⠨߃ࠄࠇࠆ‫ޔߪߡ޿ߟߦࠬࠟ߿࡞࠱ࡠࠕࠛޔߚ߹ޕ‬ㆡ↪ᄖߢ޽ࠆߣߐࠇߡ޿ࠆ‫ޕ‬18

12ߘߩઁߩ⹜㛎ᣇᴺߩ⑼ቇ⊛ߥႎ๔
TG439 ߦឝタߐࠇߡ޿ࠆၭ㙃⴫⊹ࡕ࠺࡞ EpiSkin‫ޔ‬EpiDerm‫ޔ‬SkinEthics ࠍ↪޿ߚ⊹⤏৻ᰴೝỗᕈ⹜㛎ߦ㑐ߔࠆ⺰ᢥႎ๔ߪᄙ޿‫⹜ߩࠄࠇߎޕ‬㛎ᴺߪ JaCVAM ⊹⤏ೝỗᕈ⹏ଔᆔຬળߦ߅
޿ߡ߽‫ޔ‬࿖ౝߩኾ㐷ኅߦࠃࠅ⹏ଔߐࠇ‫ޔ‬OECD TG ౝኈ߇⹺⸽ߐࠇߡ޿ࠆ 11㧕‫ޕ‬㧚⚿⺰
3 ᰴరၭ㙃⴫⊹ࡕ࠺࡞ LabCyte EPI-MODEL24 ࠍ↪޿ߚ⊹⤏ೝỗᕈ⹜㛎ᴺߪ‫ޔ‬GHS ಽ㘃ߦࠃࠆ⊹⤏ೝỗᕈߩ⼂೎ࠍߣ੍᷹ߔࠆᣇᴺߣߒߡ‫ޔ‬TG439 ᕈ⢻ᮡḰߦᓥ޿ࡃ࡝࠺࡯࡚ࠪࡦ߇ታᣉ
ߐࠇߡ߅ࠅ‫⚿ߩߘޔ‬ᨐ‫ޔ‬ᱜ⏕ᕈ‫ޔ‬ା㗬ᕈߦߟ޿ߡ߽ᅷᒰߢ޽ߞߚ‫⹜ᧄޔߡߞ߇ߚߒޕ‬㛎ᴺߪ
GHS ಽ㘃ߦࠃࠆ⊹⤏ೝỗᕈࠍ੍᷹ߔࠆ in vitro ⹜㛎ߣߒߡ᦭↪ߥᣇᴺߢ޽ࠆߣ್ᢿߒߚ‫ޕ‬

ෳ⠨ᢥ₂
1.

EC-ECVAM (2007), Statement on the validity of in vitro tests for skin irritation, issued by the
ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007. Available at:[http://ecvam.jrc.ec.europa.eu]

2.

EC-ECVAM (2007), Performance Standards for applying human skin models to in vitro skinirritation testing. Available at: [http://ecvam.jrc.ec.europa.eu]

3.

Kojima, H., Ando, Y., Idehara, K., Katoh, M., Kosaka, T., Miyaoka, E., Shinoda, S., Suzuki, T.,
Yamaguchi, Y., Yoshimura, I., Yuasa, A., Watanabe, Y. and Omori, T. (2012), Validation Study ofthe In Vitro Skin Irritation Test with the LabCyte EPI-MODEL24, Altern Lab Anim, 40, 33-50.

4. OECD (2011), Validation report for the skin irritation test method using LabCyte EPI-MODEL24,
OECD Series on Testing and Assessment No. 159, OECD, Paris. Available at:[http://www.oecd.org/env/testguidelines]
5.

OECD (2011), Peer review report of validation of the skin irritation test using LabCyte
EPI-MODEL24, OECD Series on Testing and Assessment No. 155, OECD, Paris. Available at:[http://www.oecd.org/env/testguidelines]

6.

Katoh, M. and Hata K. (2011), Refinement of LabCyte EPI-MODEL24 skin Irritation test method
for adaptation to the requirements of OECD test guideline 439, AATEX, 16, 111-122

7.

OECD (2012), In vitro skin irritation: human epidermis test method, Draft updated guideline
439 for the testing of chemicals, Paris. Available at: [http://www.oecd.org/env/testguidelines]

8.

LabCyte EPI-MODEL24 SIT SOP, Version 8.3 (June 2011), Skin irritation test using thereconstructed human model “LabCyte EPI-MODEL24” Available at: [http://jacvam.jp/]

9.

EC-ECVAM (2009), Performance Standards for in vitro skin irritation test methods based on
Reconstructed human Epidermis (RhE). Available at:[http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing] N.B. This is the current version of
the ECVAM PS, updated in 2009 in view of the implementation of UN GHS.

10. OECD (2010), In vitro skin irritation: reconstructed human epidermis test method, OECD
Guideline for the testing of chemicals No.439, Paris. Available at:[http://www.oecd.org/env/testguidelines]
11. JaCVAM: Available at: http://jacvam.jp/ (2012)

13

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Unclassified

ENV/JM/MONO(2011)39

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

08-Sep-2011___________________________________________________________________________________________English - Or. English

ENVIRONMENT DIRECTORATEENV/JM/MONO(2011)39
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYVALIDATION REPORT FOR THE SKIN IRRITATION TEST METHODUSING LABCYTE EPI-MODEL24

English - Or. English

JT03306650
Document complet disponible sur OLIS dans son format d'origine
Complete document available on OLIS in its original format

21

ENV/JM/MONO(2011)39

2

22

ENV/JM/MONO(2011)39

OECD Environment, Health and Safety Publications
Series on Testing and Assessment
No. 159

VALIDATION REPORT FOR THE SKIN IRRITATION TEST METHOD USINGLABCYTE EPI-MODEL24

Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT
Paris 2011
323

ENV/JM/MONO(2011)39
Also published in the Series on Testing and Assessment:
No. 1,
Guidance Document for the Development of OECD
Guidelines for Testing of Chemicals (1993; reformatted 1995, revised
2006)
No. 2,(1995)

Detailed Review Paper on Biodegradability Testing

No. 3,(1995)

Guidance Document for Aquatic Effects Assessment

No. 4,
Report of the OECD Workshop on Environmental
Hazard/Risk Assessment (1995)
No. 5,
Report of the SETAC/OECD Workshop on Avian Toxicity
Testing (1996)
No. 6,
Report of the Final Ring-test of the Daphnia magna
Reproduction Test (1997)
No. 7,
Guidance Document on Direct Phototransformation ofChemicals in Water (1997)
No. 8,
Report of the OECD Workshop on Sharing Information
about New Industrial Chemicals Assessment (1997)
No. 9,
Guidance Document for the Conduct of Studies ofOccupational Exposure to Pesticides during Agricultural Application(1997)
No. 10,
Report of the OECD Workshop on Statistical Analysis of
Aquatic Toxicity Data (1998)
No. 11,
Detailed Review Paper on Aquatic Testing Methods for
Pesticides and industrial Chemicals (1998)
No. 12,
Detailed Review Document on Classification Systems forGerm Cell Mutagenicity in OECD Member Countries (1998)
No. 13,
Detailed Review Document on Classification Systems for
Sensitising Substances in OECD Member Countries 1998)
No. 14,
Detailed Review Document on Classification Systems for
Eye Irritation/Corrosion in OECD Member Countries (1998)
No. 15,
Detailed Review Document on Classification Systems for
Reproductive Toxicity in OECD Member Countries (1998)
No. 16,
Detailed Review Document on Classification Systems for
Skin Irritation/Corrosion in OECD Member Countries (1998)
4

24

ENV/JM/MONO(2011)39
No. 17,
Environmental Exposure Assessment Strategies for
Existing Industrial Chemicals in OECD Member Countries (1999)
No. 18,
Report of the OECD Workshop on Improving the Use of
Monitoring Data in the Exposure Assessment of Industrial Chemicals(2000)
No. 19,
Guidance Document on the Recognition, Assessment andUse of Clinical Signs as Humane Endpoints for Experimental Animals
used in Safety Evaluation (1999)
No. 20,
Revised Draft Guidance Document for Neurotoxicity
Testing (2004)
No. 21,
Detailed Review Paper: Appraisal of Test Methods for
Sex Hormone Disrupting Chemicals (2000)
No. 22,
Guidance Document for the Performance of Out-door
Monolith Lysimeter Studies (2000)
No. 23,
Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures (2000)
No. 24,(2001)

Guidance Document on Acute Oral Toxicity TestingNo. 25,
Detailed Review Document on Hazard Classification
Systems for Specifics Target Organ Systemic Toxicity Repeated
Exposure in OECD Member Countries (2001)
No. 26,
Revised Analysis of Responses Received from Member
Countries to the Questionnaire on Regulatory Acute Toxicity Data
Needs (2001)
No 27,
Guidance Document on the Use of the Harmonised
System for the Classification of Chemicals which are Hazardous for the
Aquatic Environment (2001)
No 28,
Guidance Document for the Conduct of Skin Absorption
Studies (2004)
No 29,
Guidance Document on Transformation/Dissolution ofMetals and Metal Compounds in Aqueous Media (2001)
No 30,
Detailed Review Document on Hazard Classification
Systems for Mixtures (2001)
No 31,
Detailed Review Paper on Non-Genotoxic Carcinogens Detection: The
Performance of In-Vitro Cell Transformation Assays (2007)

525

ENV/JM/MONO(2011)39
No. 32,
Guidance Notes for Analysis and Evaluation of RepeatDose Toxicity Studies (2000)
No. 33,Harmonised Integrated Classification System for Human
Health and Environmental Hazards of Chemical Substances and
Mixtures (2001)
No. 34,
Guidance Document on the Development, Validation andRegulatory Acceptance of New and Updated Internationally Acceptable
Test Methods in Hazard Assessment (2005)
No. 35,
Guidance notes for analysis and evaluation of chronic
toxicity and carcinogenicity studies (2002)
No. 36,
Report of the OECD/UNEP Workshop on the use ofMultimedia Models for estimating overall Environmental Persistence
and long range Transport in the context of PBTS/POPS Assessment(2002)
No. 37,
Detailed Review Document on Classification Systems for
Substances Which Pose an Aspiration Hazard (2002)
No. 38,
Detailed Background Review of the Uterotrophic Assay
Summary of the Available Literature in Support of the Project of theOECD Task Force on Endocrine Disrupters Testing and Assessment(EDTA) to Standardise and Validate the Uterotrophic Assay (2003)
No. 39,
Guidance Document on Acute Inhalation Toxicity Testing(in preparation)
No. 40,Detailed Review Document on Classification in OECD
Member Countries of Substances and Mixtures Which Cause
Respiratory Tract Irritation and Corrosion (2003)
No. 41,
Detailed Review Document on Classification in OECD
Member Countries of Substances and Mixtures which in Contact with
Water Release Toxic Gases (2003)
No. 42,
Guidance Document on Reporting Summary Information
on Environmental, Occupational and Consumer Exposure (2003)
No. 43,
Guidance Document on Mammalian Reproductive
Toxicity Testing and Assessment (2008)
No. 44,
Description of Selected Key Generic Terms Used in
Chemical Hazard/Risk Assessment (2003)
No. 45,
Guidance Document on the Use of Multimedia Models for
Estimating Overall Environmental Persistence and Long-range
Transport (2004)
626

ENV/JM/MONO(2011)39
No. 46,
Detailed Review Paper on Amphibian Metamorphosis
Assay for the Detection of Thyroid Active Substances (2004)
No. 47,
Detailed Review Paper on Fish Screening Assays for the
Detection of Endocrine Active Substances (2004)
No. 48,New Chemical Assessment Comparisons and Implications
for Work Sharing (2004)
No. 49,
Report from the Expert Group on (Quantitative)
Structure-Activity Relationships [(Q)SARs] on the Principles for the
Validation of (Q)SARs (2004)
No. 50,(2005)

Report of the OECD/IPCS Workshop on Toxicogenomics

No. 51,
Approaches to Exposure Assessment in OECD Member
Countries: Report from the Policy Dialogue on Exposure Assessment inJune 2005 (2006)
No. 52,Comparison of emission estimation methods used in
Pollutant Release and Transfer Registers (PRTRs) and Emission
Scenario Documents (ESDs): Case study of pulp and paper and textile
sectors (2006)
No. 53,
Guidance Document on Simulated Freshwater Lentic
Field Tests (Outdoor Microcosms and Mesocosms) (2006)
No. 54,Current Approaches in the Statistical Analysis of
Ecotoxicity Data: A Guidance to Application (2006)
No. 55,
Detailed Review Paper on Aquatic Arthropods in Life Cycle
Toxicity Tests with an Emphasis on Developmental, Reproductive andEndocrine Disruptive Effects (2006)
No. 56,
Guidance Document on the Breakdown of Organic Matterin Litter Bags (2006)
No. 57,
Detailed Review Paper on Thyroid Hormone Disruption
Assays (2006)
No. 58,
Report on the Regulatory Uses and Applications in OECD
Member Countries of (Quantitative) Structure-Activity Relationship[(Q)SAR] Models in the Assessment of New and Existing Chemicals(2006)
No. 59,
Report of the Validation of the Updated Test Guideline
407: Repeat Dose 28-Day Oral Toxicity Study in Laboratory Rats
(2006)

7

27

ENV/JM/MONO(2011)39
No. 60,
Report of the Initial Work Towards the Validation of the
21-Day Fish Screening Assay for the Detection of Endocrine Active
Substances (Phase 1A) (2006)
No. 61,
Report of the Validation of the 21-Day Fish Screening
Assay for the Detection of Endocrine Active Substances (Phase 1B)(2006)
No. 62,
Final OECD Report of the Initial Work Towards the
Validation of the Rat Hershberger Assay: Phase-1, Androgenic
Response to Testosterone Propionate, and Anti-Androgenic Effects of
Flutamide (2006)
No. 63,

Guidance Document on the Definition of Residue (2006)

No. 64,
Guidance Document on Overview of Residue Chemistry
Studies (2006)
No. 65,
OECD Report of the Initial Work Towards the Validationof the Rodent Uterotrophic Assay - Phase 1 (2006)
No. 66,
OECD Report of the Validation of the Rodent
Uterotrophic Bioassay: Phase 2. Testing of Potent and Weak
Oestrogen Agonists by Multiple Laboratories (2006)
No. 67,
Additional data supporting the Test Guideline on the
Uterotrophic Bioassay in rodents (2007)
No. 68,
Summary Report of the Uterotrophic Bioassay Peer
Review Panel, including Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the follow
up of this report (2006)
No. 69,
Guidance Document on the Validation of (Quantitative)
Structure-Activity Relationship [(Q)SAR] Models (2007)
No. 70,
Report on the Preparation of GHS Implementation by theOECD Countries (2007)
No. 71,
Guidance Document on the Uterotrophic Bioassay Procedure to Test for Antioestrogenicity (2007)
No. 72,
Guidance Document on Pesticide Residue Analytical
Methods (2007)
No. 73,
Report of the Validation of the Rat Hershberger Assay:
Phase 3: Coded Testing of Androgen Agonists, Androgen Antagonists
and Negative Reference Chemicals by Multiple Laboratories. SurgicalCastrate Model Protocol (2007)

828

ENV/JM/MONO(2011)39
No. 74,
Detailed Review Paper for Avian Two-generation Toxicity
Testing (2007)
No. 75,
Guidance Document on the Honey Bee (Apis Mellifera L.)
Brood test Under Semi-field Conditions (2007)
No. 76,
Final Report of the Validation of the Amphibian
Metamorphosis Assay for the Detection of Thyroid Active Substances:
Phase 1 - Optimisation of the Test Protocol (2007)
No. 77,
Final Report of the Validation of the Amphibian
Metamorphosis Assay: Phase 2 - Multi-chemical Interlaboratory Study(2007)
No. 78,
Final Report of the Validation of the 21-day Fish
Screening Assay for the Detection of Endocrine Active Substances.
Phase 2: Testing Negative Substances (2007)
No. 79,Validation Report of the Full Life-cycle Test with the
Harpacticoid Copepods Nitocra Spinipes and Amphiascus Tenuiremis
and the Calanoid Copepod Acartia Tonsa - Phase 1 (2007)
No. 80,

Guidance on Grouping of Chemicals (2007)

No. 81,
Summary Report of the Validation Peer Review for theUpdated Test Guideline 407, and Agreement of the Working Group of
National Coordinators of the Test Guidelines Programme on thefollow-up of this report (2007)
No. 82,(2007)

Guidance Document on Amphibian Thyroid Histology

No. 83,
Summary Report of the Peer Review Panel on the Stably
Transfected Transcriptional Activation Assay for Detecting Estrogenic
Activity of Chemicals, and Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the
Follow-up of this Report (2007)
No. 84,
Report on the Workshop on the Application of the GHS
Classification Criteria to HPV Chemicals, 5-6 July Bern Switzerland(2007)
No. 85,
Report of the Validation Peer Review for the Hershberger
Bioassay, and Agreement of the Working Group of the National
Coordinators of the Test Guidelines Programme on the Follow-up of
this Report (2007)
No. 86,
Report of the OECD Validation of the Rodent
Hershberger Bioassay:
Phase 2: Testing of Androgen Agonists,
Androgen Antagonists and a 5 Į-Reductase Inhibitor in Dose Response
Studies by Multiple Laboratories (2008)
929

ENV/JM/MONO(2011)39
No. 87,
Report of the Ring Test and Statistical Analysis of
Performance of the Guidance on Transformation/Dissolution of Metals
and Metal Compounds in Aqueous Media (Transformation/ Dissolution
Protocol) (2008)
No. 88,Workshop on Integrated Approaches to Testing andAssessment (2008)
No. 89,Retrospective Performance Assessment of the Test
Guideline 426 on Developmental Neurotoxicity (2008)
No.90,
Background Review Document on the Rodent
Hershberger Bioassay (2008)
No. 91,
Report of the Validation of the Amphibian Metamorphosis
Assay (Phase 3) (2008)
No. 92,
Report of the Validation Peer Review for the Amphibian
Metamorphosis Assay and Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the
Follow-Up of this Report (2008)
No. 93,
Report of the Validation of an Enhancement of OECD TG
211: Daphnia Magna Reproduction Test (2008)
No. 94,
Report of the Validation Peer Review for the 21-Day Fish
Endocrine Screening Assay and Agreement of the Working Group of
the National Coordinators of the Test Guidelines Programme on the
Follow-up of this Report (2008)
No. 95,

Detailed Review Paper on Fish Life-Cycle Tests (2008)

No.96,
Guidance Document on Magnitude of Pesticide Residues
in Processed Commodities (2008)
No.97,
Detailed Review Paper on the use of Metabolising
Systems for In Vitro Testing of Endocrine Disruptors (2008)
No. 98,
Considerations Regarding Applicability of the Guidanceon Transformation/Dissolution of Metals Compounds in Aqueous
Media (Transformation/Dissolution Protocol) (2008)
No. 99,Comparison between OECD Test Guidelines and ISO
Standards in the Areas of Ecotoxicology and Health Effects (2008)
No. 100,(2009)

Report of the Second Survey on Available Omics Tools

No. 101, Report of the Workshop on Structural Alerts for the OECD
(Q)SAR Application Toolbox, 15-16 May 2008, Utrecht, theNetherlands (2009)1030

ENV/JM/MONO(2011)39
No. 102, Guidance Document for using the OECD (Q)SAR
Application Toolbox to Develop Chemical Categories According to theOECD Guidance on Grouping of Chemicals (2009)
No. 103, Detailed Review Paper on Transgenic Rodent Mutation
Assays (2009)
No. 104, Performance Assessment: Comparsion of 403 and CxTProtocols via Simulation and for Selected Real Data Sets (2009)
No. 105, Report on Biostatistical Performance Assessment of thedraft TG 436 Acute Toxic Class Testing Method for Acute Inhalation
Toxicity (2009)
No. 106, Guidance Document for Histologic Evaluation of Endocrine
and Reproductive Test in Rodents (2009)
No. 107, Preservative treated wood to the environment for wood heldin storage after treatment and for wooden commodities that are notcover and are not in contact with ground. (2009)
No. 108, Report of the validation of the Hershberger Bioassay(weanling model) (2009)
No. 109, Literature review on the 21-Day Fish Assay and the Fish
Short-Term Reproduction Assay (2009)
No. 110, Report of the validation peer review for the weanling
Hershberger Bioassay and agreement of the working of national
coordinators of the test guidelines programme on the follow-up of thisreport (2009)
No. 111, Report of the Expert Consultation to Evaluate an Estrogen
Receptor Binding Affinity Model for Hazard Identification (2009)
No. 112, The 2007 OECD List of High Production Volume Chemicals(2009)
No. 113, Report of The Focus Session on Current and Forthcoming
Approaches for Chemical Safety and Animal Welfare (2010)
No. 114, Performance Assessment of Different Cytotoxic andCytostatic Measures for the In Vitro Micronucleus Test (MNVIT):
Summary of results in the collaborative trial (2010)
No. 115, Guidance Document on the Weanling Hershberger Bioassay
in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties(2009)

1131

ENV/JM/MONO(2011)39
No. 116, Guidance Document on the Design and Conduct of Chronic
Toxicity and Carcinogenicity Studies, Supporting TG 451, 452 and 453(2010)
No. 117, Guidance Document 117 on the Current Implementation ofInternal Triggers in Test Guideline 443 for an Extended One
Generation Reproductive Toxicity Study, in the United States andCanada (2011)
No. 118, Workshop Report on OECD Countries Activities Regarding
Testing, Assessment and Management of Endocrine Disrupters Part I
and Part II (2010)
No. 119, Classification and Labelling of chemicals according to the
UN Globally Harmonized System: Outcome of the Analysis ofClassification of Selected Chemicals listed in Annex III of theRotterdam Convention (2010)
No. 120, Part 1: Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism-based
Structural Alerts for the Identification of DNA Binding Chemicals
No. 120, Part 2: Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism-based
Structural Alerts for the Identification of DNA Binding Chemicals
No. 121, Detailed review paper (DRP) on Molluscs life-cycle Toxicity
Testing (2010)
No. 122, Guidance Document on the determination of the Toxicity ofa Test Chemical to the Dung Beetle Aphodius Constans (2010)
No. 123, Guidance Document on the Diagnosis of Endocrine-related
Histopathology in Fish Gonads (2010)
No. 124,(2010)

Guidance for the Derivation of an Acute Reference DoseNo. 125, Guidance Document on Histopathology for Inhalation
Toxicity Studies, Supporting TG 412 (Subacute Inhalation Toxicity: 28Day) and TG 413 (Subchronic Inhalation Toxicity: 90-Day) (2010)
No. 126, Short Guidance on the Threshold approach for Acute Fish
Toxicity (2010)
No. 127, Peer review report of the validation of the 21-day
androgenised female stickleback screening assay (2010)
No. 128, Validation Report of the 21-day Androgenised Female
Stickleback Screening Assay (2010)12

32

ENV/JM/MONO(2011)39
No. 129, Guidance Document on using Cytotoxicity Tests toEstimate Starting Doses for Acute Oral Systemic Toxicity Tests(2010)
No. 131, Report of the Test Method Validation of Avian Acute Oral
Toxicity Test (OECD test guideline 223) (2010)
No. 132, Report of the Multi-Laboratory Validation of the H295R
Steroidogenesis Assay to Identify Modulators (2010)
No.133, Peer Review Report for the H295R Cell-Based Assay for
Steroidogenesis (2010)
No.134, Report of the Validation of a Soil Bioaccumulation Test with
Terrestrial Oligochaetes by an International ring test (2010)
No.135, Detailed Review Paper on Environmental Endocrine
Disruptor Screening: The use of Estrogen and Androgen Receptor
Binding and Transactivation Assays in Fish (2010)
No. 136, Validation Report of t he Chironomid Full Life-Cycle
Toxicity Test (2010)
No. 137, Explanatory Background Document to the OECD Test
Guideline On In Vitro Skin Irritation Testing (2010)
No. 138, Report of the Workshop on Using Mechanistic Informationin Forming Chemical Categories (2011)
No. 139, Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism Based
Structural Alerts for the Identification of Protein-binding Chemicals(2011)
No. 141, Report of the Phase 1 of the Validation of the Fish Sexual
Development Test for the Detection of Endocrine Active Substances(2011)
No. 142, Report of the Phase 2 of the Validation of the Fish Sexual
Development Test for the Detection of Endocrine Active Substances(2011)
No. 143, Peer Review Report for the Validation of the Fish Sexual
Development Test and Agreement of the Working Group of National
Co-ordinators of the Test Guideline Programme on the Follow-up of
the Peer Review (2011)
No. 144,

Validation Report for the Acute Chironomid Assay (2011)13

33

ENV/JM/MONO(2011)39
No. 145, Transgenic Rodent Somatic and Germ Cell Gene Mutation Assay:
Retrospective Performance Assessment (2011)
No. 148, Guidance Document on the Androngenised Female
Stickleback Screen (2011)
No. 152, Case Study: Assessment of an Extended Chemical Category,
the Short-chain Methacrylates, Targeted on Bioaccumulation (2011)
No. 153, Guidance Document for the Derivation of an Acute
Reference Concentration (Arfc) (2011)
No. 156,

Guidance Notes on Dermal Absorption (2011)

No. 157, Validation Report Phase 1 for the Zebrafish Embryo Toxicity
Test (2011)
No. 158, Report of Progress on the Interlaboratory Validation of theOECD Harpacticoid Copepod Development and Reproduction Test
(2011)
No. 159, Validation Report for the Skin Irritation Test Method using
Labcyte Epi-Model24(2011)© OECD 2011
Applications for permission to reproduce or translate all or part of thismaterial should be made to: Head of Publications Service,
RIGHTS@oecd.org. OECD, 2 rue André-Pascal, 75775 Paris Cedex
16, France

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ENV/JM/MONO(2011)39

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
the OECD’s work is carried out by more than 200 specialised committees and working groups composedof member country delegates. Observers from several countries with special status at the OECD, and frominterested international organisations, attend many of the OECD’s workshops and other meetings.
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 ten different series:
Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides andBiocides; 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 (www.oecd.org/ehs/).

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) wasestablished in 1995 following recommendations made by the 1992 UN Conference on Environmentand Development to strengthen co-operation and increase international co-ordination in the field ofchemical safety. The Participating Organisations are FAO, ILO, UNEP, UNIDO, UNITAR, WHO,
World Bank and OECD. UNDP is an observer. The purpose of the IOMC is to promote coordination of the policies and activities pursued by the Participating Organisations, jointly or
separately, to achieve the sound management of chemicals in relation to human health and theenvironment.

15

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ENV/JM/MONO(2011)39

This publication is available electronically, at no charge.
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/ehs/)
or contact:
OECD Environment Directorate,
Environment, Health and Safety Division
2 rue André-Pascal
75775 Paris Cedex 16
FranceFax: (33-1) 44 30 61 80 E-mail: ehscont@oecd.org
E-mail: ehscont@oecd.org

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ENV/JM/MONO(2011)39

FOREWORD
This document presents the validation report for the “Skin Irritation Test Using the LabCyte EPIMODEL24”. The project for developing a Test Guideline for an in vitro epidermal model to assess skinirritation using the LabCyte EPI-MODEL24, led by Japan, was included in the work plan of the Test
Guidelines Programme in 2009. The Working of National Coordinators of the Test Guidelines Programme
endorsed this validation at its meeting held on 12-14 April 2011. The Joint Meeting of the Chemicals
Committee and Working Party on Chemicals, Pesticides and Biotechnology (Joint Meeting) agreed to its
declassification on 5 August 2011.
A validation peer review report, accompanied by a report on additional validation work, is also expected tobe published in the Series on Testing and Assessment.
This document is published under the responsibility of the Joint Meeting.

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ENV/JM/MONO(2011)39

MEMBERS OF LABCYTE VALIDATION MANAGEMENT TEAM
Mr. Hajime Kojima, JaCVAM: Japanese Centre for the Validation of Alternative Methods
Mr. Takashi Omori; Kyoto Univ.
Mr. Kenji Idehara; Daicel Chemical Co.
Mr. Isao Yoshimura; Tokyo University of Science
Mr.Etsuyoshi Miyaoka; Tokyo University of Science
Mr. Masakazu Kato; Japan Tissue Engineering Co. Ltd.
Mr. Toshiro Yokouchi; Japan Tissue Engineering Co. Ltd.

Participant laboratoriesx

Aiken Co., Ltd. (Ms Yoko Ando and Ms Yui Asako)x

KOBAYASHI Pharmaceutical Co., Ltd. (Mr. Yoshihiro Yamaguchi and Ms Maki Nakamura)x

The Institute of Environmental Toxicology (Mr Tadashi Kosaka and Mr Koichi hayashi)x

Fancl Corp. (Ms. Tamie Suzuki and Ms Runa Izumi)x

FUJIFILM Corporation (Ms. Atsuko Yuasa, and Mr Shinichi Akimoto)x

Maruishi Pharmaceutical Co., Ltd. (Mr Yukihiko Watanabe and Mr Osamu Mitani)x

Drug Safety Testing Center Co., Ltd. (Mr Shinsuke Shinoda and Ms Saori Hagiwara)

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ENV/JM/MONO(2011)39
TABLE OF CONTENTS
1. Goal Statement21

2. Objective

21

3. Test Methods

21

3-1. Reconstructed human cultured dermal model

21

3-2. Model supplier21

4. Validation Management Structure224-1. Validation Management Team22

4-2. Chemical selection, acquisition, coding and distribution

22

4-3. Independent biostatisticians

22

4-4. Participating laboratories

23

4-5. Sponsorship24

5. Study Design24

6. Test Chemicals

246-1. Chemical selection

24

6-2. Deficit chemical

26
26

6-3. Chemical coding and distribution

26

7. Protocol
7-1. Protocol of the skin irritation test with LabCyte EPI-MODEL24
7-2. Prediction model of skin irritation

26
27

7-3. Difference between LabCyte EPI-MODEL 24 protocol and EPISKIN 27
protocol7-4. Data collection, handling, and analysis

28

7-5. Quality assurance, GLP

28

8. Results

29

8-1. 1st Phase

29

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ENV/JM/MONO(2011)39
8-1-1. Negative Control

29

8-1-2. Positive control and test chemicals
8-2. 2nd phase & 3rd phase

29
30
30

8-2-1. Comments on the Datasheets
8-2-2. Negative control

32

8-2-3. Positive control

33
34

8-2-4. Skin irritation test by cell viability
8-2-5. IL-1Į

43

8-2-6. Classification of three independent viabilities at each laboratory45

9. Discussion

47
47

9-1. Reliability
9-2. Predictivity
10. Conclusions

48
48

11. Acknowledgements

48

12. References

49

20
40

ENV/JM/MONO(2011)39
1. Goal statementx

The aim of this study was to validate in vitro skin irritation tests in a formal inter-laboratory study,
the ultimate goal of the test strategy will be to replace the regulatory Draize skin irritation test
according OECD TG 404 (OECD, 2002).

x

The primary goal of this validation study was an evaluation of the ability of the in vitro tests toreliably discriminate skin irritant (I) from non-irritant (NI) chemicals, as defined according to theOECD and United Nations proposal for Globally Harmonised System (GHS) for the classificationand labelling of skin irritation (category 1/category 2; no category; Anon., 2003) .

2. Objective
1.
The in vitro test system, employing reconstructed human epidermis model (RhE: LabCyte EPIMODEL24), has progressed through protocol optimisation as in vitro skin irritation test. The multilaboratory assessment of this system performed based on the a few ECVAM performance standards (ESAC
statement, 2007, 2008, 2009). This report shows the results of 3rd phase validation study in accordance withthe revised reference chemicals described by the new ESAC statement 2009.
2.
The present objective was to conduct a validation study to assess the reliability (reproducibility
within and between laboratories) and relevance (predictive capacity) of this test system with a challenging
set of coded 25 test chemicals for which high quality in vivo data were available. The validation study was
undertaken in accordance with the principles and criteria documented in the OECD Guidance Document on
the Validation and International Acceptance of New or Updated Test Methods for Hazard Assessment (No.
34, OECD, 2005) and according to the Modular Approach to validation (Hartung et al. 2004).
3. Test Method
3-1. Reconstructed human cultured epidermal model
3.
LabCyte EPI-MODEL24 is a new, commercially available RhE model produced by Japan Tissue
Engineering Co. Ltd. It consists of normal human epidermal keratinocytes whose biological origin isneonate foreskin. In order to expand human keratinocytes while maintaining their phenotype, they were
cultured with 3T3-J2 cells as a feeder layer (Rheinwald and Green, 1975; Green, 1978). Reconstruction ofhuman cultured epidermis is achieved by cultivating and proliferating keratinocytes on an inert filter
substrate (surface 0.3 cm2) at the air-liquid interface for 13 days with an optimized medium containing 5%
fetal bovine serum. It constructs a multilayer structure consisting of a fully differentiated epithelium withfeatures of the normal human epidermis, including a stratum corneum. LabCyte EPI-MODEL24 is embedded
in an agarose gel containing nutrient solution and shipped in 24-well plates at around 18°C (Kato, 2009).
3-2. MODEL SUPPLIER
4.
According to OECD GLP Consensus Document No.5 “Compliance of Laboratory Suppliers with
GLP Principles” the responsibility for the quality and fitness for use of equipment and materials rests
entirely with the management of the test facility (OECD, 1999).
5.
The acceptability of equipment and materials in laboratories complying with GLP-like principles
should therefore be guaranteed to any regulatory authority to which studies were submitted. In somecountries where GLP has been implemented, suppliers belong to national regulatory or voluntaryaccreditation schemes (for laboratory animals) which can provide users with additional documentary
evidence that they are using a test system of a defined quality.
6.
The audits focused on the procedures established to guarantee a defined quality of the tissue
models.

21
41

ENV/JM/MONO(2011)39
4. Validation Management structure
7.
This validation study was performed by the Japanese Society for the Alternative to Animal
Experiments (JSAAE).
The management structure of the study is shown in Figure 1.
4-1. Validation Management Group8.
The Validation Management Team (VMT), which plays a central role overseeing the conduct ofthe validation study, includes:
1) Goal statement
2) Project plan including objective3) Study protocol / amendments
4) Outcome of QC audits5) Test chemicals
6) Data management procedures
7) Timeline/ study progression8) Study interpretation and conclusions
9) Reports and publication
9.
The final decision on which laboratories participate in the validation study is the responsibility of
the VMT.
Members:
A chair (Hajime Kojima, JaCVAM: Japanese Centre for the Validation of Alternative Methods)
The sponsor representative: representatives of JSAAE (Takashi Omori; Kyoto Univ., Kenji Idehara;
Daicel Chemical Co. and Isao Yoshimura; Tokyo University of Science)
The sponsor representative, LabCyte EPI-MODEL24suppliers and lead lab (Masakazu Kato : Japan
Tissue Engineering Co., Ltd, J-TEC)
4-2. Chemical selection, acquisition, coding and distribution1) Definition of selection criteria
2) Chemical selection3) Liaise with suppliers
4) Final check of chemicals provided
5) Acquisition
6) Coding7) Distribution
MemberHajime Kojima, JaCVAM
4-3 . Independent biostatisticians
1) Approve spreadsheets
2) Collect data
3) Analyse data
Members:
Takashi Omori: Kyoto Univ., Etsuyoshi MIyaoka and Kenya Ishiyama: Tokyo University of Science2242ENV/JM/MONO(2011)39

Validation
TeamTeam
ValidationManagement
Management

Chair: JaCVAM
JaCVAM
Chair:
Biostatistics

Study Sponser
JSAAE
JSAAE

Kyoto Univ.
Kyoto Univ.
Tokyo Universityof Science
Tokyo
Universityof Science

Chemical coding
and distribution
and distribution
JaCVAM
JaCVAM
JaCVAM

Lead
laboratorylaboratory&&
Training
J-TEC
Training
J-TEC

Aiken
Aiken
Co.,
Co.
Ltd.

KOBAYASHI
PharmaceuPharmaceut
tical
Co.,
ical
Ltd.
Co.

The Institute ofEnvironmental
Environmental
Toxicology
Toxicology

Fancl㩷Corp.
Corp.

FUJIFILM
Corporation
Corporation

MaruishiPharmaceutiPharmaceuticcal Co.,al Ltd.
Co.

Drug
Drug
SafetySafetyTesting
Testing
CenterCo.,
CenterCo., Ltd.
Ltd.

Figure 1. Management structure of the JSAAE skin irritation validation study
4-4. Participating laboratories
The laboratories participating in the study are to be defined as shown in Fig. 1.
The following 6 laboratories participated in the validation study for the evaluation of the LabCyte
EPI-MODEL24 assays:
x Laboratory 1 – Aiken Co., Ltd. (Yoko Ando and Yui Asako)x

Laboratory 2 – KOBAYASHI Pharmaceutical Co., Ltd. (Yoshihiro Yamaguchi and Maki
Nakamura)x

Laboratory 3 – The Institute of Environmental Toxicology (Tadashi Kosaka and Koichi
hayashi)x

Laboratory 4 – FanclG Corp. (Tamie Suzuki and Runa Izumi)xLaboratory 5 – FUJIFILM Corporation (Atsuko Yuasa, and Shinichi Akimoto)x

This laboratory was not participated at the 3rd Phase study.
Laboratory 6 – Maruishi Pharmaceutical Co., Ltd. (Yukihiko Watanabe and Osamu Mitani)x

Laboratory 7 – Drug Safety Testing Center Co., Ltd. (Shinsuke Shinoda and Saori Hagiwara)

A lead laboratory is also identified as J-TEC (Mr. Masakazu Kato and Mr Toshihiro Yokouchi). This
laboratory was not participated in the validation study.
Each laboratory also was responsible for complying with GLP-like principles and specifying QA aspects.

2343

ENV/JM/MONO(2011)39

4-5. Sponsorship
The study was managed and finance by JSAAE and J-TEC .
1) JSAAE finance:- the management of the study (VMT meetings)- the independent statistical support (biostatistician)- the responsible for the chemicals purchase, coding and distribution to the laboratories- the independent QC audit of the data
- the publication of the study2) J-TECG finance:- the lead laboratories for the test method- training for the participating laboratories- the independent QC audit on the LabCyte EPI-MODEL24- the financial assistance for the participated laboratories
5. Study design10.
Before this validation study, the training course using LabCyte EPI-MODEL24 was performed by
J-TEC on April, 2008. All technicians from each laboratory participated at this training course.
11.
Three phases of validation studies were performed. In the 1st phase, we confirmed thetransferability of the test protocol and assessed its reproducibility, by testing three coded chemicals (ethanol,
glycerol and napthalen acetic acid) and a positive control (5% sodium lauryl sulfate solution) in sevenlaboratories between June and July of 2008.
12.
In the 2nd phase study, we confirmed the intra- and inter-laboratory reproducibility robustness, andthe correlation of test using 19 new chemicals tested in reference to the original EPISKIN performancestandards (ECVAM, 2007) . These tests were conducted by 7 laboratories between September 2008 andJanuary of 2009.
13.
In the 3rd phase study, we confirmed the intra- and inter-laboratory reproducibility robustness, andthe correlation of test using 6 chemicals tested in reference to the new EPISKIN performance standards(ESAC statement, 2009). This study was conducted by 6 laboratories, which attend the 1st and 2nd phasevalidation study between April to May, 2009.
6. Test Chemical
6-1. Chemicals Selection and list
14.
In 1st phase study, JaCVAM selected three coded chemicals (ethanol, glycerol and napthalen aceticacid) to test.
15.
According to the original ESAC Performance Standard (ESAC statement,2007) in 2nd Phase, theVMT selected 19 new chemicals to test in Table 1. One chemical, tri-isobuthyl phosphate (No. 13) on thechemical list reference for the original ECVAM Performance Standard cannot be purchased on the Japanesemarket. The VMT is responsible for the final approval of the chemicals proposed by JaCVAM. To avoid any
potential bias in the final selection, the laboratory representatives on the VMT were not party to these
discussions, nor were they informed of the final list of test chemicals for either phase of the validation study.
24
44

ENV/JM/MONO(2011)39
16.
According to the new ECVAM performance standard (ESAC statement, 2009) in 3rd phase, theVMT selected 6 new chemicals tested in Table 2. The final approval of the chemicals proposed by JaCVAM
is the responsibility of the VMT. To avoid any potential for bias in the final selection, the laboratory
representatives on the VMT did not be party to these discussions, nor were they made aware of the chemicals
finally approved for testing in either phase of the validation study.
Table 1. Reference test chemicals and codes㪥㫆㪅㪚㪿㪼㫄㫀㪺㪸㫃

㪠㫅㩷㫍㫀㫍㫆
㪚㪘㪪㪞㪟㪪㩷㫃㪸㪹㪼㫃 㫊㪺㫆㫉㪼㫅㫌㫄㪹㪼㫉
㩿㪧㪠㪠㪀㪣㪸㪹㫆㫉㪸㫋㫆㫉㫐
㪸

㪹

㪺

㪻

㪼

㪽

㪾

㪇㪈

㪈㪄㪹㫉㫆㫄㫆㪄㪋㪄㪺㪿㫃㫆㫉㫆㪹㫌㫋㪸㫅㪼 㪍㪐㪋㪇㪄㪎㪏㪄㪐㫅㫆㪇㪘㪄㪇㪈 㪙㪄㪇㪐㪐 㪚㪄㪇㪎㪎 㪛㪄㪈㪈㪌 㪜㪄㪈㪊㪊 㪝㪄㪇㪊㪈 㪞㪄㪇㪋㪐㪇㪉㪻㫀㪼㫋㪿㫐㫃㩷㫇㪿㫋㪿㪸㫃㪸㫋㪼㪏㪋㪄㪍㪍㪄㪉

㫅㫆㪇㪘㪄㪇㪉 㪙㪄㪈㪇㪇 㪚㪄㪇㪎㪏 㪛㪄㪈㪈㪍 㪜㪄㪈㪊㪋 㪝㪄㪇㪊㪉 㪞㪄㪇㪌㪇㪇㪊㪻㫀㪄㫇㫉㫆㫇㫐㫃㪼㫅㪼㩷㪾㫃㫐㪺㫆㫃 㪉㪌㪉㪍㪌㪄㪎㪈㪄㪏㫅㫆㪇㪘㪄㪇㪊 㪙㪄㪇㪏㪈 㪚㪄㪇㪎㪐 㪛㪄㪈㪈㪎 㪜㪄㪈㪊㪌 㪝㪄㪇㪊㪊 㪞㪄㪇㪌㪈㪇㪋㫅㪸㫇㪿㫋㪸㫃㪼㫅㩷㪸㪺㪼㫋㫀㪺㩷㪸㪺㫀㪻㪏㪍㪄㪏㪎㪄㪊

㫅㫆㪇㪘㪄㪇㪋 㪙㪄㪇㪏㪉 㪚㪄㪇㪏㪇 㪛㪄㪈㪈㪏 㪜㪄㪈㪊㪍 㪝㪄㪇㪊㪋 㪞㪄㪇㪌㪉㪇㪌㪸㫃㫃㫐㫃㩷㫇㪿㪼㫅㫆㫏㫐㪄㪸㪺㪼㫋㪸㫋㪼 㪎㪋㪐㪊㪄㪎㪋㪄㪌

㫅㫆

㪇㪅㪊

㪘㪄㪇㪌 㪙㪄㪇㪏㪊 㪚㪄㪇㪍㪈 㪛㪄㪈㪈㪐 㪜㪄㪈㪊㪎 㪝㪄㪇㪊㪌 㪞㪄㪇㪌㪊㪇㪍㫀㫊㫆㫇㫉㫆㫇㪸㫅㫆㫃㪍㪎㪄㪍㪊㪄㪇

㫅㫆㪇㪅㪊㪘㪄㪇㪍 㪙㪄㪇㪏㪋 㪚㪄㪇㪍㪉 㪛㪄㪈㪉㪇 㪜㪄㪈㪊㪏 㪝㪄㪇㪊㪍 㪞㪄㪇㪌㪋㪇㪎㪋㪄㫄㪼㫋㪿㫐㫃㪄㫋㪿㫀㫆㪄㪹㪼㫅㫑㪸㫃㪻㪼㪿㫐㪻㪼㪊㪋㪋㪍㪄㪏㪐㪄㪎㫅㫆㪈㪘㪄㪇㪎 㪙㪄㪇㪏㪌 㪚㪄㪇㪍㪊 㪛㪄㪈㪇㪈 㪜㪄㪈㪊㪐 㪝㪄㪇㪊㪎 㪞㪄㪇㪌㪌㪇㪏㫄㪼㫋㪿㫐㫃㩷㫊㫋㪼㪸㫉㪸㫋㪼㪈㪈㪉㪄㪍㪈㪄㪏

㫅㫆㪈㪘㪄㪇㪏 㪙㪄㪇㪏㪍 㪚㪄㪇㪍㪋 㪛㪄㪈㪇㪉 㪜㪄㪈㪋㪇 㪝㪄㪇㪊㪏 㪞㪄㪇㪌㪍㪇㪐㪸㫃㫃㫐㫃㩷㪿㪼㫇㫋㪸㫅㫆㪸㫋㪼㪈㪋㪉㪄㪈㪐㪄㪏

㫅㫆㪈㪅㪎㪘㪄㪇㪐 㪙㪄㪇㪏㪎 㪚㪄㪇㪍㪌 㪛㪄㪈㪇㪊 㪜㪄㪈㪉㪈 㪝㪄㪇㪊㪐 㪞㪄㪇㪌㪎㪈㪇㪿㪼㫇㫋㫐㫃㩷㪹㫌㫋㫐㫉㪸㫋㪼 㪌㪏㪎㪇㪄㪐㪊㪄㪐

㫅㫆

㪈㪅㪎

㪘㪄㪈㪇 㪙㪄㪇㪏㪏 㪚㪄㪇㪍㪍 㪛㪄㪈㪇㪋 㪜㪄㪈㪉㪉 㪝㪄㪇㪋㪇 㪞㪄㪇㪌㪏㪈㪈㪿㪼㫏㫐㫃㩷㫊㪸㫃㫀㪺㫐㫃㪸㫋㪼 㪍㪉㪌㪐㪄㪎㪍㪄㪊㫅㫆㪉㪘㪄㪈㪈 㪙㪄㪇㪏㪐 㪚㪄㪇㪍㪎 㪛㪄㪈㪇㪌 㪜㪄㪈㪉㪊 㪝㪄㪇㪉㪈 㪞㪄㪇㪌㪐㪈㪉㫋㪼㫉㫇㫀㫅㫐㫃㩷㪸㪺㪼㫋㪸㫋㪼㫅㫆㪉㪘㪄㪈㪉 㪙㪄㪇㪐㪇 㪚㪄㪇㪍㪏 㪛㪄㪈㪇㪍 㪜㪄㪈㪉㪋 㪝㪄㪇㪉㪉 㪞㪄㪇㪍㪇㪈㪊㪌㩿㪮㪆㪭㩷㩼㪀㩷㪪㪣㪪㪈㪋㪈㪄㪻㪼㪺㪸㫅㫆㫃㪏㪇㪄㪉㪍㪄㪉

㪘㪄㪈㪊 㪙㪄㪇㪐㪈 㪚㪄㪇㪍㪐 㪛㪄㪈㪇㪎 㪜㪄㪈㪉㪌 㪝㪄㪇㪉㪊 㪞㪄㪇㪋㪈㪈㪈㪉㪄㪊㪇㪄㪈 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪊㪘㪄㪈㪋 㪙㪄㪇㪐㪉 㪚㪄㪇㪎㪇 㪛㪄㪈㪇㪏 㪜㪄㪈㪉㪍 㪝㪄㪇㪉㪋 㪞㪄㪇㪋㪉㪈㪌㪺㫐㪺㫃㪸㫄㪼㫅㩷㪸㫃㪻㪼㪿㫐㪻㪼 㪈㪇㪊㪄㪐㪌㪄㪎 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪊㪘㪄㪈㪌 㪙㪄㪇㪐㪊 㪚㪄㪇㪎㪈 㪛㪄㪈㪇㪐 㪜㪄㪈㪉㪎 㪝㪄㪇㪉㪌 㪞㪄㪇㪋㪊㪈㪍㪈㪄㪹㫉㫆㫄㫆㪿㪼㫏㪸㫅㪼㪈㪈㪈㪄㪉㪌㪄㪈 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪎㪘㪄㪈㪍 㪙㪄㪇㪐㪋 㪚㪄㪇㪎㪉 㪛㪄㪈㪈㪇 㪜㪄㪈㪉㪏 㪝㪄㪇㪉㪍 㪞㪄㪇㪋㪋㪈㪎㱍㪄㫋㪼㫉㫇㫀㫅㪼㫆㫃㪐㪏㪄㪌㪌㪄㪌 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪉㪅㪎㪘㪄㪈㪎 㪙㪄㪇㪐㪌 㪚㪄㪇㪎㪊 㪛㪄㪈㪈㪈 㪜㪄㪈㪉㪐 㪝㪄㪇㪉㪎 㪞㪄㪇㪋㪌㪈㪏㪻㫀㪄㫅㪄㫇㫉㫆㫇㫐㫃㩷㪻㫀㫊㫌㫃㫇㪿㫀㪻㪼 㪍㪉㪐㪄㪈㪐㪄㪍 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪊㪘㪄㪈㪏 㪙㪄㪇㪐㪍 㪚㪄㪇㪎㪋 㪛㪄㪈㪈㪉 㪜㪄㪈㪊㪇 㪝㪄㪇㪉㪏 㪞㪄㪇㪋㪍㪈㪐㪹㫌㫋㫐㫃㩷㫄㪼㫋㪿㪸㪺㫉㫐㫃㪸㫋㪼㪊㪘㪄㪈㪐 㪙㪄㪇㪐㪎 㪚㪄㪇㪎㪌 㪛㪄㪈㪈㪊 㪜㪄㪈㪊㪈 㪝㪄㪇㪉㪐 㪞㪄㪇㪋㪎㪈㪈㪈㪄㪎㪈㪄㪎 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉㪋㪉㪇㪿㪼㫇㫋㪸㫅㪸㫃
1) CAS No.: Chemical abstracts service registry number.
2) PII: Primary irritation index.

㪘㪄㪉㪇 㪙㪄㪇㪐㪏 㪚㪄㪇㪎㪍 㪛㪄㪈㪈㪋 㪜㪄㪈㪊㪉 㪝㪄㪇㪊㪇 㪞㪄㪇㪋㪏㪐㪎㪄㪏㪏㪄㪈 㪚㪸㫋㪼㪾㫆㫉㫐㩷㪉

25
45

ENV/JM/MONO(2011)39
Table 2.G Test chemicals and code.
No.

ChemicalCASnumberA

Cinnamaldehyde

104-55-2B

2-Chroromethyl-3,5dimethyl-4-methoxypyridineHCl322-76821

C

Potassium hydroxide (5%aq)

168-21815

D
E
F

Benzenethiol, 5-(1,1dimethyethyl)-2-methyl
1-Methyl-3-phenyl-1piperazine
1,1,1-Torichloroethane

7340-90-1
5271-27-2
200-02463

GHS
label
no
Category
2
Category
2
Category
2
Category
2
Category
2

In
vivo
Score
2
2.7
3
3.3
3.3
4

Laboratory
a
A151

b
B176

c
C196

d
D216

f
F236

g
G256

A154

B173

C192

D211

F233

G253

A156
A153
A152
A155

B175
B172
B171
B174

C194
C191
C195
C193

D213
D214
D215
D212

F232
F234
F235
F231

G251
G254
G255
G252

1)CAS No.: Chemical abstracts service registry number.

6-2. Deficit chemical
17.
In Table1, tri-isobuthyl phosphate (No. 13) could not be used in the examination because it was notavailable in Japan. Therefore, a 5% SLS solution was used instead of tri-isobuthyl phosphate. The dataobtained with the 5% SLS solution were not used for calculating the predictivity of the test.
6-3. Chemical Coding and distribution
18.
Independent coding and distribution of chemicals were contracted out by JaCVAM to anindependent laboratory. The (company’s name) is certified according to ISO 9001, EN 4500 and GLP, andhas proven experience of reliable services. The codes were provided by JaCVAM.

7. Protocol
7-1. Protocol of the skin irritation test with LabCyte EPI-MODEL19.
In 2nd phase study, we used the SOP (ver. 5.0) and we used the SOP (ver. 6.1) in 3rd phase study.
The revised points, which make the deletion measurement of IL-1Į, revise calculating formula of viability,classification used median of 3trails and how to treat of volatile substances were shown in change trackingof the SOP (ver. 6.1). The VMT made judgments that these revise points were minor and difference with the
SOP (ver.5.0) used by 2nd phase study and this version was little in the VMT meeting on July 17, 2009.
20.
LabCyte EPI-MODEL tissues were shipped from the supplier on Mondays and delivered torecipients on Tuesdays. Upon receipt, the tissues were aseptically removed from the transport agarose
medium, transferred into 24-well plates (BD Biosciences, CA, USA) with the assay medium (0.5 mL), andincubated overnight (37°C, 5% CO2 humidified atmosphere). On the following day, the tissues weretopically exposed to the test chemicals. Liquids (25 μL) were applied with a micropipette, and solids (25mg) were applied from microtubes and moistened with 25 μL sterile water. If necessary, the mixture wasgently spread over the surface of the epidermis with a microspatula. Viscous liquids were applied using acell-saver-type tip with a micropipette. Each test chemical was applied to three tissues. In addition, threetissues serving as negative controls were treated with 25 μL distilled water, and three tissues serving as
26
46

ENV/JM/MONO(2011)39
positive controls were exposed to 5% SLS. After a 15-minute exposure, each tissue was carefully washedwith PBS (Invitrogen, CA, USA) 10 times using a washing bottle to remove any remaining test chemicalfrom the surface. The blotted tissues were then transferred to new 24-well plates containing 1 mL of fresh
assay medium.
21.
The treated and control tissues were incubated for 42 hours (37°C, 5% CO2 humidified
atmosphere). When the 42-hour post-incubation period was complete, blotted tissues were transferred to new
24-well plates containing 0.5 mL of freshly prepared MTT medium (1 mg/mL; Dojindo Co., Kumamoto,Japan) for the MTT assay and conditioned medium was collected to determine the interleukin-1 alpha (IL1Į) levels. Tissues were incubated for three hours (37°C, 5% CO2 humidified atmosphere) and thentransferred to microtubes containing 0.3 mL isopropanol, which completely immersed the tissue. Formazan
extraction was performed at room temperature, and the tissues were allowed to stand overnight.
Subsequently, 200-μL extracts were transferred to a 96-well plate. The optical density was measured at 570
nm and 650 nm as a reference absorbance, with isopropanol as a blank.
22.
The tissue viability was calculated as a percentage relative to the viability of the negative controls.
The median of three values from identically treated tissues was used to classify a chemical according to theprediction model.
23.
The amount of IL-1Į released in the conditioned medium after 42 hours was determined using an
IL-1Į ELISA kit (Invitrogen, CA, USA), following the manufacturer’s detailed instructions.

7-2. Prediction model of skin irritation
24.
In this study, the prediction model of skin irritation potential with LabCyte EPI-MODEL was set torefer to the conditions for EPISKIN described in the ECVAM Performance Standards. This prediction model
is described in Table 3. In the event that the three independent results within an individual batch were notconsistent, the result that occurred twice was used.
Acceptance criteria
1) ODNC of the negative control is greater than 0.7.
2) The viability of the positive control is less than 40%.
Table 3. Positive Criteria.
Tissue Viability (primary)
Mean tissue viability  50%
Mean tissue viability > 50%
Mean tissue viability > 50%

IL-1Į ELISA (secondary)
Mean IL-1Į release  120 pg/tissue
Mean IL-1Į release < 120 pg/tissue

Classification
Irritant
Non-irritant

7-3. Difference between LabCyte EPI-MODEL 24 protocol and EPISKIN protocol
25.
The differences between the LabCyte EPI-MODEL 24 protocol and EPISKIN protocol aresummarized in Table 3. Although the amount of medium (Table 4(A)), amount of test chemicals (Table
4(B)), and threshold of IL-1Į content (Table 4(C)) for the LabCyte EPI-MODEL 24 protocol are different
from the EPISKIN protocol, their conditions meet the descriptions of the Performance Standards.

27
47

ENV/JM/MONO(2011)39
Table 4. Differences between the LabCyte EPI-MODEL 24 protocol and EPISKIN protocol.
(A) Amount of medium.
ReasonLabCyte EPIMODEL 24
SOP

EPISKIN
SOP

Pre-incubation

0.5 mL

2 mL

Post-incubation

1 mL

2 mL

MTT assay

0.5 mL

2 mL

LabCyte EPI-MODEL 24 cultures are performed in 24-wellculture plates. A medium volume of 0.5 mL to 1 mL isappropriate to add to the 24-well culture plate. A medium
volume of 1 mL is necessary for a 42-hour culture.

These conditions meet the descriptions of the Performance Standards.
(B) Amount of test chemicals.
Test chemical

LabCyte EPI-MODEL 24
SOP

EPISKIN
SOP

ReasonLiquid

25 μL(75 μL/cm2)

10 μL(25 μL/cm2)

The lowest amount of the test chemical thatspread uniformly was applied to the test model.

Solid

25 mg+25 μL DW(75 μL/cm2)

10 mg+10 μL DW(25 μL/cm2)

These conditions meet the descriptions of the Performance Standards.
(C) Amount of test chemicals.
LabCyte EPI-MODEL 24 SOPEPISKIN SOP

Performance Standards(EPISKIN)

IL-1Į content t 120 pg/tissue(IL-1Į content t 120 pg/mL)

IL-1Į content t 100 pg/tissue(IL-1Įcontent t 50 pg/mL)

IL-1Į content t 120 pg/tissue(IL-1Į t 60 pg/mL)

The threshold of IL-1Į released in LabCyte EPI-MODEL was set based on the conditions for EPISKIN
described in the Performance Standards.
7-4. Data collection, handling, and analysis
26.
The independent biostatisticians for the study collected and organised the data using specific datacollection software (Datasheet4.0:20080910.xls in 2nd phase study and Datasheet5.0:20090430.xls in 3rdphase study). They will work in close collaboration with the biostatisticians, (Takashi Omori, Etsuyoshi
Miyaoka, and Kenya Ishiyama). After decoding the data, they will perform statistical analyses. The data
management procedures and statistical tools applied will be approved by the VMT.
7-5. Quality assurance, GLP
LABORATORIES
27.
All participating laboratories worked in the spirit of OECD GLP-like principles.
QA aspects
28.

Takashi Omori, Kenya Ishiyama and Hajime Kojima assured the quality of all the data and records.
28

48

ENV/JM/MONO(2011)39

8. Results
8-1 1st Phase
8-1-1 Negative control29.
In 1st phase data, Table 5 shows the absorbance values for the negative control. All data for thenegative control met the acceptance criteria.
Table 5. Absorbance of negative control by 1st phase study.
䎃
䎃
Lab.
a
b
c
d
e
f
g

1
Value
1.073
0.93
0.96
0.987
0.84
1.049
1.147

Exp.
2
Value
0.928
1.245
0.869
0.928
0.884
0.934
1.159

3
Value
1.007
1.042
0.761
0.939
0.973
0.968
1.074

䎃
䎃
Mean
1.003
1.072
0.863
0.951
0.899
0.984
1.127

SD
0.073
0.16
0.1
0.031
0.068
0.059
0.046

8-1-2 Positive control and test chemicals
30.
Table 6 shows the testing chemicals did not show any great score when the scores on tests wererepeated in each laboratory. Furthermore, there was no significant inter-laboratory variation. Theseexperiments suggested the feasibility of the LabCyte EPI-MODEL24 through the experiment. Alllaboratories were judged to participate at the Phase II by the validation management team.

29
49

ENV/JM/MONO(2011)39
Table 6. Viability of the positive control and three coded chemicals by 1st phase study
䎃
1
2
3
䎃
Chem. Lab. Viability Viability Viability Mean
SD
PC
a
6.35
27.55
15.67
16.52 10.63
䎃
b
3.94
3.51
3.97
3.81
0.26
䎃
c
5.45
4.81
3.49
4.58
1
䎃
d
11.74
7.22
14.08
11.02
3.49
䎃
e
31.6
9.76
38.61
26.66 15.05
䎃
f
3.1
2.89
2.93
2.97
0.11
䎃
g
4.46
7.17
2.62
4.75
2.29
P01
a
62.67
39.12
46.61
49.46 12.03
Ethanol b
41.08
50.86
86.58
59.51 23.95
䎃
c
68.13
34.13
67.31
56.53
19.4
䎃
d
68.57
40.52
33.03
47.37 18.73
䎃
e
54.19
72.08
60.55
62.27
9.07
䎃
f
.
64.16
47.98
56.07 11.44
䎃
g
4.68
5.23
6.67
5.53
1.03
P02
a
103.63104.1798.48 102.09
3.14
Glycerol b
85.5
100.5867.97
84.68 16.32
䎃c101.2499.41
104.84 101.83
2.76
䎃
d
103.3
101.35
89.73
98.13
7.34
䎃
e
101.75
98.06
99.04
99.62
1.91
䎃
f
.
97.23
96
96.62
0.87
䎃
g
94
98.16
103.6
98.59
4.82
P03
a
109.13
90.73
97.78
99.22
9.28
naphtalen b
93.96
103.91103.96 100.61
5.76
acetic acid

䎃
䎃
䎃
䎃
䎃

c
d
e
f
g

103.66102.28107.11.
92.2

102.11
98.15
104.39101.34101.04

117.3
94.56
97.36
102.07105.52

107.6998.33
102.95
101.7
99.59

8.36
3.86
5.03
0.52
6.78

8-2. 2nd phase & 3rd phase
8-2-1. Comments at the Datasheet
31.
All tests were sufficient with acceptance criteria.
There were a few comments from eachlaboratory in Tables 7 -9. By an application of Potassium hydroxide (5%aq) (B175, D213 and F232), themodel's layers were desquamated. By an application of cinnamicaldehyde (D216 and G256), the cups werediscoloured and crystallized.

30
50

ENV/JM/MONO(2011)39
Comments on the datasheets (Viability) by 2nd phase
Date
Comments
Lot
Date
Comments
Lab䇭ID Exp.No.
LCE24-081013-B
LCE24-081013-B
2008/10/20
2008/10/20aMain-䋲
This
test
was
recorded as the Main-1.
Main-2This test
recorded
LEC24-081117-B
LEC24-081117-B
2008/11/1
2008/11/1 This
This test
test was recorded
recorded as the Main-2.
Main-䋳
aMain-3
LCE24-081117-B 2008/11/22
2008/11/22 ThisaThis test
test was recorded
recorded as the Main-3.
Main-䋴
Main-4 LCE24-081117-B
LCE24-081013-B
LCE24-081013-B
2008/10/20
2008/10/20b
Main-1
LCE24-081027-B 2008.11.04
2008.11.04b
Main-2 LCE24-081027-B
LCE24-081117-B 2008/11/25
b
Main-3 LCE24-081117-B
2008/11/25
LCE24-080929-B
LCE24-080929-B
2008.10.6
c1
2008.10.6
LCE24-081020-B
LCE24-081020-B 2008/10/27
c2
2008/10/27
LCE24-081027-B
LCE24-081027-B
2008.11.3
c3
2008.11.3
LCE24-081020-B
LCE24-081020-B 2008/10/27
d81021
2008/10/27
2008/11/4
LCE24-081027-B
d81028 LCE24-081027-B
2008/11/4
LCE24-081117-B
LCE24-081117-B 2008/11/25
d81118
2008/11/25
LCE24-081006-B 2008/10/14e
Main-1 LCE24-081006-B
2008/10/14e
Main-2 LCE24-081013-B
LCE24-081013-B 2008/10/20
2008/10/20eMain-3 LCE24-081020-B
LCE24-081020-B 2008/10/27
2008/10/27
LCE24-080929-B
2008/10/6
LAB-08VAL
ff
LCE24-080929-B
LAB-08VAL
2008/10/6
LCE24-081013-B
2008/10/20
ff
MaruishiMaruishi LCE24-081013-B 2008/10/20
LAB-08VAL
ff
LCE24-081103-B 2008/11/10
LAB-08VAL LCE24-081103-B
2008/11/10
By
By anan application
application ofof G49,G53,G55,
gg
Main-1
LCE24-080929-B 2008.10.06
2008.10.06
Main-1 LCE24-080929-B
themodel's cap thewasmodel'sdiscolored.
G49,G53,G55,
cap wasByanapplication
ofG49,G53,G55,
Byan
applicationof
LCE24-081013-B
gg
Main-2
2008.10.20 the model's cap was discolored.
Main-2 LCE24-081013-B 2008.10.20
G49,G53,G55, the model's cap wasBy
By anan application
application ofof G49,G53,G55,
LCE24-081027-B
2008.11.03
gg
Main-3Main-3 LCE24-081027-B 2008.11.03 the model's cap was discolored.
G49,G53,G55, the model's cap was

Table 7.

Table 8. Comments on the datasheets (ELISA) by 2nd phase
Lab_ID
Lot
Date
Comments
Lab_ID Exp.No.
Exp.No.
Lot
DateComments
LCE24-081013-B
2008/10/20
LCE24-081013-B
2008/10/20 Thisaa
MainMain-2
This test
test was
was recorded
recordedas
asthe
the Main-1.
䋲
LEC24-081117-B
LEC24-081117-B
2008/11/1
2008/11/1 Thisaa
MainMain-3
䋳This test
test was
was recorded
recordedas
asthe
the Main-2.
LCE24-081117-B
LCE24-081117-B
2008/11/22
2008/11/22 Thisaa
MainMain-4
This test
test was
was recorded
recordedas
asthe
the Main-3.
䋴
LCE24-081013-B
LCE24-081013-B 2008/12/12
2008/12/12
Main-1bb
Main-1
LCE24-081027-B 2008/12/12
LCE24-081027-B
Main-2
2008/12/12
bb
Main-2
LCE24-081117-B 2008.12.26
LCE24-081117-B
2008.12.26
Main-3bb
Main-3
LCE24-080929-B
LCE24-080929-B
2008/10/7
cc
11
2008/10/7
LCE24-081020-B
LCE24-081020-B
2008/10/30
cc22
2008/10/30
LCE24-081027-B 2008.11.3
LCE24-081027-B
2008.11.3
cc
33
LCE24-081020-B 2008/11/11
LCE24-081020-B
2008/11/11
81021
dd
81021
LCE24-081027-B
2008/11/26
81028
LCE24-081027-B 2008/11/26
dd
81028
LCE24-081117-B
81118
2009/1/7
LCE24-081117-B
2009/1/7
dd
81118
LCE24-081006-B
2008/12/2eMain-1
LCE24-081006-B
e
Main-1
2008/12/2
LCE24-081013-B
2008/12/2
Main-2
LCE24-081013-B
ee
Main-2
2008/12/2
LCE24-081020-B
e
Main-3
2008/12/19
LCE24-081020-B
e
Main-3
2008/12/19
LCE24-081013-B 2008/11/25
2008/11/25
Maruishi LCE24-081013-B
ff
Maruishi
LCE24-081013-B
2008/11/27
f
Maruishi
LCE24-081013-B
f
Maruishi2008/11/27
LCE24-081103-B
f
LAB-08VAL
2008/12/25
f
LAB-08VAL LCE24-081103-B 2008/12/25
LCE24-080929-B 2008.10.09
2008.10.09
Main-1
LCE24-080929-B
gg
Main-1
LCE24-081013-B
2008.10.22
gMain-2
LCE24-081013-B 2008.10.22
gMain-2
LCE24-081027-B
g
Main-32008.11.05
LCE24-081027-B 2008.11.05
gMain-331
51

ENV/JM/MONO(2011)39
Table 9. Comments on the datasheets (Viability) by 3rd phase study
Lab䇭ID

a
a
a
b
b
bb
cc
cc
cc
dd
d

d
d
f
ff
f
f
f
g
g
g
g
g
g

Exp.No.
Lot
Date
Date
Comments
Comments
LCE24-090420-A
LCE24-090420-A 2009/4/27
㪥㫆㪅㪈
2009/4/27
LEC24-090511-A
LEC24-090511-A 2009/5/18
㪥㫆㪅㪉
2009/5/18
LEC24-090518-A
LEC24-090518-A 2009/5/25
㪥㫆㪅㪊
2009/5/25
20090421㪄㪈 LCE24-090420-A
LCE24-090420-A 2009/4/27
2009/4/27 By
By an
an application
applicationof
ofB175,
B175,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮
20090421㪄㪉 LEC24-090511-A
LEC24-090511-A 2009/5/20
2009/5/20 By
By an
an application
applicationof
ofB175,
B175,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮
20090421㪄㪊
LEC24-090518-A 2009/5/25
20090421㪄㪊 LEC24-090518-A
2009/5/25 By
By an
an application
applicationof
ofB175,
B175,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮
LCE24-090420-A
11
LCE24-090420-A 2009/4/27
2009/4/27
LEC24-090511-A
22
LEC24-090511-A 2009/5/18
2009/5/18
LEC24-090518-A 2009/5/25
33
LEC24-090518-A 2009/5/25
By an
an application
application ofofD213,
D213,thethemodel'smodel'slayers
layerswere
weredesquamated䋮
desquamated䋮By
By
By
LEC24-090511-A 2009/5/18
90512
LEC24-090511-A 2009/5/18 an application of D216, white crystallizations in the cup were90512
an application of D216, white crystallizations in the cup were detected.
detected.
By an application of D213, the model's layers were desquamated䋮 By
By
an application of D213, the model's layers were desquamated䋮 By
LEC24-090518-A 2009/5/25
LEC24-090518-A
2009/5/25 an application of D216, white crystallizations in the cup were90519
an application of D216, white crystallizations in the cup were detected.
detected
By an application of D213, the model's layers were desquamated䋮 By
LEC24-090525-A
90526
2009/6/1 By
an applicationof D216,
whitein thecup werean applicationof D213,the crystallizations
model's layers were
desquamated䋮
By
LEC24-090525-A
2009/6/1
90526
detected
an application of D216, white crystallizations in the cup were detected.
LAB-09VAL LCE24-090420-A 2009/4/27 By an application of F232, the model's layers were desquamated䋮
LCE24-090420-A
LAB-09VAL
LAB-09VAL LEC24-090511-A 2009/4/27
2009/5/18 By an application of F232, the model's layers were desquamated䋮
LEC24-090511-A
LAB-09VAL
2009/5/18
LAB-09VAL LEC24-090518-A 2009/5/25 By an application of F232, the model's layers were desquamated䋮
LAB-09VAL LEC24-090518-A 2009/5/25 By an application of F232, the model's layers were desquamated䋮
LCE24-090420-A 2009/4/27 By an application of G256, the model's caps were discolored䋮㽲
LCE24-090420-A 2009/4/27 By an application of G256, the model's caps were discolored䋮㽲
LCE24-090427-A
2009/5/4 By an application of G256, the model's caps were discolored䋮㽳
LCE24-090427-A
2009/5/4 By
㽳
LEC24-090511-A 2009/5/18
By an
an application
applicationof
ofG256,
G256,thethemodel'smodel'scaps
capswere
werediscolored䋮
discolored䋮㽴
LEC24-090511-A 2009/5/18 By an application of G256, the model's caps were discolored䋮㽴

8-2-2. Negative control
32.
In Table 10 and Fig.2, absorbances of negative control are shown. All data of negative control
were sufficient with acceptance criteria excluding Lab a, test1. The mean OD of lab a, test 1 is 0.59 (0.61,
0.58, 0.57). We were not accepted at this result, and accepted the results of test 2-4 re-tested at Lab a.

32
52

ENV/JM/MONO(2011)39
Table 10㩷 Absorbance of negative control

2
3

OD
Fig.2 Distribution of Absorbance on negative control
8-2-3. Positive control
33.
Table 11 and Fig.3 show three independent viabilities and statistical analysis of positive control ateach laboratory. All data were sufficient with acceptance criteria of positive control.

33
53

ENV/JM/MONO(2011)39

Table 11. Viability of positive control

2
3

Fig.3 Distribution of viability on positive control
8-2-4. Skin irritation test by cell viability
34.
The results of the skin irritation test with LabCyte EPI-MODEL 24 when it was only evaluated cell
viabilities as indicator are shown in Table 12 in 2nd phase study and Table 14 in 3rd phase study. Summary㩷statistical analysis of viability each chemical are shown in Table 13 and Fig.4 in 2nd phase study and Table
15 and Fig.5 in 3rd phase study.
35.
Invalid data obtained only Lab a, run 1. This lab performed at retesting. Therefore, the data of lab
a were accepted among run 2-4.

34
54

ENV/JM/MONO(2011)39

Table 12. Viability of chemicals at each laboratory by 2nd phase study.
䎦䏋䏈䏐䎑 䎹䏌䏙䏒 䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎓䎔

䏑䏒

䎓

䎓䎕

䏑䏒

䎓

䎓䎖

䏑䏒

䎓

䎓䎗

䏑䏒

䎓

䎓䎘

䏑䏒

䎓䎑䎖

䎓䎙

䏑䏒

䎓䎑䎖

䎓䎚

䏑䏒

䎔

䎓䎛

䏑䏒

䎔

䎓䎜

䏑䏒

䎔䎑䎚

䎔䎓

䏑䏒

䎔䎑䎚

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎖䎔䎑䎓
䎔䎔䎑䎕
䎔䎔䎑䎙
䎚䎜䎑䎛
䎚䎙䎑䎘
䎙䎘䎑䎕
䎔䎓䎜䎑䎔
䎔䎓䎖䎑䎜
䎔䎓䎓䎑䎜
䎔䎓䎙䎑䎖
䎜䎘䎑䎕
䎜䎙䎑䎘
䎚䎛䎑䎘
䎚䎛䎑䎘
䎚䎗䎑䎔
䎜䎕䎑䎘
䎚䎜䎑䎗
䎛䎕䎑䎗
䎕䎗䎑䎔
䎔䎕䎑䎙
䎔䎚䎑䎛
䎔䎔䎔䎑䎜
䎜䎓䎑䎕
䎜䎘䎑䎖
䎔䎔䎕䎑䎛
䎜䎚䎑䎔
䎔䎓䎔䎑䎔
䎔䎔䎘䎑䎜
䎔䎓䎗䎑䎔
䎛䎙䎑䎘

䏅
䎗䎚䎑䎔
䎔䎓䎑䎗
䎔䎙䎑䎔
䎙䎙䎑䎜
䎙䎔䎑䎚
䎛䎛䎑䎚
䎜䎖䎑䎖
䎜䎜䎑䎛
䎔䎓䎕䎑䎖
䎜䎗䎑䎗
䎔䎓䎓䎑䎕
䎜䎛䎑䎙
䎙䎔䎑䎚
䎚䎔䎑䎜
䎛䎗䎑䎘
䎚䎚䎑䎜
䎛䎖䎑䎘
䎛䎓䎑䎘
䎔䎓䎑䎛
䎔䎕䎑䎙
䎔䎖䎑䎕
䎛䎙䎑䎚
䎔䎓䎓䎑䎙
䎔䎓䎗䎑䎛
䎜䎙䎑䎚
䎔䎔䎓䎑䎔
䎔䎓䎜䎑䎘
䎔䎔䎘䎑䎗
䎔䎔䎓䎑䎔
䎔䎔䎔䎑䎖

䏆
䎔䎓䎑䎙
䎕䎓䎑䎖
䎔䎕䎑䎗
䎛䎛䎑䎔
䎛䎜䎑䎚
䎛䎘䎑䎛
䎜䎗䎑䎙
䎜䎖䎑䎔
䎜䎘䎑䎚
䎜䎚䎑䎔
䎜䎜䎑䎜
䎜䎚䎑䎛
䎜䎔䎑䎗
䎜䎘䎑䎕
䎛䎜䎑䎕
䎛䎔䎑䎓
䎚䎜䎑䎔
䎛䎖䎑䎙
䎕䎓䎑䎛
䎔䎙䎑䎕
䎔䎘䎑䎕
䎚䎘䎑䎖
䎛䎕䎑䎖
䎚䎚䎑䎕
䎔䎓䎙䎑䎙
䎜䎙䎑䎛
䎜䎖䎑䎘
䎔䎓䎚䎑䎘
䎔䎓䎖䎑䎙
䎔䎓䎖䎑䎚

䎯䏄䏅䎑
䏇
䎔䎗䎑䎖
䎜䎑䎔
䎜䎑䎙
䎔䎓䎕䎑䎖
䎛䎜䎑䎛
䎙䎚䎑䎙
䎔䎓䎘䎑䎔
䎔䎔䎕䎑䎛
䎔䎓䎔䎑䎗
䎔䎓䎙䎑䎔
䎔䎓䎓䎑䎜
䎜䎛䎑䎗
䎚䎜䎑䎗
䎚䎓䎑䎘
䎙䎙䎑䎔
䎜䎔䎑䎖
䎔䎓䎕䎑䎗
䎛䎕䎑䎚
䎕䎔䎑䎚
䎔䎖䎑䎛
䎔䎜䎑䎛
䎔䎓䎜䎑䎗
䎔䎓䎚䎑䎘
䎔䎓䎖䎑䎓
䎔䎓䎘䎑䎓
䎔䎓䎖䎑䎗
䎜䎛䎑䎔
䎔䎔䎗䎑䎖
䎔䎓䎛䎑䎕
䎔䎓䎘䎑䎘

䏈
䎖䎛䎑䎔
䎕䎘䎑䎕
䎖䎕䎑䎖
䎔䎓䎔䎑䎛
䎚䎙䎑䎗
䎛䎘䎑䎛
䎔䎕䎜䎑䎙
䎔䎓䎙䎑䎙
䎔䎓䎖䎑䎜
䎔䎕䎚䎑䎔
䎔䎔䎖䎑䎙
䎔䎓䎘䎑䎕
䎔䎓䎖䎑䎓
䎜䎓䎑䎖
䎛䎜䎑䎙
䎜䎚䎑䎓
䎛䎔䎑䎘
䎜䎓䎑䎚
䎔䎚䎑䎘
䎕䎕䎑䎕
䎕䎔䎑䎖
䎔䎔䎗䎑䎜
䎔䎓䎓䎑䎜
䎔䎓䎓䎑䎜
䎔䎔䎘䎑䎛
䎔䎓䎛䎑䎙
䎔䎓䎖䎑䎜
䎔䎖䎕䎑䎓
䎔䎔䎚䎑䎓
䎔䎓䎚䎑䎘

䏉
䎔䎗䎑䎖
䎔䎔䎑䎕
䎔䎓䎑䎗
䎚䎘䎑䎖
䎙䎚䎑䎕
䎚䎘䎑䎚
䎜䎗䎑䎕
䎜䎚䎑䎜
䎜䎕䎑䎘
䎔䎓䎓䎑䎔
䎜䎕䎑䎛
䎜䎕䎑䎚
䎚䎔䎑䎜
䎖䎜䎑䎖
䎘䎘䎑䎔
䎛䎚䎑䎛
䎜䎗䎑䎗
䎛䎔䎑䎔
䎔䎘䎑䎛
䎖䎔䎑䎔
䎔䎘䎑䎙
䎛䎜䎑䎚
䎜䎚䎑䎛
䎜䎙䎑䎘
䎜䎛䎑䎛
䎛䎙䎑䎘
䎜䎚䎑䎚
䎔䎓䎗䎑䎓
䎔䎓䎔䎑䎕
䎔䎓䎔䎑䎕

䏊
䎔䎓䎑䎙
䎔䎓䎑䎙
䎔䎗䎑䎓
䎜䎙䎑䎓
䎜䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎓䎑䎘
䎜䎖䎑䎗
䎔䎔䎔䎑䎔
䎔䎓䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎜䎑䎛
䎜䎙䎑䎛
䎛䎜䎑䎜
䎛䎛䎑䎗
䎛䎚䎑䎕
䎛䎔䎑䎕
䎘䎗䎑䎔
䎖䎔䎑䎘
䎕䎕䎑䎘
䎔䎜䎑䎜
䎔䎓䎔䎑䎔
䎔䎓䎓䎑䎜
䎔䎓䎜䎑䎓
䎔䎓䎕䎑䎖
䎔䎓䎖䎑䎗
䎔䎔䎕䎑䎔
䎔䎓䎚䎑䎜
䎔䎓䎛䎑䎗
䎔䎔䎖䎑䎔

35
55

ENV/JM/MONO(2011)39
Table 12.

continued

䎦䏋䏈䏐䎑

䎹䏌䏙䏒

䎔䎔

䏑䏒

䎕

䎔䎕

䏑䏒

䎕

䎶䏆䏒䏕䏈 䎨䏛䏓䎑䎔䎗䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎖䎕䎔䎘䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎖䎕䎔䎙䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎚䎕䎔䎚䎦䏄䏗䏈䏊䏒䏕䏜䎕䎑䎚䎕䎔䎛䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎔䎜䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎕䎓䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎗

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎔䎔䎖䎑䎚
䎜䎛䎑䎔
䎔䎔䎕䎑䎙
䎕䎛䎑䎕
䎔䎛䎑䎗
䎔䎘䎑䎖
䎔䎔䎑䎔
䎙䎑䎙
䎙䎑䎛
䎔䎔䎑䎔
䎚䎑䎔
䎛䎑䎕
䎙䎚䎑䎜
䎖䎕䎑䎕
䎘䎜䎑䎛
䎙䎑䎔
䎗䎑䎛
䎘䎑䎙
䎛䎕䎑䎔
䎚䎛䎑䎖
䎕䎘䎑䎖
䎔䎘䎑䎓
䎔䎜䎑䎜
䎘䎔䎑䎔
䎖䎔䎑䎔
䎜䎑䎖
䎕䎜䎑䎘

䎯䏄䏅䎑
䏅
䏆
䏇
䏈
䏉
䏊
䎔䎓䎘䎑䎓 䎔䎓䎔䎑䎓 䎔䎓䎕䎑䎗 䎔䎕䎖䎑䎔 䎔䎓䎖䎑䎔 䎔䎓䎕䎑䎛䎔䎓䎙䎑䎙 䎜䎗䎑䎙 䎔䎓䎘䎑䎛 䎔䎔䎓䎑䎗 䎜䎛䎑䎓 䎔䎓䎓䎑䎘䎔䎓䎖䎑䎚 䎜䎗䎑䎔 䎔䎓䎕䎑䎚 䎔䎓䎘䎑䎘 䎜䎗䎑䎙 䎔䎓䎜䎑䎓䎕䎗䎑䎙 䎕䎗䎑䎜 䎘䎗䎑䎖 䎘䎘䎑䎙 䎕䎚䎑䎕 䎛䎚䎑䎚䎕䎗䎑䎙 䎗䎗䎑䎛 䎚䎙䎑䎕 䎘䎚䎑䎛 䎙䎘䎑䎕 䎜䎛䎑䎓䎔䎘䎑䎜 䎕䎛䎑䎔 䎕䎚䎑䎗 䎘䎚䎑䎕 䎙䎙䎑䎓 䎔䎔䎕䎑䎙䎔䎕䎑䎔 䎔䎗䎑䎚 䎔䎓䎑䎚 䎔䎗䎑䎕 䎔䎖䎑䎔 䎔䎖䎑䎘䎛䎑䎖䎜䎑䎘 䎔䎔䎑䎚 䎔䎕䎑䎓 䎔䎙䎑䎚 䎔䎕䎑䎓䎛䎑䎛䎜䎑䎔 䎔䎓䎑䎕 䎔䎓䎑䎗 䎔䎚䎑䎓 䎔䎓䎑䎙䎜䎑䎖 䎔䎖䎑䎔䎛䎑䎓 䎔䎔䎑䎓
䎛䎑䎙䎜䎑䎕䎔䎓䎑䎕 䎔䎜䎑䎖䎛䎑䎙 䎔䎔䎑䎖䎘䎑䎜 䎕䎗䎑䎚
䎜䎑䎜
䎛䎑䎔
䎜䎑䎕
䎛䎑䎚
䎚䎑䎔
䎜䎑䎕
䎜䎕䎑䎓 䎘䎔䎑䎘 䎔䎛䎑䎔 䎜䎛䎑䎕 䎘䎜䎑䎙 䎙䎗䎑䎜䎘䎗䎑䎔 䎛䎙䎑䎖 䎚䎜䎑䎕 䎜䎓䎑䎙 䎘䎓䎑䎗 䎚䎜䎑䎙䎜䎛䎑䎖 䎛䎔䎑䎚 䎖䎚䎑䎚 䎚䎛䎑䎚 䎙䎚䎑䎘 䎛䎙䎑䎘
䎗䎑䎘
䎘䎑䎖
䎙䎑䎙
䎛䎑䎜
䎙䎑䎜
䎙䎑䎕
䎗䎑䎚
䎙䎑䎓
䎘䎑䎖
䎙䎑䎖
䎘䎑䎘
䎘䎑䎖
䎘䎑䎚
䎘䎑䎜
䎖䎑䎜
䎘䎑䎗
䎗䎑䎘
䎘䎑䎖
䎗䎙䎑䎘 䎜䎔䎑䎕 䎛䎖䎑䎚 䎜䎛䎑䎜 䎙䎜䎑䎕 䎜䎕䎑䎗䎘䎓䎑䎙 䎛䎚䎑䎖 䎙䎜䎑䎜 䎛䎚䎑䎕 䎛䎓䎑䎙 䎛䎘䎑䎜䎔䎓䎓䎑䎓 䎛䎚䎑䎘 䎘䎜䎑䎓 䎙䎜䎑䎔 䎚䎔䎑䎜 䎜䎗䎑䎗䎚䎗䎑䎙 䎔䎓䎑䎓 䎖䎓䎑䎗 䎛䎖䎑䎔 䎗䎓䎑䎔 䎖䎘䎑䎛䎔䎓䎑䎜 䎕䎕䎑䎗 䎕䎛䎑䎖 䎕䎙䎑䎔 䎛䎚䎑䎓 䎗䎗䎑䎚䎖䎕䎑䎓 䎖䎘䎑䎓 䎔䎛䎑䎕 䎙䎜䎑䎗 䎚䎔䎑䎛 䎖䎛䎑䎚䎕䎗䎑䎛 䎔䎓䎑䎗䎜䎑䎙 䎔䎓䎑䎚
䎛䎑䎔
䎛䎑䎛
䎛䎑䎓
䎚䎑䎙 䎔䎙䎑䎜
䎛䎑䎕
䎚䎑䎛
䎙䎑䎚
䎜䎑䎖
䎚䎑䎙 䎖䎓䎑䎜
䎙䎑䎕
䎛䎑䎕
䎛䎑䎙

36
56

ENV/JM/MONO(2011)39
Table 13. Summary of the statistical analysis of the viability for each chemical by 2nd phase study.
䎦䏋䏈䏐䎑 䎶䏗䏄䏗䎑䎰䏈䏄䏑䎰䏈䏇䏌䏄䏑

䎓䎔

䎓䎕

䎓䎖

䎓䎗

䎓䎘

䎓䎙

䎓䎚

䎓䎛

䎓䎜

䎔䎓

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛

䏄
䎔䎚䎑䎜
䎔䎔䎑䎙
䎔䎔䎑䎕
䎖䎔䎑䎓
䎚䎖䎑䎛
䎚䎙䎑䎘
䎙䎘䎑䎕
䎚䎜䎑䎛
䎔䎓䎗䎑䎚
䎔䎓䎖䎑䎜
䎔䎓䎓䎑䎜
䎔䎓䎜䎑䎔
䎜䎜䎑䎖
䎜䎙䎑䎘
䎜䎘䎑䎕
䎔䎓䎙䎑䎖
䎚䎚䎑䎓
䎚䎛䎑䎘
䎚䎗䎑䎔
䎚䎛䎑䎘
䎛䎗䎑䎛
䎛䎕䎑䎗
䎚䎜䎑䎗
䎜䎕䎑䎘
䎔䎛䎑䎕
䎔䎚䎑䎛
䎔䎕䎑䎙
䎕䎗䎑䎔
䎜䎜䎑䎔
䎜䎘䎑䎖
䎜䎓䎑䎕
䎔䎔䎔䎑䎜
䎔䎓䎖䎑䎚
䎔䎓䎔䎑䎔
䎜䎚䎑䎔
䎔䎔䎕䎑䎛
䎔䎓䎕䎑䎔
䎔䎓䎗䎑䎔
䎛䎙䎑䎘
䎔䎔䎘䎑䎜

䏅
䎕䎗䎑䎘
䎔䎙䎑䎔
䎔䎓䎑䎗
䎗䎚䎑䎔
䎚䎕䎑䎗
䎙䎙䎑䎜
䎙䎔䎑䎚
䎛䎛䎑䎚
䎜䎛䎑䎘
䎜䎜䎑䎛
䎜䎖䎑䎖
䎔䎓䎕䎑䎖
䎜䎚䎑䎛
䎜䎛䎑䎙
䎜䎗䎑䎗
䎔䎓䎓䎑䎕
䎚䎕䎑䎚
䎚䎔䎑䎜
䎙䎔䎑䎚
䎛䎗䎑䎘
䎛䎓䎑䎚
䎛䎓䎑䎘
䎚䎚䎑䎜
䎛䎖䎑䎘
䎔䎕䎑䎕
䎔䎕䎑䎙
䎔䎓䎑䎛
䎔䎖䎑䎕
䎜䎚䎑䎗
䎔䎓䎓䎑䎙
䎛䎙䎑䎚
䎔䎓䎗䎑䎛
䎔䎓䎘䎑䎗
䎔䎓䎜䎑䎘
䎜䎙䎑䎚
䎔䎔䎓䎑䎔
䎔䎔䎕䎑䎕
䎔䎔䎔䎑䎖
䎔䎔䎓䎑䎔
䎔䎔䎘䎑䎗

䏆
䎔䎗䎑䎗
䎔䎕䎑䎗
䎔䎓䎑䎙
䎕䎓䎑䎖
䎛䎚䎑䎛
䎛䎛䎑䎔
䎛䎘䎑䎛
䎛䎜䎑䎚
䎜䎗䎑䎘
䎜䎗䎑䎙
䎜䎖䎑䎔
䎜䎘䎑䎚
䎜䎛䎑䎕
䎜䎚䎑䎛
䎜䎚䎑䎔
䎜䎜䎑䎜
䎜䎔䎑䎜
䎜䎔䎑䎗
䎛䎜䎑䎕
䎜䎘䎑䎕
䎛䎔䎑䎕
䎛䎔䎑䎓
䎚䎜䎑䎔
䎛䎖䎑䎙
䎔䎚䎑䎗
䎔䎙䎑䎕
䎔䎘䎑䎕
䎕䎓䎑䎛
䎚䎛䎑䎖
䎚䎚䎑䎕
䎚䎘䎑䎖
䎛䎕䎑䎖
䎜䎛䎑䎜
䎜䎙䎑䎛
䎜䎖䎑䎘
䎔䎓䎙䎑䎙
䎔䎓䎗䎑䎜
䎔䎓䎖䎑䎚
䎔䎓䎖䎑䎙
䎔䎓䎚䎑䎘

䎯䏄䏅䎑
䏇
䎔䎔䎑䎓
䎜䎑䎙
䎜䎑䎔
䎔䎗䎑䎖
䎛䎙䎑䎙
䎛䎜䎑䎛
䎙䎚䎑䎙
䎔䎓䎕䎑䎖
䎔䎓䎙䎑䎗
䎔䎓䎘䎑䎔
䎔䎓䎔䎑䎗
䎔䎔䎕䎑䎛
䎔䎓䎔䎑䎛
䎔䎓䎓䎑䎜
䎜䎛䎑䎗
䎔䎓䎙䎑䎔
䎚䎕䎑䎓
䎚䎓䎑䎘
䎙䎙䎑䎔
䎚䎜䎑䎗
䎜䎕䎑䎔
䎜䎔䎑䎖
䎛䎕䎑䎚
䎔䎓䎕䎑䎗
䎔䎛䎑䎗
䎔䎜䎑䎛
䎔䎖䎑䎛
䎕䎔䎑䎚
䎔䎓䎙䎑䎙
䎔䎓䎚䎑䎘
䎔䎓䎖䎑䎓
䎔䎓䎜䎑䎗
䎔䎓䎕䎑䎕
䎔䎓䎖䎑䎗
䎜䎛䎑䎔
䎔䎓䎘䎑䎓
䎔䎓䎜䎑䎖
䎔䎓䎛䎑䎕
䎔䎓䎘䎑䎘
䎔䎔䎗䎑䎖

䏈
䎖䎔䎑䎜
䎖䎕䎑䎖
䎕䎘䎑䎕
䎖䎛䎑䎔
䎛䎛䎑䎓
䎛䎘䎑䎛
䎚䎙䎑䎗
䎔䎓䎔䎑䎛
䎔䎔䎖䎑䎖
䎔䎓䎙䎑䎙
䎔䎓䎖䎑䎜
䎔䎕䎜䎑䎙
䎔䎔䎘䎑䎖
䎔䎔䎖䎑䎙
䎔䎓䎘䎑䎕
䎔䎕䎚䎑䎔
䎜䎗䎑䎖
䎜䎓䎑䎖
䎛䎜䎑䎙
䎔䎓䎖䎑䎓
䎛䎜䎑䎚
䎜䎓䎑䎚
䎛䎔䎑䎘
䎜䎚䎑䎓
䎕䎓䎑䎖
䎕䎔䎑䎖
䎔䎚䎑䎘
䎕䎕䎑䎕
䎔䎓䎘䎑䎙
䎔䎓䎓䎑䎜
䎔䎓䎓䎑䎜
䎔䎔䎗䎑䎜
䎔䎓䎜䎑䎗
䎔䎓䎛䎑䎙
䎔䎓䎖䎑䎜
䎔䎔䎘䎑䎛
䎔䎔䎛䎑䎛
䎔䎔䎚䎑䎓
䎔䎓䎚䎑䎘
䎔䎖䎕䎑䎓

䏉
䎔䎕䎑䎓
䎔䎔䎑䎕
䎔䎓䎑䎗
䎔䎗䎑䎖
䎚䎕䎑䎚
䎚䎘䎑䎖
䎙䎚䎑䎕
䎚䎘䎑䎚
䎜䎗䎑䎛
䎜䎗䎑䎕
䎜䎕䎑䎘
䎜䎚䎑䎜
䎜䎘䎑䎕
䎜䎕䎑䎛
䎜䎕䎑䎚
䎔䎓䎓䎑䎔
䎘䎘䎑䎗
䎘䎘䎑䎔
䎖䎜䎑䎖
䎚䎔䎑䎜
䎛䎚䎑䎛
䎛䎚䎑䎛
䎛䎔䎑䎔
䎜䎗䎑䎗
䎕䎓䎑䎛
䎔䎘䎑䎛
䎔䎘䎑䎙
䎖䎔䎑䎔
䎜䎗䎑䎚
䎜䎙䎑䎘
䎛䎜䎑䎚
䎜䎚䎑䎛
䎜䎗䎑䎖
䎜䎚䎑䎚
䎛䎙䎑䎘
䎜䎛䎑䎛
䎔䎓䎕䎑䎔
䎔䎓䎔䎑䎕
䎔䎓䎔䎑䎕
䎔䎓䎗䎑䎓

䏊
䎔䎔䎑䎚
䎔䎓䎑䎙
䎔䎓䎑䎙
䎔䎗䎑䎓
䎜䎛䎑䎓
䎜䎙䎑䎓
䎜䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎔䎑䎚
䎔䎓䎓䎑䎘
䎜䎖䎑䎗
䎔䎔䎔䎑䎔
䎔䎓䎘䎑䎜
䎔䎓䎗䎑䎛
䎔䎓䎖䎑䎖
䎔䎓䎜䎑䎛
䎜䎔䎑䎚
䎛䎜䎑䎜
䎛䎛䎑䎗
䎜䎙䎑䎛
䎚䎗䎑䎕
䎛䎔䎑䎕
䎘䎗䎑䎔
䎛䎚䎑䎕
䎕䎗䎑䎙
䎕䎕䎑䎘
䎔䎜䎑䎜
䎖䎔䎑䎘
䎔䎓䎖䎑䎚
䎔䎓䎔䎑䎔
䎔䎓䎓䎑䎜
䎔䎓䎜䎑䎓
䎔䎓䎘䎑䎜
䎔䎓䎖䎑䎗
䎔䎓䎕䎑䎖
䎔䎔䎕䎑䎔
䎔䎓䎜䎑䎛
䎔䎓䎛䎑䎗
䎔䎓䎚䎑䎜
䎔䎔䎖䎑䎔

37
57

ENV/JM/MONO(2011)39

Table 13. continued.
䎦䏋䏈䏐䎑 䎶䏗䏄䏗䎑䏄䏅䎰䏈䏄䏑 䎔䎓䎛䎑䎔 䎔䎓䎘䎑䎔䎰䏈䏇䏌䏄䏑 䎔䎔䎕䎑䎙 䎔䎓䎘䎑䎓

䎔䎔

䎔䎕

䎔䎗

䎔䎘

䎔䎙

䎔䎚

䎔䎛

䎔䎜

䎕䎓

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑
䎰䏄䏛
䎰䏈䏄䏑
䎰䏈䏇䏌䏄䏑

䎰䏌䏑䎰䏄䏛䎜䎛䎑䎔 䎔䎓䎖䎑䎚䎔䎔䎖䎑䎚 䎔䎓䎙䎑䎙䎕䎓䎑䎚 䎕䎔䎑䎚䎔䎛䎑䎗 䎕䎗䎑䎙䎔䎘䎑䎖 䎔䎘䎑䎜䎕䎛䎑䎕 䎕䎗䎑䎙
䎛䎑䎕
䎜䎑䎚
䎙䎑䎛
䎛䎑䎛
䎙䎑䎙
䎛䎑䎖
䎔䎔䎑䎔 䎔䎕䎑䎔
䎛䎑䎛
䎜䎑䎛
䎛䎑䎕
䎜䎑䎜
䎚䎑䎔
䎜䎑䎖
䎔䎔䎑䎔 䎔䎓䎑䎕䎘䎖䎑䎖 䎛䎔䎑䎗䎘䎜䎑䎛 䎜䎕䎑䎓䎖䎕䎑䎕 䎘䎗䎑䎔䎙䎚䎑䎜 䎜䎛䎑䎖
䎘䎑䎘
䎗䎑䎜
䎘䎑䎙
䎗䎑䎚
䎗䎑䎛
䎗䎑䎘
䎙䎑䎔
䎘䎑䎚
䎙䎔䎑䎜 䎙䎘䎑䎚䎚䎛䎑䎖 䎘䎓䎑䎙䎕䎘䎑䎖 䎗䎙䎑䎘䎛䎕䎑䎔 䎔䎓䎓䎑䎓䎕䎛䎑䎚 䎖䎜䎑䎕䎔䎜䎑䎜 䎖䎕䎑䎓䎔䎘䎑䎓 䎔䎓䎑䎜䎘䎔䎑䎔 䎚䎗䎑䎙䎕䎖䎑䎖 䎔䎗䎑䎓
䎕䎜䎑䎘
䎜䎑䎖
䎜䎑䎖
䎛䎑䎓
䎖䎔䎑䎔 䎕䎗䎑䎛

䏆
䎜䎙䎑䎙
䎜䎗䎑䎙
䎜䎗䎑䎔
䎔䎓䎔䎑䎓
䎖䎕䎑䎙
䎕䎛䎑䎔
䎕䎗䎑䎜
䎗䎗䎑䎛
䎔䎔䎑䎔
䎜䎑䎘
䎜䎑䎔
䎔䎗䎑䎚
䎔䎖䎑䎘
䎔䎖䎑䎔
䎛䎑䎔
䎔䎜䎑䎖
䎚䎖䎑䎔
䎛䎔䎑䎚
䎘䎔䎑䎘
䎛䎙䎑䎖
䎘䎑䎛
䎘䎑䎜
䎘䎑䎖
䎙䎑䎓
䎛䎛䎑䎚
䎛䎚䎑䎘
䎛䎚䎑䎖
䎜䎔䎑䎕
䎕䎕䎑䎘
䎕䎕䎑䎗
䎔䎓䎑䎓
䎖䎘䎑䎓
䎛䎑䎙
䎚䎑䎙
䎚䎑䎙
䎔䎓䎑䎗

䎯䏄䏅䎑
䏇
䎔䎓䎖䎑䎙
䎔䎓䎕䎑䎚
䎔䎓䎕䎑䎗
䎔䎓䎘䎑䎛
䎘䎕䎑䎙
䎘䎗䎑䎖
䎕䎚䎑䎗
䎚䎙䎑䎕
䎔䎓䎑䎜
䎔䎓䎑䎚
䎔䎓䎑䎕
䎔䎔䎑䎚
䎛䎑䎙
䎛䎑䎙
䎛䎑䎓
䎜䎑䎕
䎗䎘䎑䎓
䎖䎚䎑䎚
䎔䎛䎑䎔
䎚䎜䎑䎕
䎘䎑䎖
䎘䎑䎖
䎖䎑䎜
䎙䎑䎙
䎚䎓䎑䎜
䎙䎜䎑䎜
䎘䎜䎑䎓
䎛䎖䎑䎚
䎕䎘䎑䎙
䎕䎛䎑䎖
䎔䎛䎑䎕
䎖䎓䎑䎗
䎔䎜䎑䎕
䎔䎙䎑䎜
䎜䎑䎙
䎖䎓䎑䎜

䏈
䏉
䏊
䎔䎔䎖䎑䎓 䎜䎛䎑䎙 䎔䎓䎗䎑䎔䎔䎔䎓䎑䎗 䎜䎛䎑䎓 䎔䎓䎕䎑䎛䎔䎓䎘䎑䎘 䎜䎗䎑䎙 䎔䎓䎓䎑䎘䎔䎕䎖䎑䎔 䎔䎓䎖䎑䎔 䎔䎓䎜䎑䎓䎘䎙䎑䎜 䎘䎕䎑䎛 䎜䎜䎑䎘䎘䎚䎑䎕 䎙䎘䎑䎕 䎜䎛䎑䎓䎘䎘䎑䎙 䎕䎚䎑䎕 䎛䎚䎑䎚䎘䎚䎑䎛 䎙䎙䎑䎓 䎔䎔䎕䎑䎙䎔䎕䎑䎕 䎔䎘䎑䎙 䎔䎕䎑䎓䎔䎕䎑䎓 䎔䎙䎑䎚 䎔䎕䎑䎓䎔䎓䎑䎗 䎔䎖䎑䎔 䎔䎓䎑䎙䎔䎗䎑䎕 䎔䎚䎑䎓 䎔䎖䎑䎘䎔䎓䎑䎖䎚䎑䎕 䎔䎗䎑䎗
䎔䎔䎑䎓
䎚䎑䎔
䎜䎑䎕
䎛䎑䎚
䎘䎑䎜
䎜䎑䎕
䎔䎔䎑䎖
䎛䎑䎙 䎕䎗䎑䎚
䎛䎜䎑䎔 䎘䎜䎑䎔 䎚䎚䎑䎓䎜䎓䎑䎙 䎘䎜䎑䎙 䎚䎜䎑䎙䎚䎛䎑䎚 䎘䎓䎑䎗 䎙䎗䎑䎜䎜䎛䎑䎕 䎙䎚䎑䎘 䎛䎙䎑䎘
䎙䎑䎜
䎘䎑䎙
䎘䎑䎙
䎙䎑䎖
䎘䎑䎘
䎘䎑䎖
䎘䎑䎗
䎗䎑䎘
䎘䎑䎖
䎛䎑䎜
䎙䎑䎜
䎙䎑䎕
䎛䎘䎑䎔 䎚䎖䎑䎜 䎜䎓䎑䎜䎛䎚䎑䎕 䎚䎔䎑䎜 䎜䎕䎑䎗䎙䎜䎑䎔 䎙䎜䎑䎕 䎛䎘䎑䎜䎜䎛䎑䎜 䎛䎓䎑䎙 䎜䎗䎑䎗䎘䎜䎑䎘 䎙䎙䎑䎖 䎖䎜䎑䎛䎙䎜䎑䎗 䎚䎔䎑䎛 䎗䎗䎑䎚䎕䎙䎑䎔 䎗䎓䎑䎔 䎖䎘䎑䎛䎛䎖䎑䎔 䎛䎚䎑䎓 䎗䎗䎑䎚
䎛䎑䎗
䎛䎑䎓
䎛䎑䎔
䎛䎑䎕
䎛䎑䎔
䎛䎑䎙
䎙䎑䎕
䎚䎑䎛
䎙䎑䎚
䎔䎓䎑䎚
䎛䎑䎕
䎛䎑䎛

38
58

ENV/JM/MONO(2011)39

Fig. 4. Distribution of the viability for each chemical.

39
59

ENV/JM/MONO(2011)39

Fig. 4. continued

40
60

ENV/JM/MONO(2011)39

㩷Fig. 4. continued.
Table 14. Viability of chemicals each laboratory by 3rd phase study䎦䏋䏈䏐䎑 䎹䏌䏙䏒 䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎤

䏑䏒

䎕

䎥

䎦䏄䏗䏈䏊
䎕䎑䎚
䏒䏕䏜䎃䎕

䎦

䎦䏄䏗䏈䏊
䏒䏕䏜䎃䎕

䎧

䎦䏄䏗䏈䏊
䎖䎑䎖
䏒䏕䏜䎃䎕

䎨

䎦䏄䏗䏈䏊
䎖䎑䎖
䏒䏕䏜䎃䎕

䎩

䎦䏄䏗䏈䏊
䏒䏕䏜䎃䎕

䎖

䎗

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎔䎖䎑䎖
䎔䎗䎑䎕
䎔䎗䎑䎓
䎔䎑䎘
䎖䎑䎔
䎔䎑䎘
䎓䎑䎚
䎔䎑䎖
䎓䎑䎘
䎔䎗䎑䎘
䎔䎖䎑䎙
䎔䎛䎑䎙
䎖䎑䎜
䎗䎑䎘
䎔䎑䎛
䎘䎑䎙
䎘䎑䎚
䎘䎑䎗

䏅
䎔䎔䎑䎛
䎔䎓䎑䎕
䎔䎔䎑䎔
䎕䎑䎕
䎕䎑䎕
䎕䎑䎘
䎓䎑䎚
䎔䎑䎔
䎓䎑䎛
䎕䎗䎑䎓
䎔䎙䎑䎓
䎔䎘䎑䎘
䎖䎑䎗
䎕䎑䎚
䎖䎑䎘
䎚䎑䎕
䎙䎑䎔
䎗䎑䎕

䎯䏄䏅䎑
䏆
䏇
䎔䎖䎑䎕 䎔䎖䎑䎛䎕䎕䎑䎘䎜䎑䎜䎔䎕䎑䎖 䎔䎖䎑䎕
䎕䎑䎘
䎗䎑䎓
䎕䎑䎜
䎖䎑䎓
䎖䎑䎓
䎖䎑䎜
䎓䎑䎚
䎙䎑䎜
䎔䎑䎗
䎕䎑䎓
䎔䎑䎓
䎓䎑䎛
䎔䎕䎑䎚 䎔䎓䎑䎖䎔䎕䎑䎘 䎔䎛䎑䎖䎔䎕䎑䎙 䎕䎖䎑䎓
䎖䎑䎗
䎛䎑䎕
䎖䎑䎖
䎖䎑䎜
䎖䎑䎘
䎖䎑䎚
䎙䎑䎘
䎙䎑䎗
䎙䎑䎛
䎘䎑䎗
䎙䎑䎘
䎘䎑䎗

䏉
䎔䎔䎑䎗
䎔䎔䎑䎖
䎔䎗䎑䎖
䎔䎑䎚
䎕䎑䎙
䎖䎑䎕
䎓䎑䎛
䎗䎑䎛
䎔䎑䎓
䎔䎖䎑䎛
䎛䎑䎛
䎔䎜䎑䎕
䎖䎑䎕
䎗䎑䎕
䎘䎑䎓
䎘䎑䎕
䎚䎑䎗
䎘䎑䎓

䏊
䎔䎖䎑䎚
䎛䎑䎚
䎔䎗䎑䎖
䎖䎑䎜
䎖䎑䎚
䎗䎑䎚
䎔䎑䎓
䎓䎑䎗
䎓䎑䎖
䎔䎜䎑䎖
䎔䎘䎑䎕
䎔䎗䎑䎔
䎗䎑䎔
䎖䎑䎔
䎘䎑䎔
䎚䎑䎕
䎙䎑䎛
䎚䎑䎙

41
61

ENV/JM/MONO(2011)39
Table 15㩷 Summary statistical analysis of viability each chemical by 3rd phase study䎯䏄䏅䎑䎦䏋䏈䏐䎑 䎶䏗䏄䏗䎑
䏄
䏅
䏆
䏇
䏉
䏊
䎰䏈䏄䏑
䎔䎖䎑䎛
䎔䎔䎑䎓
䎔䎙䎑䎓
䎔䎕䎑䎖
䎔䎕䎑䎖
䎔䎕䎑䎕
䎰䏈䏇䏌䏄䏑
䎔䎗䎑䎓
䎔䎔䎑䎔
䎔䎖䎑䎕
䎔䎖䎑䎕
䎔䎔䎑䎗
䎔䎖䎑䎚
䎤
䎰䏌䏑
䎔䎖䎑䎖
䎔䎓䎑䎕
䎔䎕䎑䎖
䎜䎑䎜
䎔䎔䎑䎖
䎛䎑䎚
䎰䏄䏛
䎔䎗䎑䎕
䎔䎔䎑䎛
䎕䎕䎑䎘
䎔䎖䎑䎛
䎔䎗䎑䎖
䎔䎗䎑䎖
䎰䏈䏄䏑
䎕䎑䎓
䎕䎑䎖
䎕䎑䎛
䎖䎑䎙
䎕䎑䎘
䎗䎑䎔
䎰䏈䏇䏌䏄䏑
䎔䎑䎘
䎕䎑䎕
䎕䎑䎜
䎖䎑䎜
䎕䎑䎙
䎖䎑䎜
䎥
䎰䏌䏑
䎔䎑䎘
䎕䎑䎕
䎕䎑䎘
䎖䎑䎓
䎔䎑䎚
䎖䎑䎚
䎰䏄䏛
䎖䎑䎔
䎕䎑䎘
䎖䎑䎓
䎗䎑䎓
䎖䎑䎕
䎗䎑䎚
䎰䏈䏄䏑
䎓䎑䎛
䎓䎑䎛
䎔䎑䎓
䎖䎑䎕
䎕䎑䎕
䎓䎑䎙
䎰䏈䏇䏌䏄䏑
䎓䎑䎚
䎓䎑䎛
䎔䎑䎓
䎕䎑䎓
䎔䎑䎓
䎓䎑䎗
䎦
䎰䏌䏑
䎓䎑䎘
䎓䎑䎚
䎓䎑䎚
䎓䎑䎛
䎓䎑䎛
䎓䎑䎖
䎰䏄䏛
䎔䎑䎖
䎔䎑䎔
䎔䎑䎗
䎙䎑䎜
䎗䎑䎛
䎔䎑䎓
䎰䏈䏄䏑
䎔䎘䎑䎙
䎔䎛䎑䎘
䎔䎕䎑䎙
䎔䎚䎑䎕
䎔䎖䎑䎜
䎔䎙䎑䎕
䎰䏈䏇䏌䏄䏑
䎔䎗䎑䎘
䎔䎙䎑䎓
䎔䎕䎑䎙
䎔䎛䎑䎖
䎔䎖䎑䎛
䎔䎘䎑䎕
䎧
䎰䏌䏑
䎔䎖䎑䎙
䎔䎘䎑䎘
䎔䎕䎑䎘
䎔䎓䎑䎖
䎛䎑䎛
䎔䎗䎑䎔
䎰䏄䏛
䎔䎛䎑䎙
䎕䎗䎑䎓
䎔䎕䎑䎚
䎕䎖䎑䎓
䎔䎜䎑䎕
䎔䎜䎑䎖
䎰䏈䏄䏑
䎖䎑䎗
䎖䎑䎕
䎖䎑䎗
䎘䎑䎖
䎗䎑䎕
䎗䎑䎔
䎰䏈䏇䏌䏄䏑
䎖䎑䎜
䎖䎑䎗
䎖䎑䎗
䎖䎑䎜
䎗䎑䎕
䎗䎑䎔
䎨
䎰䏌䏑
䎔䎑䎛
䎕䎑䎚
䎖䎑䎖
䎖䎑䎚
䎖䎑䎕
䎖䎑䎗
䎰䏄䏛
䎗䎑䎘
䎖䎑䎘
䎖䎑䎘
䎛䎑䎕
䎘䎑䎓
䎘䎑䎔
䎰䏈䏄䏑
䎘䎑䎘
䎘䎑䎛
䎙䎑䎙
䎘䎑䎚
䎘䎑䎜
䎚䎑䎕
䎰䏈䏇䏌䏄䏑
䎘䎑䎙
䎙䎑䎔
䎙䎑䎘
䎘䎑䎗
䎘䎑䎕
䎚䎑䎕
䎩
䎰䏌䏑
䎘䎑䎗
䎗䎑䎕
䎙䎑䎘
䎘䎑䎗
䎘䎑䎓
䎙䎑䎛
䎰䏄䏛
䎘䎑䎚
䎚䎑䎕
䎙䎑䎛
䎙䎑䎗
䎚䎑䎗
䎚䎑䎙

42
62

ENV/JM/MONO(2011)39

Fig.5 Distribution of viability each chemical
8-2-5. IL-1Į
36.
The results of the LabCyte EPI-MODEL 24 skin irritation test when IL-1Į was evaluated as anindicator are summarized in Table 16.
Table 16. IL-1Į levels from each laboratory.
䎦䏋䏈䏐䎑 䎪䎫䎶 䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎓䎔

䏑䏒

䎓

䎓䎕

䏑䏒

䎓

䎓䎖

䏑䏒

䎓

䎓䎗

䏑䏒

䎓

䎓䎘

䏑䏒

䎓䎑䎖

䎓䎙

䏑䏒

䎓䎑䎖

䎓䎚

䏑䏒

䎔

䎓䎛

䏑䏒

䎔

䎓䎜

䏑䏒

䎔䎑䎚

䎔䎓

䏑䏒

䎔䎑䎚

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䏄
䎑
䎑
䎑

䏅
䎑
䎑
䎑

䎯䏄䏅䎑
䏇

䏆
䎑
䎑
䎑

䎑
䎑
䎑

䎔䎖䎕䎑䎛
䎙䎛䎑䎔
䎜䎚䎑䎙
䎔䎕
䎚䎑䎔
䎔䎓䎑䎚
䎔䎓
䎘䎑䎖
䎙䎑䎖
䎔䎕䎕
䎖䎘䎑䎚
䎗䎗䎑䎗
䎘䎜
䎙䎕䎑䎜
䎙䎛䎑䎛

䏈
䎑
䎑
䎑

䎘䎕䎑䎜 䎘䎜䎑䎖 䎗䎔䎑䎕䎘䎙䎑䎘䎖䎚 䎛䎜䎑䎔䎗䎔䎑䎔䎚䎙 䎚䎕䎑䎗䎜䎑䎘 䎔䎘䎑䎘
䎛䎑䎙䎛䎑䎙 䎔䎔䎑䎚 䎔䎜䎑䎜䎔䎓䎑䎖 䎔䎕䎑䎜䎜䎑䎗䎙䎛 䎔䎔䎑䎚
䎛䎘䎑䎘 䎔䎖䎑䎕
䎗䎑䎚
䎚䎑䎕
䎚䎑䎜
䎜䎚䎑䎙 䎕䎗䎑䎖 䎛䎔䎑䎕䎙䎖䎑䎘 䎖䎘䎑䎔 䎔䎔䎘䎑䎖䎕䎙 䎖䎔䎑䎕 䎗䎜䎑䎗䎛䎘䎑䎚䎔䎔䎗 䎛䎘䎑䎙
䎜䎖䎑䎙 䎔䎓䎗䎑䎜 䎔䎖䎜䎑䎘䎛䎘䎑䎔 䎛䎕䎑䎜 䎙䎗䎑䎘
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎛䎑䎕
䎜䎑䎗 䎛䎗䎑䎔䎗䎑䎔䎖䎑䎙䎙䎑䎗 䎖䎔䎑䎙 䎔䎓䎑䎗䎙䎗䎑䎔 䎖䎖䎑䎔
䎘䎑䎕䎔䎓䎑䎜 䎔䎚䎑䎔 䎔䎔䎑䎕 䎗䎕䎑䎙䎔䎜䎑䎛䎛䎑䎛䎛䎑䎛 䎖䎕䎑䎕䎖䎔䎑䎖䎙䎑䎛 䎕䎓䎑䎔 䎕䎔䎑䎖䎕䎚䎑䎜䎚䎑䎗 䎖䎔䎑䎖 䎗䎔䎑䎕䎔䎚䎑䎔 䎔䎕䎑䎚䎔䎘 䎘䎓䎑䎗
䎙䎙䎑䎕 䎔䎕䎑䎕䎖䎓 䎗䎕䎑䎔

䏉
䎑
䎑
䎑

䏊
䎑
䎑
䎑

䎙䎓䎑䎚 䎙䎔䎑䎖䎜䎑䎗䎙䎛䎑䎗 䎜䎜䎑䎖䎜䎑䎙䎗䎙 䎚䎓䎑䎔 䎔䎕䎑䎙䎕䎖䎑䎕 䎔䎕䎑䎚䎛䎑䎔䎔䎓䎑䎘䎜䎑䎕 䎔䎔䎑䎜䎔䎔䎑䎖䎙䎑䎚 䎔䎘䎑䎚
䎜䎑䎘
䎕䎑䎘
䎙䎑䎖
䎔䎘䎑䎔
䎕䎑䎙
䎛䎑䎙
䎜䎑䎚
䎖䎑䎗
䎙䎑䎛
䎘䎚䎑䎚 䎔䎛䎖䎑䎘 䎔䎘䎑䎗䎖䎙䎑䎙 䎑䎕䎛䎑䎘䎖䎖 䎔䎜䎔䎑䎙 䎖䎖䎑䎕䎜䎗䎑䎗 䎙䎓䎑䎛 䎔䎔䎕䎑䎘䎛䎔䎑䎗 䎗䎛䎑䎔 䎙䎕䎑䎔䎘䎕䎑䎜 䎘䎗䎑䎛 䎔䎗䎚䎑䎔
䎑
䎑
䎑
䎑
䎑
䎑
䎙䎑䎜 䎕䎔䎑䎗
䎘䎑䎖
䎛䎑䎘
䎗䎑䎜
䎘䎑䎛
䎙䎑䎚
䎕䎑䎔
䎚䎑䎕
䎕䎜䎑䎘
䎖䎖
䎚䎑䎗
䎙䎑䎘 䎕䎘䎑䎖
䎜䎑䎚䎔䎔䎑䎕 䎕䎗䎑䎚 䎔䎓䎑䎙䎗䎙䎑䎘 䎖䎜䎑䎖䎜䎑䎛䎕䎙䎑䎚 䎕䎙䎑䎚 䎔䎗䎑䎘䎕䎙䎑䎖 䎕䎗䎑䎕 䎔䎖䎑䎕

43
63

ENV/JM/MONO(2011)39
Table 16. continued.
䎦䏋䏈䏐䎑

䎪䎫䎶

䎶䏆䏒䏕䏈 䎨䏛䏓䎑

䎔䎔

䏑䏒

䎕

䎔䎕

䏑䏒

䎕

䎔䎗

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎖

䎔䎘

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎖

䎔䎙

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎚

䎔䎚

䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎕䎑䎚

䎔䎛

䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎔䎜䎦䏄䏗䏈䏊䏒䏕䏜䎕䎖䎕䎓䎦䏄䏗䏈䏊䏒䏕䏜
䎕

䎗

䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖
䎔
䎕
䎖

䎯䏄䏅䎑
䏇
䏈
䏉
䎘
䎔䎛 䎔䎘䎑䎖 䎔䎓䎑䎗 䎔䎙䎑䎕
䎖䎑䎖
䎔䎘䎑䎛
䎔䎜
䎜䎑䎚
䎛䎑䎔
䎔䎛䎑䎕
䎛䎑䎜
䎛䎑䎚
䎛䎑䎙 䎔䎕䎑䎙䎑䎑䎑䎔䎘䎚䎑䎕 䎔䎕䎓䎑䎗 䎑䎑䎑䎑䎔䎔䎖 䎔䎔䎛䎑䎙 䎜䎓䎑䎕䎑䎑䎑䎑䎘䎛䎑䎖 䎙䎙䎑䎕
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎛䎙䎑䎜 䎙䎛䎑䎔 䎔䎕䎜䎑䎗 䎑䎔䎕䎙䎑䎛 䎔䎔䎙䎑䎘䎑䎔䎓䎓䎑䎕 䎚䎗䎑䎗 䎔䎙䎜䎑䎚 䎚䎙䎑䎔 䎔䎓䎚䎑䎘䎔䎕䎔䎑䎕 䎗䎕䎑䎘 䎛䎖䎑䎙 䎑䎚䎖䎑䎔 䎛䎚䎑䎖
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎙䎔䎑䎘 䎑
䎙䎓䎑䎙 䎜䎓䎑䎖 䎛䎙䎑䎜 䎔䎔䎗䎑䎘䎘䎚䎑䎚 䎔䎓䎗䎑䎜 䎗䎘䎑䎛 䎕䎕䎔䎑䎖 䎜䎛䎑䎚 䎚䎙䎑䎗䎑䎔䎚䎑䎕 䎘䎔䎑䎗 䎔䎖䎛䎑䎔 䎙䎖䎑䎜 䎔䎓䎕䎑䎕䎑䎘䎚䎑䎖 䎑
䎑䎔䎓䎜䎑䎕 䎑䎑䎑䎑䎑䎑䎑䎙䎜䎑䎕 䎑
䎔䎓䎕䎑䎖 䎑䎑䎑䎙䎛 䎘䎜䎑䎘 䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䎑
䏄

䏅
䎖䎔䎑䎔
䎔䎔䎑䎜
䎘

䏆

䏊
䎙䎑䎗
䎚䎑䎘
䎔䎔䎑䎜
䎖䎗䎑䎘
䎕䎚䎑䎖
䎔䎖䎑䎙

䎜䎓䎑䎛
䎚䎓䎑䎜
䎚䎜䎑䎕

䎔䎛
䎗䎘䎑䎔
䎕䎕䎑䎔

Cells highlighted in yellow indicate that the classification changed based on the IL-1Į data.

4464

ENV/JM/MONO(2011)39
8-2-6. Classification of three independent viabilities at each laboratory
37.
The classifications from mean of three independent viabilities only evaluated MTT assay wereshown in Table 17 in 2nd phase study and Table 19 in 3rd phase study. Refer to Table 18, the IL-1Į resultschanged the classification for only 3 data points. The classification of Allyl phenoxy-acetate by Lab f waschanged the misunderstood classification. The other two chemicals were changed the correct classification.
Regarding the ILĮ only a few chemicals showed different results but the overall call was that ILĮ did not
significantly contribute to the performance of the assay.

Table 17.Classification using three independent viabilities by 2nd phase study
䇸P䇹:Positive䇮䇸N䇹: Negative
㩷
Chem.
01
02
03
04
05
06
07
08
09
10
11
12
14
15
16
17
18
19
20

GHS
no
no
no
no
no
no
no
no
no
no
no
no
Category 2
Category 2
Category 2
Category 2
Category 2
Category 2
Category 2

Score
0
0
0
0
0.3
0.3
1
1
1.7
1.7
2
2
2.3
2.3
2.7
2.7
3
3
4

Lab.
a
P
N
N
N
N
N
P
N
N
N
N
P
P
P
N
P
N
P
P

b
P
N
N
N
N
N
P
N
N
N
N
P
P
P
N
P
N
P
P

c
P
N
N
N
N
N
P
N
N
N
N
P
P
P
N
P
N
P
P

d
P
N
N
N
N
N
P
N
N
N
N
N
P
P
P
P
N
P
P

e
P
N
N
N
N
N
P
N
N
N
N
N
P
P
N
P
N
N
P

f
P
N
N
N
N
N
P
N
N
N
N
N
P
P
N
P
N
N
P

g
P
N
N
N
N
N
P
N
N
N
N
N
P
P
N
P
N
P
P

Table.18. Classification of chemicals by MTT assay demolished by additional IL-1Į measurement
Lab. Classific Classific
In vivoCAS
ation by ation by
No. Chemical
GHS label score
MTT+IL
number
MTT
(PII)
-1Į
assay
N
P
f
05 Allyl phenoxy-acetate
7493-74-5 no0.3
16 1-bromohexane

111-25-1

Category 2

2.7

a

N

P

18 di-n-propyl disulphide629-19-6

Category 2

3

d

N

P

45
65

ENV/JM/MONO(2011)39
Table 19 Classification using three independent viabilities by 3rd phase study
䇸P䇹:Positive䇮䇸N䇹: Negative
㩷
Lab.
Chem.
in vivo
Score
a
b
c
d
f
P
P
P
P
P
A
no
2
B

Category 2

2.7

C

Category 2

2.7

DCategory 2

3.3

E

Category 2

3.3

F

Category 2

4

g
P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

P

Table 20.㩷 Sensitivity, specificity and accuracy on MTT assay vs GHS-EU classification in the 2nd + 3rd
Phase validation study (25 substances)䎬䏑䏇䏈䏛䎶䏈䏑䏖䏌䏗䏌䏙䏌䏗䏜䎶䏓䏈䏆䏌䏉䏌䏆䏌䏗䏜䎤䏆䏆䏘䏕䏄䏆䏜

䎯䏄䏅䎑
䏄
䏅
䏆
䏇
䏉
䏊
䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎔䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕䎛䎖䎑䎖 䎛䎖䎑䎖 䎛䎖䎑䎖 䎜䎔䎑䎙䎚䎘䎛䎖䎑䎖䎜䎒䎔䎖 䎜䎒䎔䎖 䎜䎒䎔䎖 䎔䎓䎒䎔䎖 䎔䎓䎒䎔䎖 䎔䎓䎒䎔䎖䎙䎜䎑䎕 䎙䎜䎑䎕 䎙䎜䎑䎕 䎚䎙䎑䎜 䎚䎙䎑䎜 䎚䎙䎑䎜䎔䎜䎒䎕䎘 䎔䎜䎒䎕䎘 䎔䎜䎒䎕䎘 䎕䎔䎒䎕䎘 䎔䎜䎒䎕䎘 䎕䎓䎒䎕䎘
䎚䎙
䎚䎙
䎚䎙
䎛䎗
䎚䎙
䎛䎓

Table 21.㩷 Sensitivity, specificity and accuracy on MTT assay vs GHS-EU classification in 2nd phase study(19 substances).

䎬䏑䏇䏈䏛䎶䏈䏑䏖䏌䏗䏌䏙䏌䏗䏜䎶䏓䏈䏆䏌䏉䏌䏆䏌䏗䏜䎤䏆䏆䏘䏕䏄䏆䏜

䎯䏄䏅䎑
䏄
䏅
䏆
䏇
䏈
䏉
䏊
䎘䎒䎚
䎘䎒䎚
䎘䎒䎚
䎙䎒䎚
䎗䎒䎚
䎗䎒䎚
䎘䎒䎚
䎚䎔䎑䎗 䎚䎔䎑䎗 䎚䎔䎑䎗 䎛䎘䎑䎚 䎘䎚䎑䎔 䎘䎚䎑䎔 䎚䎔䎑䎗䎜䎒䎔䎕 䎜䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕䎚䎘䎚䎘䎚䎘䎛䎖䎑䎖 䎛䎖䎑䎖 䎛䎖䎑䎖 䎛䎖䎑䎖䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎙䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎘䎒䎔䎜䎚䎖䎑䎚 䎚䎖䎑䎚 䎚䎖䎑䎚 䎛䎗䎑䎕 䎚䎖䎑䎚 䎚䎖䎑䎚 䎚䎛䎑䎜

46
66

ENV/JM/MONO(2011)39
Table 22.㩷 Sensitivity, specificity and accuracy of the MTT assay and IL-1Į vs. the GHS-EU classificationin 2nd phase study (19 substances).

䎬䏑䏇䏈䏛䎶䏈䏑䏖䏌䏗䏌䏙䏌䏗䏜䎶䏓䏈䏆䏌䏉䏌䏆䏌䏗䏜䎤䏆䏆䏘䏕䏄䏆䏜

䎯䏄䏅䎑
䏄
䏅
䏆
䏇
䏈
䏉
䏊
䎙䎒䎚
䎘䎒䎚
䎘䎒䎚
䎚䎒䎚
䎗䎒䎚
䎗䎒䎚
䎘䎒䎚
䎛䎘䎑䎚 䎚䎔䎑䎗 䎚䎔䎑䎗䎔䎓䎓䎘䎚䎑䎔 䎘䎚䎑䎔 䎚䎔䎑䎗䎜䎒䎔䎕 䎜䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕 䎔䎓䎒䎔䎕 䎜䎒䎔䎕 䎔䎓䎒䎔䎕䎚䎘䎚䎘䎚䎘䎛䎖䎑䎖 䎛䎖䎑䎖䎚䎘䎛䎖䎑䎖䎔䎘䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎚䎒䎔䎜 䎔䎗䎒䎔䎜 䎔䎖䎒䎔䎜 䎔䎘䎒䎔䎜䎚䎛䎑䎜 䎚䎖䎑䎚 䎚䎖䎑䎚 䎛䎜䎑䎘 䎚䎖䎑䎚 䎙䎛䎑䎗 䎚䎛䎑䎜

Table 23(A). Mean and range of Sensitivity, specificity and accuracy on the MTT assay using LabCyte
EPI-MODEL vs. GHS-EU classification in the 2nd + 3rd Phase validation study (25substances)
N
Mean
Min.
Max.
ECVAM criteria
Sensivitity (%)
6
83.3
75.0
91.6
80.0
Specificity (%)
6
73.1
69.2
76.9
70.0
Accuracy (%)
6
78.0
76.0
84.0
75.0

Table 23(B). Mean and range of sensitivity, specificity and accuracy of the MTT assay vs. the GHS-EU
classification in 2nd phase study (19 substances).
N
Mean
Min.
Max.
ECVAM criteria
Sensivitity (%)
7
69.4
57.1
85.7
80.0
Specificity (%)
7
79.7
75.0
83.3
70.0
Accuracy (%)
7
75.9
73.7
84.2
75.0
Table 23(C). Mean and range of sensitivity, specificity and accuracy of the MTT assay and IL-1Į vs. the
GHS-EU classification in 2nd phase study (19 substances).
N
Mean
Min.
Max.
ECVAM criteria
Sensivitity (%)
7
73.4
57.1
100.0
80.0
Specificity (%)
7
78.6
69.2
76.9
70.0
Accuracy (%)
7
76.7
68.4
89.5
75.0
9. Discussion
9-1. Reliability38.
All data of negative control and positive control each laboratory in 2nd and 3rd phase study was
sufficient with the acceptance criteria as shown in Tables 10 and 11. There were high respectabilities within
and between laboratories in this model.
39.
In all data, Invalid data obtained only one data (Lab a, run 1). This lab performed at retesting andwe accepted data of run 2-4. Therefore, the rate of invalid at this assay is 0.2% (total 1/508, 400 data: 3runs
X 7 labs X 19 chemicals+1 run in 2nd phase study & 108 data; 3 runs X 6 labs X 6 chemicals in 3rd phasestudy ). Based on a comparison of the results from the seven laboratories, the classification of 3 chemicals(No. 12, 16 and 19) should be potentially changed. However, the classifications of the remaining chemicals
were not changed. The variations of these chemicals and No.18 are larger than those of others. The IL-1Į
data changed the classification for No. 5, 16 and 18 at Lab. f (No. 5), Lab. a (No. 16), and Lab. d (No. 18).
The effect of IL-1Į on the reliability of these results is small.
47
67

ENV/JM/MONO(2011)39

9-2. Predictivity
40.
In December 2008, the EU adopted the UN Globally Harmonised System for Classification andLabelling and will implement this by means of the so-called CLP regulation (Regulation EC 1272/2008).
The new EU classification system based on UN GHS (abbreviated here as "GHS-EU") continues to use twocategories to distinguish non-irritant (no-category) from irritant (category 2) substances. However, according
to the new rules for skin irritation classification and labelling, the cut-off score to distinguish between nocategory and category 2 substances was shifted to 2.3 from a value of 2.0 (EU classification system).
Consequently substances with an in vivo score between 2.0 and 2.3 that are considered irritant under the
existing EU classification system will be considered non-irritants under the future GHS-EU classification
system, which does not use the optional UN GHS category 3.
41.
The prediction values of the LabCyte EPI-MODEL 24 skin irritation test when it was evaluated bycell viabilities (MTT) as an indicator, and the GHS-EU classifications are shown in Table 20. The sensitivity,
specificity and accuracy of this prediction model at each laboratory were 75-91.6 %, 69.2-76.9 %, and 7684 %, respectively. These predictivities were similar with each laboratory. The mean and range of prediction
values of the skin irritation test with LabCyte EPI-MODEL 24 when it was only evaluated by MTT as anindicator and the GHS-EU classification are shown in Table 23(A). The mean sensitivity, specificity andaccuracy of this prediction model are 83.3%, 73.1%, and 78.0%, respectively. Some deviations from the
ESAC Performance standard (sensitivity of 80%, specificity of 70% and an accuracy of 75%) that were
specific adaptations for the Japanese model.The effect of IL-1Į on the predictivity was small compared withresults in Tables 21,22, 23 (B) and 23(c).
G
10. Conclusions
42.
Based on the GHS-EU classification, 12 irritants and 13 non-irritants in the ECVAM PerformanceStandards(2007,2009) were tested by the 7 labs using LabCyte EPI-MODEL. The assay demonstrated highreliability within and between laboratories, and acceptable reliability of the positive control (100%) andaccuracy (77.5% overall accuracy, 82.3% overall sensitivity, 72.6% overall specificity) on the MTT assay for
use as a stand-alone assay to distinguish between skin irritants and non-irritants.
This report summarized at JSAAE 1st report and 2nd report on this validation study.
11. Acknowledgement
43.

This validation study has supported by the Health and Labour Sciences Research Grant, Japan.

48
68ENV/JM/MONO(2011)39

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Organisation de Coopération et de Développement Économiques
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07-Dec-2011___________________________________________________________________________________________English - Or. English
ENVIRONMENT DIRECTORATEENV/JM/MONO(2011)44
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THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYPEER REVIEW REPORT OF THE VALIDATION OF THE SKIN IRRITATION TEST USINGLABCYTE EPI-MODEL 24
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Detailed Review Paper on Biodegradability Testing

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Guidance Document on Direct Phototransformation ofChemicals in Water (1997)
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Report of the OECD Workshop on Sharing Information
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Guidance Document for the Conduct of Studies ofOccupational Exposure to Pesticides during Agricultural Application(1997)
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Report of the OECD Workshop on Statistical Analysis of
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Detailed Review Paper on Aquatic Testing Methods for
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Detailed Review Document on Classification Systems forGerm Cell Mutagenicity in OECD Member Countries (1998)
No. 13,
Detailed Review Document on Classification Systems for
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No. 14,
Detailed Review Document on Classification Systems for
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No. 15,
Detailed Review Document on Classification Systems for
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Detailed Review Document on Classification Systems for
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474

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Environmental Exposure Assessment Strategies for
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Report of the OECD Workshop on Improving the Use of
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Guidance Document on the Recognition, Assessment andUse of Clinical Signs as Humane Endpoints for Experimental Animals
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No. 20,
Revised Draft Guidance Document for Neurotoxicity
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No. 21,
Detailed Review Paper: Appraisal of Test Methods for
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Guidance Document for the Performance of Out-door
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Guidance Document on Aquatic Toxicity Testing ofDifficult Substances and Mixtures (2000)
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Guidance Document on Acute Oral Toxicity TestingNo. 25,
Detailed Review Document on Hazard Classification
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Revised Analysis of Responses Received from Member
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Guidance Document on the Use of the Harmonised
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Guidance Document for the Conduct of Skin Absorption
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Guidance Document on Transformation/Dissolution ofMetals and Metal Compounds in Aqueous Media (2001)
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Detailed Review Paper on Non-Genotoxic Carcinogens Detection:
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Guidance Notes for Analysis and Evaluation of RepeatDose Toxicity Studies (2000)
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No. 33,Harmonised Integrated Classification System for Human
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Guidance Document on the Development, Validation andRegulatory Acceptance of New and Updated Internationally Acceptable
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Guidance notes for analysis and evaluation of chronic
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Report of the OECD/UNEP Workshop on the use ofMultimedia Models for estimating overall Environmental Persistence
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Detailed Review Document on Classification Systems for
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Detailed Background Review of the Uterotrophic Assay
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Guidance Document on Acute Inhalation Toxicity Testing(in preparation)
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Detailed Review Document on Classification in OECD
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Guidance Document on Reporting Summary Information
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No. 43,
Guidance Document on
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Guidance Document on the Use of Multimedia Models for
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Detailed Review Paper on Amphibian Metamorphosis
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Detailed Review Paper on Fish Screening Assays for the
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No. 48,New Chemical Assessment Comparisons and Implications
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No. 49,
Report from the Expert Group on (Quantitative)
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No. 50,(2005)

Report of the OECD/IPCS Workshop on Toxicogenomics

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No. 52,Comparison of emission estimation methods used in
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Guidance Document on Simulated Freshwater Lentic
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No. 54,Current Approaches in the Statistical Analysis of
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Detailed Review Paper on Aquatic Arthropods in Life Cycle
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Detailed Review Paper on Thyroid Hormone Disruption
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Report of the Validation of the Updated Test Guideline
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Report of the Initial Work Towards the Validation of the
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Report of the Validation of the 21-Day Fish Screening
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Final OECD Report of the Initial Work Towards the
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Guidance Document on the Definition of Residue (2006)

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Guidance Document on Overview of Residue Chemistry
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OECD Report of the Initial Work Towards the Validationof the Rodent Uterotrophic Assay - Phase 1 (2006)
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OECD Report of the Validation of the Rodent
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Additional data supporting the Test Guideline on the
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Summary Report of the Uterotrophic Bioassay Peer
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Guidance Document on the Validation of (Quantitative)
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Guidance Document on Pesticide Residue Analytical
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Detailed Review Paper for Avian Two-generation Toxicity
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Guidance Document on the Honey Bee (Apis Mellifera L.)
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No. 76,
Final Report of the Validation of the Amphibian
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Final Report of the Validation of the Amphibian
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Final Report of the Validation of the 21-day Fish
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Guidance on Grouping of Chemicals (2007)

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Summary Report of the Validation Peer Review for theUpdated Test Guideline 407, and Agreement of the Working Group of
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ENV/JM/MONO(2011)44
No. 88,Workshop on Integrated Approaches to Testing andAssessment (2008)
No. 89,Retrospective Performance Assessment of the Test
Guideline 426 on Developmental Neurotoxicity (2008)
No.90,
Background Review
Hershberger Bioassay (2008)

Documentonthe

RodentNo. 91,
Report of the Validation of the Amphibian Metamorphosis
Assay (Phase 3) (2008)
No. 92,
Report of the Validation Peer Review for the Amphibian
Metamorphosis Assay and Agreement of the Working Group of the
National Coordinators of the Test Guidelines Programme on the
Follow-Up of this Report (2008)
No. 93,
Report of the Validation of an Enhancement of OECD TG
211: Daphnia Magna Reproduction Test (2008)
No. 94,
Report of the Validation Peer Review for the 21-Day Fish
Endocrine Screening Assay and Agreement of the Working Group of
the National Coordinators of the Test Guidelines Programme on the
Follow-up of this Report (2008)
No. 95,

Detailed Review Paper on Fish Life-Cycle Tests (2008)

No.96,
Guidance Document on Magnitude of Pesticide Residues
in Processed Commodities (2008)
No.97,
Detailed Review Paper on the use of Metabolising
Systems for In Vitro Testing of Endocrine Disruptors (2008)
No. 98,
Considerations Regarding Applicability of the Guidanceon Transformation/Dissolution of Metals Compounds in Aqueous
Media (Transformation/Dissolution Protocol) (2008)
No. 99,Comparison between OECD Test Guidelines and ISO
Standards in the Areas of Ecotoxicology and Health Effects (2008)
No. 100,(2009)

Report of the Second Survey on Available Omics Tools

No. 101, Report of the Workshop on Structural Alerts for the OECD
(Q)SAR Application Toolbox, 15-16 May 2008, Utrecht, theNetherlands (2009)
No. 102, Guidance Document for using the OECD (Q)SAR
Application Toolbox to Develop Chemical Categories According to theOECD Guidance on Grouping of Chemicals (2009)10
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ENV/JM/MONO(2011)44
No. 103, Detailed Review Paper on Transgenic Rodent Mutation
Assays (2009)
No. 104, Performance Assessment: Comparsion of 403 and CxTProtocols via Simulation and for Selected Real Data Sets (2009)
No. 105, Report on Biostatistical Performance Assessment of thedraft TG 436 Acute Toxic Class Testing Method for Acute Inhalation
Toxicity (2009)
No. 106, Guidance Document for Histologic Evaluation of Endocrine
and Reproductive Test in Rodents (2009)
No. 107, Preservative treated wood to the environment for wood heldin storage after treatment and for wooden commodities that are notcover and are not in contact with ground. (2009)
No. 108, Report of the validation of the Hershberger Bioassay(weanling model) (2009)
No. 109, Literature review on the 21-Day Fish Assay and the Fish
Short-Term Reproduction Assay (2009)
No. 110, Report of the validation peer review for the weanling
Hershberger Bioassay and agreement of the working of national
coordinators of the test guidelines programme on the follow-up of thisreport (2009)
No. 111, Report of the Expert Consultation to Evaluate an Estrogen
Receptor Binding Affinity Model for Hazard Identification (2009)
No. 112, The 2007 OECD List of High Production Volume Chemicals(2009)
No. 113, Report of The Focus Session on Current and Forthcoming
Approaches for Chemical Safety and Animal Welfare (2010)
No. 114, Performance Assessment of Different Cytotoxic andCytostatic Measures for the In Vitro Micronucleus Test (MNVIT):
Summary of results in the collaborative trial (2010)
No. 115, Guidance Document on the Weanling Hershberger Bioassay
in Rats: A Short-term Screening Assay for (Anti) Androgenic Properties(2009)
No. 116, Guidance Document on the Design and Conduct of Chronic
Toxicity and Carcinogenicity Studies, Supporting TG 451, 452 and 453(2010)
No. 117, Guidance Document 117 on the Current Implementation ofInternal Triggers in Test Guideline 443 for an Extended One

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Generation Reproductive Toxicity Study, in the United States andCanada (2011)
No. 118, Workshop Report on OECD Countries Activities Regarding
Testing, Assessment and Management of Endocrine Disrupters Part I
and Part II (2010)
No. 119, Classification and Labelling of chemicals according to the
UN Globally Harmonized System: Outcome of the Analysis ofClassification of Selected Chemicals listed in Annex III of theRotterdam Convention (2010)
No. 120, Part 1: Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism-based
Structural Alerts for the Identification of DNA Binding Chemicals
No. 120, Part 2: Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism-based
Structural Alerts for the Identification of DNA Binding Chemicals
No. 121, Detailed review paper (DRP) on Molluscs life-cycle Toxicity
Testing (2010)
No. 122, Guidance Document on the determination of the Toxicity ofa Test Chemical to the Dung Beetle Aphodius Constans (2010)
No. 123, Guidance Document on the Diagnosis of Endocrine-related
Histopathology in Fish Gonads (2010)
No. 124,(2010)

Guidance for the Derivation of an Acute Reference DoseNo. 125, Guidance Document on Histopathology for Inhalation
Toxicity Studies, Supporting TG 412 (Subacute Inhalation Toxicity: 28Day) and TG 413 (Subchronic Inhalation Toxicity: 90-Day) (2010)
No. 126, Short Guidance on the Threshold approach for Acute Fish
Toxicity (2010)
No. 127, Peer review report of the validation of the 21-day
androgenised female stickleback screening assay (2010)
No. 128, Validation Report of the 21-day Androgenised Female
Stickleback Screening Assay (2010)
No. 129, Guidance Document on using Cytotoxicity Tests toEstimate Starting Doses for Acute Oral Systemic Toxicity Tests(2010)
No. 131, Report of the Test Method Validation of Avian Acute Oral
Toxicity Test (OECD test guideline 223) (2010)1282

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No. 132, Report of the Multi-Laboratory Validation of the H295R
Steroidogenesis Assay to Identify Modulators (2010)
No.133, Peer Review Report for the H295R Cell-Based Assay for
Steroidogenesis (2010)
No.134, Report of the Validation of a Soil Bioaccumulation Test with
Terrestrial Oligochaetes by an International ring test (2010)
No.135, Detailed Review Paper on Environmental Endocrine
Disruptor Screening: The use of Estrogen and Androgen Receptor
Binding and Transactivation Assays in Fish (2010)
No. 136, Validation Report of t he Chironomid Full Life-Cycle
Toxicity Test (2010)
No. 137, Explanatory Background Document to the OECD Test
Guideline On In Vitro Skin Irritation Testing (2010)
No. 138, Report of the Workshop on Using Mechanistic Informationin Forming Chemical Categories (2011)
No. 139, Report of the Expert Consultation on Scientific andRegulatory Evaluation of Organic Chemistry Mechanism Based
Structural Alerts for the Identification of Protein-binding Chemicals(2011)
No. 141, Report of the Phase 1 of the Validation of the Fish Sexual
Development Test for the Detection of Endocrine Active Substances(2011)
No. 142, Report of the Phase 2 of the Validation of the Fish Sexual
Development Test for the Detection of Endocrine Active Substances(2011)
No. 143, Peer Review Report for the Validation of the Fish Sexual
Development Test and Agreement of the Working Group of National
Co-ordinators of the Test Guideline Programme on the Follow-up of
the Peer Review (2011)
No. 144,

Validation Report for the Acute Chironomid Assay (2011)

No. 145, Transgenic Rodent Somatic and Germ Cell Gene Mutation
Assay: Retrospective Performance Assessment (2011)
No. 148, Guidance Document on the Androngenised Female
Stickleback Screen (2011)
No. 152, Case Study: Assessment of an Extended Chemical Category,
the Short-chain Methacrylates, Targeted on Bioaccumulation (2011)1383

ENV/JM/MONO(2011)44
No. 153, Guidance Document for the Derivation of an Acute
Reference Concentration (Arfc) (2011)
No. 155, Peer Review Report of the validation of the “Skin Irritation
Test using LabCyte EPI-MODEL24 (2011)© OECD 2011
Applications for permission to reproduce or translate all or part of thismaterial should be made to: Head of Publications Service,
RIGHTS@oecd.org. OECD, 2 rue André-Pascal, 75775 Paris Cedex
16, France

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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
the OECD’s work is carried out by more than 200 specialised committees and working groups composedof member country delegates. Observers from several countries with special status at the OECD, and frominterested international organisations, attend many of the OECD’s workshops and other meetings.
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 ten different series:
Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides andBiocides; 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 (www.oecd.org/ehs/).

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, UNEP, UNIDO, UNITAR, WHO, World Bank and OECD.
UNDP is an observer. The purpose of the IOMC is to promote co-ordination of the policies and activities
pursued by the Participating Organisations, jointly or separately, to achieve the sound management ofchemicals in relation to human health and the environment.

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This publication is available electronically, at no charge.
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/ehs/)
or contact:
OECD Environment Directorate,
Environment, Health and Safety Division
2 rue André-Pascal
75775 Paris Cedex 16
FranceFax: (33-1) 44 30 61 80 E-mail: ehscont@oecd.org

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FOREWORD

This document presents the Peer Review Report of the validation of the “Skin Irritation Test using LabCyte
EPI-MODEL24”, as a “me-too” development following the Performance Standards of Test Guideline 439:
In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method, including four annexes. Additional
information was generated after the peer review because the first validation study was on-going when
OECD experts were discussing draft Performance Standards (PS) for TG 439.
The LabCyte test method has been considered as similar to the Reconstructed Human Epidermis Test
Method that is the basis of TG 439, and has therefore been subjected to an additional validation study: a“me-too” validation study based on the PS of TG 439. This document also presents:x

Appendix 1: JaCVAM Re-analysis of the initial validation data according to the
Performance Standards of TG 439

x

Appendix 2: Background Document for a modified skin irritation test using LabCyte EPIMODEL24x

Appendix 3: Standard Operating Procedure for the Skin irritation test using the LabCyte
EPI-MODEL24 for the additional validation studyxAppendix 4: additional validation study: “me-too” validation study according to peer
review report.

Finally, the report includes the agreement of the Working Group of National Coordinators of the Test
Guidelines Programme (WNT) on the follow-up to the peer review.
The project for developing a Test Guideline for the in vitro epidermal model LabCyte EPIMODEL24 toassess skin irritation was proposed by Japan and included in the work plan of the Test Guidelines
Programme in 2009. A validation report was published as No. 147 in the Series on Testing andAssessment. An OECD Peer Review was performed in December 2009 and the final Peer Review Panel(PRP) Report was available on 17th of March 2010.
This document was endorsed by the WNT at its meeting held on 12-14 April 2011. The Joint Meeting of
the Chemicals Committee and Working Party on Chemicals, Pesticides and Biotechnology (Joint Meeting)agreed to its declassification on 5 October 2011.
This document is published under the responsibility of the Joint Meeting.

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Agreement of the Working Group of National Coordinators of the Test Guidelines Programme on
the follow-up to the Peer Review Report

The peer review report of the validation of the modified skin irritation test method using the LabCyte EPIModel24 as a TG 439 “me-too” test was submitted to the Working Group of National Coordinators of the
Test Guidelines Programme (WNT) at its meeting held on 12-14 April 2011, for endorsement.
Consideringx

the issues raised in the peer review report regarding the performance of the assay including within
and between laboratory reproducibility, variability between
replicates and most notably, themisclassification of the strong irritant reference chemical, 1-bromohexane,

xAdditional information provided by Japan to address the issues raised in thereport,peer

review

The WNT agreed that before adding the “me too test” to TG 439, the Expert Group on Skin Irritation andCorrosion should review all existing data of the LabCyte test method, and submit a recommendation for
WNT approval of the test method as a “me-too” test method to TG 439, or for further work if necessary. In
case the Expert Group agrees on that the LabCyte test method is validated and should be added to TG 439,
this approval would be requested from the WNT by a written procedure.

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SUMMARY REPORT OF THE PEER REVIEW PANELON LABCYTE EPI-MODEL 24 IN VITRO TEST METHODFOR THE ASSESSMENT OF SKIN IRRITATION POTENTIAL OF CHEMICALS

prepared forEnvironment Directorate
Organisation for Economic Co-operation and Development Paris

March 17, 2010

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PREAMBLE
This document presents the summary report of the assessment by an independent Peer Review Panel on the
LabCyte EPI-MODEL 24 in vitro test method for the assessment of skin irritation potential of chemicals.
Until today, for regulatory purposes the skin irritation potential of chemicals has been investigated by the
Draize skin test. In this in vivo experiment, the chemical is applied for usually four hours to the skin of
rabbits. Based on a scoring of the two endpoints erythema and oedema formation and the reversibility ofsuch effects, the skin irritation potential is assessed.
However, the advancement of in vitro methods has brought forward reconstructed human epidermis
models. These models are based on non-transformed human-derived epidermal keratinocytes. The
keratinocytes are cultured to form a multilayered, highly differentiated model of the human epidermis.
After topical exposure to a neat test chemical, cell viability is measured by the amount MTT [(3-4,5dimethyl thiazole 2-yl) 2,5-diphenyltetrazoliumbromide] being reduced by dehydrogenase. The relative
reduction of viability is then related to the skin irritation potential of the chemical.
After several years of evaluation and optimisation, two models underwent formal validation by theEuropean Centre for the Validation of Alternative Methods (ECVAM) resulting in a statement on thescientific validity of the tests by ECVAM Scientific Advisory Board (ESAC). As a consequence, theOrganisation for Economic Co-operation and Development (OECD) is currently in the process of drafting
the respective OECD test guideline ‘In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test
Method’.
Anticipating that in future further reconstructed human epidermis models will be developed, the draft test
guideline includes the annex ‘Performance standards for assessment of proposed similar or modified invitro reconstructed human epidermis (RhE) test methods for skin irritation’. These performance standards
can be used to assess other analogous test methods (colloquially referred to as “me-too” tests) that are
based on similar scientific principles and measure or predict the same biological or toxic effect.
The LabCyte EPI-MODEL 24 in vitro test method is considered to potentially be such a test method, towhich the performance standards apply. The method was validated in a study employing several Japaneselaboratories. The documentation of this study together with some additional information has been provided
to the OECD and constitutes the basis for this peer review.

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Summary Report of the Peer Review Panelon the LabCyte EPI-MODEL 24 in vitro test method for the assessment of skin irritation potential ofchemicals

The peer review process
1. The Peer Review Panel (Panel) was constituted in December 2009, to provide an independent review ofthe validation of the LabCyte EPI-MODEL 24 in vitro test method for the assessment of skin irritation
potential of chemicals. The test method has been considered as a similar Reconstructed Human Epidermis(RhE) Test Method and underwent therefore a validation study referring to the respective performancestandards. In this respect it has to be noted that the validation of the LabCyte EPI-MODEL 24 took place
while the OECD test guideline ‘In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test
Method’ including the annex on performance standards was still in the drafting process. The work of the
Panel was coordinated by a panel chair contracted by the OECD. In addition to experts invited by the
Secretariat, potential Panel members were nominated by the Working Group of the National Coordinatorsof the Test Guidelines Programme (WNT) and then approached by the chair. From the originally fiveassigned panel members, one resigned from the assignment in January 2010, but was immediately
replaced. The members of the Panel are listed in Annex 1.
Furthermore, an independent contact person who is familiar with the LabCyte EPI-MODEL 24 wasnominated to support the panel in case of open issues needing clarification.
2. The Panel was asked to evaluate the data collected on the test method, and to answer specific charge
questions. These questions were proposed by the panel chair and agreed by the (OECD). Panel members
were asked to base their review on nine documents, which have been provided by the OECD. These
documents contained information relevant to the validation of the LabCyte EPI-MODEL 24.
As background information, they were also provided with the three following documents:
OECD
Guidance
Documentonthe
Validation
andInternational
Acceptance
of
New
or
Updated
MethodsforHazardAssessment,
Serieson Testing and Assessment, Number 34, 2005
(last access on March 08, 2010, under
http://www.olis.oecd.org/olis/2005doc.nsf/LinkTo/NT00002EAE/$FILE/JT00188291.pdf)- OECD GUIDELINE FOR THE TESTING OF CHEMICALS (DRAFT PROPOSAL FOR A NEW
GUIDELINE) - In Vitro Skin Irritation: Reconstructed Human Epidermis (RhE) Test Method(last access on March 08, 2010, under http://www.oecd.org/dataoecd/1/59/43664841.pdf)- European Commission: Explanatory Background Document to the OECD
Draft Test Guideline on in vitro Skin Irritation Testing(last access on March 08, 2010, under http://ecvam.jrc.ec.europa.eu/ft_doc/ECVAM-BfROECDv3.0_OECD_post_ECM2c.pdf)

Two further documents, one describing the content of the review documents, and the other providing acomplete version of Table 16 of the file ‘D3 LabCyte_skin_irritation_testprotocol_ver_7.2 NC’, wereadded by the PRP chair to facilitate the review. All documents, identified by their file names as provided to
the PRP, are listed in Annex 2.
The charge to the Panel was to assess to what extent the eight OECD validation criteria set out in theOECD Guidance Document had been met and to evaluate whether the test method complies with theperformance standards of the draft OECD Test Guideline on Skin Irritation. It has to be noted that these21

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two sets of charge questions are not to be considered independent as they address similar aspects. Alleleven charge questions are listed in Annex 3. A summary of the Panel’s responses to the individualquestions is presented in paragraphs 4 to 40 below. For transparency, the individual comments from thePanel members are provided in Annex 4.
3. During the evaluation process, the Panel held two teleconferences which were organised and coordinatedby the chair. Subsequently, each Panel member provided written responses on the charge questions to the
PRP chair by February 15, 2010. Based on these responses, a draft report was compiled by the chair andprovided to the PRP for review and comments (February 22, 2010). In this draft all aspects raised by atleast one reviewer were included. Furthermore, it contained some clarifications on specific issues added by
the chair. The PRP commented on the draft report until March 02, 2010. Accounting for this feedback andresolving remaining open issues, the final report was drafted by the chair and send to the PRP for approvalon March 09, 2010. This report presents the resulting approved responses of the Panel to each of the charge
questions.
General Panel responses
4. The Panel stated that the presentation of the review information could have been more structured andfocused, e.g. by referencing to the performance standards and/or to the charge questions. Therefore, it
proposes to facilitate future reviews by aligning the presentation of information with the review charge
questions.
All information in the review documents on IL-1Į has not been considered by the Panel because this endpoint has not been previously declared as formally validated and there are no performance standards
available
Panel responses to the charge questions - part I: The eight OECD principles and criteria for test
method validation
5. The PRP reached consensus regarding the charge questions relating to the eight OECD principles andcriteria for test method validation.
Charge question 1: A rationale for the test method should be available, including a clear statement ofscientific need and regulatory purpose.
6. The Panel agreed that this criterion had been fully met. The rationale for the test method is clearly statedwith regard to the scientific basis and regulatory purpose. In addition, as the LabCyte EPI-MODEL 24
refers to performance standards of presumably similar methods, which are in the process of OECD
adoption, the general need and regulatory purpose as defined in the draft guideline applies as well.
Charge question 2: The relationship between the test method endpoint(s) and the biological effect and to
the toxicity of interest should be addressed, describing limitations of the test methods.
7. The Panel agreed that this criterion had been partly met.
8. In particular, it has not been met with regard to the description of limitations. In the provided SOP this
topic is only touched, but not sufficiently covered. For example, substances sticking to the tissue might
create problems. Respective information might be deductible from the literature of the validated test
methods, e.g. from paragraph 7 of the respective OECD draft test guideline.

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9. Although, in general acceptable, the relationship of the method endpoint and the biological effect could
be described in more detail. As a potential me-too method this relationship has been described earlier forthe already validated methods and does thus apply also to the LabCyte EPI-MODEL 24. However, additionof information as given in paragraph 11 of the OECD Draft guideline (Version 7.6) would be helpful.
Charge question 3: A detailed protocol for the test method should be available
10. The Panel agreed that this criterion had been partly met.
11. In particular, a measure of variability between tissue replicates has not been defined.
12. Furthermore, as already addressed under paragraph 8, limitations are not sufficiently covered.
13. Particular protocol changes have been proposed by the panel. The most important suggestion was toexplain the prediction model in more detail. Minor suggestions were- to better define the storage temperature of the MTT solution and the positive control substance- to better and more objectively define changes considered significant for chemicals that stain the tissue- to change the instruction ‘...must be repeated three times...’ to ‘...must be performed three times in total...’- to include instructions for the case, when only two tissues provide measurements, e.g. when one tissue isdamagedCharge question 4: Within- and between-laboratory reproducibility of the test method should bedemonstrated
14. The Panel agreed that this criterion had been partly met.
15. Most importantly, within- and between-laboratory reproducibility have not been calculated. All
required data for the calculation have been provided, but appropriate statistical analysis and documentation
is missing and should be included. In particular, these should be addressed by an appropriate measure ofvariability between runs viabilities, e.g. the standard deviation, and the concordance of classifications
based on run. Results should be discussed, especially with regard to less reproducible chemicals
16. Regarding intra-assay reproducibility, a measure of variability between tissues has not been defined,
nor calculated (see also paragraph 11.)
Charge question 5: Demonstration of the test method’s performance should be based on testing ofrepresentative, preferably coded reference chemicals
17. The Panel agreed that this criterion had been met.
18. All chemicals currently proposed by the OECD draft test guideline have been tested, with one
acceptable exception, in a coded manner.
Charge question 6: The performance of test methods should have been evaluated in relation to existing
relevant toxicity data as well as information from the relevant target species.
19. The Panel agreed that this criterion had been met.

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20. As only chemicals which have been included in other validation activities leading to the OECD draft
test guideline, were tested, the relation to existing toxicity data from the relevant target species wasestablished.
Charge question 7: All data supporting the assessment of the validity of the test method should beavailable for expert review
21. The Panel agreed that this criterion had been partly met.
22. As mentioned before, within- and between-laboratory reproducibility have not been calculated anddocumented (see paragraph 15).
23. Regarding intra-assay reproducibility, a measure of variability between tissues has not been defined,
nor calculated (see also paragraph 11).
Charge question 8: Ideally, all data supporting the validity of a test method should have been obtainedin accordance with the principles of Good Laboratory Practice (GLP)
24. The Panel agreed that this criterion had been partly met. It has to be noted that this optional criterion
initially led to different individual interpretation by the PRP members.
25. In general, the conduct of the study in the spirit of GLP is acceptable. For example, also in the original
validation study, which led to the draft OECD test guideline, not all laboratories were GLP compliant.
However, the panel agreed that the adherence to GLP principles could have been described in more detail.
If available, documentation on this issue generated in the frame of the validation study of the LabCyteEPIMODEL 24 should be provided.
Panel responses to the charge questions - part II: Performance Standards of the draft Test Guideline
on Skin Irritation
26. The PRP reached consensus regarding the charge questions relating to the Performance Standards ofthe draft Test Guideline on Skin Irritation.
Charge question 9: Adherence to the essential test method components should be demonstrated
27. The Panel agreed that this criterion had been partly met. This charge question refers to the test method
components as specified in the draft OECD test guideline. The respective components will be addressedindividually.
28. Functional conditions – Viability: An upper acceptance limit for the negative control tissues, e.g.
defined by a maximum optical density (OD), is missing. Although the data base for defining this might besmall, it should nevertheless be defined if possible. Furthermore, it has not been defined what is considered
as an acceptable variation of viabilities from replicate tissues.
29. Functional conditions – Barrier function: Further data on lipid composition could be added if available.
30. Functional conditions – Morphology: Acceptance criteria required for batch acceptance should be
defined.

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31. Functional conditions – Reproducibility: A measure of variability between tissues has not been defined,
nor calculated (see also paragraph 11).
32. Functional conditions – Quality control (QC): It is not entirely evident how the model producer intends
to ensure and demonstrate that each batch of the RhE model used meets defined production release criteria.
Charge question 10: Reliability and accuracy should be demonstrated by using at least therecommended reference chemicals
33. The Panel agreed that this criterion had been met.
Charge question 11: The test should have been assessed based on the defined reliability and accuracy
values
34. The Panel agreed that this criterion had not been met. When addressing this question, the panel agreed
to exclude data of ‘Lab e’ from analysis according to data ‘Rule 2’ of paragraph 14 of annex 2 of theOECD draft test guideline, which addresses the performance standards. Therefore, the data of ‘Lab e’ havenot been considered.
35. Most importantly, the specific restriction of the performance standards that only two in vivo Category 2
substances, 1-decanol and di-n-propyl disulphide, may be misclassified as No Category by more than one
participating laboratory, has not been met. In particular, 1-bromohexane, Category 2, has been
misclassified as No Category by five of six participating laboratories (excluding ‘Lab e’ data). In addition,
but without consequence, butyl methacrylate, Category 2, has been misclassified as No Category in one ofsix laboratories (excluding ‘Lab e’ data, which also misclassified butyl methacrylate). As a potential
solution optimisation of the protocol either regarding the application time (prolongation beyond 30
minutes, with the risk of a decrease in specificity and overall accuracy) or regarding the test substanceapplication (re-spreading of the material during application) has been proposed.
36. The panel agreed that the within- and between laboratory reproducibility has not been assessed withregard to the requirements of the performance standards. A detailed extended evaluation of these
reproducibility values addressing the performance standards requirements should be presented (see also
paragraph 15).
37. The panel anticipated that one result of a detailed analysis of the within- and between laboratory
reproducibility would be that two laboratories might have a within-laboratory reproducibility of lower than90%. This would not comply with the requirement of paragraph 14 of annex 2 of the OECD draft test
guideline that ‘...within-laboratory variability should show a concordance of classifications (GHS Category
2/No Category) obtained in different, independent test runs of the 20 Reference Chemicals within one
single laboratory equal or higher () than 90%’.
38. In contrast to the performance standards, the median viability instead of the mean viability, as requiredin ‘Rule 2’ of paragraph 9 of annex 2 of the OECD draft test guideline, which addresses the performancestandards, has been used for deriving a final classification for a complete run sequence of a given
laboratory. The panel expects that this will result in only minor differences in results, but suggests to reanalyse the data and document the results accordingly.
39. Regarding the predictive values, the panel agreed that the requirements, i.e. sensitivity  80%,
specificity  70% and accuracy  75%, were met, when considering all 25 tested chemicals. It was noted,

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however, that the overall sensitivity would be  80% when considering only the 19 chemicals tested in thesecond phase of the study.

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Recommendations
40. The Panel agrees that this report provides a summary of their views on the status of the validation of
the LabCyte EPI-MODEL 24 in vitro test method for the assessment of skin irritation potential ofchemicals, as detailed in the responses to the questions posed to the Panel and based on the information
related to the test method validation provided to the Panel.
41. The report of the Panel, along with the provided review documents on the LabCyte EPI-MODEL 24
should form the basis for decisions on whether the validation meets the OECD principles for validation andthe performance standards of the draft OECD Test Guideline ‘In Vitro Skin Irritation: Reconstructed
Human Epidermis (RhE) Test Method’. The Panel recommends that the OECD considers the Panel reportas guidance for recommending additional work required to fully meet all OECD principles andperformance standards.
42. Future work should focus especially on the following aspects. Most importantly, the issue of
misclassifying 1-bromohexane should be resolved. Furthermore, an extensive analysis of the within- andbetween reproducibility referring to the performance standards of the draft OECD Test Guideline should
be carried out and appropriately documented. It is also recommended to assess variability between
replicate tissues and to define a respective acceptance criterion. In order to comply better with theperformance standards, analyses using the mean instead of the median for deriving a final classification for
a complete run sequence of a given laboratory should be carried out. Finally, appropriate documentation
describing and demonstrating the adherence to GLP principles should be provided.

27

97

ENV/JM/MONO(2011)44

ANNEX 1
MEMBERS OF THE PEER REVIEW PANEL

Panel member
Craig Blackstock
Penny Jones
Kristina Kejlová
Albrecht PothKlaus Rudolf Schröder

98

Affiliation
Charles River, Edinburgh, UK
Unilever, Bedford, UK
National Institute of Public Health, Prague, Czech
Republic
Harlan Cytotest Cell Research GmbH, Rossdorf,
Germany
BioMed- zet Life Science GmbH, Linz, Austria (until
31.12.2009 Henkel AG & Co. KGaA, Germany)28

ENV/JM/MONO(2011)44

ANNEX 2
List of all documents provided to the PRP(identified by file name)• A1 _J_ToxicolSci(2009).pdf• A4 LabCyte_skin_irritation_testprotocol_ver_6.01 NC.pdf• A6 Draft 2nd report LabCyte0907____.pdf• Appendix7.pdf• D1 A suitable exposure time for SIT with LabCyte EPI-MODEL24.pdf• D2 LabCyte EPI-MODEL background data document.pdf• D3 LabCyte_skin_irritation_testprotocol_ver_7.2 NC.pdf• LabCyte report 091008.pdf• D3 Table 16.pdf• List of J-TEC reference document.pdf• Content of review documents.docx• 3rd TG SKIN IRR_V.7.6_9 Sept_09_Clean.pdf• GD No.34.pdf• SKIN IRR BACKGROUND DOC (V.3 0) 14 August 2009.pdf

2999ENV/JM/MONO(2011)44

ANNEX 3Charge questions

PRP Charge questions - part I:
The eight OECD principles and criteria for test method validation
1. A rationale for the test method should be available, including a clear statement of scientific need andregulatory purpose.
2. The relationship between the test method endpoint(s) and the biological effect and to the toxicity ofinterest should be addressed, describing limitations of the test methods.
3. A detailed protocol for the test method should be available.
4. Within- and between-laboratory reproducibility of the test method should be demonstrated.
5. Demonstration of the test method’s performance should be based on testing of representative, preferably
coded reference chemicals.
6. The performance of test methods should have been evaluated in relation to existing relevant toxicity data
as well as information from the relevant target species.
7. All data supporting the assessment of the validity of the test method should be available for expert
review.
8. Ideally, all data supporting the validity of a test method should have been obtained in accordance withthe principles of Good Laboratory Practice (GLP)PRP Charge questions - part II:
Performance Standards of the draft Test Guideline on Skin Irritation
9. Adherence to the essential test method components should be demonstrated (see also question 3).
10. Reliability and accuracy should be demonstrated by using at least the recommended reference
chemicals (see also question 5).
11. The test should have been assessed based on the defined reliability and accuracy values (see alsoquestions 4 and 6

100

30

101

1. Rationale for the test
methodVALIDATION CRITERIAThe overall impression of the Panel is that the LabCyte EPI-MODEL 24 has been properly evaluated invalidation activities. Consensus has been reached for all charge questions.

General comments

31

The Panel agreed that this criterion has been met.

The Panel agreed that the rationale for the test method is clearly stated, while the scientific basis and theregulatory purpose are well described. The test is intended to replace the Draize skin irritation test for
regulatory purposes as currently defined in OECD Test Guideline 404.

Regarding the additional three charge questions referring to the performance standards of the draft Test
Guideline on Skin Irritation, the Panel members agreed that the specific restriction that only two in vivo
Category 2 substances, 1-decanol and di-n-propyl disulphide, may be misclassified as ‘No Category’ by morethan one participating laboratory, has not been met. In particular, 1-bromohexane, Category 2, has been
misclassified as ‘No Category’ by five of six participating laboratories.

Regarding the eight OECD principles and criteria for test method validation, several issues still need to beaddressed more thoroughly. The major criticism from all Panel members was that the within-and betweenlaboratory reproducibility has not been properly analysed and documented. Furthermore, a measurement forvariability between identically treated tissues needs still to be defined together with a respective acceptancecriterion.

COMMENTS AND RECOMMENDATIONSCOLLATED COMMENTS FROM THE PEER REVIEW PANEL
ON THELABCYTE EPI-MODEL24 IN VITRO TEST METHODFOR THE ASSESSMENT OF SKIN IRRITATION POTENTIAL OF CHEMICALSISSUESANNEX 4ENV/JM/MONO(2011)44

102

4. Demonstration of withinand between-laboratory
reproducibility

3. Availability of a detailed
test method protocol

2. Relationship between the test
method endpoint(s) and thebiological effect and to thetoxicity of interest

ENV/JM/MONO(2011)44

32

The Panel agreed that this criterion had been partly met.

The Panel agreed that the presented information on within- and between-laboratory reproducibility is not
sufficient. As the generated data would allow a comprehensive evaluation of this aspect, the panel recommends
to conduct and document a detailed statistical analysis of within- and between-laboratory reproducibility,
including an evaluation of variability between replicate tissues.

The Panel agreed that this criterion had been partly met.

The panel evaluated the provided protocol (version 7.2). Although largely appropriate, the panel recommendsto:- elaborate on the test method limitations- include a measurement of variability between replicate tissues including a respective
acceptance criterion- explain the prediction model in more detail- better define the storage temperature of the MTT solution and the positive control
substance- better and more objectively define changes considered significant for chemicals that stain
the tissue- change the instruction ‘...must be repeated three times...’ to ‘...must be performed threetimes in total...’- include instructions for the case, when only two tissues provide measurements, e.g. when
one tissue is damaged.

The Panel agreed that this criterion had been partly met.

The panel agreed that the relevance of using chemically induced cytotoxicity measured by MTT to a humanskin model to assess skin irritation potential has been well established already in the evaluation of the models
leading to draft OECD Test Guideline. However, it is recommended to describe this more explicitly, e.g. as
done in the draft guideline (Version 7.6) in paragraph 11.
Furthermore, the panel agreed that the limitations of the model, e.g. regarding certain properties of test
chemicals, should be described in more detail as for example done in paragraph 7 the draft guideline (Version
7.6).

103

9. Test method components

PERFORMANCE STANDARDS

8. GLP (ideally)

7. Availability of all relevant
data for expert review

The panel agreed that the data from the test method have been compared with relevant toxicity data, namely
with Draize skin irritation test data which it is designed to replace. Furthermore, all chemicals tested have beenused previously in validation studies of similar models.

6. Test methods evaluation
related to existing
relevant toxicity data

33

The panel agreed that some detailed information to comprehensively address the test method components was
missing.
Regarding ‘Functional conditions – Viability’, an upper acceptance limit for the negative control tissues, e.g.

The Panel agreed that this criterion had been partly met.

Initially, the optional nature of this criterion led to diverging interpretations. While some reviewers considered
the statement that the validation study was carried out in the spirit of GLP as sufficient to fulfil this criterion,
other reviewers requested evidence substantiating this statement and considered the criterion not fulfilled. Aftera discussion of this issue during the review process of this report, the understanding and interpretation of thiscriterion could be harmonised. As a result, the Panel agreed that the carrying out the validation in the spirit of
GLP is acceptable. Nevertheless, the Panel recommended to better document how this was established and toprovide respective documentation, if available.

The Panel agreed that this criterion has been met.

The panel assumed that all relevant data has been provided. Furthermore, it noted that the standard protocol isavailable to the public.

The Panel agreed that this criterion has been met.

The Panel agreed that this criterion has been met.

5. Demonstration of the test
method’s performancebased on testing ofrepresentative reference
chemicals

The Panel agreed that the test chemicals, which were tested coded, was representative and appropriate for thedemonstration of the test method’s performance. In particular, all reference chemicals currently proposed in thedraft OECD Test Guideline have been tested with the acceptable exception of tetrachloroethylene.

ENV/JM/MONO(2011)44

104

11. Test assessment basedon defined reliability andaccuracy values

10. Demonstration ofreliability and relevanceusing reference
chemicals

ENV/JM/MONO(2011)44

34

The panel agreed on several crucial issues. Most importantly and the main reason for failing this criterion, thespecific requirement of paragraph 12, Annex 2, of the draft OECD Test guideline (‘...further specific restriction
applies to the sensitivity of the proposed in vitro test method inasmuch as only two in vivo Category 2
substances, 1-decanol and di-n-propyl disulphide, may be misclassified as No Category by more than one
participating laboratory.’) was not met. The chemical 1-bromohexane, which is an in vivo Category 2
substance, has been classified as ‘negative’ by five out of six laboratories in the validation exercise (excluding
laboratory e).
Furthermore, within-and between laboratory reproducibility have not been appropriately evaluated. In thisregard, the Panel anticipated that, while the between-laboratory reproducibility might fulfil the respective
requirements, two laboratories (a and f) might not meet the requirement that the within-laboratory variability
should show a concordance of classifications (GHS Category 2/No Category) obtained in different, independent
test runs of the 20 Reference Chemicals within one single laboratory equal or higher () than 90%.
Related to this, the Panel recommends additional analyses based on final classifications for a complete
run sequence of a given laboratory derived from the mean viabilities (instead of median viabilities) as specified

The Panel agreed that this criterion has been met.

After clarification that a lot of review comments to this charge question would be equally well or better suitedfor charge question 11, the panel agreed that, with one acceptable exception, the currently recommendedreference chemicals have been used to demonstrate reliability and relevance of the test method. In addition, six
further chemicals, which have been used in the validation of the models leading to the draft OECD Test
Guideline, have been tested.

The Panel agreed that this criterion had been partly met.

defined by a maximum optical density (OD), is missing. Although the data base for defining this might besmall, it should nevertheless be defined if possible. Furthermore, it has not been defined what is considered as
an acceptable variation of viabilities from replicate tissues.
Regarding ‘Functional conditions – Morphology’, an acceptance criteria required for batch acceptance should
be defined.
Regarding ‘Functional conditions – Reproducibility’: A measure of variability between tissues should be
defined.
Regarding ‘Functional conditions – Quality control (QC)’, it was not entirely evident to the Panel how themodel producer intends to ensure and demonstrate that each batch of the RhE model used meets defined
production release criteria.

105

35

The Panel agreed that this criterion has not been met.

in the performance standards.
Regarding the predictive values, the panel agreed that the requirements, i.e. sensitivity  80%, specificity 
70% and accuracy  75%, were met, when considering all 25 tested chemicals. It was noted, however, that theoverall sensitivity would be  80% when considering only the 19 chemicals tested in the 2nd phase of the study.

ENV/JM/MONO(2011)44

ENV/JM/MONO(2011)44

Appendix 1

JaCVAM Re-Analysis of initial validation data according the Performance Standards of TG 439
August 23, 2010
During our validation studies, the draft OECD performance standards (PS) that is based on the ECVAM
performance standards had been on the table by OECD international experts. We performed our studies
using the reference chemical list in the original or revised ECVAM performance standards (ECVAM 2007),in accordance with the validation plan. On the other hand, we did not follow other rules of the performancestandard that were not included in the plan. Therefore, we could not calculate within- and betweenlaboratory reproducibility or analyze our data according to the rules of the performance standard (annex2)
in the OECD draft test guideline.
With instructions related to our reanalyzing the data to OECD peer review panel, in this report I haveanalyzed our data according to the OECD draft performance standards.
With reference to definitions of the rules for reliability and accuracy values, the validation data were set inorder as shown below.
1) Invalid data obtained only from Lab a, during run 1 (data not shown). This is because the negative OD
detected during run 1 was lower than the acceptable limit. This lab carried out retesting. Therefore, the data
Lab a, runs 2-4, were accepted as complete run sequences. All run sequences were completed in all otherlaboratories.
2) Twenty chemicals from the reference chemical (RC) list in the OECD performance standard (table 1)
are used in this report.
3) With the exclusion of the data from Lab e, fully complete data from six laboratories were analyzed withthree complete run sequences using all 20 RCs.
Below are our answers to questions regarding data analysis.
1. To questions 3, 4, 7 and 9 in the summary report of the LabCyte peer review panel:
A measure of variability between tissue triplicates was defined in the SOP and calculated in Table 2, anddetailed data is presented in the attached files.
2. To questions 4, 7 and 11 in the summary report of the LabCyte peer review panel:Within- and between-laboratory reproducibility were calculated according to annex 2.
As shown in Tables 3-1 and 3-2, there was no difference in median and mean variability among tissue
triplicates.
Within-laboratory reproducibility: equal or higher than 90% in all laboratories as shown in Table 4. 2

106

36

ENV/JM/MONO(2011)44

Between-laboratory reproducibility: equal or higher than 80% in all laboratories as shown in Table 4.
As shown in Tables 5 and 6, accuracy of the required predictive values was sufficient relative to the
ECVAM criteria.
Table 1: Reference test chemicals according to OECD performance standards (PS)No.

Validationcode

Chemical

CAS number

GHS label

In vivo score
(PII)

1

01

1-bromo-4-chlorobutane

6940-78-9

no

0

2

02

diethyl phthalate

84-66-2

no

0

3

04

naphtalen acetic acid

86-87-3

no

0

4

05

allyl phenoxy-acetate

7493-74-5

no

0.3

5

06

isopropanol

67-63-0

no

0.3

6

07

4-methyl-thio-benzaldehyde

3446-89-7

no

1

7

08

methyl stearate112-61-8

no

1

8

10

heptyl butyrate

5870-93-9

no

1.7

9

11

hexyl salicylate6259-76-3

no

2

10

A

Cinnamaldehyde

104-55-2

no

2

11

14

1-decanol

112-30-1

Category 2

2.3

12

15

cyclamen aldehyde

103-95-7

Category 2

2.3

13

16

1-bromohexane

111-25-1

Category 2

2.7

14

B

2-Chroromethyl-3,5-dimethyl-4-methoxypyridine HCl

322-76821

Category 2

2.7

15

C

Potassium hydroxide (5%aq)

168-21815

Category 2

3

16

18

di-n-propyl disulphide629-19-6

Category 2

3

17

D

Benzenethiol, 5-(1,1-dimethyethyl)-2-methyl

7340-90-1

Category 2

3.3

18

E

1-Methyl-3-phenyl-1-piperazine

5271-27-2

Category 2

3.3

19

20

heptanal

111-71-7

Category 2

4

20

F

1,1,1-Torichloroethane

200-02463

Category 2

4

37

107

ENV/JM/MONO(2011)44

Table 2: Mean viability at each test for each chemical according to OECD PS

108

No.

Code

Vivo

Score

01

01

no

0

02

02

no

0

3

04

no

0

4

05

no

0.3

5

06

no

0.3

6

07

no

1

7

08

no

1

8

10

no

1.7

9

11

no

2

10

A

No

2

11

14

Category 2

2.3

12

15

Category 2

2.3

13

16

Category 2

2.7

14

B

Category 2

2.7

15

C

Category 2

3

16

18

Category 3

3

17

D

Category 2

3.3

18

E

Category 2

3.3

19

20

Category 3

4

20

F

Category 2

4

Exp.
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

a
31.0
11.2
11.6
79.8
76.5
65.2
106.3
95.2
96.5
78.5
78.5
74.1
92.5
79.4
82.4
24.1
12.6
17.8
111.9
90.2
95.3
115.9
104.1
86.5
113.7
98.1
112.6
13.3
14.2
14
11.1
6.6
6.8
11.1
7.1
8.2
67.9
32.2
59.8
1.5
3.1
1.5
0.7
1.3
0.5
82.1
78.3
25.3
14.5
13.6
18.6
3.9
4.5
1.8
31.1
9.3
29.5
5.6
5.7
5.4

b
47.1
10.4
16.1
66.9
61.7
88.7
94.4
100.2
98.6
61.7
71.9
84.5
77.9
83.5
80.5
10.8
12.6
13.2
86.7
100.6
104.8
115.4
110.1
111.3
105.0
106.6
103.7
11.8
10.2
11.1
12.1
8.3
8.8
9.3
10.2
9.9
92.0
54.1
98.3
2.2
2.2
2.5
0.7
1.1
0.8
46.5
50.6
100.0
24
16
15.5
3.4
2.7
3.5
24.8
8.0
9.3
7.2
6.1
4.2

38

Lab.
c
10.6
20.3
12.4
88.1
89.7
85.8
97.1
99.9
97.8
91.4
95.2
89.2
81.0
79.1
83.6
20.8
16.2
15.2
75.3
82.3
77.2
107.5
103.6
103.7
101.0
94.6
94.1
13.2
22.5
12.3
14.7
9.5
9.1
13.1
19.3
8.1
51.5
86.3
81.7
2.5
2.9
3
0.7
1.4
1
91.2
87.3
87.5
12.7
12.5
12.6
3.4
3.3
3.5
10.4
7.6
7.6
6.5
6.8
6.5

d
14.3
9.1
9.6
102.3
89.8
67.6
106.1
100.9
98.4
79.4
70.5
66.1
91.3
102.4
82.7
21.7
13.8
19.8
109.4
107.5
103.0
114.3
108.2
105.5
102.4
105.8
102.7
13.8
9.9
13.2
10.7
11.7
10.2
8.0
8.6
9.2
18.1
79.2
37.7
4
3
3.9
6.9
2
0.8
83.7
69.9
59.0
10.3
18.3
23
8.2
3.9
3.7
9.6
16.9
30.9
6.4
5.4
5.4

f

14.3
11.2
10.4
75.3
67.2
75.7
100.1
92.8
92.7
71.9
39.3
55.1
87.8
94.4
81.1
15.8
31.1
15.6
89.7
97.8
96.5
104.0
101.2
101.2
103.1
98.0
94.6
11.4
11.3
14.3
13.1
16.7
17.0
8.6
5.9
7.1
59.6
50.4
67.5
1.7
2.6
3.2
0.8
4.8
1
69.2
80.6
71.9
13.8
8.8
19.2
3.2
4.2
5
8.1
7.8
8.2
5.2
7.4
5

g
10.6
10.6
14.0
96.0
94.8
103.3
104.8
103.3
109.8
96.8
89.9
88.4
87.2
81.2
54.1
31.5
22.5
19.9
101.1
100.9
109.0
107.9
108.4
113.1
102.8
100.5
109.0
13.7
8.7
14.3
13.5
12.0
10.6
9.2
24.7
9.2
64.9
79.6
86.5
3.9
3.7
4.7
1
0.4
0.3
92.4
85.9
94.4
19.3
15.2
14.1
4.1
3.1
5.1
8.8
6.7
8.6
7.2
6.8
7.6

ENV/JM/MONO(2011)44

Table 3-1: Mean viability at three tests for each chemical according to OECD PS
䎃
NO.
Code
a
b
c
d
f
GHS label
1
17.9
24.5
14.4
11.0
12.0
01
no
2
73.8
72.4
87.8
86.6
72.7
02
no
3
99.3
97.8
98.2
101.8
95.2
04
no
4
77.0
72.7
91.9
72.0
55.4
05
no
5
84.8
80.7
81.2
92.1
87.8
06
no
6
18.2
12.2
17.4
18.4
20.8
07
no
7
99.1
97.4
78.3
106.6
94.7
08
no
8
102.1
112.2
104.9
109.3
102.1
10
no
9
108.1
105.1
96.6
103.6
98.6
11
no
10
no
13.8
11.0
16.0
12.3
12.3
A
11
8.2
9.7
11.1
10.9
15.6
14
Category 2
12
8.8
9.8
13.5
8.6
7.2
15
Category 2
13
53.3
81.4
73.1
45.0
59.1
16
Category 2
14
2.0
2.3
2.8
3.6
2.5
B
Category 2
15
0.8
0.8
1.0
3.2
2.2
C
Category 2
16
61.9
65.7
88.7
70.9
73.9
18
Category 2
17
15.6
18.5
12.6
17.2
13.9
D
Category 2
18
3.4
3.2
3.4
5.3
4.2
E
Category 2
19
23.3
14.0
8.6
19.2
8.0
20
Category 2
20
5.5
5.8
6.6
5.7
5.9
F
Category 2

g
11.7
98.0
105.9
91.7
74.2
24.6
103.7
109.8
104.1
12.2
12.0
14.4
77.0
4.1
0.6
90.9
16.2
4.1
8.1
7.2

Table 3-2: Median viability at three tests for each chemical according to OECD PS
䎃
NO.
Code
a
b
c
d
f
GHS label
1
11.6
16.1
12.4
9.6
11.2
01
no
2
76.5
66.9
88.1
89.8
75.3
02
no
3
96.5
98.6
97.8
100.9
92.8
04
no
4
78.5
71.9
91.4
70.5
55.1
05
no
5
82.4
80.5
81.0
91.3
90.7
06
no
6
17.8
12.6
16.2
19.8
21.3
07
no
7
95.3
100.6
77.2
107.5
100.9
08
no
8
104.1
111.3
103.7
108.2
101.2
10
no
9
112.6
105.0
94.6
102.7
98.0
11
no
10
no
14.0
11.1
13.2
13.2
11.4
A
11
6.8
8.8
9.5
10.7
16.7
14
Category 2
12
8.2
9.9
13.1
8.6
7.1
15
Category 2

g
10.6
96.0
104.8
89.9
81.2
22.5
101.1
108.4
102.8
13.7
12.0
9.2

39

109

ENV/JM/MONO(2011)44

13
14
15
16
17
18
19
20

16
B
C
18
D
E
20
F

Category 2
Category 2
Category 2
Category 2
Category 2
Category 2
Category 2
Category 2

59.8
1.5
0.7
78.3
14.5
3.9
23.3
5.6

92.0
2.2
0.8
50.6
16.0
3.4
14.0
6.1

81.7
2.9
1.0
87.5
12.6
3.4
8.6
6.5

37.7
3.9
2.0
69.9
18.3
3.9
19.2
5.4

59.6
2.6
1.0
71.9
13.8
4.2
8.0
5.2

79.6
3.9
0.4
92.4
15.2
4.1
8.1
7.2

Table 4: Reproducibility according to OECD PS using mean viabilities

Lab.

Index

a
b
c
d
f
g
18/20 19/20 20/20 19/20 20/20 20/20
90
95
100
95
100
100
19/20
95.0

Withinlaboratory
Betweenlaboratory

Table 5:㩷㩷Sensitivity, specificity and accuracy on MTT assay vs GHS-EU classification according
to OECD PS

Lab.

Index

a
b
c
d
f
g
8/10 8/10 8/10 9/10 8/10 8/10
80
80
80
90
80
80
7/10 7/10 7/10 7/10 7/10 7/10
70
70
70
70
70
70
15/20 15/20 15/20 16/20 15/20 15/20
75
75
75
80
75
75

Sensitivity
Spescificity
Accuracy
Table 6:

Mean and range of sensitivity, specificity and accuracy on the MTT assay using
LabCyte EPI-MODEL vs. GHS-EU classification according to OECD PS

Sensivitity (%)
Specificity (%)
Accuracy (%)

110

N

Mean

Min.

Max.

6
6
6

81.7
70.0
76.7

80.0
70.0
75.0

90.0
70.0
80.0

40

ECVAM criteria
80.0
70.0
75.0

ENV/JM/MONO(2011)44

Appendix 2

Background document for the modified skin irritation test using LabCyte EPI-MODEL24 (LabCyte
EPI-MODEL24 SIT)
Japan Tissue Engineering Co., Ltd. R&D

TABLE OF CONTENTS
1.

Abstract2.

Purpose of the Document

3. Introduction
4. Description of LabCyte EPI-MODEL24
4.1.
Condition of the functional reconstructed human epidermis, LabCyte EPI-MODEL24
4-2.
Quality control for LabCyte EPI-MODEL24
4-2a.
Quality control procedures for LabCyte EPI-MODEL24
4-2b.
Batch control information for LabCyte EPI-MODEL24
4-3.
Shipment of LabCyte EPI-MODEL24 out of Japan
4-3a.
Purpose4-3b.
Quality check of the exported LabCyte EPI-MODEL24
4-3c.
Shipment schedule
4-3d.
Test facility
4-3e.
Results and discussion
5.

Optimization of the LabCyte EPI-MODEL24 SIT and assessment of the original SIT(OPTIMIZATION STAGE)
5-1.
Summary of the OPTIMIZATION STAGE
5-2.
Materials and methods
5-2a.
Test chemicals
5-2b.
Experimental protocol for a suitable exposure period for the LabCyte EPI-MODEL24 SIT
5-2c.
Protocol for the original LabCyte EPI-MODEL24 SIT
5-3.
Results
5-3a.
Investigation of a suitable exposure period for the LabCyte EPI-MODEL24 SIT
5-3b.
Assessment of the original LabCyte EPI-MODEL24 SIT by intra-laboratory study
5-4.
Discussion

6.

Modification of the original LabCyte EPI-MODEL24 SIT and assessment of the modified SIT(MODIFICATION STAGE)
6-1.
Summary of the MODIFICATION STAGE

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ENV/JM/MONO(2011)44

6-2.
Materials and methods
6-2a.
Reference chemicals described in OECD TG 439
6-2b.
Wide-range of test chemicals
6-2c.
Test Protocol for modification of the original LabCyte EPI-MODEL24 SIT
6-2d.
Protocol of the modified LabCyte EPI-MODEL24 SIT (SOP ver.8.2)
6-2e.
Detecting chemical interference with MTT endpoints and correction procedures
6-3.
Results
6-3a.
Modification of the original LabCyte EPI-MODEL24 SIT
6-3b.
Assessment of the modified LabCyte EPI-MODEL24 SIT based on the OECD TG 439 forintra-laboratory study
6-3c.
Assessment of the modified LabCyte EPI-MODEL24 SIT using 54 test chemicals over awide range of classification
6-4.
Discussion
7.

Conclusion

8.

Acknowledgement

9.

Abbreviations

10. Attachments (not included in this document)
11. References

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ENV/JM/MONO(2011)44

1.

Abstract
A validation study of an in vitro skin irritation test method using a reconstructed human epidermal
model for replacement of the Draize test was conducted by the European Centre for the Validation of
Alternative Methods (ECVAM), and a protocol using EpiSkinTM (SkinEthic, France) was approved as avalidated reference method (VRM) in April, 2007. Structural and performance test criteria for skinmodels are defined in the ECVAM Performance Standards. We have performed several evaluations of
our reconstructed human epidermal model LabCyte EPI-MODEL24 (Japan Tissue Engineering Co., Ltd,
Japan), and have confirmed that it is suitable for skin irritation tests defined by the ECVAM PerformanceStandards.
The original study for the development of a test method showed that test chemicals could be classified
into non-irritants and irritants using the LabCyte EPI-MODEL24 (ORIGINAL STAGE). The VRM
consists of a 15 minute exposure period, followed by a rinsing step and a 42 hour post-incubation period
before quantitative measurement of cell viability using an MTT reaction. First, we examined theexposure period when the test chemicals are applied and found that the optimal exposure period is 15
minutes. After that, the performance of the original LabCyte EPI-MODEL24 SIT, which included an
optimization study, was confirmed in intra-laboratory study and then evaluated on relevance and ability
to meet minimum criteria described in the ECVAM performance standards for phase 1, 2 and 3
validation studies. The results of the validation studies for the original LabCyte EPI-MODEL24 SIT
were summarized in JSAAE REPORTS and then these reports were assessed by a peer review panel inOECD, according to the OECD draft test guideline for skin irritation testing, which was presented in
2009.
However, the OECD peer review panel pointed out that the performance of the original LabCyte EPIMODEL24 SIT did not meet the criteria shown in OECD draft test guideline for skin irritation testing,because the prediction results for the chemical 1-bromohexane, which is an in vivo Category 2 substance,
were shown as negative in the original LabCyte EPI-MODEL24 SIT. Various modifications of theoriginal LabCyte EPI-MODEL24 SIT have been examined by the kit supplier in order to solve the 1bromohexane problem, with the result that they were able to solve the problem by modifying thewashing protocol for the testing chemicals (the modified LabCyte EPI-MODEL24 SIT). When thepredictive potency of the modified LabCyte EPI-MODEL24 SIT was evaluated with the 20 test
chemicals which were listed in the new OECD TG 439 for in vitro skin irritation testing, adopted in July,
2010, the sensitivity, the specificity and the overall accuracy were 90 %, 70 %, and 80%, respectively.. It
was thought that these results met the acceptance criteria described in OECD TG 439. In order toconfirm the performance of the modified LabCyte EPI-MODEL24 SIT according to OECD TG 439, a
final validation study was executed from August to November, 2010. Results demonstrated highreliability and acceptable accuracy in the MTT assay, for use as a stand-alone assay to distinguishbetween skin irritants and non-irritants. Details of the results of the validation study will be described ina Japanese Center for the Validation of Alternative Methods (JaCVAM) final report.

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2.

Purpose of this document
The principle of in vitro skin irritation testing using a reconstructed human epidermis (RhE) model as
described in the new OECD test guideline 439 (OECD TG 439; Attachment 1) for in vitro skin irritation
testing, is to detect the initiating events in the following cascade of skin irritation. Chemical-induced skinirritation, manifested by erythema and oedema, is the result of a cascade of events beginning withpenetration of the stratum corneum and damage to the underlying layers of keratinocytes. The dying
keratinocytes release mediators that begin the inflammatory cascade which acts on the cells in the dermis,
particularly the stromal and endothelial cells. It is the dilation and increased permeability of theendothelial cells that produce the observed erythema and oedema. The RhE-based test methods measure
the initiating events in the cascade.
The main purpose of this document is to propose a new in vitro skin irritation test method using
LabCyte EPI-MODEL24 (Japan Tissue Engineering Co., Ltd, Japan; the LabCyte EPI-MODEL24 SIT)
as a “me-too” test based on the validation test method (VRM) for EpiSkinTM (SkinEthic, France)
according to OECD TG 439.
This document is composed of the following categories:
1. Description of the LabCyte EPI-MODEL24: It is confirmed that quality control for the LabCyte
EPI-MODEL24 quality standards and/or the characteristics thereof meet with the criteria describedin OECD TG 439.
2. Optimization of the LabCyte EPI-MODEL24 SIT and assessment of the original SIT(OPTIMIZATION STAGE): The optimal exposure period for the LabCyte EPI-MODEL24 SIT
was investigated as a method based on VRM, according to the ECVAM performance standard (1).
The reliability performance of the original (optimized) SIT was evaluated based on the OECD
draft test guideline for skin irritation testing (2).
3. Modification of the original LabCyte EPI-MODEL24 SIT and assessment of the modified SIT(MODIFICATION STAGE): The original LabCyte EPI-MODEL24 SIT was modified because theoriginal LabCyte EPI-MODEL24 SIT did not meet the criteria shown in the OECD draft test
guideline (2). The performance of the modified SIT was evaluated with respect to reliability andaccuracy, according to OECD TG 439.
In the end, through the above research stages it was confirmed that the performance of the LabCyte
EPI-MODEL24 SIT meets the acceptance criteria shown in OECD TG 439 as a “me-too” test method
similar to VRM.

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3.

Introduction
Trials to replace the Draize skin irritation test on rabbits in vivo, carried out according to the OECD
TG 404 test guidelines (3), have been underway for many years worldwide, especially in the European
Union (EU). These efforts have been accelerated by the enforcement of the 7th Amendment to the
Cosmetics Directive and of EU regulations for the registration, evaluation, and authorization ofchemicals (REACH). Investigation of various in vitro/ex vivo test systems showed that the best results
were achieved by a system using a three-dimensional reconstructed human epidermal model (4, 5). The
European Centre for the Validation of Alternative Methods (ECVAM) therefore evaluated two
reconstructed human epidermal models, EpiSkinTM (SkinEthics, Nice, France) and EpiDermTM (MatTek,
MA, USA), during a prevalidation study of in vitro skin irritation tests during 2000–2001 (5, 6).
However, since the predictive performance of the two models in the ECVAM prevalidation study did notmeet the acceptance criteria set by the Management Team, further investigations were required to
improve the test protocols (6). Subsequently, during 2003–2004 a common protocol for the in vitro
evaluation of skin irritation potential was developed, optimized, and allowed to proceed to a formal
ECVAM validation study (7, 8). As a result of the study, an in vitro skin irritation test (SIT) using the
EpiSkinTM reconstructed human epidermal model (EpiSkinTM test method) has been scientificallyvalidated as a stand-alone method of distinguishing skin irritants from non-irritants according to the EUclassification (9). Furthermore, the ECVAM performance standards for applying a new humanepidermis model to in vitro skin irritation have been documented based on the VRM (1). The ECVAM
performance standards can then be used to evaluate the accuracy and reliability of other analogous test
methods, also known as “me-too” tests, either based on similar scientific principles and measures or to
predict the same biological or toxic effect.
Various other available in vitro three-dimensional epidermis equivalents have been developed, such as
the LabCyte EPI-MODEL24 (Japan Tissue Engineering Co. Ltd., Japan). The LabCyte EPI-MODEL24
is a new reconstructed human epidermis model, which is grown for 13 days in a chemically defined
medium using normal human keratinocytes (10). The tissue model consists of a fully differentiatedepidermis, including a basal cell layer, stratum spinosum, stratum granulosum and stratum corneum (10,11). Since the launch of commercially released kits in 2005, the LabCyte EPI-MODEL24 has mainly
been used for skin irritation studies (11-15) or for skin corrosion (16), but it is also commonly used for
UV-related experiments (17-19), for DNA microarray (20,21) and for various experiments about skinfunction (22-26).
In order to develop a test method according to the VRM (EpiSkinTM test methods), which is able todiscriminate between non-irritant versus irritant test substances using the LabCyte EPI-MODEL24, theoptimal exposure period for LabCyte EPI-MODEL24 was determined before the assessment of its
performance. Then, the performance of the original LabCyte EPI-MODEL24 SIT (15 minute exposure)
after the optimization study was evaluated for its relevance and its ability to meet ECVAM minimum
performance standards criteria.
The validation study of the original LabCyte EPI-MODEL24 SIT was executed from 2008 to 2009.
The results for validation studies of the original LabCyte EPI-MODEL24 SIT were summarized in
JSAAE REPORTS (Attachment 2, 3).
During the validation study of the original LabCyte EPI-MODEL24 SIT, two important decisions were
presented by ECVAM. One of them changed the test chemical exposure period for the modified
EpiDermTM SIT, from 15 minutes to 60 minutes, while the other stated that the SIT using SkinEthic RHE(SkinEthics, Nice, France), which was a new reconstructed human epidermal model, was regarded by
ECVAM as sufficiently similar in comparison with the VRM and was admitted as a validation study on
the basis of the ECVAM performance standards (27). Furthermore, the ECVAM performance standards

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were revised (the ECVAM performance standards (updated)); 28,29) in 2009. It was stated that the invivo classification of test chemicals would be changed from referring to the EU DSD (European
Classification System based on the Dangerous Substance Directive) to the UN GHS (United Nations
Globally Harmonized System of Classification and Labelling of Chemicals). This change was reflectedin the OECD draft test guideline for skin irritation testing (2).
It should be noted that, the JSAAE REPORTS (Attachment 2, 3) about the validation study for theoriginal LabCyte EPI-MODEL24 SIT were assessed by a peer review panel in OECD according to theacceptance criteria as shown in the OECD draft test guideline. Unfortunately, the OECD peer review
panel pointed out that the performance of the original LabCyte EPI-MODEL24 SIT did not meet thecriteria shown in the OECD draft test guideline for skin irritation testing (2), because the prediction
results of the chemical 1-bromohexane, which is an in vivo Category 2 substance, was shown as negativein the original LabCyte EPI-MODEL24 SIT by five out of six laboratories in the validation exercise. In
order to solve the problem where 1-bromohexane showed a false-negative, various modification trials for
LabCyte EPI-MODEL24 SIT were examined by the kit supplier. From the results of their examination, it
was found that the problem could be solved by the improvement of the washing protocol (the modified
LabCyte EPI-MODEL24 SIT). Using the 20 test chemicals listed in the OECD TG 439 (Attachment 1),
which was adopted as the new guideline for the in vitro skin irritation testing by OECD in July, 2010, thepredictive potency of the modified LabCyte EPI-MODEL24 SIT was evaluated, with the result that thesensitivity, specificity and overall accuracy were 90%, 70% and 80%, respectively. It was thought thatthese results met the acceptance criteria described in the OECD TG 439. In order to confirm theperformance of the modified LabCyte EPI-MODEL24 SIT according to OECD TG 439, the final andformal validation study of the modified LabCyte EPI-MODEL24 SIT was performed from August to
November, 2010.

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4.

Description of LabCyte EPI-MODEL24

4-1. Condition of the functional reconstructed human epidermis, LabCyte EPI-MODEL24
LabCyte EPI-MODEL24 is a new, commercially available reconstructed human cultured epidermal
model produced by Japan Tissue Engineering Co. Ltd. It consists of normal human epidermal
keratinocytes whose biological origin is neonate foreskin. In order to expand the human keratinocytes
while maintaining their phenotype, they are cultured with 3T3-J2 cells as a feeder layer (30,31).
Reconstruction of human cultured epidermis is achieved by cultivating proliferating keratinocytes on an
inert filter substrate (surface 0.3 cm2) at the air-liquid interface for 13 days, with an optimized medium
containing 5% fetal bovine serum. The result is a multilayer structure consisting of a fully differentiatedepithelium with features of the normal human epidermis, including a stratum corneum (Fig.4-1). The
LabCyte EPI-MODEL24 is embedded in an agarose gel containing a nutrient solution and shipped in 24well plates at around 18°C.

Fig.4-1.

Histological cross-sectional views of the LabCyte EPI-MODEL24 with
H&E staining (original magnification: ×40).

Histological examination shows a completely stratified epidermis containing all major epidermal
layers, including a stratum basal (SB), stratum spinosum (SS), stratum granulosum (SG), and stratum
corneum (SC) (Fig.4-1). In addition, specific epidermal differentiation markers and basement membrane
constituents are expressed in the appropriate regions, as seen in human skin (Fig.4-2).

(A)

(B)

100P
Pm

100P
Pm

Fig.4-2.

Histologicalcross-sectional

views

47

of

LabCyteEPI-MODEL24

with

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ENV/JM/MONO(2011)44

immunohistochemical staining using Laminin (a) and Transglutaminase (b).

(a)

(b)

K

m

(c)

Fig.4-3.
(a)
(b)
(c)
(d)

(d)

Transmission electron micrographs of the LabCyte EPI-MODEL24:
Basement membrane (original magnification; ×10,000). Note anchoring
filament (arrows).
Keratohyalin granules in SG cells which are connected by desmosome(original magnification; ×5000). Note keratohyalin granules (K), desmosome(arrows).
Lipid lamella bodies in the interface between SG and SC (original
magnification; ×30,000).
Note lipid lamellar bodies (arrow head) and their extrusion (arrows).
Lipid bilayer at SC (original magnification; ×100,000). Additional ruthenium
tetroxide fixation was used.

Ultra-structurally, it was possible to observe a fully developed basement membrane zone, consisting ofa highly developed lamina densa, lamina lucida, and anchoring filaments. Extrusion of lamellar bodies
was observed at the interface between the SG and SC (Fig.4-3). Lipid lamellae, showing a characteristic
electron dense and electron lucent pattern, were present. Keratohyalin granules were ubiquitously present
in the granular cells at the SG (Fig.4-3).
Their histological evaluation was summarized in a scientific report (10).
The synthesis of specific lipids, including ceramides, which are known to be responsible for the water
barrier of the stratum corneum, was detected in the LabCyte EPI-MODEL (22).
4-2. Quality control for LabCyte EPI-MODEL24
4-2a. Quality control procedures for LabCyte EPI-MODEL24
LabCyte EPI-MODEL24 is manufactured according to a defined standard operation procedure (SOP).
All batches of the LabCyte EPI-MODEL24 production are checked for their viability, barrier function
and morphology.
The product is released following stringent quality control procedures.

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ENV/JM/MONO(2011)44

The quality of the final product is assessed by the following protocol and decision criteria;
1. Tissue viability: MTT assay
Three LabCyte EPI-MODEL24 tissues were subjected to an MTT assay as follows. Tissues were
put in the wells of 24-well plates containing 0.5 ml of MTT medium (0.5 mg/ml; Dojindo Co.,
Kumamoto, Japan) and were incubated for three hours (37°C, 5% CO2, humidified atmosphere).
Formazan produced in the tissues was extracted with isopropanol (300 l) and the extract (200 l)
was measured at 570 nm and at 650 nm as a reference absorbance, with isopropanol as a blank.
The mean of the OD values indicates tissue viability for each LabCyte EPI-MODEL24 batch.
QC acceptance criteria: OD  0.8
2. Barrier function: 50% inhibitory concentration (IC50) assay.
To evaluate whether the stratum corneum in the LabCyte EPI-MODEL24 tissue resists the rapid
penetration of the cytotoxic marker chemical sodium lauryl sulphate (SLS), the viability of theepidermis tissue was estimated in terms of the half maximal inhibitory concentration (IC50).
Various concentrations (0.1, 0.2, 0.3, and 0.4%(w/v)) of SLS (25 ȝl) were applied to the LabCyte
EPI-MODEL24, and cell viability was measured after 18 hours using an MTT assay. All
experiments were performed in triplicate. The acceptable range for the LabCyte EPI-MODEL24 isshown in Table 4-1.
Table 4-1: QC acceptable range of barrier function
LabCyte EPI-MODEL24
IL50(18 hours SLS)

Lower limit
0.14 (w/v)%(1.4mg/ml)

Mean
0.26 (w/v)%(2.6mg/ml)

Upper limit
0.40 (w/v)%(4.0mg/ml)

The test protocol for the barrier function of the LabCyte EPI-MODEL24 was established in May,
2008. In order to set an acceptable QC range, data for a barrier function database was collectedfrom June to December, 2008. From June 2008 to October 2010, there was no batch with results
outside the QC acceptance criteria.
3. Morphology.
A piece of LabCyte EPI-MODEL24 was fixed with 4% parafolmaldehyde and 2% sucrose in 0.1M
phosphate buffered saline (PBS) (pH 7.4) for more than three hours and processed for embedding
in paraffin. Five-micrometer vertical sections were cut and stained with hematoxylin and eosin forlight-microscopic examination.
QC acceptance criteria: Confirmation of the formation of multilayered epidermis-like tissue
containing a stratum corneum.
Since the start of the commercial release of the product until October 2010, no batch has beenoutside the QC acceptance criteria..
4-2b. Batch control information for LabCyte EPI-MODEL24
Quality control data for the tissue viability and barrier function of each LabCyte EPI-MODEL24 batchis shown in Table 4-2.
Table 4-2: Batch information for LabCyte EPI-MODEL24
Tissue viability: October, 2005 – October, 2010
Barrier function: June, 2008 – October, 2010

49

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ENV/JM/MONO(2011)44

Year
2005 OctoberNovember
December

120

Month

Tissue viability
Mean±SD
CV1)
(OD)
(%)
1.03±0.18
17.6
1.21±0.1512.7
1.23±0.12
10.0

50

Barrier function
Mean±SD
CV1)
(%)
(%)

ENV/JM/MONO(2011)44

Table 4-2:

Continued

Year
2006

2007

2008

2009

Month
January
February
March
April
May
June
July
AugustSeptember
OctoberNovember
DecemberJanuary
February
March
April
May
June
July
AugustSeptember
OctoberNovember
DecemberJanuary
February
March
April
May
June
July
AugustSeptember
OctoberNovember
DecemberJanuary
February
March
April
May
June
July
AugustSeptember

Tissue viability
Mean±SD
CV1) (%)(OD)
1.34±0.06
4.3
1.14±0.1613.7
1.24±0.26
21.3
1.48±0.138.7
1.49±0.1811.9
1.37±0.1712.4
1.41±0.1510.6
1.43±0.07
5.2
1.52±0.128.1
1.93±0.09
4.6
1.98±0.199.7
1.68±0.137.6
1.70±0.08
4.7
1.62±0.09
5.3
1.67±0.116.9
1.51±0.09
6.1
1.52±0.09
6.1
1.66±0.10
6.3
1.50±0.22
15.0
2.07±0.40
19.3
1.48±0.18
12.4
1.70±0.15
8.6
1.92±0.199.7
1.70±0.05
3.2
1.74±0.20
11.4
1.37±0.23
17.0
1.35±0.1813.7
1.42±0.1510.8
1.33±0.1410.7
1.28±0.03
2.0
1.32±0.03
2.3
1.26±0.04
3.5
1.31±0.06
4.6
1.55±0.06
3.7
1.54±0.07
4.2
1.51±0.09
6.1
1.23±0.29
23.8
1.41±0.128.7
1.45±0.09
6.1
1.44±0.2315.7
1.60±0.1710.8
1.34±0.10
7.4
1.36±0.07
4.9
1.47±0.08
5.4
1.40±0.11
8.0

51

Barrier function
Mean±SD
CV1)
(%)
(%)

0.23±0.02
0.24±0.04
0.27±0.01
0.28±0.03
0.26±0.01
0.26±0.00
0.26±0.02
0.25±0.01
0.25±0.01
0.28±0.01
0.23±0.02
0.24±0.01
0.26±0.04
0.25±0.03
0.24±0.02
0.24±0.01

10.1
17.9
5.2
9.1
4.7
1.1
6.8
4.1
5.0
4.7
6.6
4.2
15.9
12.7
9.0
4.7

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ENV/JM/MONO(2011)44

OctoberNovember
December

122

1.54±0.11
1.58±0.05
1.52±0.12

52

6.9
3.4
8.0

0.29±0.04
0.26±0.02
0.24±0.02

13.6
6.1
6.8

ENV/JM/MONO(2011)44

Table 4-2.
Year

Continued
Month

2010

January
February
March
April
May
June
July
AugustSeptember
October
TissueOctober, 2005 to October,viability
2010
June, 2008 to October,
Barrier2010
function
1) Coefficient Variation.

Tissue viability
Mean±SD
CV1) (%)(OD)
1.51±0.159.91.60±0.127.5
1.52±0.08
5.0
1.44±0.138.9
1.63±0.03
1.9
1.58±0.12
7.6
1.58±0.08
4.8
1.49±0.106.8
1.47±0.128.1
1.42±0.08
5.5

Barrier function
Mean±SD
CV1)
(%)
(%)
0.26±0.00
1.6
0.25±0.01
3.4
0.28±0.03
9.4
0.25±0.01
4.6
0.25±0.01
5.1
0.24±0.02
8.9
0.25±0.01
4.0
0.24±0.01
5.2
0.25±0.01
4.1
0.25±0.02
8.8

1.49±0.23

0.25±0.02

15.5

9.2

As shown in Table 4-2, the mean SD of tissue viability and barrier function (IC50) from the evaluation ofcontinuous batches of LabCyte EPI-MODEL24 (with tissue viability data taken from October, 2005 to
October, 2010 and barrier function data taken from June, 2008 to October, 2010) were 1.49 ± 0.23% and0.25 ± 0.02%, respectively. The tissue viability and barrier function of LabCyte EPI-MODEL24 tissue
have remained constant, indicating reproducibility (low coefficient variation (CV): 15.5% and 9.2%,
respectively) and monthly consistency is high.
4-3. Shipment of LabCyte EPI-MODEL24 out of Japan
4-3a. Purpose
To examine whether LabCyte EPI-MODEL24 is exportable, it was sent to China and viability and barrier
function tests were performed.
4-3b. Quality check of the exported LabCyte EPI-MODEL24
1. Viability
Three LabCyte EPI-MODEL24 tissues were subjected to an MTT assay as follows. Tissues were
put in the wells of 24-well plates containing 0.5 ml of MTT medium (0.5 mg/ml) and wereincubated for three hours (37°C, 5% CO2, humidified atmosphere). Formazan produced in theμlμl
and at 650 nm as a reference absorbance, with isopropanol as a blank.
The mean OD value indicates the tissue viablility for each LabCyte EPI-MODEL24 batch.
Acceptance criteria:
The mean OD  0.8
2. Barrier function
The following test is performed using 15 tissue models.
To evaluate whether the stratum corneum in the LabCyte EPI-MODEL24 tissue resists the rapid
penetration of the cytotoxic marker chemical sodium lauryl sulphate (SLS), the viability of theepidermis tissue was estimated in terms of the half maximal inhibitory concentration (IC50).

53

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ENV/JM/MONO(2011)44

Various concentrations (0.1, 0.2, 0.3, and 0.4%(w/v)) of SLS (25 ȝl) are applied to the LabCyte
EPI-MODEL24, and cell viability is measured after 18 hours by an MTT assay. All experiments
are performed in triplicate. The MTT assay is performed using three tissue models and their
viabilities are determined.
Acceptance criteria:
0.14%  IC50  0.4%
LabCyte EPI-MODEL24 was exported three times and it was confirmed that results were within theacceptance criteria in all batches.
4-3c. Shipment schedule
1st shipment:
2nd shipment:
3rd shipment:

August 24, 2009.
August 31, 2009.
September 14, 2009

4-3d. Test facility
Shanghai Entry-Exit Inspection and Quarantine Bureau of the People’s Republic of China
Technical Center For Animal, Plant And Food Inspection and Quarantine Head of Animal andToxicology Lab. (Shanghai, China)
4-3e. Results and discussion
Shipment results are shown in Table 4-3.
Table 4-3: Results of Shipments to China
Lot No.

Shipping date

Delivery date

Transportation period

LCE24-090824-A
LCE24-090831-A
LCE24-090914-A

Aug. 24, 2009
Aug. 31, 2009
Sep. 14, 2009

Aug. 26, 2009
Sep. 2, 2009
Sep. 16, 2009

2 days2 days2 daysThe QC results for the exported LabCyte EPI-MODEL24 from the Shanghai Entry-Exit Inspection andQuarantine Bureau of the People’s Republic of China are shown in Table 4-4.
Table 4-4: QC results for LabCyte EPI-MODEL24 exported to China
Lot No.
LCE24-090824A
LCE24-090831A
LCE24-090914A

Starting datefor
QC testing
Aug. 26,
2009
Sep. 2, 2009
Sep. 16,2009

Viability
Blank(570nm)0.040±0.000

Fold
21.8

BarrierfunctionΰIC50(%)α
0.32

1.011±0.033

0.037±0.000

27.1

0.27

Pass

1.054±0.034

0.038±0.000

28.0

0.25

Pass

Tissue(570nm/650nm)
0.871±0.035

QC
resultPass

All three batches of the LabCyte EPI-MODEL24 shipped to China were within the QC acceptancecriteria as described in the Test Protocol, suggesting that the quality of the LabCyte EPI-MODEL24 wasmaintained during the overseas shipment.

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ENV/JM/MONO(2011)44

5.

Optimization of the LabCyte EPI-MODEL24 SIT and assessment of the original SIT(OPTIMIZATION STAGE)

5-1. Summary of the OPTIMIZATION STAGE
The aim of the optimization study was to develop a test method able to discriminate non-irritant versus
irritant test chemicals using the LabCyte EPI-MODEL24 as a “me-too“ test based on the VRM(EpiSkinTM test method) according to the ECVAM performance standard (1). The VRM consists of a 15
minute exposure period of test chemicals, followed by a rinsing step and a 42 hour post-incubation
period before quantitative measurement of cell viability using an MTT reaction.
First of all, we examined a suitable exposure period for the LabCyte EPI-MODEL24 SIT and the
optimized exposure period was set at 15 minutes. The performance of the original LabCyte EPIMODEL24 SIT, which was reflected in the results of the optimization study, has been confirmed byintra-laboratory study.
After that, the original LabCyte EPI-MODEL24 SIT was evaluated for relevance and the ability tomeet minimum criteria described in the ECVAM performance standards in the phase 1, 2 and 3 formal
validation studies. The results of validation studies for the original LabCyte EPI-MODEL24 SIT weresummarized in JSAAE REPORTS (Attachments 2,3) and then their reports were assessed by a peer
review panel in OECD according to the OECD draft test guideline for skin irritation testing which waspresented in 2009 (2).
5-2. Materials and methods
5-2a. Test chemicals
Nineteen test chemicals shown in Table 5-1 were selected among the twenty reference chemicals of the
ECVAM performance standard (1). By definition, reference chemicals are used to determine if theperformance of a new in vitro human skin model system for skin irritation testing is comparable to thatof the VRM in the ECVAM performance standard.
Unfortunately, tri-isobuthyl phosphate (No.13) was not used in the examination because it was
unavailable in Japan.
Table 5-1: Selected test chemicals for the optimization of a suitable exposure period.
No.
1
2
3
4
5
6
7
8
9
10
11
12
(13)

Name
1-bromo-4-chlorobutane
diethyl phthalate
di-propylene glycol
naphthalen acetic acidallyl phenoxyacetate
isopropanol
4-methyl-thio-benzaldehyde
methyl stearate
allyl heptanoate
heptyl butyrate
hexyl salicylate
terpinyl acetate(tri-isobutyl phosphate)

Test chemicals
CAS
In vivonumber1)class2)6940-78-9
NI
84-66-2
NI
25265-71-8
NI
86-87-3
NI7493-74-5
NI
67-63-0NI
3446-89-7
NI
112-61-8
NI
142-19-8
NI
5870-93-9
NI6259-76-3
I
80-26-2
I
126-71-6
I

55

PII3)

Supplier

0
0
0
0
0.3
0.3
1.0
1.0
1.7
1.7
2.0
2.0
2.0

Wako chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical
Kanto chemical
Wako chemical
AldrichSigma Fluka
Alfa Aesar
Unavailable in
Japan

125

ENV/JM/MONO(2011)44

14 cyclamen aldehyde
103-95-7
15 1-decanol112-30-1
16 1-bromohexane
111-25-1
17 Į-terpineol
98-55-5
18 di-n-propyl disulphide629-19-6
19 butyl methacrylate
97-88-1
20 heptanal
111-71-7
1) CAS No.: Chemical abstracts service registry number.
2) I: irritant, NI: non irritant
3) PII: primary irritation index.

I
I
I
I
I
I
I

2.3
2.3
2.7
2.7
3.0
3.0
3.3

Wako chemical
Wako chemical
Wako chemical
Kanto chemical
Wako chemical
Wako chemical
Kanto chemical

5-2b. Experimental protocol for a suitable exposure period for the LabCyte EPI-MODEL24 SIT
LabCyte EPI-MODEL24 tissues were shipped from the supplier on Mondays and delivered torecipients on Tuesdays. Upon receipt, the tissues were aseptically removed from the transport agarose
medium, transferred into 24-well plates (BD Biosciences, CA, USA) with the assay medium (0.5 ml) andincubated overnight (37°C, 5%, CO2, humidified atmosphere). On the next day, the tissues were topically
exposed to the test chemicals. Liquids (25 ȝl) were applied with a micropipette, and solids (25 mg) wereapplied from microtubes and moistened with 25 ȝl sterile water. If necessary, the mixture was gently
spread over the surface of the epidermis with a microspatula. Viscous liquids were applied by using acell-saver-type tip with a micropipette. Each test chemical was applied to three tissues. In addition, threetissues serving as negative controls were treated with 25 ȝl distilled water, and three tissues serving as
positive controls were exposed to 5% SLS. After 10, 15, 20 or 30 minutes of exposure, each tissue wascarefully rinsed with PBS (Invitrogen, CA, USA) ten times using a washing bottle to remove any
remaining test chemical from the surface. The blotted tissues were then transferred to new wells on 24well plates containing 1 ml of fresh assay medium.
The treated and control tissues were post-incubated for 42 hours (37°C, 5%, CO2, humidified
atmosphere). When the 42-hour post-incubation period was is completed, blotted tissues weretransferred to new wells on 24-well plates containing 0.5 ml of freshly prepared MTT medium (0.5mg/ml) for the MTT assay. Tissues were incubated for three hours (37°C, 5% CO2, humidified
atmosphere) and were then transferred to microtubes containing 300 ȝl isopropanol, completely
immersing the tissue. Formazan extraction was performed at room temperature and the tissues wereallowed to stand overnight. Subsequently, 200 ȝl extracts were transferred to a 96-well plate. The opticaldensity was measured at 570 nm and at 650 nm as a reference absorbance, with isopropanol as a blank.
The tissue viability was calculated as a percentage relative to the viability of negative controls. The
mean of the three values from identically treated tissues was used to classify a chemical according to theprediction model.
In this study, the prediction model for skin irritation potential with LabCyte EPI-MODEL24 was set
with reference to the conditions for the VRM (EpiSkinTM test method) described in the ECVAM
performance standards. This prediction model is described in Table 5-2. In the event that the three
independent results within an individual batch were not in agreement, the result that occurred twice wasused.
Table 5-2: Prediction models for the LabCyte EPI-MODEL24 SIT
Cell viability
mean  50%
mean > 50%

Judgment
Irritant
Non irritant

5-2c. Protocol for the original LabCyte EPI-MODEL24 SIT
The test protocol for the LabCyte EPI-MODEL24 SIT is described in section 5-2b based on the

126

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ENV/JM/MONO(2011)44

EpiSkinTM VRM. From the result of the investigation of a suitable exposure period (see section 5-2b and5-3b), the exposure period in the original LabCyte EPI-MODEL24 SIT was set at 15 min. The overall
test protocol is described in the original SOP for the LabCyte EPI-MODEL24 SIT (ver. 4.0).
Three independent tests were performed on different batches of the LabCyte EPI-MODEL24, withthree tissues per test chemical. After the 42-hour post-incubation period was completed, conditioned
medium was collected for analysis of interleukin-1 alpha (IL-1D) release according to the VRM.
The tissue viability was calculated as a percentage relative to the viability of negative controls. The
mean of the three values from identically treated tissues was used to classify a chemical according to theprediction model.
The amount of IL-1D release in the conditioned medium collected after 42 hours was determined using
the IL-1D ELISA kit (Invitrogen, CA, USA), according to the detailed instructions of the manufacturer.
In this study, the prediction model for skin irritation potential with the LabCyte EPI-MODEL24 was
set in reference to the conditions for the VRM (EpiSkinTM test method) described in the ECVAM
performance standards. This prediction model is described in Table 5-4. In the event that the three
independent results within an individual batch were not in agreement, the result that occurred twice wasused.
Table 5-4: Prediction models for the original LabCyte EPI-MODEL24 SIT
Cell viability (1st)mean  50%
mean > 50%
mean > 50%

Judgment

IL-1D ELISA (2nd)

Irritant

IL-1Į content (mean)  120 pg/tissueIL-1Į content (mean) < 120 pg/tissue

Non irritant

5-3. Results
5-3a. Investigation of a suitable exposure period for the LabCyte EPI-MODEL24 SIT
The results of the investigation of a suitable exposure period for the LabCyte EPI-MODEL24 SIT were
evaluated by cell viability, as shown in Table 5-5.
Table 5-5: Results obtained from the experiment with various exposure periods
Test chemical

Exposure period (minutes)
15
20

10
In
vitro
class

viability(%)

1-bromo-4-chlorobutane

NI44.0±12.8I49.6±5.0I

25.3±7.5

I

diethyl phthalate

NI93.3±8.4

NI

108.0±3.9NI118.5±1.6NI

di-propylene glycol
naphthalen acetic acidallyl phenoxy-acetate
isopropanol
4-methyl-thiobenzaldehyde

NI
NI
NI
NI

90.3±2.892.4±7.787.5±11.3
83.5±6.3

NI
NI
NI
NI

108.1±6.4
103.3±1.8
100.6±5.4102.1±1.6

NI
NI
NI
NI

95.3±5.7
100.8±7.0
90.7±6.4
68.7±11.9

NI
NI
NI
NI

14.5±6.5
92.4±12.
690.5±3.3
84.9±4.2
91.6±2.8
9.6±1.1

NI

15.0±2.7I

17.2±1.6I

10.9±2.6I

15.5±2.6

1)

1)

viability
(%)

30

In
vitro
class

In vivoclass1)

Name

viability
(%)

In
vitro
class

1)

methyl stearate

NI94.4±2.0NI

113.0±2.6

NI89.6±7.0NIallyl heptanoate

NI92.9±3.1

NI

115.9±8.6NI83.3±3.6

NI

heptyl butyrate

NI95.2±9.5

NI

126.3±1.2NI

90.9±4.6

NIhexyl salicylate

I97.5±5.6

NI

107.4±4.8NI87.6±2.4

NI

terpinyl acetate
cyclamen aldehyde
1-decanol

I
I
I

37.7±11.8
16.9±0.3
16.1±1.0I
I
I

52.3±16.3
16.0±3.2
19.2±1.3NII
I

22.4±3.3
13.6±2.3
14.2±0.9

I
I
I

57

viability(%)

117.2±4.
0117.2±5.
4121.2±5.
3113.4±3.
522.3±3.7
14.6±1.915.7±2.4

In
vitro
class1)
I
NI
NI
NI
NI
I
I
NI
NI
NI
NI
I
I
I

127

ENV/JM/MONO(2011)44

1-bromohexaneI

92.2±4.0

NI

102.0±5.9NIĮ-terpineol
2-chloromethyl-3,5dimethyl-4methoxypyridine HC
di-n-propyl disulfide
butyl methacrylate
HeptanalI

7.9±1.5I

10.7±0.2

I

103.5±16.
3
10.0±0.7I70.8±15.
88.5±1.4I

5.8±0.2I

5.6±0.3I

4.3±0.7I

5.1±0.7

I

I
I
I

98.0±3.573.2±7.522.2±5.5NINI
I

96.7±6.0
19.3±3.2
12.6±4.5NII
I

93.4±8.0
14.6±0.6
11.3±1.8NII
I

90.7±4.0
15.3±1.5
12.1±0.7

NI
I
I

NI

NI
I

In the case of a 10 minute exposure period, butyl methacrylate was evaluated as a non-irritant, though
it had been classified as an irritant in vivo (shown in red). Because the sensitivity of the prediction with a
10 minute exposure period was lower than that for the group with an exposure period of 15 minutes or
longer, it was thought that a 10 minute exposure period was not long enough.
With the 30 minute exposure period, on the other hand, isopropanol was indicated as an irritant, though
it had been classified as non-irritant in vivo (shown in blue). It was decided that the 30 minute exposure
period was too long and unsuitable, because the specificity of prediction was lower than that for groups
with exposure periods of 20 minutes or less.
In the case of a 15 minute exposure period, 1-bromo-4-chlorobutane (non-irritant in vivo class) andterpinyl acetate (irritant in vivo class) resulted in cell viabilities of around 50%, while both of them wereapparently indicated as an irritant in the 20 minute exposure. In vivo skin irritancy of 1-bromo-4chlorobutane is very low, as its primary irritation index is 0 and it was thought that the 15 minuteexposure period was better than the 20 minute exposure period because the cell viability for the 15
minute exposure period was higher. Also, in vivo skin irritancy of terpinyl acetate is at the borderline
between non-irritant and irritant, and it was thought that the 15 minute exposure period was better thanthe 20 minute exposure because cell viability was around 50%.
From the above considerations, it was concluded that the suitable exposure period was 15 minutes. The
original LabCyte EPI-MODEL24 SIT, which reflected the optimization study for a suitable exposure
period, was referred to in the SOP for the ver.4.0 of the LabCyte EPI-MODEL24 SIT.
5-3b. Assessment of the original LabCyte EPI-MODEL24 SIT by intra-laboratory study
Nineteen test chemicals were tested using the LabCyte EPI-MODEL24 and the common protocol
described in the ECVAM performance standard. Three independent runs were performed on different
batches of LabCyte EPI-MODEL24, with three tissues per test chemical (Table 5-1). When only cell
viability is used as an indicator, negative predictions for three chemicals, hexyl salicylate (No. 11), 1bromohexane (no. 16), and di-n-propyl disulphide (no. 18), were not concordant with the results of the invivo classification in the group of nine irritants and also two chemicals, 1-bromo-4-chlorobutane (no. 1)
and 4-methyl-thio-benzaldehyde (no. 7), were classified as positive in the group of ten non-irritants,(Table 5-6).
Table 5-6. Results of skin irritation tests by cell viability and IL-1D release evaluation.
The original SIT using LabCyte EPI-MODEL24

Test chemical
No.

128

Name

1

1-bromo-4chlorobutane

2diethyl phthalate

Cell viability (%)
Run

1
2
3
1
2
3

Mean

44.8
34.6
25.1
95.4
100.7
102.5

±

±
±
±
±
±
±

SD

6.7
11.8
7.6
3.2
7.7
2.5

In vitro
prediction
by cellviability
only1)

Judgment1)

I
I
I
NI
NI
NI

I

NI

58

In vitro

IL-1D(pg/tissue)
Mean

±

SD

Judgment1)

prediction bycell viability
and IL-1a
release1)

90.5
129.5
178.6
29.2
15.5
51.7

±
±
±
±
±
±

4.7
13.7
17.4
33.0
5.0
10.4

NI
I
I
NI
NI
NI

I
I
I
NI
NI
NI

I

NI

ENV/JM/MONO(2011)44

Table 5-6.

Continued
The original SIT using LabCyte EPI-MODEL24

Test chemical
No.

Cell viability (%)

Name

Run

Mean

±

SD

In vitro
prediction

Judgement1)

Mean

IL-1D(pg/tissue)±SDby onlyJudge

In vitro prediction

ment1)

by cell viability andIL-1a release1)

cell
viability1)3

di-propylene glycol

4naphthalenacid

5allyl phenoxy-acetate6

isopropanol

74-methyl-thio-benzaldehyde

8methyl stearate

9

allyl heptanoate

10

heptyl butyrate

11

hexyl salicylate

12

terpinyl acetate

acetic14

1-decanol

15

cyclamen aldehyde

161-bromohexane

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

95.4
103.9
99.0
96.8
97.0
101.8
87.7
75.1
99.2
86.2
86.1
94.5
25.7
22.7
20.9
107.2
110.4
107.1

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

1

45.9

±

2

93.7

±

3
1
2
3
1
2
3

118.9
97.2
109.8
99.3
103.1
110.8
101.1

±
±
±
±
±
±
±

1

18.0

±

2
3
1
2
3
1
2
3
1

36.3
39.1
15.5
21.6
17.2
19.7
21.8
17.0
16.9

±
±
±
±
±
±
±
±
±

2

59.0

±

17

Į-terpineol

3
1
2
3

84.1
12.2
14.5
7.3

±
±
±
±

18

di-n-propyl
disulphide

1

93.9

±

2
3

103.3
104.2

±
±

1

18.1

±

2
3
1
2
3

44.9
32.7
11.1
14.3
9.8

±
±
±
±
±

19

20

butyl methacrylate

heptanal

2.8
8.4
5.1
5.2
2.0
2.5
5.3
4.8
2.3
2.1
1.4
2.6
2.4
5.4
1.2
3.2
1.8
3.0
42.
4
14.
4
5.8
4.1
2.7
3.7
4.4
3.2
4.1
10.
0
6.5
8.1
0.8
1.5
1.8
1.8
1.4
3.6
0.9
10.
0
7.9
1.3
0.3
0.7
10.
7
4.3
9.0
11.
3
2.1
2.2
2.1
1.6
0.8

NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
I
I
NI
NI
NI

NI

6.5
16.7
16.0
2.9
5.7
12.0
36.3
115.0
27.8
57.7
94.7
99.8
93.9
119.3
102.9
6.7
7.9
5.6

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

3.6
6.3
12.1
1.5
2.9
8.2
22.8
20.3
6.8
9.9
8.8
19.8
10.5
29.1
2.9
0.8
1.7
1.2

NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI

NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
I
I
NI
NI
NI

NI

I

NI

495.8

±

257.3

I

I

NI

NI

86.3

±

99.4

NI

NI

NI
NI
NI
NI
NI
NI
NI

11.4
8.8
7.6
13.2
10.6
4.9
13.5

±
±
±
±
±
±
±

3.3
3.4
1.8
3.1
4.7
2.2
9.6

NI
NI
NI
NI
NI
NI
NI

NI
NI
NI
NI
NI
NI
NI

293.4

±

30.3

I

I

233.6
233.6
175.9
166.5
164.4
144.2
110.4
173.2
309.3

±
±
±
±
±
±
±
±
±

83.5
83.5
13.8
27.2
21.9
5.8
13.3
38.1
96.5

I
I
I
I
I
I
NI
I
I

I
I
I
I
I
I
I
I
I

164.8

±

33.3

I

I

39.0
216.8
167.7
239.0

±
±
±
±

36.7
20.3
4.8
53.4

NI
I
I
I

NI
I
I
I

19.1

±

16.6

NI

NI

6.5
21.4

±
±

2.6
15.0

NI
NI

NI
NI

I

130.5

±

39.6

I

I

I

I

112.3
148.9
154.6
136.6
189.5

±
±
±
±
±

11.5
10.0
10.3
3.5
24.8

NI
I
I
I
I

I
I
I
I
I

I

NI

NI

NI

I

NI

NI

NI

I

I

I
I
I
I
I
I
I
I
I

I

I

NI

NI
NI
I
I
I

I

NI

NI

NI
NI
I
I
I
I
I
I

NI

NI

NI

I

NI

NI

NI

I

I

I

I

I

NI

1) I: irritant, NI: non irritant

59

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ENV/JM/MONO(2011)44

Dermal irritation is the production of reversible damage with inflammation. IL-1D is a key cytokine
released in the inflammation process of skin irritation. In order to evaluate whether the IL-1D release
could be used as an assay to predict the irritation potential of the test chemicals, we measured the IL-1D
content in the conditioned medium from tissues onto which the chemicals were applied. The results of
the IL-1D assay were used as a second endpoint to support the classification based on the MTT assay
results. In this assay, 1-bromohexane (no. 16) was classified as an irritant based on the complementaryevaluation of IL-1D release, although it was classified as a non-irritant in the MTT assay (Table 5-6).
Summarizing the above data, the sensitivity and specificity of predictions improved to 77.8% and 80%(Table 5-7). The overall accuracy was 78.9% (Table 5-7).
D release evaluation
Table 5-7: Contingency table for skin irritation tests by cell viability and IL-1D

In vitro prediction

Irritant
72
9

Irritant
Non-irritantTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

In vivo classification
Non-irritant
2
8
10

Total
9
10
19

77.8
80.0
78.9

5-4. Discussion
The first purpose of the optimization stage is to set a suitable exposure period for the LabCyte EPIMODEL24 SIT similar to VRM, as a “me-too” test. From the results of examining various exposure
periods ranging from 10 to 30 min, it was judged that the exposure period of 15 minutes was the optimal
period for the LabCyte EPI-MODEL24 SIT. The original LabCyte EPI-MODEL24 SIT, which reflected
an optimization study of a suitable exposure period, was referred to in the SOP for ver.4.0 of the
LabCyte EPI-MODEL24 SIT.
The second aim of this study was to evaluate whether the commonly used irritation protocols describedin the ECVAM performance standards (1) were applicable to the original LabCyte EPI-MODEL24 SIT.
To compare the performances of the original LabCyte EPI-MODEL24 SIT and VRM, we tested with the
19 test chemicals described in the ECVAM performance standards. Only the prediction for 1bromohexane differed between the original LabCyte EPI-MODEL24 SIT and VRM, when viability was
the endpoint used as an indicator for prediction. This chemical was erroneously predicted as a nonirritant by the LabCyte original EPI-MODEL24 SIT, whereas VRM classified it in concordance with thein vivo class. As demonstrated in a previous study (32), the IL-1D assay can improve prediction andsupport the viability assay. In this study of the original LabCyte EPI-MODEL24 SIT, the prediction for
1-bromohexane was improved, so that it was truly shown as an irritant through the combination of the
MTT and IL-1D assays. As a result of this improvement, the sensitivity and the specificity of thisprediction model using the LabCyte EPI-MODEL24 fulfilled both conditions described in the ECVAM
performance standards: the sensitivity of a “me-too” test must be equal to or higher than 70% and thespecificity equal to or higher than 80%. The ECVAM performance standard must demonstrate similarity
and/or equivalence to the original LabCyte EPI-MODEL24 SIT in a formal inter-laboratory study, on thebasis of the VRM. From these considerations, the original LabCyte EPI-MODEL24 SIT was evaluated ina validation study under blind conditions and supported by the Japanese Society for Alternatives toAnimal Experiments (JSAAE).
The phase 1 (confirmation of technical transfer) and phase 2 (multisite ) validation studies for theoriginal SIT using the LabCyte EPI-MODEL24 were performed according to the ECVAM performance

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standards. During the phase 1 and phase 2 validation studies, assay acceptance criteria regarding
negative and positive control for the original LabCyte EPI-MODEL24 SIT were set based on the resultsof the phase 1 validation study. The acceptance criteria are referred to in the SOP for ver.5.0 of the
LabCyte EPI-MODEL24 SIT. Details of the results of the validation study are described in the JSAAE
report “VALIDATION STUDY OF IN VITRO SKIN IRRITATION TEST USING LABCYTE EPIMODEL24” (Attachment 2). The conclusion described in the validation report is as follows:
Based on the EU classification, 9 irritants (one skin irritant could not be purchased in Japan) and 10
non-irritants in the ECVAM Performance Standards were tested by the same 7 labs. The assay
demonstrated acceptable reliability of the positive control (100%) and accuracy (71% overall accuracy,
64% overall sensitivity, 79% overall specificity) in the MTT assay for use as a stand-alone assay to
distinguish between skin irritants and non-irritants. In addition, the IL-1Į endpoint was determined to beunnecessary.
Because it was decided that the IL-1Į endpoint was not necessary after the phase 2 validation study,
this conclusion was reflected in the SOP for ver.6.01 of the LabCyte EPI-MODEL24 SIT (Attachment 4).
In the ECVAM performance standards (updated) (28) that were revised in 2009, the in vivo
classification of test chemicals was changed from referring to the EU DSD (European Classification
System based on the Dangerous Substance Directive) to referring to the UN GHS (United Nations
Globally Harmonised System of Classification and Labelling of Chemicals). This change was reflectedin the OECD draft test guideline for skin irritation (2). With the changes for in vivo criteria, the set ofreference chemicals described in the performance standard (updated) or the OECD draft test guideline
was updated, and 6 chemicals were replaced. In line with these changes, the phase 3 validation study ofthe original SIT using LabCyte EPI-MODEL24 was performed with newly replaced 6 reference
chemicals. Details of the results for the phase 3 validation study are described in the JSAAE report“VALIDATION STUDY OF IN VITRO SKIN IRRITATION TEST USING LABCYTE EPI-MODEL24(2ND REPORT)” (Attachment 3). The content of the conclusion described in the validation report is asfollows:
Based on the GHS-EU classification, 5 irritants and 1 non-irritant in the new ECVAM PerformanceStandards were tested by 6 labs using LabCyte EPI-MODEL24. The assay demonstrated high reliability
with and without laboratories in the MTT assay, for use as a stand-alone assay to distinguish between
skin irritants and non-irritants.
The validation reports (Attachment 2,3) were assessed by a peer review panel in OECD. Unfortunately,
the OECD peer review panel pointed out that the prediction results of the chemical 1-bromohexane,
which is an in vivo Category 2 substance, in the original LabCyte EPI-MODEL24 SIT, did not meet the
specific requirement of paragraph 12, Annex 2 of the OECD draft test guideline (2). Miss-classificationof 1-bromohexane was not allowed in the OECD draft test guideline. However, the chemical 1bromohexane was classified as a false-negative by five out of six laboratories in the validation exercise
for the original LabCyte EPI-MODEL24 SIT. Therefore, a trial for the improvement of the original
LabCyte EPI-MODEL24 SIT to solve this problem became indispensable.

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6.

Modification of the original LabCyte EPI-MODEL24 SIT and assessment of the modified SIT(MODIFICATION STAGE)

6-1. Summary of the MODIFICATION STAGE
As recommended by the OECD peer review panel, improvement of the original LabCyte EPIMODEL24 SIT to solve the problem that 1-bromohexane was shown as a false-negative is indispensable
in enabling its use as a “me-too” test similar to VRM (see Section 5). It is surmised that one reason 1bromohexane showed a false negative in the original LabCyte EPI-MODEL24 SIT (SOP ver.6.01) isbecause 1-bromohexane did not remain in the reconstructed human epidermis tissues at the postincubation for 42 hours. In order to solve the 1-bromohexane problem, we tried to find a condition where
the 1-bromohexane would be retained in the stratum corneum of the LabCyte EPI-MODEL24 tissues atthe post incubation. More concretely, the following item were examined.
a. Applied amount of test chemical.
b. Incubation temperature at exposure to the test chemicals.
c. Washing protocol for test chemicals on the tissue surface.
The 1-bromohexane problem was not solved by modifying chemical application, such as changing theamount of test chemicals applied or changing the temperature of incubation at exposure to the test
chemicals. Only by modifying the washing protocol, were we able to solve the 1-bromohexane problem.
The modified washing protocol that solved the 1-bromohexane problem was reflected in the SOP forver.8.2 of the LabCyte EPI-MODEL24 SIT.
When the predictive potency of the modified LabCyte EPI-MODEL24 SIT was evaluated with the 20
test chemicals which were listed in the new OECD TG 439 for in vitro skin irritation testing, adopted in
July, 2010, the sensitivity, the specificity and the overall accuracy were 90 %, 70 %, and 80%,
respectively. It was thought that these results met the acceptance criteria described in the OECD TG 439.
Furthermore, a catch-up validation study was performed with the modified LabCyte EPI-MODEL24 SIT.
Results demonstrated high reliability and acceptable accuracy in the MTT assay, for use as a stand-alone
assay to distinguish between skin irritants and non-irritants.
6-2. Materials and methods
6-2a. Reference chemicals described in OECD TG 439
Twenty reference test chemicals shown in Table 6-1 were selected from the list in OECD TG 439(Attachment 1). Reference chemicals are used to determine the reliability and accuracy of a proposed
similar or modified test method, proven to be sufficiently similar to the VRM, both structurally andfunctionally.

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Table 6-1: Reference chemicals list in the OECD TG 439
No.

Name

1
2
3
4
5
6
7
8
9
10
11
12
13
14

Test chemicalCAS
In vivo1)
numberclass2)6940-78-9
NI
84-66-2
NI
86-87-3
NI7493-74-5
NI
67-63-0NI
3446-89-7
NI
112-61-8
NI
5870-93-9
NI6259-76-3
NI
104-55-2
NI
112-30-1
I
103-95-7
I
111-25-1
I86604-75I
3
629-19-6
I
1310-58-3
I
7340-90-1I

1-bromo-4-chlorobutane
Diethyl phthalate
naphthalen acetic acidallyl phenoxy-acetate
isopropanol
4-methyl-thio-benzaldehyde
methyl stearate
heptyl butyrate
hexyl salicylate
Cinnamaldehyde
1-decanol
Cyclamen aldehyde
1-bromohexane
2-chloromethyl-3,5-dimethyl-4methoxypyridine HCl
15
di-n-propyl disulphide
16
Potassium Hydroxide 5%
17
Benzene thiol 5-(1,1dimethylethyl)-2-methyl
181-methyl-3-phenyl-1-piperazine
5271-27-2
19heptanal
111-71-720
1,1,1 Trichloroethane
71-55-6
1) CAS No.: Chemical abstracts service registry number.
2) I: irritant, NI: non irritant
3) PII: primary irritation index.

I
I
I

PII3)

Supplier

0
0
0
0.3
0.3
1.0
1.0
1.7
2.0
2.0
2.3
2.3
2.7
2.7

Wako chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical
Kanto chemical
AldrichSigma Fluka
AldrichWako chemical
Wako chemical
Wako chemical
Wako chemical

3.0
3.0
3.3

Wako chemical
Wako chemicalTokyo Chemical

3.3
3.3
4.0

Tokyo Chemical
Kanto chemical
Wako chemical

6-2b. Wide-range of test chemicals
Fifty-four test chemicals over a wide range of classes were used for evaluation of the Modified
LabCyte EPI-MODEL SIT, as shown in Table 6-2. These chemicals were referred to in the report ofKandárová et. Al (33). This selected test set consists of chemicals tested in EpiDerm and EPISKIN
optimization studies preceding the ECVAM skin irritation validation study and also includes chemicals
from the ECVAM validation study (32). Available information on irritation potential and classificationaccording to the UN GHS systems are provided in Table 6-2.

63

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ENV/JM/MONO(2011)44

Table 6-2: Fifty-four test chemicals over a MTT of classes
No.
1
2
3
4
5

1-bromo-4-chlorobutane
diethyl phthalate
di-propylene glycol
naphthalen acetic acid
3-chloronitrobenzene

67

3,3-dithiodipropionic acid
4,4-methylenebis(2,6-di-tertbuthylphenol)
4-amino-1,2,4-triazole
benzyl benzoate
sodium bicarbonate
erucamide
1,5-hexadiene
Polyeth䌹lene glycol 400
glycerol3,3-dimethylpentane
allyl phenoxy-acetate
isopropanol
benzyl salicylate
lauric acid
4-methyl-thio-benzaldehyde
methyl stearate
benzyl acetatehydroxycitronellal
isopropyl myristateisopropyl palmitate
n-buthyl propionate
sodium bisulphite
benzyl alcohol
allyl heptanoate
heptyl butyrate

8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

134

Name

Test chemicals
CAS
In vivo1)
numberclass2)6940-78-9
No Cat.
84-66-2
No Cat.
25265-71-8
No Cat.
86-87-3
No Cat.
121-73-3
No Cat.

PII3)

Supplier

0
0
0
0
0

1119-62-6
118-82-1

No Cat.
No Cat.

00

Wako chemical
Wako chemical
Wako chemical
Wako chemical
LGC
STANDARDS
Wako chemicalTokyo Chemical

584-13-4
120-51-4
144-55-8
112-84-5
592-42-7
25322-68-3
56-81-5
562-49-2
7493-74-5
67-63-0
118-58-1
143-07-7
3446-89-7
112-61-8
140-11-4
107-75-5
110-27-0
142-91-6
590-01-2
7631-90-5
100-51-6
142-19-8
5870-93-9

No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.

0
0
0
0
0
0
0
0
0.3
0.3
0.3
0.3
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.3
1.7
1.7

Wako chemical
Wako chemical
Kanto chemical
Wako chemical
AldrichWako chemical
Wako chemical
AldrichWako chemical
Wako chemical
Wako chemical
Kanto chemical
Wako chemical
Kanto chemical
Wako chemical
Wako chemical
AldrichWako chemical
Wako chemical
Kanto chemical
AldrichWako chemical
Aldrich64

ENV/JM/MONO(2011)44

Table 6-2.
No.

Continued

Name

31
32

2-ethoxy ethyl methacrylate
hexyl salicylate

33

linalyl acetate

34

terpinyl acetate

35

Linalool36

cinnamaldehyde

37

eugenol

38
39
40
41
42
43

Test chemicals
CAS
In vivonumber1)class2)2370-63-0
No Cat.
6259-76-3
No Cat.
(Cat. 3)111-95-7
No Cat.
(Cat. 3)80-26-2
No Cat.
(Cat. 3)78-70-6
No Cat.
(Cat. 3)
104-55-2
No Cat.
(Cat. 3)97-53-0
No Cat.
(Cat. 3)103-95-7
Cat. 2
112-30-1
Cat. 2111-25-1
Cat. 2
98-55-5
Cat. 2110-53-2
Cat. 286604-75-3
Cat. 2cyclamen aldehyde
1-decanol
1-bromohexane
Į-terpineol
1-bromopentane
2-chloromethyl-3,5-dimethyl-4methoxypyridine HC
44butyl methacrylate
97-88-1
Cat. 245
di-n-propyl disulphide629-19-6
Cat. 246
potassium hydroxide 5%1310-58-3
Cat. 247
heptanal
111-71-7
Cat. 248
benzene thiol, 5-(1,17340-90-1
Cat. 2dimethylethyl)-2-methyl
491-methyl-3-phenyl-1-piperazine
5271-27-2
Cat. 250
SLS (20% aq)
151-21-3
Cat. 251
1,1,1 trichloroethane
71-55-6
Cat. 2
52
tetrachlotroethylene
127-18-4
Cat. 253
capric acid (decanoic acid)334-48-5
Cat. 254
SLS (5% aq)
127-18-4
Cat. 2
1) CAS No.: Chemical abstracts service registry number.
2) No Cat.: No Category, Cat. 2: Category 2, Cat. 3: Category 3
3) PII: primary irritation index.

PII3)

Supplier

1.7
2.0

Wako chemical
Sigma Fluka

2.0

Wako chemical

2.0

Alfa Aesar

2.0

Kanto chemical

2.0

Aldrich2.0

Wako chemical

2.3
2.3
2.7
2.7
2.7
2.7

Wako chemical
Wako chemical
Wako chemical
Kanto chemical
Kanto chemical
Wako chemical

3.0
3.0
3.0
3.3
3.3

Wako chemical
Wako chemical
Wako chemical
Kanto chemicalTokyo Chemical

3.3
4.0
4.0
4.0
4.0

Tokyo Chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical
Wako chemical

6-2c. Test Protocol for modification of the original LabCyte EPI-MODEL24 SIT
The test protocol for modification of the original LabCyte EPI-MODEL24 SIT is described in section
5-2b. From the conclusion that an IL-1Į endpoint was determined to be unnecessary in the validation
studies, the content of IL-1Į was not analyzed in this study. Three independent tests were performed on
different batches of LabCyte EPI-MODEL24, with three tissues per test chemical. The overall test
protocol is described in the original SOP for the LabCyte EPI-MODEL24 SIT (ver. 6.01).
6-2d. Protocol of the modified LabCyte EPI-MODEL24 SIT (SOP ver.8.2)
Prediction of the skin irritation potential of test chemicals by the modified LabCyte EPI-MODEL24
SIT was performed according to the modified protocol described in the SOP for ver.8.2 and described as

65

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ENV/JM/MONO(2011)44

follows. LabCyte EPI-MODEL24 tissues were aseptically removed from the transport agarose medium,transferred into 24-well plates (BD Biosciences, CA, USA) with the assay medium (0.5 ml) andincubated overnight (37°C, 5%, CO2, humidified atmosphere). On the next day, the tissues were topically
exposed to the test chemicals. Liquids (25 ȝl) were applied with a micropipette, and solids (25 mg) wereapplied from microtubes and moistened with 25 ȝl sterile water. If necessary, the mixture was gently
spread over the surface of the epidermis with a microspatula. Viscous liquids were applied using a cellsaver-type tip with a micropipette. Each test chemical was applied to three tissues. In addition, threetissues serving as negative controls were treated with 25 ȝl distilled water, and three tissues serving as
positive controls were exposed to 5% SLS. After 15 minutes, of exposure, each tissue was carefullyrinsed with PBS at least ten times or more with a washing bottle to completely remove remaining test
chemical from the surface. Furthermore, the leftover PBS outside the culture insert was gently removed
with a sterile cotton bud, but the residue of PBS inside the insert was not touched. The blotted tissues
were then transferred to new wells on 24-well plates containing 1 ml of fresh assay medium.
The treated and control tissues were post-incubated for 42 hours (37°C, 5% CO2, humidified
atmosphere). When the 42-hour post-incubation period was completed, blotted tissues were transferred
to new wells on 24-well plates containing 0.5 ml of freshly prepared MTT medium (0.5 mg/ml) for the
MTT assay. Tissues were incubated for three hours (37°C, 5% CO2, humidified atmosphere) and werethen transferred to microtubes containing 300 ȝl isopropanol, completely immersing the tissue.
Formazan extraction was performed at room temperature and the tissues were allowed to stand overnight.
Subsequently, 200 ȝl extracts were transferred to a 96-well plate. The optical density was measured at
570 nm and at 650 nm as a reference absorbance, with isopropanol as a blank.
Three independent tests were performed on different batches of LabCyte EPI-MODEL24, with threetissues per a test chemical. The prediction model is shown in Table 6-3. In the event that the three
independent results within an individual batch were not in agreement, the result that occurred twice wasused.
Table 6-3: The prediction models for the LabCyte EPI-MODEL24 SIT
Cell viability
mean  50%
mean > 50%

Judgment
Irritant
Non irritant

6-2e. Detecting chemical interference with MTT endpoints and correction procedures
A possible limitation of this skin irritation protocol described in OECD TG 439 might be due to theeffect of a small amount of test chemicals on the MTT endpoints directly. The following two types oftest chemicals can interfere with the MTT assay.
A. Chemicals that stain epidermis tissue.
B. Chemicals that can directly reduce MTT.
Test chemicals that stain the epidermis tissue could transfer from the epidermis tissue to the extraction
solution and affect the optical density (OD) measurements. Test chemicals that can directly reduce MTT
can also affect the optical density (OD) measurements, if the test chemical is present in the epidermis
tissues when the MTT viability test is performed. The overall test protocol is described in the original
SOP for the LabCyte EPI-MODEL24 SIT (ver. 8.2).
A. Detection of chemicals that stain the tissue
Step 1 (preliminary test)
Twenty-five ȝl (Liquid) or 25mg (Solid) of the test chemical was added to wells on a 24-wellassay plate preliminarily filled with 0.5mL of distilled water. Untreated distilled water is used as acontrol. The mixture was incubated in a CO2 incubator for 15 minutes. After incubation, the

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mixture was evaluated for staining of the distilled water macroscopically. If the colour of thesolution changes significantly, the test chemical is presumed to have the potential to stain thetissue and a functional check on viable tissues (Step2) should be performed. If the colour of thesolution does not change significantly, it is determined that the test chemical has no potential tostain the tissue.
Step 2 (Functional check on viable tissue)
Twenty-five ȝl (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color ofthe distilled water (Step 1), was added onto the surface of the epidermis tissues. Distilled water isused as a negative control. All procedures of the original SIT using LabCyte EPI-MODEL24
described in section 6-2d were performed. However, the tissue was incubated for 3 hours in a
culture medium without MTT instead of incubating in a medium containing MTT, in order toevaluate the staining of the epidermis tissues. The ratio of staining by a test chemical comparedwiththenegative
controlwascalculated.
If the ratio of staining by a test chemical is <5%, correction of the results is not necessary. If theratio is between 5% and 30%, the corrected MTT OD is calculated.
If the ratio of staining by a test chemical is >30%, the test chemical must be considered
incompatible with the test. However, when the cell viability (%) is <50%, the test chemical isdetermined as an irritant. Therefore, correction of the results or determination of incompatibility ofthe test chemical is not necessary in this case.
B. Detection of chemicals that directly reduce MTT
Step 3 (preliminary test)
Twenty-five ȝl (Liquid) or 25mg (Solid) of the test chemical was added to wells on a 24-wellassay plate preliminarily filled with 0.5mL of MTT medium. Untreated MTT medium is used as acontrol. The mixture was incubated in a CO2 incubator for 1 hour. After incubation, the mixture
was evaluated for the staining of the MTT medium macroscopically. If the MTT medium turns
significantly blue/purple, the test chemical can reduce MTT and an additional functional check(Step 4) must be performed. When the color of the solution does not change significantly, the test
chemicalisdetermined
not
to
have
the
potential
toreduceMTT.
Step 4 (Functional check on viable tissue)
Twenty-five ȝl (Liquid) or 25mg (Solid) of a test chemical, which clearly changed the color of the
MTT medium to blue/purple (Step3), was added onto the surface of the epidermis tissues. Distilledwater is used as a negative control. All procedures of the original SIT using LabCyte EPIMODEL24 described in section 6-2d were performed. However, epidermis tissues that have been
freeze-killed at -20 °C or lower for more than 24 hours were used instead of viable epidermis
tissues. The ratio of staining by a test chemical compared with the negative control was calculated.
When the ratio of staining by a test chemical is <30%, the corrected MTT OD is calculated. Whenthe ratio of staining by a test chemical is >30%, the test chemical must be considered incompatible
with the test. However, when the cell viability (%) is <50%, the test chemical is determined as an
irritant. Therefore, correction of the results or determination of incompatibility of the test chemical
is not necessary in this case.

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6-3. Results
6-3a. Modification of the original LabCyte EPI-MODEL24 SIT

Viability 䋨 % of Negative Control 䋩First of all, in order to promote thepenetration of 1-bromohexane to the stratum
150
corneum, an increase in the applied amount of
test chemical was examined. The appliedamount of test chemical was 25ȝL in theoriginal LabCyte EPI-MODEL24 SIT (SOP100
ver.6.01). Unfortunately, when the appliedamount of 1-bromhexane was increased from25ȝL to 50ȝL or 100ȝL, the cell viability ofeach tissue did not change at all (Fig.6-1).
50
From these results, it was concluded that the
1-bromohexane problem could not be solved
by changing the applied amount of test
chemical.
0
For a purpose similar to the experiment withthe applied amount, changing the temperature
1-bromohexane (PL)of incubation at exposure of the test chemical
was examined. The incubation temperature atchemical exposure was room temperature in
the original LabCyte EPI-MODEL24 SIT
Fig.6-1
Influence of applied amount of chemical(SOP ver.6.01). 1-boromohexane was judgedoncell viability of LabCyte EPIas a skin irritant with incubation at 37Ԩ for
MODEL24chemical exposure (Fig.6-2). However,isopropanol, which is a non irritant chemical
in UN-GHS, showed a false-positive under the same condition (Fig.6-2). In the trial with the change inincubation temperature to 37Ԩ at exposure to the test chemicals, the 1-bromohexane problem wassolved, but test chemicals that showed a false-positive increased, and the specificity of the LabCyte EPIMODEL24 SIT decreased.
Therefore, it was thought that the 1-bromohexane problem could not be solved by changing the
incubation temperature at exposure to the test chemicals.
25 (Ver.7.1)

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50

100

ENV/JM/MONO(2011)44

B. 37㷄

A. Room temparature (SOP ver.6.01)

150

Viability 䋨 % of Negative Control 䋩Viability 䋨 % of Negative Control 䋩

150

100

50

0

50

0
diethyl phthalate allyl phenoxyacetate

Not irritant

isopropanol

1-bromohexane

diethyl phthalate allyl phenoxyacetate

Irritant

Not irritantIn vivo class

Fig.6-2100

isopropanol

1-bromohexane

Irritant
In vivo class

Influence of change in incubation temperature on the cell viability of LabCyte
EPI-MODEL24

On the other hand, in order to retain chemical penetration into the stratum corneum, we modified thewashing protocol as follows:
1. The PBS stream from the washing bottle doesn't hit directly on the tissue surface, but it hits thewall of the cell culture insert and the tissue surface is washed by the PBS current.
2. PBS inside the cell culture insert may be removed by gently tapping only once at the top of the
beaker after each rinsing if necessary.
3. When removing the leftover PBS from the culture insert with a cotton bud, the inner side of thecell culture insert is not used, in order to prevent test chemicals that have penetrated the stratum
corneum from being extracted by the cotton pad.
With the modification of the washing protocol, 1-bromohexane was then judged as a skin irritant(Fig.6-3). On the other hand, diethyl phthalate, allyl phenoxy-acetate, and isopropanol, which are all non
irritant chemicals in the UN-GHS classification, were judged as non irritants under the same conditions(Fig.6-3).

69

139

ENV/JM/MONO(2011)44

B. Modified protocol

A. Original protocol (SOP ver.6.01)

150

Viability 䋨 % of Negative Control 䋩Viability 䋨 % of Negative Control 䋩

150

100

50

0

100

50

0
diethyl phthalate allyl phenoxyacetate

Not irritant

isopropanol

1-bromohexane

diethyl phthalate allyl phenoxyacetate

Irritant

Not irritantIn vivo class

Fig.6-3

isopropanol

1-bromohexane

Irritant
In vivo class

Influence of change to washing protocol on cell viability of LabCyte EPIMODEL24

From these results, it was suggested that the 1-bromohexane problem could be solved by modifying thewashing protocol.
Therefore, the modified washing protocol that where 1-bromohexane could be judged as a skin irritant
was reflected in the SOP for ver.8.2 of the LabCyte EPI-MODEL24 SIT (Attachment 7).
Points in the washing protocol from the SOP for ver.6.01 which were modified in the SOP for ver.8.2
are shown in Table 6-3.
Table 6-4: Washing protocol modifications from the ver. 6.01 SOP to the ver.8.2 SOP
Modifications
1. The PBS stream
from the washingbottle2. Removal of PBS
by tapping
3. How to use thecotton pad

SOP ver.6.01 (Original SIT)
There were no instructions about the
PBS stream.

SOP ver.8.2 (Modified SIT)
It was instructed that the PBS stream
must avoid hitting directly on thetissue surface.
Instructions stated that one must tap
It was instructed to tap only once if
as much as possible after each rinsing. necessary
Instructions stated that the cotton pad It was instructed to use the cotton
should be used on the inner and outer pad only on the outer side of the cellsides of the cell culture insert.
culture insert.

6-3b. Assessment of the modified LabCyte EPI-MODEL24 SIT based on the OECD TG 439 forintra-laboratory study
For 10 irritant chemicals (no.11 to no.20) in vivo, the results for di-n-propyl disulphide (no. 15) were
not concordant with those of the in vivo classification (Table 6-4). 1-bromohexane (no.13) was judged
as an irritant as shown by the modified LabCyte EPI-MODEL24 SIT (Table 6-4). On the other hand, for

140

70

ENV/JM/MONO(2011)44

10 non-irritants (from no.1 to no.10), 3 chemicals, 1-bromo-4-chlorobutane (no. 1), 4-methyl-thiobenzaldehyde (no. 6) and cinnamaldehyde (no. 10) were classified as positive by the modified LabCyte
EPI-MODEL24 SIT (Table 6-4).
Table 6-5:
Result of the modified LabCyte EPI-MODEL24 SIT with 20 test chemicals
listed in OECD TG 439.
The modified LabCyte EPI-MODEL24 SIT
Cell viability (%)
In vitro
class
Mean ± SD
Decision17.3 ± 4.4
II23.7 ± 1.5
I18.6 ± 2.1
I104.3 ± 4.4
NINI103.1 ± 1.5
NI55.4 ± 13.8
NI
92.7 ± 1.2
NINI107.8 ± 2.0
NI98.5 ± 3.8
NI94.7 ± 1.8
NINI96.3 ± 3.6
NI56.0 ± 3.8
NI83.5 ± 5.6
NINI87.7 ± 2.8
NI82.7 ± 8.5
NI24.6 ± 5.8
II21.4 ± 1.1
I21.8 ± 0.8
I98.6 ± 4.8
NINI113.1 ± 4.5
NI101.5 ± 4.9
NI105.9 ± 2.0
NINI108.8 ± 10.4
NI
109.7 ± 1.3
NI88.4 ± 8.2
NINI119.5 ± 2.0
NI112.2 ± 2.2
NI21.4 ± 4.1
II30.1 ± 5.9
I35.6 ± 4.6
I24.9 ± 3.8
II29.8 ± 6.5
I42.1 ± 2.9
I30.1 ± 11.2
II
24.2 ± 7.7
I36.0 ± 4.5
I36.3 ± 8.2
II40.3 ± 10.8
I
43.0 ± 3.8
I

Test chemical
no.
1

Name
1-bromo-4-chlorobutane

2diethyl phthalate

3naphthalen acetic acid4

allyl phenoxy-acetate5

isopropanol

64-methyl-thio-benzaldehyde

7methyl stearate

8

heptyl butyrate

9hexyl salicylate

10cinnamaldehyde

11

1-decanol

12cyclamen aldehyde

131-bromohexane

Run
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

71

141

ENV/JM/MONO(2011)44

Table 6-5.

Continued
Test chemical

no.
14

Name
2-chloromethyl-3,5-dimethyl-4methoxypyridine HC

15

di-n-propyl disulphide

16

Potassium hydroxide (5% aq.)17

benzene thiol, 5-(1,1dimethylethyl)-2-methyl

181-methyl-3-phenyl-1-piperazine

19

Heptanal

20

1,1,1-trichloroethane

Run
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

The modified LabCyte EPI-MODEL24 SIT
Cell viability (%)
In vitro
class
Mean ± SD
Decision10.7 ± 1.5
II11.0 ± 1.7
I16.4 ± 2.5
I74.9 ± 8.6
NINI77.1 ± 8.9
NI63.2 ± 7.6
NI
3.3 ± 0.4II
2.5 ± 0.9I
3.4 ± 1.4
I
22.2 ± 8.3
II23.3 ± 2.4
I37.2 ± 16.8
I
12.1 ± 4.5
II20.5 ± 5.7
I13.0 ± 4.4
I13.7 ± 9.9
Ii18.2 ± 1.8
I19.9 ± 2.5
I13.4 ± 0.6
II12.0 ± 1.1
I10.1 ± 4.3
I

Summarizing the data, the sensitivity and specificity of predictions by the modified LabCyte EPIMODEL24 SIT were 90.0 % and 70.0 %, respectively (Table 6-5). The overall accuracy was 80.0%(Table 6-5). We believe that these results meet the acceptance criteria described in OECD TG 439(Attachment 5).
Table 6-6: Prediction model for the modified LabCyte EPI-MODEL24 SIT according to the SOP
for ver.8.2.

In vitro prediction

Irritant
91
10

Irritant
Non-irritantTotal

Sensitivity (%)
Specificity (%)
Accuracy (%)

In vivo classification
Non-Irritant
3
7
10

Total
12
8
20

90.0
70.0
80.0

6-3c. Assessment of the modified LabCyte EPI-MODEL24 SIT using 54 test chemicals over a wide
range of classification
First, 54 chemicals were examined to detect interference with MTT endpoints. Of the 54 tested

142

72

ENV/JM/MONO(2011)44

chemicals, there were no chemicals which directly stained epidermis tissue (data not shown). On theother hand, in the experiment for direct MTT reduction (6-2e, STEP 3), 4-methyl-thio-benzaldehyde (no.
20), benzyl alcohol (no. 28), cinnamaldehyde (no. 37), eugenol (no. 38), cyclamen aldehyde (no. 39),
heptanal (no. 48) and benzene thiol 5-(1,1-dimethylethyl)-2-methyl (no. 49) were detected as potential
MTT reducers (Fig. 6-4).

4-methylthio
benzylcinnamaldeheugenolalcoholydebenzaldehydeFig.6-4
Direct MTT reduction ability (STEP 3).

cyclamenaldehyde

heptanal

5-(1,1dimethylethyl)-2-methyl

Table 6-7: Detection of MTT reducer in 54 test chemicals.
Positive chemicals in step 3
4-methyl-thio-benzaldehyd
benzyl alcohol
cinnamaldehyde

Cell viability(%: not corrected)
21
13
23

Staining Ratio(% /NC1))
13
14
80

34

145

26

90

26
33

16
56

eugenol
cyclamen aldehyde
heptanalbenzene thiol 5-(1,1-dimethylethyl)-2methyl

Corrected
viability (%)
20
12incompatible(16)
incompatible(21)
incompatible(19)
25
incompatible(28)

Therefore, we examined whether these substances reduced MTT using freeze-killed tissue instead ofliving tissue. As a result, the staining ratio (compared with a negative control) for 4-methyl-thiobenzaldehyde, benzyl alcohol, and heptanal were between 5% and 30 % (Table 6-7). Therefore corrected
cell viabilities for these test chemicals had to be calculated (Table 6-7). However, the staining ratios for
cinnamaldehyde, eugenol, cyclamen aldehyde, and benzene thiol 5-(1,1-dimethylethyl)-2-methyl werehigher than 30 % and the cell viabilities of all potential MTT inducers were lower than 50%, so thesechemicals were finally determined as irritants.
Next, the performance of the modified LabCyte EPI-MODEL24 SIT was evaluated with the 54 test
chemicals. Results obtained with each chemical are given in 6-8. Sodium bisulphite (no. 27) and 2ethoxy ethyl methacrylate (no. 31) showed different predictions in three independent test runs.
Concordance with classifications obtained within-laboratory had a sufficient level of reproducibility forthe modified LabCyte EPI-MODEL24 SIT at 96% (52/54).
As for 17 irritants (no.39 to no.54), the results for di-n-propyl disulphide (no. 46) were not concordant
with those of the in vivo classification (Table 6-8). With regard to 37, non-irritants (from no.1 to no.37),
11 chemicals, 1-bromo-4-chlorobutane (no. 1), 4-methyl-thio-benzaldehyde (no. 20), benzyl acetate (no.

73

143

ENV/JM/MONO(2011)44

22), hydroxycitronellal (no. 23), n-buthyl propionate (no. 26),, benzyl alcohol (no. 28), 2-ethoxy ethyl
methacrylate (no. 31), terpinyl acetate (no. 34), linalol (no.35), cinnamaldehyde (no. 36) and eugenol (no.
37) were classified as positive by the modified LabCyte EPI-MODEL24 SIT (Table 6-8).
In summary, based on the mean of the three independent runs (Table 6-8), 26 out of 37 non-irritants
and 16 out of 17 irritants in the GHS classification were classified correctly by the modified LabCyte
EPI-MODEL24 SIT. The statistical parameters describing assay performance are displayed in Table 6-9.
Sensitivity and specificity of predictions by the modified LabCyte EPI-MODEL24 SIT were 94.1% and70.3%, respectively. Overall accuracy was 77.8% (Table 6-9).

144

74

ENV/JM/MONO(2011)44

Table 6-8: Results for the modified LabCyte EPI-MODEL24 SIT tested with 54 chemicals.
Test chemical

The modified LabCyte EPI-MODEL24 SIT (SOPver.8.2)
Cell viability (%)

No.

Name

1

1-bromo-4-chlorobutane

2diethyl phthalate

3

di-propylene glycol

4naphthalen acetic acid

5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22

3-chloronitrobenzene
3,3-dithiodipropionic acid
4,4-methylenebis(2,6-di-tert-buthylphenol)
4-amino-1,2,4-triazole
benzyl benzoate
sodium bicarbonate
Erucamide
1,5-hexadienepolyethlene glycol 400
Glycerol3,3-dimethylpentane
allyl phenoxy-acetate
isopropanol
benzyl salicylate
lauric acid
4-methyl-thio-benzaldehyde
methyl stearate
benzyl acetate

23hydroxycitronellal

24

isopropyl myristate25

isopropyl palmitate

Run

Mean

±

SD

Judgment

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

17.3
23.7
18.6
104.3
103.1
55.4
109.9
100.2
92.9
92.7
107.8
98.5
104.3
98.0
95.2
100.0
98.9
89.9
103.9
100.0
100.0
101.0
97.4
98.8
105.7
99.6
100.3
99.6
100.0
100.3
102.5
92.7
90.0
85.6
92.5
95.6
106.6
98.2
103.2
125.7
98.1
103.3
72.3
76.3
90.8
94.7
96.3
56.0
83.5
87.7
82.7
93.6
98.2
99.9
94.0
109.2
110.0
24.6
21.4
21.8
98.6
113.1
101.5
11.3
29.8
37.4
19.8
20.4
32.3
107.9
97.3
97.2
104.1
102.5
115.9

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

4.4
1.5
2.1
4.4
1.5
13.8
2.0
10.2
12.2
1.2
2.0
3.8
4.4
2.2
6.9
5.6
2.5
10.1
10.6
3.5
9.9
6.9
6.7
8.4
2.0
3.2
10.1
3.7
5.5
12.4
2.2
2.8
11.0
12.2
7.2
3.8
8.7
5.4
4.3
9.0
6.5
6.5
3.2
9.7
1.9
1.8
3.6
3.8
5.6
2.8
8.5
16.4
3.6
9.7
4.0
7.4
6.3
5.8
1.1
0.8
4.8
4.5
4.9
2.7
4.7
8.0
11.2
1.8
6.0
3.7
5.2
12.4
10.6
1.3
2.2

I
I
I
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
I
I
I
NI
NI
NI
I
I
I
I
I
I
NI
NI
NI
NI
NI
NI

75

VRM

EpiDermTM
SIT

I

I

I

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

In vitro
class

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

NI

I

I

NI/I

I

NI

NI

NI

I

I

NI

I

NI

I

NI

NI

NI

NI

NI

NI

145

ENV/JM/MONO(2011)44

Table 6-8. Continued
Test chemical

The modified LabCyte EPI-MODEL24 SIT (SOP forver.8.2)
Cell viability (%)

no.
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40

146

Name
n-buthyl propionate
sodium bisulphite
benzyl alcohol
allyl heptanoate
heptyl butyrate
2-ethoxy ethyl methacrylate
hexyl salicylate
linalyl acetate
terpinyl acetate
Linaloolcinnamaldehyde
Eugenol
cyclamen aldehyde
1-decanol
1-bromohexane41

Į-terpineol

421-bromopentane

432-chloromethyl-3,5-dimethyl

44butyl methacrylate

45

di-n-propyl disulphide46

potassium hydroxide 5%

47

heptanal

48

benzene thiol, 5-(1,1-dimethylethyl)-2methyl

491-methyl-3-phenyl-1-piperazine-4-methoxypyridine HC

Run

Mean

±

SD

Judgment

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

23.1
41.2
45.9
11.1
61.2
74.7
5.6
17.5
17.8
95.6
108.9
104.6
105.9
108.8
109.7
29.3
43.1
74.4
88.4
119.5
112.2
101.2
86.8
92.6
26.2
33.6
36.3
7.9
16.6
25.4
21.4
30.1
35.6
18.5
30.4
32.2
30.1
24.2
36.0
24.9
29.8
42.1
36.3
40.3
43.0
13.6
17.4
33.0
24.3
17.7
23.7
10.7
11.0
16.4
28.6
24.5
33.6
74.9
77.1
63.2
3.3
2.5
3.4
13.7
18.2
19.9
22.2
23.3
37.2
12.1
20.5
13.0

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

4.7
6.4
6.4
5.5
6.8
9.7
1.3
4.9
2.0
5.7
10.7
3.3
2.0
10.4
1.3
5.2
5.9
3.6
8.2
2.0
2.2
1.1
6.7
12.6
4.1
3.3
10.9
0.7
1.9
4.0
4.1
5.9
4.6
2.2
4.4
7.1
11.2
7.7
4.5
3.8
6.5
2.9
8.2
10.8
3.8
1.3
1.6
5.8
15.1
3.9
5.7
1.5
1.7
2.5
6.1
4.1
11.8
8.6
8.9
7.6
0.4
0.9
1.4
9.9
1.8
2.5
8.3
2.4
16.8
4.5
5.7
4.4

I
I
I
I
NI
NI
I
I
I
NI
NI
NI
NI
NI
NI
I
I
NI
NI
NI
NI
NI
NI
NI
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
NI
NI
NI
I
I
I
I
I
I
I
I
I
I
I
I

76

VRM

EpiDermTM
SIT

I

I

I

NI

NI

NI

I

NI

I

NI

NI

NI

NI

NI

NI

I

NI/I

I

NI

NI

NI

NI

NI/I

NI

I

NI/I

NI

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

NI

I/NI

NI

I

I/NI

I

I

I/NI

I

I

I

I

I

I

I

In vitroclassENV/JM/MONO(2011)44

Table 6-8. Continued
Test chemical

The modified LabCyte EPI-MODEL24 SIT (SOPver.8.2)
Cell viability (%)

no.

Name

50

SLS (20% aq)

51

1,1,1 trichloroethane52

tetrachlotroethylene

53

capric acid (decanoic acid)54

SLS (5% aq)

Run

Mean

±

SD

Judgment

1
2
3
1
2
3
1
2
3
1
2
3
1
2
3

9.4
13.2
13.4
13.4
12.0
10.1
11.1
17.1
22.6
6.1
9.8
17.6
14.5
11.4
14.4

±
±
±
±
±
±
±
±
±
±
±
±
±
±
±

0.9
1.3
4.2
0.6
1.1
4.3
1.3
0.9
2.1
2.8
0.4
3.4
1.9
1.0
0.9

I
I
I
I
I
I
I
I
I
I
I
I
I
I
I

VRM

EpiDermTM
SIT

I

NI

I

I

I

I

I

I

I

In vitro
class

I

I

I

I

I

Source of VRM and EpiDermTM SIT data (33)

Table 6-9. Prediction model for skin irritation test by cell viability evaluation with 54 test
chemicals and based on the SOP for ver.8.2.

In vitro prediction
Sensitivity (%)
Specificity (%)
Accuracy (%)

Irritant
Non-irritant
Total

Irritant
16
1
17

In vivo classification
Non-Irritant
11
26
37

Total
27
27
54

94.1
70.3
77.8

6-4. Discussion
As recommended by the OECD peer review panel, the original LabCyte EPI-MODEL24 SIT had to be
improved in order to solve the problem where 1-bromohexane was being shown as a false-negative. We
hypothesized that it was important to retain 1-bromohexane in the LabCyte EPI-MODEL24 tissue at thepost incubation. Therefore, we modified the conditions for the application of test chemicals, such as theapplied amount of test chemical and the temperature at exposure to test chemicals. However,
unfortunately, the 1-bromohexane problem was not solved by changing conditions under which test
chemicals were applied. In the end, it was found that the problem concerning 1-bromohexane could be
solved by changing the washing protocol. By changing the washing protocol, 1-bromohexane penetration
into the stratum corneum could be sufficiently retained. The judgments for the other 19 reference
chemicals listed in OECD TG 439 excluding 1-bromohexane did not change between the original
LabCyte EPI-MODEL24 SIT and the modified LabCyte EPI-MODEL24 SIT. With regard to thepredictive potency of the modified LabCyte EPI-MODEL24 SIT with the 20 test chemicals listed in theOECD TG 439, the sensitivity, specificity and overall accuracy were 90 %, 70 %, and 80%, respectively.
It was thought that these results met the acceptance criteria described in the OECD TG 439.
In the evaluation of the performance of the modified LabCyte EPI-MODEL24 SIT with the wide-rangeof 54 test chemicals, sensitivity, specificity and overall accuracy were 94.1 %, 70.3 %, and 77.8%,

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respectively. Prediction potency was thought to be almost equal to the VRM (EpiSkinTM test method) or
EpidermTM SIT (33).
OECD TG 439 requires demonstration of similarity and/or equivalence to the SIT using the newreconstructed human epidermal model in a formal inter-laboratory study, based on the VRM. It wasconsidered that the performance of the modified LabCyte EPI-MODEL24 SIT needed to be evaluated bya formal validation study under blind conditions, as stipulated by OECD TG 439. Therefore, a catch-up
validation study of the modified LabCyte EPI-MODEL24 SIT was performed from September toNovember, 2010, supported by the Japanese Center for the Validation of Alternative Methods (JaCVAM).
Results demonstrated high reliability and acceptable accuracy on the MTT assay for use as a stand-alone
assay to distinguish between skin irritants and non-irritants. Details of the results of the validation study
will be described in a JaCVAM final report.
7.

Conclusion
Through a final, formal validation study based on OECD TG 439, this document demonstrates that themodified LabCyte EPI-MODEL24 SIT is a useful in vitro methodology for classification of skinirritation potential.

8.

Acknowledgement
We would like to thank Ms. Lu Qiu at the Shanghai Entry-Exit Inspection and Quarantine Bureau of
the People’s Republic of China for her technical support with the QC experiments for LabCyte EPIMODEL24 after the export of shipments of LabCyte EPI-MODEL24 out of Japan. We would also like tothank Mr. Yoshihiro Yamaguchi and Ms. Maki Nakamura in Kobayashi Pharmaceuticals Co., Ltd. fortheir gracious support of this work.

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9.

Abbreviations

ECVAM

European Centre for the Validation of Alternative Methods

EU DSD

European Classification System based on the Dangerous Substance Directive (DSD)

GHS

Globally harmonised system on the classification and labelling of chemicals

IL-1Į

Interleukin-1 alphaJaCVAM

Japanese Center for the Validation of Alternative Methods

JSAAE

Japanese Society for Alternatives to Animal Experiments

J-TEC

Japan Tissue Engineering Co. Ltd.

MTT

3-(4,5-Dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide. A vital dye used to assess
cell viability via a colorimetric assay. Cell viability is used as a proxy for predicting theskin irritancy potential of xenobiotic substances in human reconstructed epidermis
models.

OECD

Organisation for Economic Cooperation and Development

QC

Quality control

PBS

Phosphate buffer saline

RhE

Reconstructed human epidermis

SDStandard deviation

SIT

Skin irritation test(ing)

SLS

Sodium lauryl sulphateTG

Test guideline

UN GHSThe GHS system for skin irritation as applicable to all authorities, i.e. using one irritantcategory.

VRM

Validated reference method

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10. Attachments (not in this OECD Report)
Attachment 1.

OECD TG 439: OECD guideline for the testing of chemicals. In vitro Skin Irritation:
Reconstructed
Human
Epidermis
Test
Method.
(2010)
Website: http://browse.oecdbookshop.org/oecd/pdfs/browseit/9743901E.PDF

Attachment 2.

LABCYTE VALIDATION MANAGEMENT TEAM: JSAAE DRAFT REPORT:VALIDATION STUDY OF IN VITRO SKIN IRRITATION TEST USING LABCYTEEPI-MODEL24. APRIL 15, 2009.

Attachment 3.

LABCYTE VALIDATION MANAGEMENT TEAM: JSAAE REPORT: VALIDATIONSTUDY OF IN VITRO SKIN IRRITATION TEST USING LABCYTE EPI-MODEL24(2ND REPORT). JULY 22, 2009

Attachment 4.

Skin irritation test protocol using the reconstructed human model “LabCyte EPIMODEL24” Ver.6.01

Attachment 5.

Skin irritation test protocol using the reconstructed human model “LabCyte EPIMODEL24” Ver.8.2

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11. References
1.

ECVAM SIVS, 2007.: Performance standards for applying human skin models to in vitro skinirritation
testing.
(2007)
Website: http://ecvam.jrc.it/indexs.html

2.

OECD
GUIDELINEFORTHETESTINGOF
CHEMICALSDRAFT PROPOSAL FOR A NEW GUIDELINE In vitro Skin Irritation: Reconstructed Human
Epidermis
(RhE)
Test
Method.
(2009)
Version
7.6
Website: http://ecvam.jrc.it/indexs.html

3.

OECD Test Guideline 404.: OECD guideline for the testing of chemicals. Acute dermal
irritation/corrosion.
(2002)
Website:http://www.oecd.org/document/55/0,3746,en_2649_34377_2349687_1_1_1_1,00.html

4.

Botham, P.A., et al.: Alternative methods for skin irritation testing: the current status. ECVAM skinirritation task force report 1. Altern. Lab. Anim., 26, 195-211, (1998)

5.

Fentem, J.H., et al.: A prevalidation study on in vitro tests for acute skin irritation: results andevaluation by the management team. Toxicol. in vitro., 15, 57-93, (2001)

6.

Zuang, V., et al.: Follow-up to the ECVAM prevalidation study on in vitro tests for acute skinirritation. ECVAM skin irritation taskforce report 2. Altern. Lab. Anim., 30, 109-129, (2002)

7.

Cotovio, J., et al.: The in vitro acute skin irritation of chemicals: Optimization of the EpiSkin
prediction model within the framework of the ECVAM validation process. Altern. Lab. Anim., 33,
329-349, (2004)

8.

Kandárová, H., et al.: Optimisation of the EpiDerm test protocol for the upcoming ECVAM
validation study on in vitro skin irritation test. ALTEX, 21, 107-114, (2004)

9.

ESAC statement, 2007.: Statement on the validity of in vitro tests for skin irritation.
Website: http://ecvam.jrc.it/indexs.html

10.

Katoh, M., et al.: Morphological Characterization of a reconstructed, cultured human epidermal
model (LabCyte EPI-MODEL24) as an alternative to in vivo models. AATEX, 14, 873-878, (2009)11.

Katoh, M., et al.: Assessment of human epidermal model LabCyte EPI-MODEL24 for in vitro skinirritation testing according to European centre for the validation of alternative methods (ECVAM)Validated Protocol. J. Toxicol. Sci., 34, 327-334, (2009)12.

Kojima, H., et al.: Dose-response evaluation using an epidermal model, an alternative to skinirritation testing. AATEX, 11, 177-184, (2006)

13.

Morinaga, M., et al.: Study on the prediction of human lip irritation from cosmetics materials using
HeLa-MNTT assay. AATEX, 14, 415-416, (2008)

14.

Watanabe, E., et al.: Test Method for Skin Damage of Titania Photocatalyst Nanoparticles in vitro.
Materials Sci. Forum, 569, 9-12, (2008)15.

Moniruzzaman, M., et al.: Ionic liquid-in-oil microemulsion as a potential carrier of sparingly
soluble drug: Characterization and cytotoxicity evaluation. Int. J. Pharmaceu., 400, 243-250, (2010)16

Katoh, M., et al.: Assessment of the human epidermal model LabCyte EPI-MODEL24 for in vitro
skin corrosion testing according to the OECD test guideline 431. J. Toxicol. Sci., 34, 411-417,(2010)

17.

Kobayashi, K., et al.: The development of a filter to enhance the efficacy and safety of excimer light(308 nm) therapy. Photodermatol. Photoimmunol. Photomed., 25, 30-36, (2009)

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18.

Nin, M., et al.: Dichotomous effect of ultraviolet B on the expression of corneodesmosomal enzymes
in human epidermal keratinocytes. J. Dermatol. Sci., 54, 17-24, (2009)19.

Noborio, R., et al.: Preferential induction of endothelin-1 in a human epidermal equivalent model bynarrow-band ultraviolet B light sources. Photodermatol. Photoimmunol. Photomed., 26, 159-16,(2010)

20.

Niwa, M., et al.: Evaluation of the skin irritation using a DNA microarray on a reconstructed humanepidermal Model. Biol. Pharm. Bull., 32, 203-208, (2009)

21.

Kobori, M., et al.: Evaluation of Anti-inflammatory and Anti-allergic Effects of Food Components
Using DNA Microarray Analysis. JARQ, 44, 243-248, (2010)22

Tokudome, Y., et al.: Effect of topically applied sphingomyelin-based liposomes on the ceramide
level in a three-dimensional cultured human skin model. J. Liposome Res., 20, 49-54, (2010)

23.

Li, Y., et al.: Trypsin as a novel potential absorption enhancer for improving the transdermal deliveryof macromolecules. J. Pharm. Pharmaco., 61, 1005-1012, (2009)24.

Iwai, I., et al.: Change in optical properties of stratum corneum included by protein carbonylation invitro. Int. J. of Cosmetic Sci., 30, 41-46, (2008)

25

Sugiura, K., et al.: LEDGF/DFS70, a major autoantigen of atopic dermatitis, is a component of
keratohyalin granules. J. Invest. Dermatol., 127, 75-80, (2007)

26.

Sugiura, K., et al.: The unfolded protein response is activated in differentiating epidermal
keratinocytes. J. Invest. Dermatol., 129, 2126-2135, (2009)27.

ESAC statment, 2008.: Statement on the scientific validity of in vitro tests for skin irritation testing.
Website: http://ecvam.jrc.it/indexs.html

28.

ESAC statement, 2009.: Statement on the scientific validity of in vitro tests for skin irritation testing.
Website: http://ecvam.jrc.it/indexs.html

29.

ECVAM Performance Standards for Skin Irritation Testing (updated), 2009.: Performance standards
for in vitro skin irritation test methods based on reconstructed human epidermis (RhE).
Website: http://ecvam.jrc.it/indexs.html

30.

Green, H.: Cyclic AMP in relation to proliferation of the epidermal cell: new view. Cell, 15, 801-811,(1978)

31.

Rheinwald, J.G., et al.: Serial cultivation of strains of human epidermal keratinocytes: the formationof keratinizing colonies from single cells. Cell, 6, 331-344, (1975)

32.

Spielmann, H., et al.: The ECVAM international validation study on in vitro tests for acute skinirritation: Report on the validity of the EPISKINTM and EpiDerm assays and on the skin integrity
function test. Altern. Lab. Anim., 35, 559-601, (2007)

33.

Kandárová, H., et al.: In vitro skin irritation test: Improving the sensitivity of the EpiDerm skinirritation test protocol. Altern Lab Anim. 37, 671-689, (2009)

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Appendix 3-JSAAE Skin Irritation Test Validation StudySKIN IRRITATION TESTUSING THE RECONSTRUCTED HUMAN MODEL “LABCYTE EPI-MODEL 24”
Ver. 8.2

LabCyte EPI-MODEL24 SKIN IRRITATION TEST-42 HOURS

S.O.P.

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TABLE OF CONTENTS
1. RATIONAL AND BACKGROUND
1.1
LabCyte EPI-MODEL 24 SKIN IRRITATION TEST (SIT using LabCyte EPI-MODEL24)1.2
BACKGROUND OF SIT using LabCyte EPI-MODEL24
1.3
BASIS OF THE METOD
1.3.1
TEST SYSTEM DESCRIPTION
1.3.2
ASSAY QUALITY CONTROL
1.4
LIMITATION OF THE METHOD
1.5
BRIEF BASIC PROCEDURE
1.6
DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)
2. MATERIALS
2.1 LabCyte EPI-MODEL 24
2.1.1
LabCyte EPI-MODEL 24 KIT COMPONENTS
2.1.2
SHIPMENT OF LabCyte EPI-MODEL24
2.1.3
INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24
2.2
TEST CHEMICALS
2.3
CONSUMABLES
2.4
OTHERS
2.4.1
EQUIPMENT / INSTRUMENTS
2.4.2
CONSUMMABLE ITEMS
3. TEST METHOD
3.1 PREPARATIONS
3.1.1
MTT SOLUTION
3.1.2
POSITICE CONTROL SUBSTANCE
3.1.3
NEGATIVE CONTROL SUBSTANCE
3.1.4
POLY WASH BOTTLE FOR PBS
3.2 TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT
3.2.1
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE
3.2.2
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT
3.3
EXECUTION OF THE TEST
3.3.1
PREPARATION OF LabCyte EPI-MODEL 24 (DAY -1)
3.3.2
APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0)
3.3.3
POST TREATMENT INCUBATION (DAY 0~2)
3.3.4
MTT ASSAY (DAY 2)
3.3.5
FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2~3)
4. ASSESSMENT
4.1
CONDITIONS FOR A SUCCESSFUL STUDY
4.2
ASSAY CRITERIAMDS 1: RECEIPT OF LABCYTE EPI-MODEL 24

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MDS 2: PRE-INCUBATION OF LABCYTE EPI-MODEL 24 (Section 3.3.1)
MDS 3-1: APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section
3.1.2, 3.3.2 ~ 3.3.3)
MDS 3-2: APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section
3.3.2 ~ 3.3.3)
MDS 4: MTT ASSAY (Section 3.3.4)
MDS 5: FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.5)

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1. RATIONAL AND BACKGROUND
1.1

SKIN IRRITATION TEST using LabCyte EPI-MODEL 24 (SIT using LabCyte EPI-MODEL24)

The SIT using LabCyte EPI-MODEL24 is designed for the prediction of acute skin irritation ofchemicals by measurement of its cytotoxic effect, as reflected in the MTT assay, on the Reconstructed
Human Epidermis (RHE) model. The SIT using LabCyte EPI-MODEL24 is not a kit; LabCyte EPIMODEL24 tissues are commercially available per tissues item (with a minimum of 24 LabCyte EPIMODEL24 tissues per order).
1.2

BACKGROUND OF SIT using LabCyte EPI-MODEL24

Performance standards for applying human skin models to in vitro skin irritation testing were also
defined based on the validated test EpiSkinTM test method (ECVAM SIVS, 2007). These performancestandards can be then used to evaluate the accuracy and reliability of other analogous test methods (alsoreferred to as “me-too” tests) that are based on similar scientific principles and measure or predict the same
biological or toxic effect.
Based on the GHS-EU classification, 12 irritants and 13 non-irritants in the draft performance standards(ECVAM 2007) and the statement by ESAC (ESAC2009) were performed the validation study through the
7 labs SIT using LabCyte EPI-MODEL24. Results were summarized at JSAAE 1st report and 2nd reporton this validation study.
1.3

BASIS OF THE METHOD

Chemical-induced skin irritation, manifested by erythema and oedema, is the results of a cascade ofevents beginning with penetration of the stratum corneum and damage to the underlying layers ofkeratinocytes. The dying keratinocytes release mediators that begin the inflammatory cascade which actson the cells in the dermis, particularly the stromal and endothelial cells. It is the dilation and increased
permeability of the endothelial cells that produce the observed erythema and oedema. The RhE-based test
methods measure the initiating events in the cascade.
The relative viability of the treated tissues was measured at the end of the treatment exposure (15minutes) followed by a post-exposure period (42 hours) using MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5diphenyltetrazoliumbromide] assay. A cutoff value of 50% viability of the negative control value wasconsidered and used to classify test substances as irritant (I) or non irritant (NI). The culture environment
might allow the detection of very small quantities of cytokines secreted by the epidermis in response to
topical application of test substances.
1.3.1

TEST SYSTEM DESCRIPTION

LabCyte EPI-MODEL24 is a new, commercially available RhE model produced by Japan Tissue
Engineering Co. Ltd. It consists of normal human epidermal keratinocytes whose biological origin isneonate foreskin. In order to expand human keratinocytes while maintaining their phenotype, they were
cultured with 3T3-J2 cells as a feeder layer (Rheinwald and Green, 1975; Green, 1978). Reconstruction ofhuman cultured epidermis is achieved by cultivating and proliferating keratinocytes on an inert filter
substrate (surface 0.3 cm2) at the air-liquid interface for 13 days with an optimized medium containing 5%
fetal bovine serum. It constructs a multilayer structure consisting of a fully differentiated epithelium withfeatures of the normal human epidermis, including a stratum corneum. LabCyte EPI-MODEL24 isembedded in an agarose gel containing nutrient solution and shipped in 24-well plates at around 18°C.

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1.3.1.1

Quality control of the test system

The LabCyte EPI-MODEL24 is manufactured according to defined quality assurance procedures. Eachbatch production was provided with quality controls such as storage conditions, RHE instructions for use,
lot number and origin, histology (demonstration of human epidermis-like structure with multilayered
stratum corneum), cell viability, barrier function integrity (0.14  IC50  0.4).
1.3.1.2

Precautions

The epidermal cells are taken from healthy donor negative to HIV, and Hepatitis. Nevertheless, handlingprocedures for biological materials should be followed:a) It is recommended to wear gloves during handling with the skin and kit components.
b) After use, the epidermis, the material and all media in contact with it should be decontaminated prior
to disposal (e.g. using special containers or autoclaving).
1.3.2

ASSAY QUALITY CONTROL

1.3.2.1
Assay Acceptance Criterion 1: Negative Control
The absolute OD of the negative control (NC) tissues (treated with sterile DPBS) in the MTT assay is anindicator of tissue viability obtained in the testing laboratory after shipping and storing procedures andunder specific conditions of use.
0.7  Mean OD (A570/650) measured value  2.5
1.3.2.2

Assay Acceptance Criterion 2: Positive Control

A 5% SDS (in H2O) solution (see 7.6.3) is used as positive control (PC) and tested concurrently with thetest chemicals. Concurrent means here the PC has to be tested in each assay, but not more than one PC isrequired per testing day. Viability of positive control should be within 95±1 % confidence interval of thehistorical data.
Mean tissue viability  40%
1.3.2.3

Assay Acceptance Criterion 3: Standard Deviation (SD)

Since in each test skin irritancy potential is predicted from the mean viability determined on 3 single
tissues, the variability of tissue replicates should be acceptably low.
Standard Deviation (SD) of tissue viability of 3 identically treated replicates for negative control
and positive control  18 %1.4

LIMITATION OF THE METHOD

One limitation of this assay method is a possible interference of the test substance with the MTT
endpoint. A colored test substance or one that directly reduces MTT (and thereby mimics dehydrogenase
activity of the cellular mitochondria) may interfere with the MTT endpoint. However, these test substance
are a problem only if at the time of the MTT test (i.e. 42 hours after test substance exposure) sufficientamounts of the test substance are still present on (or in) the tissues. In case of this unlikely event, the (true)
metabolic MTT reduction and the contribution by a colored test material or (false) direct MTT reduction by
the test material can be quantified by a procedure described in Section 3.2.
The method is not designed for testing of highly volatile test substances, gases and aerosols.

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1.5

BRIEF BASIC PROCEDURE

On the day of receipt, LabCyte EPI-MODEL24 tissues are conditioned by incubation to releasetransportstress related compounds and debris overnight. After pre-incubation, tissues are topically exposedto the test chemicals for 15 minutes. Preferably, three tissues are used per test chemical (TC) and for the
positive control (PC) and negative control (NC). Tissues are then thoroughly rinsed, blotted to remove thetest substances, and transferred to fresh medium. After 42 hr incubation period, the MTT assay is
performed by transferring the tissues to the well containing MTT medium (0.5 mg/ml). After 3 hr MTT
incubation, the blue formazan salt formed by cellular mitochondria is extracted with 0.3 mL/tissue of
isopropanol and the optical density of the extracted formazan is determined using a spectrophotometer at
570 nm and 650 nm as reference. Relative cell viability is calculated for each tissue as % of the mean of
the negative control tissues. Skin irritation potential of the test material is predicted if the remaining
relative cell viability is below 50%.
1.6

DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)

According to the GHS classification (Category 2 or no label), an irritant is predicted if the mean relative
tissue viability of three individual tissues exposed to the test substance is reduced below 50% of the mean
viability of the negative controls.
In vitro results
Tissue viability is  50%
Tissue viability is > 50%

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2. MATERIALS
2.1

LabCyte EPI-MODEL 24

2.1.1
LabCyte EPI-MODEL 24 KIT COMPONENTS
LabCyte EPI-MODEL 24 kit components are shown in Table 1.
Table 1 - LabCyte EPI-MODEL24 Kit Components
Component
Qty
Description
LabCyte EPI-MODEL 24 plate
Contains 24 culture inserts with tissues fixed in
1 platenutritive agar medium for transport (usable area:
0.3cm2).
Assay Medium
Basic medium for incubation (30mL). Store at
1 bottle
refrigeration temperature.
24-well plate
Blank plate for use in assay. Store at room1 plate
temperature.
2.1.2
SHIPMENT OF LabCyte EPI-MODEL24
LabCyte EPI-MODEL 24 is packed in a special container (Icompo/NIPPON EXPRESS CO., LTD) anddelivered by NIPPON EXPRESS CO., LTD. After the Icompo is delivered, examine the contents andmake sure that all kit components (LabCyte EPI-MODEL24 plate, assay medium, and 24-well assay plate)
are included in the package. Confirm lot numbers and expiration dates also. Record details in the Methods
Documentation Sheet (MDS) 1.
NIPPON EXPRESS will pick up the Icompo at a later date (generally, the day after the date of delivery),
and we ask that you return it with a slip documenting receipt, as well as the insulating materials.
2.1.3
INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24
Begin incubating all of the culture inserts after opening the package. Do not store the culture inserts
again after opening.
The human epidermis cells used in LabCyte EPI-MODEL 24 originate from a normal donor and areHIV-, HBV-, HCV-, and HPV-negative. However, handle them with enough care and in accordance withthe laboratory biosafety guidelines since they contain raw materials of human origin.
2.2 TEST CHEMICALS
Coded test chemicals are delivered to each laboratory.
2.3 CONSUMABLES
The following consumables are required.
* The described quantities are necessary so that 1 to 6 samples can be assayed once.
x Assay Medium, 100mL (J-TEC: 402250)
1 bottle
x MTT, 25mg (J-TEC: 403026)
1 bottle
x Wide orifice cell saver tips for micro-pipettes (sterile) 96 tips
1 box
x 24-well assay plate (Becton,Dickinson and Company: 353047)
7 platesx 96-well plate (Becton,Dickinson and Company: 353072)
1 platesx Phosphate buffered saline (PBS) 500mL (Invitrogen: 14190-144)
2 bottles
x Isopropanol 500mL (Wako Pure Chemical Industries: 164-08335)
1 bottlex SLS 25g (SIGMA:L4390)
1 bottlex Sterile distilled water 20mL (Otsuka Pharmaceutical: 36A1X00001)
1 bottles
x Sterile cotton buds (JAPAN COTTON BUDS: 10A754D)
1 box

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2.4 OTHERS
2.4.1
EQUIPMENT / INSTRUMENTS
x Safety cabinet (or clean bench)x Water bath (37 °C)
x CO2 incubator (37 °C, 5%CO2, capable of maintaining high humidity)x Autoclave
x 96-well multi-plate reader (required filters: 450nm, 570nm, 650nm)
x Precision balance (0.1mg)
x Aspirator
x Stop-watchesx Adjustable micro-pipette (10-200ȝL, 200-1000ȝL)x Sharp-edged forceps (sterile)x Micro spatula (sterile)x Beaker (1~2L: sterile)x Sterilizable poly wash bottle (500~1000mL: sterile)
2.4.2

CONSUMMABLE ITEMSx Micro-pipette tips (sterile: 10~200ȝL, 200~1000ȝL)x Microtubes (1.5mL)
x Scalpel (KEISEI MEDICAL INDUSTRIAL: Keisei Scalpel 11A)

3. TEST METHOD*Perform operations in Section 3.1.1~3.1.4 and Section 3.3.1~3.3.2 aseptically in a safety cabinet (orclean bench).
*Operations other than above do not need to be performed with an aseptic technique. For these
operations, refer to Section 2.1.3 INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24
3.1

PREPARATIONS

3.1.1
MTT SOLUTION(1) Dissolve MTT in the assay medium to prepare the MTT medium (final concentration: 0.5mg/mL)
Use ultrasonic cleaning equipment or a vortex mixer as necessary in order to completely dissolve
the MTT.
*Store in a dark, cold place and use it within 24 hours.
(2) Record details of step (1) above in the MDS 4.
3.1.2
POSITIVE CONTROL SUBSTANCE(1) Weigh 500mg of SLS precisely.
(2) To prepare a positive control solution, put the SLS into a graduated cylinder or measuring flask anddilute to 10mL with distilled water (final concentration: 5% w/v)]* Store in a dark, cold place and use it within 24 hours.
(3) Record details of steps (1) and (2) above in the MDS 3.
3.1.3
NEGATIVE CONTROL SUBSTANCE(1) Use distilled water.
3.1.4
POLY WASH BOTTLE FOR PBS(1) Sterilize poly wash bottle using an autoclave.
(2) Fill the sterilized poly wash bottle aseptically with sterile PBS.

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3.2
TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT
There are two kinds of test chemicals that interfere with the MTT assay as follows.
(a) Chemical that stains epidermis tissues.
(b) Chemical that is able to directly reduce MTT.
Test chemical that stains the epidermis tissues has a possibility to transfer from the epidermis tissues tothe extraction solution and to affect the optical density (OD) measurements.
Test chemical that is able to directly reduce MTT can affect the optical density (OD) measurements, if
the test chemical is present in the epidermis tissues when the MTT viability test is performed. Detection
procedure of these test chemicals is described below.
3.2.1
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE
3.2.1.1
STEP1 (PRELIMINARY TEST)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical into wells of 24-well assay plate
preliminarily filled with 0.5mL of distilled water. Untreated distilled water is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 15 minutes.
(3) After incubation, shake the mixture gently and evaluate the staining of the distilled water
macroscopically.
(4) When the color of the solution changes significantly, the test chemical is presumed to have thepotential to stain the tissue and a functional check on viable tissues (Step2) should be performed.
When the color of the solution does not change significantly, the test chemical is determined not tohave a potential to stain the tissue.
3.2.1.2
STEP2 (FUNCTIONAL CHECK ON VIABLE TISSUE)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color of the
distilled water (Step1), onto the surface of the epidermis tissues. Distilled water is used as negative
control.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST. However,incubate the tissue for 3 hours in culture media without MTT instead of incubating in media
containing MTT to evaluate the staining of the epidermis tissues.
(3) Calculate ratio of staining by test chemical from the following formula.
Ratio of staining by test
chemical (%) =OD test chemical – OD negative control
OD negative control

×100(4) When the ratio of staining by test chemical is <5%, correction of the results is not necessary. Whenthe ratio is between 5% and 30%, the corrected MTT OD is calculated using the following
formula.test chemical
Corrected MTT OD =OD stained tissue (MTT assay)-OD stained tissue (no MTT
assay)

When the ratio of staining by test chemical is >30%, the test chemical must be considered
incompatible with the test. However, when the Cell viability (%), which is calculated according to
the procedures described in this SOP Section 3.3.5.2, is <50%, the test chemical is determined as
irritant. Therefore correction of the results or determination of incompatibility of the test chemical is
not necessary.
3.2.2
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT
3.2.2.1
STEP3 (PRELIMINAY TEST)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical into wells of 24-well assay plate

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ENV/JM/MONO(2011)44

preliminarily filled with 0.5mL of MTT medium. Untreated MTT medium is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 1 hour.
(3) After incubation, shake the mixture gently and evaluate the staining of the MTT medium
macroscopically.
(4) When the MTT medium turns blue/purple significantly, the test chemical can reduce MTT and
additional functional check (Step4) must be performed.

5% SLS

1-bromohexane1,1,15-(1,1cinnamaldehy
eugenoltetrachloroeth dimethylethyldeane)-2-methyl
Photo 1 – Example of test for direct MTT reduction ability (STEP 3). Test substances 5-(1,1dimethylethyl)-2-methyl, cinnamaldehyde and eugenol and have directly reduced MTT. In
these cases, Step 4 must be performed.
3.2.2.2
STEP4 (FUNCTIONAL CHECK ON VIABLE TISSUE)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color of the
MTT medium into blue/purple (Step3), onto the surface of the epidermis tissues. Distilled water isused as negative control.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST. However,
use the epidermis tissues that has been freeze-killed at -20 °C or lower for more than 24 hours
instead of viable epidermis tissues.
(3) Calculate ratio of staining by test chemical from the following formula.
Ratio of staining by test
chemical (%) =OD test chemical – OD negative control
OD negative control

×100(4) When the ratio of staining by test chemical is <30%, correct OD data using the following formula.
Corrected OD =OD (viable tissue) test chemical – [OD (freeze-killed tissue) test
chemical - OD (freeze-killed tissue) negative control]

When the ratio of staining by test chemical is >30%, the test chemical must be considered
incompatible with the test. However, When the Cell viability (%), which is calculated according to
the procedures described in this SOP Section 3.3.5.2, is <50%, the test chemical is determined as
irritant. Therefore correction of the results or determination of incompatibility of the test chemical is
not necessary.

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3.3

EXECUTION OF THE TEST

3.3.1 PREPARATION OF LabCyte EPI-MODEL
24 (DAY -1)
Figure 1(1) Pre-warm the assay medium for 30 minutes to
37 °C using a water bath.
1 row(2) Fill 3 wells of the 1st row of each 24-well assay (substance application)plate with the pre-warmed assay medium 2 row(0.5mL/well).
3 row
ĺ Figure 1(post-incubation)(3) Open the LabCyte EPI-MODEL 24 aluminum
4 row
package.
(MTT assay)(4) Open the LabCyte EPI-MODEL 24 plate lid
and pick up the culture inserts using sterile forceps.
*Do not touch the epidermis surface of culture inserts.
*Use forceps to remove agar medium sticking to the outside of the culture inserts.
(5) Transfer the culture inserts into assay medium
filled wells of the 1st row using sterile forceps. Figure 2
ĺ Figure 2*Avoid air bubble formation under the tissue 1 row(substance application)
inserts.
2 row(6) Place the plate (lid on) in a CO2 incubator.
(7) Incubate overnight (15~30 hours) until Section 3 row(post-incubation)
3.2.2 “APPLICATION OF TEST CHEMICALSAND RINSING.”
4 row(MTT assay)(8) Record details of steps (1) - (7) above in the
MDS 2.

Assay Plate

st

nd

rd

th

Assay Plate

st

nd

rd

th

3.3.2

APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0)

3.3.2.1
PREPARATION OF WELLS FOR POST-INCUBATION (3RD ROW)
(1) Pre-warm the assay medium for 30 minutes to 37 °C using a water bath.
(2) Remove the assay plate from the CO2 incubator.
(3) Open the lid of the assay plate, and fill 3 wells
Figure 3of the 3rd row with the pre-warmed assay
medium (1.0mL/well) using a micropipette.
1 row
ĺ Figure 3(substance application)(4) Place the plate (lid on) in a CO2 incubator.
2 row(5) Incubate until application of test chemicals
3 row(0~12 hours).
(post-incubation)(6) Record details of steps (1) – (5) above in the
4 row
MDS 3.
(MTT assay)

Assay Plate

st

nd

rd

th

3.3.2.2
APPLICATION OF TEST CHEMICALS(1) Remove the assay plate from the CO2 incubator.
(2) Apply test chemicals onto the surface of epidermis tissues in the 1st row of the assay plate. Use 3
wells per test chemical (N=3).
FOR LIQUIDs: Carefully apply 25ȝL of the test chemical onto the central part of each epidermis using
a micropipette. After applied, close the lid of the assay plate and tap the side of the plate outside thesafe cabinet (or clean bench) in order for the liquid to spread out over the entire epidermis surface. If
necessary, use a micro spatula to coat the unapplied surface with liquids. Do not push the epidermis

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Photo 2 - Pipette tips for viscous
liquids
surface too hard with the spatula.
*Use wide orifice cell saver tips for viscous liquids.
ĺ Photo 2
Use a pipette, etc. to familiarize yourself with thenature of the test chemicals in advance.
FOR SOLIDs: Weigh out 25mg (±1mg) of the solid
chemical with a precision balance in advance. Apply
first 25ȝL of distilled water and then the weighed test
chemical onto the epidermis surface. Use a micro
spatula if necessary to gently spread the test chemical.
ĺ Photo 3*One 24-well assay plate should be used to assay
only one test chemical.
ĺ Figure 4(1 samples x 3(n) = 3 (culture inserts))(3) Apply test chemical onto each well at 1~3-minuteintervals.
(4) Incubate each well for 15 minutes in the cabinet(lid on between the intervals).
*Close the lid of the assay plate at all times
except when applying samples. It might affectthe amount of test sample if the lid is kept open,
due to air circulation in the safe cabinet (or clean
bench).
(5) Record details of steps (1) - (4) above in the
MDS 3.

Photo 3 – Applying a solid substance
Figure 4st

1 row(substance application)
2nd row

3rd row(post-incubation)
4th row(MTT assay)

3.3.2.3 REMOVAL OF THE TEST CHEMICALS(1) 15 minutes (±30 seconds) after applying a chemical,
open the assay plate and pick up a culture insert withsterile forceps.
(2) Fill the culture insert with PBS using a PBS filled
poly wash bottle. Hit the PBS stream from thewashing bottle on the side-wall of the culture insert
and wash on the tissue surface by the PBS current.
ĺ Photo 4
Attention: Must not to hit the PBS stream on thetissue surface directly. Be careful not todamage the tissue surface.
(3) Discard the PBS into a beaker by tilting the insert. If
necessary, remove the PBS inside the culture insert bytapping it above the beaker only once.
ĺ Photo 5(4) Repeat steps (2) and (3) at least 10 times or more as
much as possible, and remove all residual test
chemical on the tissue surface almost completely.
(5) Gently remove the leftover PBS outside the culture
insert with a sterile cotton bud. But don’t touch inside
the culture insert by a cotton bud.

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Assay Plate
Test substancePhoto 4 - Rinse 1Photo 5 - Rinse 2

ENV/JM/MONO(2011)44

ĺ Photo 6
Attention: Even if residues of washing PBS remainon the tissue surface, don’t do at all
because it is not necessary to remove them.

95

Photo 6 - Rinse 3

165

ENV/JM/MONO(2011)44(6) If test material remains on the epidermis
surface, repeat steps (2) ~ (5) again.
(7) Transfer the blotted culture insert to a well inthe 3rd row of the same column (for postincubation).
ĺ Figure 5*Avoid air bubble formation under the culture
inserts.
(8) Repeat steps (1) ~ (7) for all the culture inserts
at 1~3-minute intervals.
(9) Record details of steps (1) – (8) above in the
MDS 3.

Figure 5

Assay Plate
Test substance

st

1 row
(substance application)
2nd row

3rd row(post-incubation)
4th row(MTT assay)

3.3.3
POST TREATMENT INCUBATION (DAY 0~2)
(1) Close the lid of the assay plate and place it in a CO2 incubator.
(2) Incubate for 42 hours.
3.3.4
MTT ASSAY (DAY 2)
3.3.4.1
PREPARATION OF WELLS FOR MTT ASSAY(1) Pre-warm MTT medium for 30 minutes to 37°C
using a water bath.
Figure 6(2) Remove the assay plate from the CO2 incubator.
(3) Open the lid of the assay plate, and fill each 1 row
well of the 4th row with the pre-warmed MTT (substance application)
2 row
medium (0.5mL/well) using a micropipette.
ĺ Figure 6
row(4) Close the lid of the assay plate and place it in 3(post-incubation)
the CO2 incubator.
4 row(MTT assay)(5) Incubate until starting MTT assay (about 0 ~ 12hours).
(6) Record details of steps (1) – (5) above in the MDS 4.

Assay Plate
Test substance

st

nd

rd

th

3.3.4.2
MTT ASSAY(1) Remove the assay plate from the CO2 incubator
after 42 hours (±1 hour) of post-incubation.
Figure 7(2) Transfer each culture insert from the 3rd row to
the 4th row of the corresponding column.
1 row
ĺ Figure 7(substance application)*Avoid dripping from the base end surface of the 2 row
culture insert into other wells.
row*Avoid air bubble formation under the culture 3(post-incubation)
inserts.
4 row(3) Close the lid of the assay plate and place it in the(MTT assay)
CO2 incubator.
(4) Incubate for 3 hours.
(5) Record details of steps (1) – (4) above in the MDS 4.
st

nd

rd

th

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96

Assay Plate
Test substanceENV/JM/MONO(2011)44

3.3.5
FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2~3)
3.3.5.1
FORMAZAN EXTRACTION(1) Remove the assay plate(s) from the CO2 incubator 3 hours (±5 minutes) after the MTT assay.
(2) Open the lid of the assay plate and pinch thecultured epidermis from each culture insert of the Photo 7 - Detachment of
4th row with forceps.
epidermis
ĺ Photo 7*Use a micro spatula to scratch up the epidermis or a
scalpel to cut the membrane filter on the base of theculture insert if the cultured epidermis cannot be
pinched due to damage from a test chemical.
(3) Transfer the epidermis tissue into a 1.5mL microtube.
(4) Add 300ȝL of isopropanol to the micro tubes andsoak the entire epidermis tissue in the isopropanol.
(5) Incubate the micro tubes in a dark cold place (orrefrigerator) overnight (more than 15 hours) inorder to completely extract pigments.
*Tighten the micro tube seal.
*Periodically shaking the micro tubes will contribute to a more
efficient extraction.
(6) Shake the micro tubes to mix the solution.
*If split epidermis tissues are suspended, wait until they sink or
gently centrifuge them (if a centrifuge is available).
(7) Transfer 200ȝL of the solution in each micro tube into each well on a 96-well plate.
*One well of 200ȝL of isopropanol should be set as a blank.
*Figure 8 shows an example of allocation in a 96-well plate.
Figure 8 – Allocation for a 96-well plate
A

1
Blank

B

DW-1

C

DW-2

D
E
F
G

DW-3
5% SLS1
5% SLS2
5% SLS3

2

3

4

5

6

7

8

9

10

11

Sample1-1
Sample1-2
Sample1-3
Sample2-1
Sample2-2
Sample2-3

Sample3-1
Sample3-2
Sample3-3Sample4-1
Sample4-2
Sample4-3

Sample5-1
Sample5-2
Sample5-3
Sample6-1
Sample6-2
Sample6-3

Sample7-1
Sample7-2
Sample7-3
Sample8-1
Sample8-2
Sample8-3

Sample9-1
Sample9-2
Sample9-3
Sample10-1
Sample10-2
Sample10-3

Sample11-1
Sample11-2
Sample11-3
Sample12-1
Sample12-2
Sample12-3

Sample13-1
Sample13-2
Sample13-3
Sample14-1
Sample14-2
Sample14-3

Sample15-1
Sample15-2
Sample15-3
Sample16-1
Sample16-2
Sample16-3

Sample17-1
Sample17-2
Sample17-3
Sample18-1
Sample18-2
Sample18-3

Sample19-1
Sample19-2
Sample19-3
Sample20-1
Sample20-2
Sample20-3

12H(8) Record details of steps (1) – (7) above in the MDS 5.
3.3.5.2
OPTICAL DENSITY MEASUREMENTS OF THE EXTRACTS(1) Using a 96-well plate reader, measure optical densities (OD) at 570nm and 650nm and determine
the measured OD by subtracting the 570nm OD from the 650nm OD.
The equation is shown below:
Measured OD = [570nm ODsample – 570nm ODblank] – [650nm ODsample – 650nm ODblank]*Set the plate reader-calculated value as the measured OD if the 96-well plate reader performs
automatic calculations.
(2) Calculate the cell viability of a sample using the equation below. Furthermore, calculate thevariability (SD) of tissue replicates.
(3) Record details of steps (1) and (2) above in the MDS 5.

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ENV/JM/MONO(2011)44

Cell Viability (%)㧩Measured ODsample
Mean Measured ODNC

×100

4. ASSESSMENT
4.1 CONDITIONS FOR A SUCCESSFUL STUDY
The skin irritation test should be considered successful if both of the following criteria have been met.
x Tissue viability: 0.7  mean OD (A570/650) measured value for negative control  2.5.
x Positive control: mean tissue viability for 5% SLS (positive control)  40%.
x SD: SD (negative control and positive control) of tissue viability of 3 identically treated replicates 
18 %4.2 ASSAY CRITERIA
The criteria for in vitro interpretation are shown below.
The test must be performed 3 times per a sample in total. Sort the tissue viabilities obtained from therepeated tests in ascending order, and classify the irritancy based on the median of those tissue viabilities.
Tissue Viability (primary)Classification
Tissue viability is  50%
Irritant
Tissue viability is > 50%
Non Irritant[FLOWCHART] ASSESSMENT FLOWCHART(1) Tissue viability in negative control ĺ (either criterion is not met) ĺ Assay Failure
0.7  Mean OD measured value  2.5
Positive control (5% SLS) result should be “irritant”
Mean tissue viability  40%
SD”
SD (negative control and positive control) of tissue viability of 3 identically treated replicates 
18 %Ļ
(All criteria are met)Ļ(2) Assessment of test samples (3-time repeated tests: all tests satisfy (1))
The median of the 3 tissue viabilities (%)  50% ĺ (Yes) ĺ Classified as irritant
Ļ(No)ĻClassified as non irritant

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MDS 1:RECEIPT OF LABCYTE EPI-MODEL 24
Laboratory name:1.

Test name:

Test No. :LabCyte EPI-MODEL 24
Date received㧦
Lot No.㧦
Exouration date㧦(MM/DD/YYYY)
Accessories㧦

Assay medium, 30mL Ƒ 㧔Lot No.㧦(MM/DD/YYYY)
24 well assay plate ƑExpiration date㧦

㧕

Note

2.

Assay medium
Date received㧦
Lot No.㧦
Expiration date㧦(MM/DD/YYYY)
Note

Operator:
Date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Secretariat

Name:

Check date:(MM/DD/YYYY)

99

Study director:

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ENV/JM/MONO(2011)44

MDS 2:
PRE-INCUBATION OF LABCYTE EPI-MODEL 24 (Section 3.3.1)
Laboratory name:1.

Test name:

Test No. :Warm up the assay medium and add 0.5mL of the assay medium to the wells of the 1st row on the
24-well assay plate.
Expiration date㧦㧔Lot No.㧦    (MM/DD/YYYY)
Warm for 30 min.
Ƒ
Add 0.5mL of assay medium to each wellƑ
Number of plates㧦
Assay medium㧦

2.

3.
.

Transfer culture inserts to wells in the 1st row on the 24-well assay plate.
LabCyte EPI-MODEL 24㧦㧔Lot No.㧦    Expiration date㧦(MM/DD/YYYY)
Time/date executed㧦
(MM/DD/YYYY HH:MM)㧕

Confirm that there are no bubbles under the cell culture insert.

Ƒ

LabCyte EPI-MODEL 24 is cultured in CO2 incubator overnight.
Time/date of culture start㧦
(MM/DD/YYYY HH:MM)
Planned time/date of exposure to test chemical㧦
(MM/DD/YYYY HH:MM)
Note

Operator:
Date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Secretariat

Name:

170

㧕

Check date:(MM/DD/YYYY)

100

Study director:

ENV/JM/MONO(2011)44

MDS 3-1:
APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section 3.1.2, 3.3.2 ~3.3.3)
Test name:

Laboratory name:1.

Test No.:

Preparation of positive control.
Preparation vol.
 mL
Weight of SLS
mg(MM/DD/YYYY HH/MM)

Operation date㧦2.

Warm up the assay medium and add 1.0mL of the assay medium to the wells of the 3rd row on the
24-well assay plate.
Expiration date㧦㧕
Assay medium㧦㧔Lot No.㧦    (MM/DD/YYYY)
Warm for 30 min.
Ƒ
Add 1.0mL of assay medium.
ƑTime/date executed㧦
(MM/DD/YYYY HH/MM)

3.

Apply test chemicals to the LabCyte EPI-MODEL 24.
Time/date completed㧦
Time/date execution started㧦(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)

4.

After exposure to test chemical for 15 min., wash out the LabCyte EPI-MODEL 24 and transfer theculture inserts to the 3rd row on the 24-well assay plate.
PBS㧦㧔Lot No.㧦    Expiration date㧦㧕(MM/DD/YYYY)
Time/date completed㧦
Time/date execution started㧦(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)
Confirm that there are no bubbles under the cell culture insert.

5.

Ƒ

Test chemical information
Test chemical code No.
Distilled Water(Negative control)
5%SLS(Positive control)

Lot
No.

Physicalstate

Test chemicalvol.(weight)(measured weight)

Time ofapplication

Exposuretime
㧔15min.㧕

Liquid25ȝL㧦ƑLiquid25ȝL㧦Ƒ25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

Liquid,viscous,solid
Liquid,viscous,solid
Liquid,viscous,solid
Liquid,viscous,101

171

ENV/JM/MONO(2011)44

solid
Liquid,viscous,solid
Liquid,viscous,solid
Liquid,viscous,solid
Liquid,viscous,solid
Liquid,viscous,solid
Liquid,viscous,solid

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

25ȝL, (  mg,  mg, 
mg)

㧦

Ƒ

Operator:
Date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Secretariat

Name:

172

Check date:(MM/DD/YYYY)

102

Study director:

ENV/JM/MONO(2011)44

MDS 3-2:
APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION(Section 3.3.2~3.3.3)
Test name:

Laboratory name:5.

Test chemical information (continued)
Test chemical code
No.

Lot
No.

Physical state
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid
Liquid,viscous, solid

6.

Test No. :Exposure
Test chemicalTime oftime
vol.(weight)
application(measured weight)
㧔15min.㧕25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)25ȝL, (  mg,  mg, Ƒ㧦mg)Culture LabCyte EPI-MODEL 24 in CO2 incubator for 42 hrs.
Time/date post-incubation started㧦(MM/DD/YYYY HH:MM)
Time/date post-incubation completed㧦(MM/DD/YYYY HH:MM)
Note

Operator:
Date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Secretariat

Name:

Check date:103

Study director:

173

ENV/JM/MONO(2011)44(MM/DD/YYYY)

174

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ENV/JM/MONO(2011)44

MDS 4:MTT ASSAY (Section 3.3.4)
Laboratory name:1.

2.

Test name:

Test No. :Preparation of MTT medium
Lot No.     Time/date executed㧦
Preparation vol.    mL
(MM/DD/YYYY HH/MM)
Warm up the MTT medium and add 0.5mL of the MTT medium to the wells in the 4th row on the
24-well assay plate.
Expiration date㧦       㧕
MTT medium.㧦㧔Lot No.㧦   (MM/DD/YYYY)
Warm for 30 min. Ƒ Add 0.5mL of the MTT medium. Ƒ Time/date executed㧦
(MM/DD/YYYY HH/MM)

3.

After post-incubation, the LabCyte EPI-MODEL24 transfer to wells of 4th row of 24-well assay
plate.
Time/date started㧦(MM/DD/YYYY HH/MM)

Time/date completed㧦(MM/DD/YYYY HH/MM)

Confirm that there are no bubbles under the cell culture insert.
4.

ƑStore LabCyte EPI-MODEL 24 culture overnight in CO2 incubator for 42 hrs.

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ENV/JM/MONO(2011)44

Information on MTT reaction time
Test chemicalcode No.

Lot
No.

Distilled Water(Negative
control)
5%SLS (Positive
control)

Time
MTT
when
reactionMTT
startreaction
time
ends
㧦
㧦

Test chemicalcode No..

Lot
No.

Time
MTT
when
reactionMTT
startreaction
time
ends
㧦
㧦

㧦

㧦

㧦

㧦

㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦

㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦

㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦

㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦
㧦

Note
Operator:
Date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Secretariat

Name:

176

Check date:(MM/DD/YYYY)

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ENV/JM/MONO(2011)44

MDS 5:FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.5)
Laboratory name:1.

Test name:

Test No. :After MTT reaction, use forceps to pick up the cultured epidermis from the cell culture insert and put
it in a 1.5mL microtube.
Did you use a scalpel to cut out the cultured epidermis?
Date of execution㧦
(MM/DD/YYYY)2.

Ƒ

Add isopropanol (300ȝL) to microtube so that the cultured epidermis is completely immersed in
isopropanol.
To add isopropanol (300ȝL)Isopropanol Lot No.
Immersion of the cultured epidermis in isopropanol.
Date of execution㧦
(MM/DD/YYYY)

3.
4.

Ƒ
Ƒ

For MTT formazan extraction, allow micro tube to stand in a cold and dark space.
Place micro tube in a cold and dark space.
Ƒ
Extract solution (200mL) is transferred to each well on the 96-well plate.
Transfer to the 96-well plate.
Time/date executed㧦
(MM/DD/YYYY HH/MM)
Sample location on 96-well plate.
1
2
3
4
A
blank
Distilled
B
Water-1
DistilledC
Water-2
Distilled
D
Water-3
5% SLSE1
5% SLSF
2
5% SLSG
3
H

Ƒ

5

6

7

107

8

9

10

11

12

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ENV/JM/MONO(2011)44

5.

Analyze extract OD at 570nm and 650nm, and calculate the OD(570nm-650nm).
Analyze OD at 570nm and 650nm.
Calculate the OD(570nm-650nm).
Calculate cell viability and SD.
Cell viability and SD are recorded on a separate data sheet.
The data sheet is attached to the back of this sheet.
Check for input errors.
Time/date executed㧦
(MM/DD/YYYY HH/MM)
Note

Operator:
Date:(MM/DD/YYYY)

Check date:(MM/DD/YYYY)

Secretariat

Name:

178

Check date:(MM/DD/YYYY)

108

Study director:

Ƒ
Ƒ
Ƒ
Ƒ
Ƒ
Ƒ

ENV/JM/MONO(2011)44

REVISION HISTORY
Rev.

Content

Ver.1
Ver.2
Ver.3

1) First version
1) Revised clerical error.
1) Revised the post-incubation time and assessment criteria incompliance with the EpiSkin method described in “PerformanceStandards for Applying Human Skin Models to in vitro Skin
Irritation Testing”
2) Added photos and figures for instruction.
1) Added MDS 1~6.
2) Added instruction and operational steps regarding the IL-1Į ELISA
kit.
3) Added subsections “Delivery of LabCyte EPI-MODEL24” and“Instruction For Use of LabCyte EPI-MODEL24” to Section 2.
4) Added the description regarding test chemicals to Section 2.
5) To Section 2, added the description of materials provided by J-TEC
separately from other materials.
6) Stated the specific calculation procedures in Section 3.2.5.2“OPTICAL DENSITY MEASUREMENTS OF EXTRACTS”.
1) Moved scalpel from Section 2.4 “MATERIALS PROVIDED BY JTEC” to Section 2.5 “MATERIALS NOT PROVIDED WITH THE
J-TEC KITS”.
2) Removed the description regarding how to execute procedures
alone.
3) Moved IL-1Į ELISA reagents from Section 3.1“PREPARATIONS” to Section 3.2 “TEST METHOD”.
4) Added a flowchart for the IL-1Į ELISA procedures.
5) Changed from “in a cold dark place” to “in a cold dark place (orrefrigerator)” regarding formazan extraction.
6) Added the description of “ultrasonic cleaning equipment or vortex
mixer” as an example of an MTT dissolution method.
7) Changed the exposure time column from entering actual time tocheckboxes on the MDS 3.
1) Corrected typing errors in the section number for IL-1Į ELISA
reagents.
2) Removed the space for SLS lot numbers on the MDS 3.
3) Removed the space for PBS lot numbers on the MDS 3.
4) Added the space for isopropanol lot numbers on the MDS 5.
5) Added a checkbox about using a scalpel when removing tissues in
the MDS 5.
6) Added the space for IL-1Į ELISA kit lot numbers on the MDS 6.
7) Changed the applicable parts of product codes and kit components
in Section 2.2, with the change of IL-1Į ELISA kit types to a 96
well test only.
8) Decreased the volume by half to 10mL and changed the storage
condition from within 1 month to within 24 hours in Section 3.1.2“POSITIVE CONTROL SUBSTANCE”.
9) Added the manufacturers and product codes of the 24-well plate and
96-well plate in Section 2.4 “MATERIALS PROVIDED BY J-

Ver.4

Ver.4.1

Ver.5.0

109

Date
Revised27/02/2008
28/02/2008
17/03/2008

15/05/2008

21/05/2008

27/08/2008

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ENV/JM/MONO(2011)44

Ver.
6.0

Ver.
6.01

180

TEC”.
10) Added specific time frames for incubation or culturing.
11) Added the conditions for a successful study in Section 4“ASSESSMENT”
12) Changed the specific method of applying liquids in Section 3.2.2.2“APPLICATION OF TEST CHEMICALS”.
13) Added descriptions in English on the MDS Sheets.
14) Changed the application time interval from 1 minute to
1~3minute(s).
15) Numbered figures and flowcharts.
16) Increased the size of spaces for lot numbers on the MDS Sheets.
17) Changed spaces for dates from MM/DD to MM/DD/YYYY.
18) Added director check date, study director, secretariat check dateand name at the end of each MDS.
19) Changed the size of matrixes for sample allocation to a 96-wellplate in the MDS 5 & 6.
20) Changed the test chemical name to test chemical code in the MDS
3 & 4.
21) Divided the MDS 3 into MDS 3-1 and 3-2, and added spaces for
date, operator, check date, study director at the end of the MDS 3-1,
and spaces for laboratory name, test name and test no. at the
beginning of the MDS 3-2.
1) Removed the descriptions regarding the measurement of IL-1Į
production, since the validation committee decided to use cell
viabilities only as an index for the skin irritancy test at the meetingin 2009.
2) Revised the expression “the materials provided by J-TEC” for thevalidation study to that for a standard skin irritancy test preparation.
3) Clearly stated the cell viability equation to use the mean of measured
values.
4) Clearly stated to use the median of cell viabilities from the threetime repeated tests as assay criteria.
1) In order to avoid the possible influence of volatile test chemicals on
the results of other test chemicals, the types of test chemicals perplate was changed from 2 chemicals to just 1 chemical.

27/02/2009

23/03/2009

Ver.
7.01

1) Test for detecting chemicals that interfere with MTT endpoint wasadded to Section 3.2.

03/07/2009

Ver.
7.2
Ver.
8.1

1) Revised clerical error.

30/09/2009

1) Added the description about rational and background as following
chapters in Section 1.
LabCyte EPI-MODEL 24 SKIN IRRITATION TEST (SIT using
LabCyte EPI-MODEL24)BACKGROUND OF SIT using LabCyte EPI-MODEL24
BASIS OF THE METOD
LIMITATION OF THE METHODBRIEF BASIC PROCEDUREDATA INTERPRETATION PROCEDURE (PREDICTION

30/06/2010

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ENV/JM/MONO(2011)44

Ver.8.2

MODEL)
2) Added photo about chemicals that directly reduce MTT in Section
3.
3) Added the washing protocol more detail in Section 3.
4) Added assessment about SD.
1) Changed description about the washing protocol in Section 3.
2) Changed unit of consumable reagents and vessels from per avalidation study to per a test.

111

17/08/2010

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Appendix 4

JaCVAM Report:
Me-too Validation Study of in vitro Skin Irritation Test using LabCyte EPI-MODEL24(LabCyte EPI-MODEL24 SIT)

February 28, 2011

LabCyte Validation Management Team

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Members of LabCyte Validation Management TeamMr. Hajime Kojima, JaCVAM: Japanese Centre for the Validation of Alternative Methods
Mr. Masakazu Katoh, Japan Tissue Engineering Co. Ltd.
Mr. Toshiro Yokouchi, Japan Tissue Engineering Co. Ltd.
Biostatistian group
Mr. Takashi Omori, Doshisya Univ.
Participant laboratories
KOBAYASHI Pharmaceutical Co., Ltd. (Mr. Yoshihiro Yamaguchi and Ms. Maki Nakamura)
Fancl Corp. (Ms. Tamie Suzuki and Ms. Runa Izumi)
Drug Safety Testing Center Co., Ltd. (Mr. Shinsuke Shinoda and Ms. Saori Hagiwara)

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Abbreviations
CAS: Chemical Abstracts Service
ECVAM: European Centre for the Validation of Alternative Methods
ESAC: ECVAM Scientific Advisory Committee
GHS: Globally Harmonised System
GLP: Good Laboratory PracticeISO: International Organization for Standardization
JaCVAM: Japanese Centre for the Validation of Alternative Methods
JSAAE: Japanese Society for Alternatives to Animal Experiments
J-TEC: Japan Tissue Engineering Co. Ltd.
NIHS: National Institute of Health Sciences
OECD: Organisation for Economic Co-operation and Development
QC: Quality control
PBS: Phosphate buffered saline
PS: Performance Standard
RhE: Reconstructed human epidermis
SD: Standard deviation
SLS: Sodium lauryl sulphate
SPSF: Standard Project Submission Form
TG: Test Guideline
UN: United Nations
VMT: Validation management team
VRM: Validated reference method
WNT: National Coordinators of the Test Guideline Project

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Contents
1. Goal Statement
2. Objective   
3. Background
4. Test methods 
4-1. Reconstructed human cultured dermal model
4-2. Model supplier
5. Validation management structure
5-1. Validation management team
5-2. Chemical selection, acquisition, coding and distribution
5-3. Independent biostatisticians
5-4. Participating laboratories
5-5. Sponsorship
6. Study design
7. Test chemicals
7-1. Chemical selection
7-2. Chemical coding and distribution
8. Protocol
8-1. Protocol of the skin irritation test with LabCyte EPI-MODEL24
8-2. Prediction model of skin irritation
8-3. Data collection, handling, and analysis
8-4. Quality assurance, GLP
9. Results
9-1. Comments on the datasheets
9-2. Negative control
9-3. Positive control
9-4. Viability of chemicals
9-5. Classification of three independent viabilities at each laboratory
10. Discussion
10-1. Reliability
10-2. Predictivity
11. Conclusions
12. Acknowledgements
13. References

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Appendices:
1. Validation study of in vitro skin irritation test using LabCyte EPI-MODEL24 (FINAL report)
2. Summary report of the peer review panel on LabCyte EPI-MODEL24 in vitro test method for theassessment of skin irritation potential of chemicals
3. Additional validation of the RhE tests: LabCyte EPI-MODEL24 acute skin irritation prediction
4. Skin Irritation test protocol using the reconstructed human model “LabCyte EPI-MODEL24” (ver.8.2)
5. Original data in the catch-up validation study
6. Test records and data sheet in KOBAYASHI Pharmaceutical Co., Ltd. (example: in Japanese)
7. Result of QC for each lot of LabCyte EPI-MODL24
8. Detailed review documents on the “LabCyte EPI-MODEL24”
9. Masakazu Katoh, Fumiyasu Hamajima, Takahiro Ogasawara, and Ken-ichiro Hata (2009) Assessmentof the Human Epidermal Model LabCyte EPI-MODEL for In Vitro Skin Irritation Testing According to
the ECVAM-Validated Protocol , Journal of Toxicological Science, 34(3) 327-334.
10. OECD TG 439, In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

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1. Goal statementxx

The ultimate goal of the test strategy is to replace the regulatory Draize skin irritation test to meet
the OECD (Organisation for Economic Co-operation and Development) TG (Test Guideline) 404(OECD, 2002).
The primary goal of this catch-up validation study is to evaluate the ability of in vitro tests toreliably discriminate skin irritant (I) from non-irritant (NI) chemicals, as defined by the OECD and
UN (United Nations) proposal for GHS (Globally Harmonised System) for the classification andlabelling of skin irritation (category 1/category 2; no category; Anon., 2003).

2. Objective
The OECD Working group of the National Coordinators of the Test Guideline Project (WNT)
accepted the TG No.439: in vitro skin irritation test guideline in March 2010. This TG addresses thehuman health endpoint of skin irritation. Three validated test methods currently adhere to this TG.
Prevalidation, optimization and validation studies have been completed for an in vitro test method that uses
a Reconstructed human epidermis (RhE) model. This method is commercially available as EpiSkin™ andhas been designated as the Validated Reference Method (VRM). Two other commercially available in vitro
skin irritation RhE test methods, namely the EpiDerm™ SIT (EPI-200) and SkinEthic™ RHE test methods,
have shown similar results to the VRM according to Performance Standard (PS) - based validation.
On the other hand, another in vitro test system that employs a RhE model (LabCyte EPI-MODEL24)
has progressed through protocol optimization as a skin irritation test. A multi-laboratory assessment of thissystem was performed according to several ECVAM (European Centre for the Validation of Alternative
Methods) performance standards (ESAC: ECVAM Scientific Advisory Committee statement, 2007, 2008,2009). The present objective added the Japanese RhE and other similar models to adhere to the OECD TG
439. A me-too validation study was conducted to assess the reliability (reproducibility within and between
laboratories) and relevance (predictive capacity) of this test system. The study included a challenging set of
20 test chemicals that would meet the performance standard set forth in the TG No.439. The validation
study was undertaken in accordance with the principles and criteria documented in the OECD GuidanceDocument on the Validation and International Acceptance of New or Updated Test Methods for Hazard
Assessment (No. 34, OECD, 2005) and according to the Modular Approach to validation (Hartung et al.
2004).
ΗBackground3Η
Researchers in Japan aimed to include the Japanese RhE (LabCyte EPI-MODEL24: LabCyte EPIMODEL24 SIT) in vitro skin irritation test in the TG as an addition to other similar models in the SPSF
(Standard Project Submission Form) that were submitted to OECD WNT by EU delegate in April 2008.
The validation study described herein was performed between April 2008 and January 2009 by the
Validation Management Team (VMT), with financial support from the Japanese Society for Alternative toAnimal Experiments (JSAAE).
The study conducted by VMT referenced the original ECVAM performance standard (ECVAM 2007),
in which a range of appropriate models was described as one of the acceptance criteria. After completion of
the first phase of the study in August 2008, the VMT discussed the criteria for the Labcyte EPI MODEL24
SIT. The VMT decided that the criteria were not set because there was not enough data to define this kindof range at that time. Furthermore, the pre-specification was considered to have too narrow a range and thedraft OECD TG came under review at the time of the discussions. As a result, the reliability of the model
was considered to be high. Therefore, the VMT decided that criteria for the range may not be needed forthis model, while the check for variation should be done.
Based on validation results of the Labcyte EPI-MODEL24 SIT, a member of the Japanese WNTsubmitted an SPSF of it to OECD in January 2009 and the OECD WNT accepted this assay in its working

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plan in May 2009. The VMT submitted the first validation report to the OECD secretary in July 2009.
On the other hand, we confirmed an additional validation study in reference to the new ECVAM
performance standards (ESAC statement, 2009) to be revised for the TECD TG between April and May of2009 and submitted the second validation report and Background Review Documents to the OECDsecretary in August 2009.
Using these documents, OECD performed a peer review and we received the peer review report from theOECD secretary in March 2010. The OECD peer review on the LabCyte EPI-MODEL24 SIT in vitro test
method for the assessment of skin irritation potential of chemicals was performed with the validation reportand background review documents. In the summary report, the peer review panel indicated that the issue of
misclassifying 1-bromohexane should be resolved.
To resolve this issue, the protocol was revised by Japan Tissue Engineering (J-TEC). To confirm
general versatility with the revised protocol, we planned an additional validation study according to theOECD performance standard.
4. Test methods
4-1. Reconstructed human cultured epidermal model
LabCyte EPI-MODEL24 is a new, commercially available RhE model produced by J-TEC. It consistsof normal human epidermal keratinocytes whose biological origin is neonate foreskin. In order to expand
human keratinocytes while maintaining their phenotype, the cells are cultured with 3T3-J2 cells as a feeder
layer (Rheinwald and Green, 1975; Green, 1978). Reconstruction of a human cultured epidermis isachieved by cultivating and proliferating keratinocytes on an inert filter substrate (surface area 0.3 cm2) atthe air-liquid interface for 13 days with an optimized medium containing 5% fetal bovine serum. The
process generates a multilayer structure consisting of a fully differentiated epithelium with features of thenormal human epidermis, including a stratum corneum. LabCyte EPI-MODEL24 is embedded in an
agarose gel containing nutrient solution and shipped in 24-well plates at around 18 °C (Kato, 2009:
Appendix 4).
4-2. MODEL supplier
According to OECD Good Laboratory Practice (GLP) Consensus Document No.5 “Compliance ofLaboratory Suppliers with GLP Principles”, responsibility for the quality and fitness for use of equipment
and materials rests entirely with the management of the test facility (OECD, 1999).
The acceptability of equipment and materials in laboratories complying with GLP must therefore be
guaranteed to any regulatory authority to which studies are submitted. In some countries where GLP has
been implemented, suppliers belong to national regulatory or voluntary accreditation schemes (forlaboratory animals) that can provide users with additional documentation proving that they are using a test
system of defined quality.
Audits performed during the study focused on procedures established to guarantee a defined quality ofthe tissue models.
5. Validation management structure
The management structure of the study is shown in Figure 1.
5-1. Validation management team
The VMT played a central role in overseeing the conduct of the validation study, including
implementation of the following aspects of the study:1) Goal statement

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2) Project plan including objective3) Study protocol / amendments
4) Outcome of QC (Quality Control) audits5) Test chemicals
6) Data management procedures
7) Timeline / study progression8) Data collection and analysis
9) Study interpretation and conclusions10) Reports and publications
The VMT made the final decision on which laboratories would participate in the validation study.
Responsible VMT members:
Chair (Hajime Kojima, JaCVAM: Japanese Centre for the Validation of Alternative Methods)
The sponsor representative, LabCyte EPI-MODEL 24 suppliers and lead lab (Masakazu Katoh: J-TEC)
5-2. Chemical selection, acquisition, coding, and distribution1) Definition of selection criteria
2) Chemical selection3) Liaise with suppliers
4) Final check of chemicals provided
5) Acquisition
6) Coding7) Distribution
Responsible VMT member: Hajime Kojima, JaCVAM
5-3 . Independent biostatisticians
1) Approve spreadsheets
Responsible VMT member: Takashi Omori: Doshisya Univ.

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Validation Management Team

Chair: JaCVAM
Study Sponsor
JSAAE

Biostatistics
Doshisya Univ.

Lead
laboratory&
Training
J-TEC

Chemical coding
and distribution
JaCVAM
JaCVAM

KOBAYASHI
Pharmaceutical
Co.

Fancl㩷
Corp.

Drug Safety Testing Center
Co., Ltd.

Fig. 1. Management structure of me-too validation study on the LabCyte EPI-MODEL24 SIT
5-4. Participating laboratories
The laboratories participating in the study are shown in Fig. 1.
The following three laboratories participated in the validation study for the evaluation of the LabCyte
EPI-MODEL 24 assays:
x Laboratory a –– KOBAYASHI Pharmaceutical Co., Ltd. (Yoshihiro Yamaguchi and Maki
Nakamura)x Laboratory b –– Fancl Corp. (Tamie Suzuki and Runa Izumi)
x Laboratory c –– Drug Safety Testing Center Co., Ltd. (Shinsuke Shinoda and Saori
Hagiwara)
A lead laboratory was also identified as J-TEC (Mr. Masakazu Kato and Mr Toshihiro Yokouchi).
This laboratory did not participate in the validation study.
Each laboratory was responsible for complying with GLP principles and specifying QC aspectsof the study.
5-5. Sponsorship
The study was managed and financed by JaCAM and J-TEC.
1) JaCVAM financially supported the following activities:- management of the study (VMT meetings)- provision of independent statistical support (VMT meetings)- purchase, coding, and distribution of chemicals to the laboratories

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ENV/JM/MONO(2011)44- independent QC audit of the data
- publication of the study results2) J-TEC supported the following aspects of the study:- the lead laboratories for the test method- training of the participating laboratories- independent QC audit of the LabCyte EPI-MODEL24 SIT- financial assistance of the participated laboratories
6. Study design and test period
Before initiation of the validation study, J-TEC delegates conducted a training course on using LabCyte
EPI-MODEL24 SIT with a revised protocol (ver.8.1) at the National Institute of Health Sciences (NIHS)
on July 27, 2010. All technicians from each laboratory participated in this training course. Furthermore,
all laboratories participated in preliminary testing. After this preliminary test, the protocol was revised tover.8.2 to include detailed descriptions of the washing protocol. Three laboratories attended an additional
validation study after one laboratory was not able to obtain a positive test result with 1-bromohexane.
The preliminary test was conducted by three laboratories between August and September of 2010. The
duration of validation study was between September and November of 2010.
7. Test chemicals
7-1. Chemical selection
To meet the OECD performance standard, the VMT selected 20 chemicals for testing (Table 1). In
the reference chemicals of OECD Test guideline No. 439, tetrachloroethylene was not selected. Instead ofit, No.20, 1,1,1-trichloroethane was tested. Because tetrachloroethylene was changed from 1,1,1trichloroethane at the final step before approving as the test guideline, the VMT escaped the attention of
conclusive confirmation. The class of their chemicals is at the same level and the VMT judged not tobecome a serious problem in this validation study. The final approval of the chemicals proposed by
JaCVAM was the responsibility of the VMT. To avoid any potential for bias in the final selection,
laboratory representatives on the VMT did not participate in these discussions, nor were they made awareof the chemicals finally approved for testing in the validation study.

Table 1.

No.

Minimum List of Reference Chemicals for Determination of Accuracy and Reliability
Values for Similar or Modified RhE Skin Irritation Test Methods and Codes
UN GHS
Chemical codein vivo Cat. Storage㩷 Name
CAS number
Lab a
Lab b
Lab c

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1

1-bromo-4chlorobutane

6940-78-92diethyl phthalate

84-66-2

3naphthalene acetic acid

86-87-3

4 allyl phenoxy-acetate

7493-74-5

5 isopropanol

67-63-0

4-methylthiobenzaldehyde

3446-89-7

7 methyl stearate112-61-8

8 heptyl butyrate

5870-93-99 hexyl salicylate6259-76-3

6

10

cinnamaldehyde

104-55-2

11

1-decanol

112-30-1

12cyclamen aldehyde

103-95-7

13

1-bromohexane

11-25-114

2-chloromethyl-3,5dimethyl-4methoxypyridine HCl

86604-75-3

15di-n-propyl disulphide629-19-6

16

potassium hydroxide
5%1310-58-3

17

benzynethiol,5-(1,1dimethylethyl)-2methyl

7340-90-1

18

1-methyl-3-phenyl-1piperazine

5271-27-2

19heptanal

111-71-720

1,1,1-trichloroethane

71-55-6

No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
No Cat.
(OptionalCat. 3)
No Cat.
(OptionalCat. 3)
No Cat.
(OptionalCat. 3)
Cat.2
Cat.2
Cat.2

B-261

D-281

G-301

RT

B-262

D-282

G-302

RT

B-263

D-283

G-303

RT

B-264

D-284

G-304

RT

B-265

D-285

G-305

RT

B-266

D-286

G-306

RT

B-267

D-287

G-307

RT

B-268

D-288

G-308

RT

B-269

D-289

G-309

2-8C

B-270

D-290

G-310

RT

B-271

D-291

G-311

RT

B-272

D-292

G-312

RT

B-273

D-293

G-313

RT

B-274

D-294

G-314

RT

B-275

D-295

G-315

RT

B-276

D-296

G-316

RT

B-277

D-297

G-317

RT

B-278

D-298

G-318

RT

B-279

D-299

G-319

RT

B-280

D-300

G-320

Cat.2

Cat.2
Cat.2
Cat.2

Cat.2
Cat.2
Cat.2

1) CAS No.: Chemical abstracts service registry number.

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ENV/JM/MONO(2011)44

7-2. Chemical coding and distribution
Independent coding and distribution of chemicals were contracted out by JaCVAM to an independent
laboratory. The undeciphered information on this code was confirmed by the VMT after the validation
study because this chemical code list was too easy and simple. The certification of chemicals wasaccording to ISO (International Organization for Standardization) 9001 and GLP, and has provenexperience of reliable services. The codes were provided by JaCVAM.
8. Protocol
8-1. Protocol of the skin irritation test with LabCyte EPI-MODEL24 SIT
According to the suggestion of the OECD peer review panel, J-TEC resolved the false-negative issues of 1bromohexane. The SOP (Standard Operating Procedure: ver.8.1) included a modified washing protocol ina revision dated June 30, 2010. Modifications to the washing protocol are shown in Table 2 (Detailed
process described in Appendix 7). Using the revised SOP, the validation study was performed to show
clear data and addressed comments of the OECD peer review panel.
Table 2. Modification points of washing protocol between SOP ver.7.1 and SOP ver.8.2.
Modification points
SOP ver.7.1
SOP ver.8.2
1. Handling the PBS stream
from washing bottle

It was not defined.

The revision specified to avoid hitting thetissue surface directly with the PBS tream.

2. Removal of PBS by tapping

It was not defined.

It was briefly defined.

3. Correct use of the cottonpad

It was not defined.

It was defined to avoid touching the tissue
surface directly with the cotton pad.

LabCyte EPI-MODEL 24 tissues were shipped from the supplier on Mondays and delivered torecipients on Tuesdays. Upon receipt, the tissues were aseptically removed from the transport agarose
medium, transferred into 24-well plates (BD Biosciences, CA, USA) with the assay medium (0.5 mL), andincubated overnight (37°C, 5% CO2 humidified atmosphere). On the following day, the tissues weretopically exposed to the test chemicals. Liquids (25 ȝL) were applied with a micropipette, and solids (25mg) were applied from microtubes and moistened with 25-ȝL sterile water. If necessary, the mixture wasgently spread over the surface of the epidermis with a microspatula. Viscous liquids were applied using acell-saver-type tip with a micropipette. Each test chemical was applied to three tissues. In addition, threetissues serving as negative controls were treated with 25-ȝL distilled water, and three tissues serving as
positive controls were exposed to 5% SLS (sodium lauryl sulphate). After a 15-minute exposure, eachtissue was carefully washed with PBS (phosphate buffered saline, Invitrogen, CA, USA) 10 times using awashing bottle to remove any remaining test chemical from the surface. The blotted tissues were thentransferred to new 24-well plates containing 1 mL of fresh assay medium.
The treated and control tissues were incubated for 42 hours (37 °C, 5% CO2 humidified atmosphere).
When the 42-hour incubation period was complete, blotted tissues were transferred to new 24-well plates
containing 0.5 mL of freshly prepared MTT medium (1 mg/mL; Dojindo Co., Kumamoto, Japan) for the
MTT assay. Tissues were incubated for 3 hours (37 °C, 5% CO2 humidified atmosphere) and thentransferred to microtubes containing 0.3 mL isopropanol, which completely immersed the tissue. Formazan
extraction was performed at room temperature, and the tissues were allowed to stand overnight.

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Subsequently, 200-ȝL extracts were transferred to a 96-well plate. The optical density was measured at 570
nm and 650 nm as a reference absorbance, with isopropanol as a blank.
The tissue viability was calculated as a percentage relative to the viability of the negative controls. The
median of three values from identically treated tissues was used to classify a chemical according to theprediction model.

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8-2. Prediction model of skin irritation
In this study, the prediction model (acceptability criteria and positive criteria) of skin irritation potential
with LabCyte EPI-MODEL24 SIT was set to refer to the conditions for the OECD TG 439 and its
Performance Standards.
8-2-1) Acceptance criteria on the RhE test method components
According to paragraph 27; acceptability criteria in the OECD TG 439, tissues treated with the negative
controls and positive controls, i.e. 5% aqueous SLS, should reflect their ability to respond to an irritantchemical under the conditions of the test method. Associated and appropriate measures of variability
between tissue replicates should be defined.
1) ODNC of the negative control is greater than 0.7.
2) The viability of the positive control (5% aqueous SLS) is less than 40%.
3) If standard deviations (SDs) are used they should be within the one-sided 95% tolerance interval
calculated from historical data; for the VRM SD < 18%.
8-2-2) Positive criteria
The OD values obtained with each test sample can be used to calculate the percentage of viability
normalized to the No Category, which is set to 100%. The cut-off value for percentage of cell viability
distinguishing irritant from non-classified test chemicals and the statistical procedure(s) used to evaluate
the results and identify irritant chemicals, should be clearly defined, documented, and proven to beappropriate. The cut-off values for the prediction of irritation are given below:
The test chemical is considered to be an irritant to skin in accordance with GHS category 2 if the tissue
viability after exposure and post-treatment incubation is less than or equal () to 50%.
Depending on the country and regional regulatory requirements, the test chemical may be considered as a
No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.
8-2-3) Study acceptance criteria
It is possible that one or several tests with one or more test chemicals do not meet test acceptancecriteria for the test and control chemicals or are not acceptable for other reasons. To complement missing
data, a maximum number of two additional tests for each test chemical is admissible ("retesting"). Because
retesting requires concurrent testing with a positive control and negative control, a maximum number of
two additional runs may be conducted for each test chemical.
It is conceivable that even after retesting, the minimum number of three valid runs required for eachtested chemical is not obtained for every Reference Chemical in every participating laboratory, leading toan incomplete data matrix. In such cases the following three criteria should all be met in order to consider
the datasets acceptable:
1. All 20 Reference Chemicals should have at least one complete run sequence.
2. In each of at least three participating laboratories, a minimum of 85% of the run sequences need to becomplete (for 20 chemicals, three invalid run sequences are allowed in a single laboratory).
3. A minimum of 90% of all possible run sequences from at least three laboratories need to be complete(for 20 chemicals tested in three laboratories, a total of six invalid run sequences are allowed).
8-2-4) Rules
The calculation of the reliability and accuracy values of the proposed test method should be done
considering all four criteria below, ensuring that values for reliability and relevance are calculated in apredefined and consistent manner:
1. Only data of runs from complete run sequences qualify for calculation of within- and betweenlaboratory variability and predictive capacity (accuracy) of the test method.
2. The final classification for each Reference Chemical in each participating laboratory should be obtainedby using the mean value of viability over the different runs of a complete run sequence.
3. Only data obtained for chemicals that have complete run sequences in all participating laboratories
qualify for calculation of between-laboratory variability of the test method.
4. Calculation of the accuracy values should be done on the basis of individual laboratory predictions

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ENV/JM/MONO(2011)44

obtained for the 20 Reference Chemicals by the different participating laboratories.
In this context, a run sequence consists of three independent runs from one laboratory for one test
chemical. A complete run sequence is a run sequence from one laboratory for one test chemical where all
three runs are valid. This means that any single invalid run invalidates an entire run sequence of three runs.

196

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ENV/JM/MONO(2011)44

Within-laboratory reproducibility
An assessment of within-laboratory reproducibility should show that the concordance of classifications(UN GHS Category 2 and No Category) obtained in different, independent test runs of 20 Reference
Chemicals within one single laboratory is equal to or higher than () 90%.
Between-laboratory reproducibility
An assessment of between-laboratory reproducibility is not essential if the proposed test method is to
be used in a single laboratory only. For methods to be transferred between laboratories, the concordance ofclassifications (UN GHS Category 2 and No Category) obtained in different, independent test runs of 20
Reference Chemicals between preferentially a minimum of 3 laboratories should be equal or higher than() 80%.
Predictive capacity (accuracy)
The accuracy (sensitivity, specificity and overall accuracy) of the proposed similar or modified test
method should be comparable or better to that of the VRM, taking into consideration information relating
the species of interest (Table 3). The sensitivity should be equal to or higher than () 80%. However, an
additional restriction applies to the sensitivity of the proposed in vitro test method; only two in vivo
Category 2 chemicals, 1-decanol and di-n-propyl disulphide, may be misclassified as a No Category bymore than one participating laboratory. The specificity should be equal to or higher than () 70%. No
restrictions with regard to specificity of the proposed in vitro test method were applied; any participating
laboratory may misclassify any in vivo No Category chemical as long as the final specificity of the test
method is within the acceptable range. The overall accuracy should be equal to or higher than () 75%.
Although the sensitivity of the VRM calculated for the 20 Reference Chemicals listed in Table 1 is equal to90%, the defined minimum sensitivity value required for any similar or modified test method to beconsidered valid is set at 80% because both 1-decanol (a borderline chemical) and di-n-propyl disulphide(a false negative of the VRM) are known to be non-irritant chemicals in humans, although they have been
identified as irritants in the rabbit test. Since RhE models are based on cells of human origin, they may
predict these chemicals as non-irritant (UN GHS No Category).

Table3: Required predictive values for sensitivity, specificity and overall accuracy for any
similar or modified test method to be considered valid
Sensitivity
 80%Specificity
 70%

Overall Accuracy
 75%

8-3. Data collection, handling, and analysis
The independent biostatistician for the study collected and organized the data using specific datacollection software (Datasheet5.0:20090430.xls). They worked in close collaboration with JaCVAM(Hajime Kojima). After decoding the data, JaCVAM performed statistical analyses. The data management
procedures and statistical tools applied were approved by the VMT.
8-4. Quality assurance, GLP Laboratories
All participating laboratories conducted research following OECD GLP-like principles.
QC aspects

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ENV/JM/MONO(2011)44

JaCVAM (Hajime Kojima) assured the quality of all the data and records. After the validation study,
all study documents were submitted to the chairperson of VMT and only data sheets were forwarded by email to the biostatistician. All data sheets from one participating laboratory, KOBAYASHI PharmaceuticalCo., Ltd. are provided as an example in Appendix 6. The chairperson reviewed the contents of the study
documents and clarified illegible or unclear content by contacting each group by e-mail or telephone.

198

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ENV/JM/MONO(2011)44

9. Results
9-1 Comments in the datasheets
A few comments from each laboratory are listed in Table 4. Application of potassium hydroxide(5%aq) (B276, B296, and B288) caused the model's layers to be desquamated. Upon application of
B301, B304, B306, and B310, the cups were discoloured and crystallized. The VMT judged that theseoccurrences had no effect on the results of the study.
Table 4 Comments on the datasheets (Viability)

9-2 Negative control
Table 5 shows the absorbance values for the negative control. All data for the negative control met theacceptance criteria.

Table 5 Viability of negative control
Laboratory

Lab a

Average OD/
triplicate tissues
0.88

Average, SD at
all OD

0.91±0.05

0.87
0.92
0.98

Lab b

1.03

1.03±0.06

1.02
1.13
0.98
0.98

Lab c

1.09

1.07±0.09

0.98
1.01
1.06
1.20

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ENV/JM/MONO(2011)44

9-3 Positive control
Table 6 shows the absorbance values for the positive control. All data for the positive control met theacceptance criteria.

200

130

ENV/JM/MONO(2011)44

Table 6 Viability of the positive control
Laboratory

Average OD/
triplicate tissues

Average, SD at
all OD

Lab a

2.29

2.65±0.68

3.62
2.59
2.10

Lab b

4.98

3.87±0.84

3.22
4.50
3.02
3.62

Lab c

2.87

2.69±0.49

3.37
2.64
2.55
2.02

9-4 Viability of chemicals
Table 7 shows the mean viability of testing chemicals at each tissue. Two data points at Lab a, eightdata points at Lab b, and four data points at Lab c showed a SD > 18% and did not meet the acceptancecriteria. Instead of generating insufficient data, each laboratory re-tested up to two additional runs. At Lab
b, No. 15 resulted in a single invalid run, thereby invalidating an entire run sequence of three runs. In
addition, the VMT did not accept all data from the fourth or fifth runs. The original data are shown inAppendix 5.
All study acceptance criteria were met as shown below.
1. All 20 Reference Chemicals had at least one complete run sequence at each laboratory.
2. In each of three participating laboratories, at least 95% of the run sequences were complete (One invalid
run sequence was allowed in Lab b).
3. 99.4% of all possible run sequences from the three laboratories were complete (for 20 chemicals tested
in three laboratories, a total of one invalid run sequence is allowed).
These experiments confirmed the feasibility of the LabCyte EPI-MODEL24 SIT test method.
Table 7.

Mean viability of chemicals at each laboratory

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䎔䎚䎑䎗

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9-5. Classification of three independent viabilities at each laboratory
The classifications from individual viabilities and the mean of three independent viabilities are shownin Table 8. Lab a misclassified two data points (No. 4 and 15 at the first test), Lab b misclassified two
data points (No. 15 at the first and second tests), and Lab c missed no classifications. As previously
discussed, the third data point of the test with No. 15 at Lab b induced a single invalid run, thereby
invalidating the entire run sequence of three runs. Therefore, the VMT judged “not detected” in theclassification of No.15.
Table 8: Classification using three independent viabilities
P: Positive, N: Negative, F: Final detemination by median, ND: Not detected.

UN
No GHS invivo Cat.

Lab a
1

Lab b
2

3

F

1

Lab c
2

3

F

1

2

3

F

1

No

P

P

P

P

P

P

P

P

P

P

P

P

2

No

N

N

N

N

N

N

N

N

N

N

N

N

3

No

N

N

N

N

N

N

N

N

N

N

N

N

4

No

P

N

N

P

N

N

N

N

N

N

N

N

5

No

N

N

N

N

N

N

N

N

N

N

N

N

6

No

P

P

P

P

P

P

P

P

P

P

P

P

7

No

N

N

N

N

N

N

N

N

N

N

N

N

8

No

N

N

N

N

N

N

N

N

N

N

N

N

9

No

N

N

N

N

N

N

N

N

N

N

N

N

10

No

P

P

P

P

P

P

P

P

P

P

P

P

11

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

12

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

13

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

14

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

15

Cat.2

P

N

N

N

P

P

ND

ND

N

N

N

N

16

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

17

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

18

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

19

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

20

Cat.2

P

P

P

P

P

P

P

P

P

P

P

P

133

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ENV/JM/MONO(2011)44

10. Discussion
10-1. Reliability
Within-laboratory reproducibility
An assessment of within-laboratory reproducibility should show a concordance of classifications (UNGHS Category 2 and No Category) obtained in different, independent test runs of the 20 Reference
Chemicals at each laboratory. As shown in Table 8 above, Lab a missed two classifications (No. 4 and 15)
and the rate of within-laboratory reproducibility was 90.0% (18/20). Lab b missed one data point (No. 15)
and the rate of reproducibility was 95.0% (19/20). Lab c missed no classifications and had areproducibility rate of 100%. Therefore, results of all laboratories were sufficient, having a reproducibility
rate equal to or higher than () 90%.
Between-laboratory reproducibility
For methods to be transferred between laboratories, the concordance of classifications (UN GHS
Category 2 and No Category) obtained in different, independent test runs of the 20 Reference Chemicals
between three laboratories was evaluated. As shown in Table 8, all laboratories missed more than fourclassifications and the rate of between-laboratory reproducibility was 95.0% (19/20). Therefore, alllaboratories had a sufficient between-laboratory reproducibility that was equal to or higher than () 80%.
10-2. Predictivity
The accuracy (sensitivity, specificity, and overall accuracy) of the LabCyte EPI-MODEL24 SIT skinirritation test was evaluated by cell viabilities (MTT) as an indicator, and the UN-GHS classifications areshown in Table 9. The sensitivity, specificity, and accuracy of this prediction model at each laboratory
were 90–100%, 60-70%, and 75–84.2%, respectively. Some deviations from the OECD Performancestandard (sensitivity of 80%, specificity of 70%, and accuracy of 75%; shown in Table 3) were specificadaptations for the LabCyte EPI-MODEL excluding 60% of specificity at Lab a. Unfortunately, one ofthree laboratories is insufficient with acceptance criteria at the OECD Performance standard. Two of threelaboratories, however, are sufficient with acceptance criteria at the OECD Performance standard and the
VMT considered that this assay had acceptable reliability of accuracy.
Table 9. 2x2 tables
Lab a

In vitro prediction

204

In vivo classification
Irritant

Non-Irritant

Total

Irritant

94

13

Non-irritant

1

6

7

Total

10

10

20

Sensitivity (%)

90.0

Specificity (%)

60.0

Accuracy (%)

75.0

134

ENV/JM/MONO(2011)44

Lab b

In vitro prediction

In vivo classification
Irritant

Non-Irritant

Total

Irritant

93

12

Non-irritant

0

7

7

Total

9

10

19

Sensitivity (%)

100.0

Specificity (%)

70.0

Accuracy (%)

84.2

Lab c

In vivo classification

In vitro prediction

Irritant

Non-Irritant

Total

Irritant

93

12

Non-irritant

1

7

8

Total

10

10

20

Sensitivity (%)

90.0

Specificity (%)

70.0

Accuracy (%)

80.0

11. Conclusions
Based on the reference list in the OECD Performance Standards, a catch-up validation of the LabCyte
EPI-MODEL24 SIT by three labs was performed. The assay demonstrated high reliability within andbetween laboratories, and acceptable reliability of accuracy (75–84.2% overall accuracy, 90–100% overallsensitivity, and 60-70% overall specificity) on the MTT assay excluding 60% of specificity at one
laboratory. Two of three laboratories are sufficient with acceptance criteria at the OECD Performancestandard and the VMT considered that this assay had acceptable reliability of accuracy for use as a standalone assay to distinguish between skin irritants and non-irritants.
12. Acknowledgement
This validation study was supported by the Health and Labour Sciences Research Grant, Japan.

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13. References
ECVAM : Performance standards for applying human skin models to in vitro skin irritation testing (2007)
EPISKIN®-MTTreduction
scientifically validated as replacement to the Draize skin irritation test (2007)
ESAC statement on the validity of in-vitro tests for skin irritation (2007)
ESAC Statement on the Scientific Validity of in-vitro Tests for Skin Irritation Testing (2008)
ESAC Statement on the Performance under UN GHS of Three in-vitro Assays for Skin Irritation Testing
and the Adaptation of the Reference Chemicals and Defined Accuracy Values of the ECVAM Skin
Irritation Peformance Standards (2009)
Globally Harmonised System of Classification and Labelling of Chemicals (GHS). Part 3: Health andEnvironmental Hazards, pp. 107–228.New York, NY, USA, and Geneva, Switzerland: United
Nations Organisation(2003).
Green H. (1978): Cyclic AMP in relation to proliferation of the epidermal cell: new view. Cell, 15, 801811.
Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Gribaldo, L., Halder, M.,
Hoffmann, S., Roi A.J., Prieto, P., Sabbioni, E., Scott, L., Worth, A. and Zuang. V. (2004) A
Modular Approach to the ECVAM Principles on Test Validity. ATLA 32, 467-72.
Masakazu Katoh, Fumiyasu Hamajima, Takahiro Ogasawara, and Ken-ichiro Hata (2009) Assessment of
the Human Epidermal Model LabCyte EPI-MODEL for In Vitro Skin Irritation Testing According to
the ECVAM-Validated Protocol , Journal of Toxicological Science, 34(3) 327-334.
OECD (1999) OECD series on principles of good laboratory practice and compliance monitoring No 5,
Compliance of Laboratory Suppliers with GLP Principles. Paris, France: Organisation for Economic
Cooperation and Development.
OECD (2002) OECD Guidelines for the Testing of Chemicals No. 404: Acute Skin Irritation/Corrosion.
Paris, France: Organisation for Economic Cooperation and Development.
OECD (2010) OECD Guidelines for the Testing of Chemicals No. 439: In vitro Skin Irritation assay.
Paris, France: Organisation for Economic Cooperation and Development.
[http://www.oecd.org/document/22/0,2340,en_2649_34377_1916054_1_1_1_1,00.html/].
OECD (2005) Guidance Document on the Validation and International Acceptance of New or Updated
Test Methods for Hazard Assessment. Environmental Health and Safety Monograph Series on Testing andAssessment No. 34. Available at:
http://www.oecd.org/document/30/0,3343,en_2649_34377_1916638_1_1_1_1,00.html. Accessed on
12.02.2008.
Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on
classification, labelling and packaging of substances and mixtures, amending and repealing
Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006)
Rheinwald J.G. and Green H. (1975): Serial cultivation of strains of human epidermal keratinocytes: theformation of keratinizing colonies from single cells. Cell, 6, 331-344

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Adopted:26 July 2013

OECD GUIDELINES FOR THE TESTING OF CHEMICALSIn Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

INTRODUCTION
1.
Skin irritation refers to the production of reversible damage to the skin following the applicationof a test chemical for up to 4 hours [as defined by the United Nations (UN) Globally Harmonized Systemof Classification and Labelling of Chemicals (GHS)](1). This Test Guideline (TG) provides an in vitro
procedure that may be used for the hazard identification of irritant chemicals (substances and mixtures) inaccordance with UN GHS Category 2 (1) (2). In member countries or regions that do not adopt the optional
UN GHS Category 3 (mild irritants), this Test Guideline can also be used to identify non-classified
chemicals. Therefore, depending on the regulatory framework and the classification system in use, thisTest Guideline may be used to determine the skin irritancy of chemicals either as a stand-alone
replacement test for in vivo skin irritation testing or as a partial replacement test within a tiered testing
strategy (4).
2.
The assessment of skin irritation has typically involved the use of laboratory animals [OECD TG
404; adopted in 1981 and revised in 1992 and 2002] (4). In relation to animal welfare concerns, TG 404 inits supplement recommended a tiered testing strategy for the determination of skin corrosion/irritation,using validated in vitro and ex vivo test methods, thus avoiding pain and suffering of animals. Threevalidated in vitro test methods have been adopted as OECD TGs 430, 431 and 435 (5) (6) (7), to be used
for the corrosivity part of the tiered testing strategy recommended in supplement to TG 404 (4).
3.
This Test Guideline addresses the human health endpoint skin irritation. It is based on the in vitro
test system of reconstructed human epidermis (RhE), which closely mimics the biochemical andphysiological properties of the upper parts of the human skin, i.e. the epidermis. The RhE test system uses
human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model withrepresentative histology and cytoarchitecture. Performance Standards (PS) developed by EC-ECVAM (8)(9) are available to facilitate the validation and assessment of similar and modified RhE-based test
methods, in accordance with the principles of Guidance Document No. 34 (10) (See Annex 4).
4.
Pre-validation, optimisation and validation studies have been completed for four commerciallyavailable in vitro test methods (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (35) (36)(37) (38) (39) based on the RhE test system. These four test methods are included in this TG and are listed
in Annex 2, which also provides information on the type of validation study used to validate the respective
test methods. As noted in Annex 2, three of these methods have been used to develop the present TG
including the Performance Standards (Annex 4) and are, in Annex 2 and 4, referred to as Validated
Reference Methods (VRM).
5.
Mutual Acceptance of Data will only be guaranteed for test methods, validated according to the
Performance Standards (Annex 4), if these test methods have been reviewed and adopted by OECD. The
1© OECD, (2013)
You are free to use this material for personal, non-commercial purposes without seeking prior consent fromthe OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written
permission from the OECD.

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test methods included in this TG can be used indiscriminately to address countries’ requirements for test
results from in vitro test method for skin irritation, while benefiting from the Mutual Acceptance of Data.
6.

Definitions of terms used in this document are provided in Annex 1.

INITIAL CONSIDERATIONS AND LIMITATIONS
7.
A limitation of the Test Guideline, as demonstrated by the full prospective validation study
assessing and characterising RhE test methods (17), is that it does not allow the classification of chemicals
to the optional UN GHS Category 3 (mild irritants) (1). Thus, the regulatory framework in member
countries will decide how this Test Guideline will be used. When employed as a partial replacement test,
follow-up in vivo testing may be required to fully characterize skin irritation potential (4). It is recognizedthat the use of human skin is subject to national and international ethical considerations and conditions.
8.
This Test Guideline addresses the in vitro skin irritation component of the tiered testing strategy
recommended in supplement to TG 404 on dermal corrosion/irritation (4). While this Test Guideline does
not provide adequate information on skin corrosion, it should be noted that OECD TG 431 on skincorrosion is based on the same RhE test system, though using another protocol (6). This Test Guideline isbased on RhE-models using human keratinocytes, which therefore represent in vitro the target organ of thespecies of interest. It moreover directly covers the initial step of the inflammatory cascade/mechanism ofaction (cell and tissue damage resulting in localised trauma) that occurs during irritation in vivo. A wide
range of chemicals has been tested in the validation underlying this Test Guideline and the database of thevalidation study amounted to 58 chemicals in total (17) (19) (24). The Test Guideline is applicable tosolids, liquids, semi-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; noother pre-treatment of the sample is required. Gases and aerosols have not been assessed yet in a validation
study (25). While it is conceivable that these can be tested using RhE technology, the current Test
Guideline does not allow testing of gases and aerosols. It should also be noted that highly coloured
chemicals may interfere with the cell viability measurements and need the use of adapted controls forcorrections (see paragraphs 24-26).
9.
A single testing run composed of three replicate tissues should be sufficient for a test chemical
when the classification is unequivocal. However, in cases of borderline results, such as non-concordant
replicate measurements and/or mean percent viability equal to 50 ± 5%, a second run should be considered,as well as a third one in case of discordant results between the first two runs.
PRINCIPLE OF THE TEST10.
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 multilayered,highly differentiated model of the human epidermis. It consists of organized basal, spinous and granularlayers, and a multilayered stratum corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo.
11.
Chemical-induced skin irritation, manifested mainly by erythema and oedema, is the result of acascade of events beginning with penetration of the chemicals through the stratum corneum where they
may damage the underlying layers of keratinocytes and other skin cells. The damaged cells may either
release inflammatory mediators or induce an inflammatory cascade which also acts on the cells in thedermis, particularly the stromal and endothelial cells of the blood vessels. It is the dilation and increased
permeability of the endothelial cells that produce the observed erythema and oedema (25). Notably, the2© OECD, (2013)

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RhE-based test methods, in the absence of any vascularisation in the in vitro test system, measure theinitiating events in the cascade, e.g. cell / tissue damage (17) (18), using cell viability as readout.
12.
Cell viability in RhE models is measured by enzymatic conversion of the vital dye MTT [3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1], into a
blue formazan salt that is quantitatively measured after extraction from tissues (25). Irritant chemicals are
identified by their ability to decrease cell viability below defined threshold levels (i.e.  50%, for UN GHS
Category 2). Depending on the regulatory framework and applicability of the Test Guideline, chemicals
that produce cell viabilities above the defined threshold level, may be considered non-irritants (i.e. > 50%,
No Category).
DEMONSTRATION OF PROFICIENCY
13.
Prior to routine use of any of the four validated test methods that adhere to this Test Guideline(Annex 2), laboratories should demonstrate technical proficiency, using the ten Proficiency Chemicals
listed in Table 1.
14.
As part of the proficiency testing, it is recommended that users verify the barrier properties of thetissues after receipt as specified by the RhE model producer. This is particularly important if tissues areshipped over long distance/time periods. Once a test method has been successfully established andproficiency in its use has been acquired and demonstrated, such verification will not be necessary on aroutine basis. However, when using a test method routinely, it is recommended to continue to assess thebarrier properties at regular intervals.
Table 1: Proficiency Chemicals1
Chemical

CAS NR

In vivo score2

Physical state

UN GHS
Category

0
0.3
1
1.7

Solid
LiquidSolid
LiquidNo Cat.
No Cat.
No Cat.
No Cat.
(Optional Cat. 3)3
No Cat.
(Optional Cat. 3)3

NON-CLASSIFIED CHEMIALS
naphthalene acetic acid
86-87-3
isopropanol
67-63-0
methyl stearate112-61-8
heptyl butyrate

5870-93-92
Liquidhexyl salicylate6259-76-3
CLASSIFIED CHEMICALS
cyclamen aldehyde
103-95-7
2.3
LiquidCat. 2
1-bromohexane
111-25-1
2.7
LiquidCat. 2
potassium hydroxide (5% aq.)
1310-58-3
3
LiquidCat. 2
1-methyl-3-phenyl-1-piperazine
5271-27-2
3.3
Solid
Cat. 2heptanal
111-71-7
3.4
LiquidCat. 2
1
The Proficiency Chemicals are a subset of the chemicals used in the validation study.
2
In vivo score in accordance with the OECD Test Guideline 404 (4).
3
Under this Test Guideline, the UN GHS optional Category 3 (mild irritants) (1) is considered as No
Category.

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OECD/OCDEPROCEDURE
15.
The following is a description of the components and procedures of a RhE test method for skinirritation assessment (See also Annex 3 for parameters related to each test method). Standard Operating
Procedures (SOPs) for the four test methods complying with this TG are available (27) (28) (29) (40).
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. Stratum corneum should be multilayered 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. The containment properties of the RhE model should preventthe 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 by bacteria, viruses,
mycoplasma, or fungi.
Functional conditions
Viability
17.
The assay used for determining the magnitude of viability is the MTT-assay (26). The RhE model
users should ensure that each batch of the RhE model used meets defined criteria for the negative control(NC). The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD< 0.1.
An acceptability range (upper and lower limit) for the negative control OD values (in the Skin Irritation
Test Method conditions) are established by the RhE model developer/supplier. Acceptability ranges for the
4 validated test methods are given in Table 2. It should be documented that the tissues treated with NC are
stable in culture (provide similar viability measurements) for the duration of the test exposure period.
Table 2: Acceptability ranges for negative control OD values of the test methods included in this TG
TM

EpiSkin (SM)
EpiDerm™ SIT (EPI-200)
SkinEthic™ RHE
LabCyte EPI-MODEL24 SIT

Lower acceptance limit
 0.6
 0.8
 0.8
 0.7

Upper acceptance limit
 1.5
 2.8
 3.0
 2.5

Barrier function
18.
The stratum corneum and its lipid composition should be sufficient to resist the rapid penetrationof cytotoxic benchmark chemicals, e.g. SDS or Triton X-100, as estimated by IC50 or ET50 (Table 3).

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Morphology
19.
Histological examination of the RhE model should be provided demonstrating human epidermislike structure (including multilayered stratum corneum).
Reproducibility
20.
The results of the positive and negative controls of the test method should demonstrate
reproducibility over time.
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 should
be provided to the test method users, so that they are able to include this information in the test report. An
acceptability range (upper and lower limit) for the IC50 or the ET50 should be established by the RhE model
developer/supplier. Only results produced with qualified tissues can be accepted for reliable prediction of
irritation classification. The acceptability ranges for the four test methods included in this TG are given in
Table 3.
Table 3: QC batch release criteria of the test methods included in this TG
TM

EpiSkin (SM)(18 hours treatment with SDS) (27)
EpiDerm™ SIT (EPI-200)(1% Triton X-100) (28)
SkinEthic™ RHE(1% Triton X-100) (29)
LabCyte EPI-MODEL24 SIT(18 hours treatment with SDS) (40)

Lower acceptance limit
IC50 = 1.0 mg/ml

Upper acceptance limit
IC50 = 3.0 mg/ml

ET50 = 4.0 hr

ET50 = 8.7 hr

ET50 = 4.0 hr

ET50 = 10.0 hr

IC50 = 1.4 mg/ml

IC50 = 4.0 mg/ml

Application of the Test and Control Chemicals
22.
At least three replicates should be used for each test chemical and for the controls 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. ranging from 26 to 83 PL/cm2 or mg/cm2(see Annex 3), should be used. For solid chemicals, the epidermis surface should be moistened with
deionised or distilled water before application, to improve contact between the test chemical and theepidermis surface. Whenever possible, solids should be tested as a fine powder. A nylon mesh may be used
as a spreading aid in some cases (see Annex 3). At the end of the exposure period, the test chemical should
be carefully washed from the epidermis surface with aqueous buffer, or 0.9% NaCl. Depending on the RhE
test methods used, the exposure period ranges between 15 and 60 minutes, and the incubation temperature
between 20 and 37°C. These exposure periods and temperatures are optimized for each individual RhE test
method and represent the different intrinsic properties of the test methods (e.g. barrier function) (seeAnnex 3).

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23.
Concurrent NC and positive controls (PC) should be used in each run to demonstrate that
viability (with the NC), barrier function and resulting tissue sensitivity (with the PC) of the tissues arewithin a defined historical acceptance range. The suggested PC chemical is 5% aqueous SDS. The
suggested NC chemicals are water or phosphate buffered saline (PBS).
Cell Viability Measurements
24.
According to the test procedure, it is essential that the viability measurement is not performed
immediately after exposure to the test chemical, but after a sufficiently long post-treatment incubation
period of the rinsed tissue in fresh medium. This period allows both for recovery from weak cytotoxic
effects and for appearance of clear cytotoxic effects. A 42 hours post-treatment incubation period wasfound optimal during test optimisation of two of the RhE-based test methods underlying this TG (12) (13)(14) (15) (16).
25.
The MTT assay is a validated quantitative method which should be used to measure cell viability
under this Test Guideline. It is compatible with use in a three-dimensional tissue construct. The tissue
sample is placed in MTT solution of appropriate concentration (e.g. 0.3 - 1 mg/mL) for 3 hours. The MTT
is converted into blue formazan by the viable cells. The precipitated blue formazan product is thenextracted from the tissue using a solvent (e.g. isopropanol, acidic isopropanol), and the concentration offormazan is measured by determining the OD at 570 nm using a filter band pass of maximum ± 30 nm.
26.
Optical properties of the test chemical or its chemical action on MTT (e.g. chemicals may
prevent or reverse the colour generation as well as cause it) may interfere with the assay leading to a false
estimate of viability. This may occur when a specific test chemical is not completely removed from thetissue by rinsing or when it penetrates the epidermis. If a test chemical acts directly on the MTT(e.g. MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls
should be used to detect and correct for test chemical interference with the viability measurement
technique. Detailed description of how to correct direct MTT reduction and interferences by colouring
agents is available in the SOPs for the four validated test methods included in this Test Guideline (27) (28)(29) (40).
Acceptability Criteria
27.
For each test method using valid RhE model batches (see paragraph 21), tissues treated with the
NC should exhibit OD reflecting the quality of the tissues that followed shipment, receipt steps and all
protocol processes. Control OD values should not be below historically established boundaries. Similarly,
tissues treated with the PC, i.e. 5% aqueous SDS, should reflect their ability to respond to an irritantchemical under the conditions of the test method (see Annex 3 and for further information SOPs of the four
test methods included in this TG (27) (28) (29) (40)). Associated and appropriate measures of variability
between tissue replicates, i.e., standard deviations (SD) should fall within the acceptance limits established
for the test method used (see Annex 3).
Interpretation of Results and Prediction Model
28.
The OD values obtained with each test chemical can be used to calculate the percentage of
viability normalised to NC, which is set to 100%. The cut-off value of percentage cell viability
distinguishing irritant from non-classified test chemicals and the statistical procedure(s) used to evaluate
the results and identify irritant chemicals should be clearly defined, documented, and proven to beappropriate (see SOPs of the test methods for information). The cut-off values for the prediction of
irritation are given below:
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x

The test chemical is considered to be irritant to skin in accordance with UN GHS
Category 2 if the tissue viability after exposure and post-treatment incubation is less thanor equal () to 50%.

x

Depending on the regulatory framework in member countries, the test chemical may beconsidered as non-irritant to skin in accordance with UN GHS No Category if the tissue
viability after exposure and post-treatment incubation is more than (>) 50%.

DATA AND REPORTING
Data
29.
For each run, data from individual replicate tissues (e.g. OD values and calculated percentage cell
viability data for each test chemical, including classification) should be reported in tabular form, including
data from repeat experiments as appropriate. In addition means ± SD for each run should be reported.
Observed interactions with MTT reagent and coloured test chemicals should be reported for each tested
chemical.
Test Report
30.

The test report should include the following information:
Test and Control Chemicals:- Chemical name(s) such as CAS name and number, if known;- Purity and composition of the chemical (in percentage(s) by weight);- Physical-chemical properties relevant to the conduct of the study (e.g. physical state,stability, volatility, pH and water solubility if known);- Treatment of the test/control chemicals prior to testing, if applicable (e.g. warming,
grinding);- Storage conditions;
Justification of the RhE model and protocol used
Test Conditions:- Cell system used;- Complete supporting information for the specific RhE model used including its performance.
This should include, but is not limited to;i) viabilityii) barrier functioniii) morphologyiv) reproducibility and predictivityv) Quality controls (QC) of the model- Details of the test procedure used;- Test doses used, duration of exposure and post treatment incubation period;- Description of any modifications to the test procedure;- Reference to historical data of the model. This should include, but is not limited to:i) acceptability of the QC data with reference to historical batch dataii) acceptability of the positive and negative control values with reference to positive andnegative control means and ranges- Description of evaluation criteria used including the justification for the selection of the cutoff point(s) for the prediction model;- Indication of controls used for direct MTT-reducers and/or colouring test chemicals;
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Results:- Tabulation of data from individual test chemical for each run and each replicate
measurement together with the mean, SD and overall classification;- Results of controls used for direct MTT-reducers and/or colouring test chemicals;- Description of other effects observed;
Discussion of the results
Conclusion
LITERATURE
1.
UN (2009), United Nations Globally Harmonized System of Classification and Labelling ofChemicals (GHS), Third revised edition, UN New York and Geneva. Available at:[http://www.unece.org/trans/danger/publi/ghs/ghs_rev03/03files_e.html]
2.
EC-ECVAM (2009), Statement on the “Performance under UN GHS of three in vitro assays forskin irritation testing and the adaptation of the Reference Chemicals and Defined Accuracy Values of the
ECVAM skin irritation Performance Standards”, issued by the ECVAM Scientific Advisory Committee(ESAC30), 9 April 2009. Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]3.
EC (2008), REGULATION (EC) No 1272/2008 of the European Parliament and of the Councilof 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending andrepealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006. Official
Journal of the European Union L353, 1-1355.
4.
OECD (2004), Acute Dermal Irritation/Corrosion, OECD Guideline for the Testing ofChemicals No. 404, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
5.
OECD (2004), In Vitro Skin Corrosion: Transcutaneous Electrical Resistance (TER), OECD
Guideline for the Testing of Chemicals No. 430, OECD, Paris. Available at:[http://www.oecd.org/env/testguidelines]
6.
OECD (2004), In Vitro Skin Corrosion: Human Skin Model Test, OECD Guideline for the
Testing of Chemicals No. 431, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
7.
OECD (2006), In Vitro Membrane Barrier Test Method for Skin Corrosion, OECD Guideline for
the Testing of Chemicals No. 435, OECD, Paris. Available at: [http://www.oecd.org/env/testguidelines]
8.
EC-ECVAM (2009), Performance Standards for in vitro skin irritation test methods based on
Reconstructed human Epidermis (RhE). Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/altanimal-testing] N.B. This is the current version of the ECVAM PS, updated in 2009 in view of theimplementation of UN GHS.
9.
EC-ECVAM (2009), ESAC Statement on the Performance Standards (PS) for in vitro skinirritation testing using Reconstructed human Epidermis, issued by the ECVAM Scientific Advisory
Committee (ESAC31), 8 July 2009. Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/alt-animaltesting]
10.
OECD (2005), Guidance Document on the Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment, OECD Series on Testing and Assessment No. 34, OECD,
Paris. Available at: [http://www.oecd.org/env/testguidelines]
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11.
Fentem, J.H., Briggs, D., Chesné, C., Elliot, G.R., Harbell, J.W., Heylings, J.R., Portes, P.,
Roguet, R., van de Sandt, J.J. M. and Botham, P. (2001), A prevalidation study on in vitro tests for acuteskin irritation, Results and evaluation by the Management Team, Toxicol. in Vitro 15, 57-93.
12.
Portes, P., Grandidier, M.-H., Cohen, C. and Roguet, R. (2002), Refinement of the EPISKIN
protocol for the assessment of acute skin irritation of chemicals: follow-up to the ECVAM prevalidation
study, Toxicol. in Vitro 16, 765–770.
13.
Kandárová, H., Liebsch, M., Genschow, E., Gerner, I., Traue, D., Slawik, B. and Spielmann, H.
(2004), Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro
skin irritation tests, ALTEX 21, 107–114.
14.
Kandárová, H., Liebsch, M., Gerner, I., Schmidt, E., Genschow, E., Traue, D. and Spielmann, H.
(2005), The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation
tests – An assessment of the performance of the optimised test, ATLA 33, 351-367.
15.
Cotovio, J., Grandidier, M.-H., Portes, P., Roguet, R. and Rubinsteen, G. (2005), The in vitro
acute skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of
the ECVAM validation process, ATLA 33, 329-349.
16.
Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliot, G.R., Fentem,
J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van De Sandt, J.J.M., Wiemann, C. and Worth,A. (2002), Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation, The
European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2, ATLA 30,
109-129.
17.
Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet, R.,
Cotovio, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H., Gamer, A., Remmele,
M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang, V. (2007), The ECVAM international
validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and
EpiDerm assays and on the skin integrity function test, ATLA 35, 559-601.
18.
Hoffmann, S. (2006), ECVAM skin irritation validation study phase II: Analysis of the primary
endpoint
MTT
and
the
secondary
endpoint
IL1-Į.
Availableat:[http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]19.
Eskes, C., Cole, T., Hoffmann, S., Worth, A., Cockshott, A., Gerner, I. and Zuang, V. (2007),The ECVAM international validation study on in vitro tests for acute skin irritation: selection of test
chemicals, ATLA 35, 603-619.
20.
Cotovio, J., Grandidier, M.-H., Lelièvre, D., Roguet, R., Tinois-Tessonneaud, E. and Leclaire, J.
(2007), In vitro acute skin irritancy of chemicals using the validated EPISKIN model in a tiered strategy Results and performances with 184 cosmetic ingredients, AATEX, 14, 351-358.
21.
EC-ECVAM (2007), Statement on the validity of in vitro tests for skin irritation, issued by the
ECVAM Scientific Advisory Committee (ESAC26), 27 April 2007. Available at:[http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]
22.
EC-ECVAM (2007), Performance Standards for applying human skin models to in vitro skinirritation testing. Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing] N.B. These are9© OECD, (2013)

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the original PS used for the validation of two test methods. These PS should not be used any longer as an
updated version (8) is now available.
23.
EC-ECVAM (2008), Statement on the scientific validity of in vitro tests for skin irritation testing,issued by the ECVAM Scientific Advisory Committee (ESAC29), 5 November 2008. Available at:[http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]24.
OECD (2010), Explanatory background document to the OECD draft Test Guideline on in vitro
skin irritation testing. Published in OECD Series on Testing and Assessment, No. 137, OECD, Paris.
Available at:
http://www.oecd.org/officialdocuments/displaydocumentpdf/?cote=env/jm/mono(2010)36&doclanguage=en25.
Welss, T., Basketter, D.A. and Schröder, K.R. (2004), In vitro skin irritation: fact and future.
State of the art review of mechanisms and models, Toxicol. in Vitro 18, 231-243.
26.
Mosmann, T. (1983), Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J. Immunol. Methods 65, 55-63.
27.
EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study:
Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation ofchemicals. Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]
28.
EpiDerm™ SOP, Version 7.0 (Revised March 2009), Protocol for: In vitro EpiDerm™ skinirritation test (EPI-200-SIT), For use with MatTek Corporation's reconstructed human epidermal model
EpiDerm (EPI-200). Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]29.
SkinEthic™ RHE SOP, Version 2.0 (February 2009), SkinEthic skin irritation test-42bis test
method for the prediction of acute skin irritation of chemicals: 42 minutes application + 42 hours postincubation. Available at: [http://ihcp.jrc.ec.europa.eu/our_activities/alt-animal-testing]
30.
Harvell, J.D., Lamminstausta, K., and Maibach, H.I. (1995), Irritant contact dermatitis, In:
Practical Contact Dermatitis, pp 7-18, (Ed. Guin J. D.). Mc Graw-Hill, New York.
31.
EC (2001), Commission Directive 2001/59/EC of 6 August 2001 adapting to technical progress
for the 28th time Council Directive 67/548/EEC on the approximation of laws, regulations andadministrative provisions relating to the classification, packaging and labelling of dangerous substances,
Official Journal of the European Union L225, 1-333.
32.
Basketter, D.A., York, M., McFadden, J.P. and Robinson, M.K. (2004), Determination of skinirritation potential in the human 4-h patch test. Contact Dermatitis 51, 1-4.
33.
Jirova, D., Liebsch, M., Basketter, D., Spiller, E., Kejlova, K., Bendova, H., Marriott, M. andKandarova, H. (2007), Comparison of human skin irritation and photo-irritation patch test data withcellular in vitro assays and animal in vivo data, AATEX, 14, 359-365.
34.
Jírová, D., Basketter, D., Liebsch, M., Bendová, H., Kejlová, K., Marriott, M. and Kandárová, H.
(2010), Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal
data, Contact Dermatitis, 62, 109-116.

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35.
Katoh, M., Hamajima, F., Ogasawara, T. and Hata K. (2009), Assessment of human epidermal
model LabCyte EPI-MODEL for in vitro skin irritation testing according to European centre for thevalidation of alternative methods (ECVAM)-Validated Protocol, J Toxicol Sci, 34, 327-334
36.
Katoh, M. and Hata K. (2011), Refinement of LabCyte EPI-MODEL24 skin Irritation test
method for adaptation to the requirements of OECD test guideline 439, AATEX, 16, 111-122
37.
OECD (2011), Validation report for the skin irritation test method using LabCyte EPIMODEL24, OECD Series on Testing and Assessment No. 159, OECD, Paris. Available at:[http://www.oecd.org/env/testguidelines]
38.
OECD (2011), Peer review report of validation of the skin irritation test using LabCyte EPIMODEL24, OECD Series on Testing and Assessment No. 155, OECD, Paris. Available at:[http://www.oecd.org/env/testguidelines]
39.
Kojima, H., Ando, Y., Idehara, K., Katoh, M., Kosaka, T., Miyaoka, E., Shinoda, S., Suzuki, T.,
Yamaguchi, Y., Yoshimura, I., Yuasa, A., Watanabe, Y. and Omori, T. (2012), Validation Study of the In
Vitro Skin Irritation Test with the LabCyte EPI-MODEL24, Altern Lab Anim, 40, 33-50.
40.
LabCyte EPI-MODEL24 SIT SOP, Version 8.3 (June 2011), Skin irritation test using thereconstructed human model “LabCyte EPI-MODEL24” Available at: [http://jacvam.jp/]

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OECD/OCDEANNEX 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 (10).
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 (10).
ET50: Can be estimated by determination of the exposure time required to reduce cell viability by 50%
upon application of the marker chemical at a specified, fixed concentration, see also IC50.
EU CLP (European Commission Regulation on the Classification, Labelling and Packaging of
Substances and Mixtures): Implements in the European Union (EU) the UN GHS system for theclassification of chemicals (substances and mixtures) (3).
GHS (Globally Harmonized System of Classification and Labelling of Chemicals by the United
Nations (UN)): A system proposing the classification of chemicals (substances and mixtures) according tostandardized types and levels of physical, health and environmental hazards, and addressing corresponding
communication elements, such as pictograms, signal words, hazard statements, precautionary statements
and safety data sheets, so that to convey information on their adverse effects with a view to protect people(including employers, workers, transporters, consumers and emergency responders) and the environment(1).
IC50: Can be estimated by determination of the concentration at which a marker 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.
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. Interchangeably used with similar test method (10).
Mixture: means a mixture or a solution composed of two or more substances in which they do not react.
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 selected
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from among the chemicals used to demonstrate the acceptable performance of the validated test method;and (iii) the comparable levels of accuracy and reliability, 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 (10).
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 should
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 (10).
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method (10).
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 (10).
Replacement test: A test which is designed to substitute for a test that is in routine use and accepted forhazard identification and/or risk assessment, and which has been determined to provide equivalent or
improved protection of human or animal health or the environment, as applicable, compared to theaccepted test, for all possible testing situations and chemicals (10).
Sensitivity: The proportion of all positive/active test chemicals that are correctly classified by the test. It isa measure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method (10).
Skin irritation: The production of reversible damage to the skin following the application of a test
chemical for up to 4 hours. Skin irritation is a locally arising reaction of the affected skin tissue andappears shortly after stimulation (30). It is caused by a local inflammatory reaction involving the innate(non-specific) immune system of the skin tissue. Its main characteristic is its reversible process involving
inflammatory reactions and most of the clinical characteristic signs of irritation (erythema, oedema, itchingand pain) related to an inflammatory process.
Specificity: The proportion of all negative/inactive test chemicals that are correctly classified by the test. 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 (10).
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
Tiered testing strategy: Testing which uses test methods in a sequential manner; the test methods selectedin each succeeding level are determined by the results in the previous level of testing (10).
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OECD/OCDEANNEX 2TEST METHODS INCLUDED IN THIS TG

Nr.
1

2

3
4

Test method name
EpiSkin™

Validation study type
Full prospective validation study (20032007). The test method components of thismethod were used to define the essential
test method components of the original andupdated ECVAM PS (8) (9) (22)*.
Moreover, the method's data relating to
identification of non-classified vs classified
substances formed the main basis fordefining the specificity and sensitivity
values of the original PS*.
EpiDerm™ SIT (EPI- EpiDerm™ (original): Initially the test
method underwent full prospective
200)
validation together with Nr. 1. from 20032007. The test method components of thismethod were used to define the essential
test methods components of the original andupdated ECVAM PS (8) (9) (22)*.
EpiDerm™ SIT (EPI-200): A
modification of the original EpiDerm ™
was validated using the original ECVAM
PS (22) in 2008*
Validation study based on the original
SkinEthic™ RHE
ECVAM Performance Standards (22) in
2008*.
LabCyteEPI- Validation study (2011-2012) based on the
Performance Standards (PS) of OECD TG
MODEL24 SIT
439 which are based on the updated
ECVAM PS* (8) (9).

References(2) (8) (9) (11) (12)(15) (16) (17) (18)(19) (20) (21) (22)(24) (27)(2) (8) (9) (11) (13)(14) (16) (17) (18)(19) (21) (22) (24)(28)(2) (22) (23) (24)(28)(2) (22) (23) (24)(29)(8) (9) (35) (36)(37) (38) (39) (40)and PS of this TG**) The original ECVAM Performance Standards (PS) (22) were developed in 2007 upon completion of theprospective validation study (17) which had assessed the performance of test methods Nr 1 and 2 inreference to the classification system as described in the 28th amendment to the EU Dangerous Substances
Directive (31). In 2008 the UN GHS was introduced (1) (3), effectively shifting the cut-off value for
distinguishing non-classified from classified substances from an in vivo score of 2.0 to 2.3. To adapt to thischanged regulatory requirement, the accuracy values and reference chemical list of the ECVAM PS were
updated in 2009 (2) (8) (9). As the original PS, also the updated PS were largely based data from methods
Nr. 1 and 2 (17), but additionally used data on reference chemicals from method Nr. 3. In 2010, theupdated ECVAM PS were used for stipulating the PS as presented in this TG (Annex 4). As methods Nos.
1, 2 and 3 [i.e. EpiSkin™, EpiDerm™ SIT (EPI-200) and SkinEthic™ RHE] have served to define this TG
including the PS, they are considered as Validated Reference Methods (VRM) (Annex 4). Detailed
information on the validation studies, a compilation of the data generated as well as background to the14© OECD, (2013)220

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necessary adaptations of the PS as a consequence of the UN GHS implementation can be found in the
ECVAM/BfR explanatory background document to this OECD TG (24).
SIT: Skin Irritation Test
RHE: Reconstructed Human Epidermis

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PROTOCOL PARAMETERS SPECIFIC TO EACH OF THE TEST METHODSINCLUDED IN THIS TG
The RhE methods do show very similar protocols and notably all use a post-incubation period of 42 hours(27) (28) (29). Variations concern mainly three parameters relating to the different barrier functions of thetest methods and listed here: A) pre-incubation time and volume, B) Application of test chemicals andC) Post-incubation volume.
TM

EpiDerm
SIT (EPI-200)

SkinEthic
RHETM

LabCyteEPIMODEL24
SIT

18-24 hours
0.9mL< 2 hours
0.3mL

15-30 hours
0.5mL

10ȝL
(26ȝL/cm2)
10mg(26mg/cm2)+ DW (5ȝL)
Not used30ȝL
(47ȝL/cm2)
25mg(39mg/cm2)+ DPBS (25ȝL)If necessary16ȝL(32ȝL/cm2)
16mg(32mg/cm2)+ DW (10ȝL)Applied25ȝL
(83ȝL/cm2)
25mg(83mg/cm2)+ DW (25ȝL)
Not used

15 minutes

60 minutes

42 minutes

15 minutes

RTa) at RT for 25
minutesb) at 37ºC for
35 minutes

RTRT

Medium volume
2 mLD) Maximum acceptable variability

0.9mL x 2

2 mL

1 mL

Standard
SDd18
deviation
between tissue
replicates
RT: Room temperature
DW: distilled water
DPBS: Dulbecco’s Phosphate Buffer Saline

SDd18

SDd18

SDd18

TM

EpiSkin(SM)A) Pre-incubation

Incubation time18- 24 hours
Medium volume
2mLB) Chemical application
For liquids
For solids
Use of nylon
meshTotal applicationtime
Application
temperatureC) Post-incubation volume

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ANNEX 4PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIEDIN VITRO RECONSTRUCTED HUMAN EPIDERMIS (RhE) TEST METHODS FOR SKIN
IRRITATION(Intended for the developers of new or modified similar test methods)
1.
Generally, the purpose of Performance Standards (PS) is to communicate the basis on which new
test methods, both proprietary (i.e. copyrighted, trademarked, registered) and non-proprietary can be
determined to have sufficient accuracy and reliability for specific testing purposes. The following PS were
defined on the basis of three validated and accepted reference methods using RhE; the PS can be used toevaluate the reliability and accuracy of other analogous test methods (colloquially referred to as “me-too”
tests) that are based on similar scientific principles and measure or predict the same biological or toxic
effect (10).
2.
Prior to adoption of modified test methods, i.e. proposed potential improvements to an approved
test method, there should be an evaluation to determine the effect of the proposed changes on the test
performance and the extent to which such changes affect the information available for the othercomponents of the validation process. Depending on the number and nature of the proposed changes, thedata generated and the supporting documentation for those changes, they should either be subjected to thesame validation process as described for a new test, or, if appropriate, to a limited assessment of reliability
and relevance using established PS (10).
3.
Methods considered similar (me-too) to the Validated Reference Methods (VRM, see Annex 2)
used to define the present Performance Standards or modifications of validated RhE methods should be
evaluated prior to their inclusion in the Test Guideline to determine their reliability and accuracy usingchemicals representing the full range of the Draize irritancy scores. When evaluated using the 20
recommended Reference Chemicals of the PS (Table 1), the proposed similar or modified test methods
should have reliability and accuracy values which are comparable or better than those derived from theVRM (Table 2 of this Annex) (2) (17). The reliability and accuracy values that should be achieved areprovided in paragraphs 8 to 12 of this Annex. Non-classified chemicals (UN GHS No Category) andclassified chemicals (UN GHS Category 2) (1), representing different chemical classes are included. The
reliability of the test method, as well as its ability to correctly identify UN GHS Category 2 irritantchemicals and, depending on the regulatory framework in member countries, also its ability to correctly
identify UN GHS No Category chemicals (for member countries that do not adopt optional UN GHS
Category 3), should be determined prior to its use for testing new test chemicals.
4.
These PS are based on the EC-ECVAM PS (8), updated according to the UN GHS systems onclassification and labelling (1) (2) (9). The original PS (22) were defined upon completion of the validation
study (17) and were based on the EU classification system as described in the 28 th amendment to theDangerous Substances Directive (31). Due to the adoption of the UN GHS system for classification andlabelling in EU (EU CLP) (3), which took place between the finalisation of the validation study and thecompletion of this Test Guideline, the PS have been updated (8) (9). This update concerned: i) thecomposition of the PS Reference Chemicals and ii) the defined reliability and accuracy values (2) (9) (24).
5.

The PS comprises the following three elements (10):I) Essential Test Method Components
II) Minimum List of Reference Chemicals
III) Defined Reliability and Accuracy Values
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OECD/OCDEI) Essential Test Method Components
6.
These consist of essential structural, functional, and procedural elements of a validated test methodthat 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 assurethat a similar or modified proposed test method is based on the same concepts as the validated test methods
used to define the PS (10). The essential test method components are described in detail in paragraphs 16
to 21 of the Test Guideline:
The general conditions (paragraph 16)
The functional conditions, which include:- viability (paragraph 17);- barrier function (paragraph 18);
- morphology (paragraph 19);
- reproducibility (paragraph 20); and,- quality control (paragraph 21)
For specific parameters (e.g. for Tables 2 and 3), adequate values should be provided for any new similar
or modified test method; these specific values may vary depending on the specific test method.
II) Minimum List of Reference Chemicals
7.
Reference Chemicals are used to determine if the performance (reliability and accuracy) of a
proposed similar or modified test method is comparable or better than that of the VRM (2) (8) (9) (17)(24). An evaluation on the basis of these reference chemicals can be performed only for methods proven to
be structurally and functionally sufficiently similar in reference to element I) of the PS, or representing aminor modification of one of the validated test methods used to define the present PS. The
20 recommended Reference Chemicals listed in Table 1 of this Annex include chemicals representing
different chemical classes (i.e. chemical categories based on functional groups), and are representative of
the full range of Draize irritancy scores (from non-irritant to strong irritant). The chemicals included in thislist comprise 10 UN GHS Category 2 chemicals and 10 non-categorised chemicals, of which 3 are optional
UN GHS Category 3 chemicals. Under this Test Guideline, the optional Category 3 is considered as No
Category. The chemicals listed in Table 1 are selected on the basis of data from the VRM and relate tochemicals used for the prospective validation study (17) as well as chemicals used in the optimisation
phases following Pre-validation. Due regard has been given to chemical functionality and physical state
when composing this list (15) (19). The Reference Chemicals represent the minimum number of chemicals
that should be used to evaluate the accuracy and reliability of a proposed similar or modified test method,but should not be used for the development of new test methods. In situations where a listed chemical isunavailable, other chemicals for which adequate in vivo reference data are available could be used,primarily from the chemicals used in the optimisation phase following pre-validation or the validation
study of the VRM. If desired, additional chemicals representing other chemical classes and for whichadequate in vivo reference data are available may be added to the minimum list of Reference Chemicals tofurther evaluate the accuracy of the proposed test method.

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Table 1: Minimum List of Reference Chemicals for Determination of Accuracy andReliability Values for Similar or Modified RhE Skin Irritation Test Methods1
ChemicalCAS
NumberNON-CLASSIFIED CHEMICALS
1-bromo-4-chlorobutane
6940-78-9
diethyl phthalate
84-66-2
naphthalene acetic acid
86-87-3allyl phenoxy-acetate
7493-74-5
isopropanol
67-63-0
4-methyl-thio3446-89-7
benzaldehyde
methyl stearate112-61-8

Physicalstate

In vivo
score

VRM*
Cat.
based onin vitro

UN GHS Cat.
based on in vivo
results

LiquidLiquidSolid
LiquidLiquid

0
0
0
0.3
0.3

Cat. 2
No Cat.
No Cat.
No Cat.
No Cat.

No Cat.
No Cat.
No Cat.
No Cat.
No Cat.

Liquid1Cat. 2

No Cat.

Solid

1

No Cat.

No Cat.
No Cat.
(Optional Cat. 3)
No Cat.
(Optional Cat. 3)
No Cat.
(Optional Cat. 3)

heptyl butyrate

5870-93-9

Liquid

1.7

No Cat.

hexyl salicylate6259-76-3

Liquid2

No Cat.

cinnamaldehyde

104-55-2

Liquid2Cat. 2

CLASSIFIED CHEMICALS
1-decanol2
112-30-1
Liquid2.3
Cat. 2
Cat. 2cyclamen aldehyde
103-95-7
Liquid2.3
Cat. 2
Cat. 2
1-bromohexane
111-25-1
Liquid2.7
Cat. 2
Cat. 22-chloromethyl-3,5dimethyl-486604-75-3
Solid
2.7
Cat. 2
Cat. 2
methoxypyridine HCl
di-n-propyl disulphide2
629-19-6
Liquid3
No Cat.
Cat. 2
potassiumhydroxide
1310-58-3
Liquid3Cat. 2
Cat. 2(5% aq.)benzenethiol,
5-(1,17340-90-1
Liquid3.3
Cat. 2
Cat. 2dimethylethyl)-2-methyl
1-methyl-3-phenyl-15271-27-2
Solid
3.3
Cat. 2
Cat. 2
piperazine
heptanal
111-71-7
Liquid3.4
Cat. 2
Cat. 2
tetrachloroethylene
127-18-4
Liquid4Cat. 2
Cat. 2*) VRM = validated reference methods (Annex 2)
1
The chemical selection is based on the following criteria; (i), the chemicals are commerciallyavailable; (ii), they are representative of the full range of Draize irritancy scores (from non-irritant tostrong irritant); (iii), they have a well-defined chemical structure; (iv), they are representative of thechemical functionality used in the validation process; and (v), they are not associated with anextremely toxic profile (e.g. carcinogenic or toxic to the reproductive system) and they are notassociated with prohibitive disposal costs.
2
Chemicals that are irritant in the rabbit but for which there is reliable evidence that they are nonirritant in humans (32) (33) (34).

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III) Defined Reliability and Accuracy Values
8.
For purposes of establishing the reliability and relevance of proposed similar or modified test
methods to be transferred between laboratories, all 20 Reference Chemicals in Table 1 should be tested in
at least three laboratories. However, if the proposed test method is to be used in a single laboratory only,
multi-laboratory testing will not be required for validation. It is however essential that such validation
studies are independently assessed by internationally recognised validation bodies, in agreement withinternational guidelines (10). In each laboratory, all 20 Reference Chemicals should be tested in three
independent runs performed with different tissue batches and at sufficiently spaced time points. Each run
should consist of a minimum of three concurrently tested tissue replicates for each included test chemical,
NC and PC.
9.
The calculation of the reliability and accuracy values of the proposed test method should be done
considering all four criteria below together, ensuring that the values for reliability and relevance arecalculated in a predefined and consistent manner:
1. Only the data of runs from complete run sequences qualify for the calculation of the test
method within, and between-laboratory variability and predictive capacity (accuracy).
2. The final classification for each Reference Chemicals in each participating laboratory
should be obtained by using the mean value of viability over the different runs of acomplete run sequence.
3. Only the data obtained for chemicals that have complete run sequences in allparticipating laboratories qualify for the calculation of the test method between-laboratory
variability.
4. The calculation of the accuracy values should be done on the basis of the individuallaboratory predictions obtained for the 20 Reference Chemicals by the different
participating laboratories.
In this context, a run sequence consists of three independent runs from one laboratory for one test
chemical. A complete run sequence is a run sequence from one laboratory for one test chemical where all
three runs are valid. This means that any single invalid run invalidates an entire run sequence of three runs.
Within-laboratory reproducibility
10.
An assessment of within-laboratory reproducibility should show a concordance of classifications(UN GHS Category 2 and No Category) obtained in different, independent test runs of the 20 Reference
Chemicals within one single laboratory equal or higher () than 90%.
Between-laboratory reproducibility
11.
An assessment of between-laboratory reproducibility is not essential if the proposed test method
is to be used in a single laboratory only. For methods to be transferred between laboratories, theconcordance of classifications (UN GHS Category 2 and No Category) obtained in different, independent
test runs of the 20 Reference Chemicals between preferentially a minimum of three laboratories should beequal or higher () than 80%.
Predictive capacity
12.
The predictive capacity (sensitivity, specificity and accuracy) of the proposed similar or modified
test method should be comparable or better to that of the VRM, taking into consideration additional
information relating to relevance in the species of interest (Table 2 of this Annex). The sensitivity shouldbe equal or higher () than 80% (2) (8) (9) (24). However, a further specific restriction applies to the20© OECD, (2013)

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sensitivity of the proposed in vitro test method in as much as only two in vivo Category 2 reference
chemicals, 1-decanol and di-n-propyl disulphide, may be misclassified as No Category by more than one
participating laboratory. The specificity should be equal or higher () than 70% (2) (8) (9) (24). There is nofurther restriction with regard to the specificity of the proposed in vitro test method, i.e. any participating
laboratory may misclassify any in vivo No Category chemical as long as the final specificity of the test
method is within the acceptable range. The accuracy should be equal or higher () than 75% (2) (8) (9)(24). Although the sensitivity of the VRM calculated for the 20 Reference Chemicals listed in Table 1 isequal to 90%, the defined minimum sensitivity value required for any similar or modified test method to beconsidered valid is set at 80% since both 1-decanol (a borderline chemical) and di-n-propyl disulphide (afalse negative of the VRM) are known to be non-irritant in humans (32) (33) (34), although being
identified as irritants in the rabbit test. Since RhE models are based on cells of human origin, they may
predict these chemicals as non-irritant (UN GHS No Category).
Table 2: Required predictive values for sensitivity, specificity andaccuracy for any similar or modified test method to be considered valid
Sensitivity
 80%Specificity
 70%

Accuracy
 75%

Study Acceptance Criteria
13.
It is possible that one or several tests pertaining to one or more test chemicals does/do not meet thetest acceptance criteria for the test and control chemicals or is/are not acceptable for other reasons. To
complement missing data, for each test chemical a maximum number of two additional runs are admissible("retesting"). More precisely, since in case of retesting also PC and NC have to be concurrently tested, amaximum number of two additional runs may be conducted for each test chemical.
14.
It is conceivable that even after retesting, the minimum number of three valid runs required foreach tested chemical is 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 toconsider the datasets acceptable:
1. All 20 Reference Chemicals should have at least one complete run sequence;
2. In each of at least three participating laboratories, a minimum of 85% of the runsequences need to be complete (for 20 chemicals; i.e. 3 invalid run sequences are allowed
in a single laboratory);
3. A minimum of 90% of all possible run sequences from at least three laboratories need
to be complete (for 20 chemicals tested in 3 laboratories; i.e. 6 invalid run sequences areallowed in total).

21© OECD, (2013)

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Page 1 of 29-JSAAE Skin Irritation Test Validation Study-

SKIN IRRITATION TESTUSING THE RECONSTRUCTED HUMAN MODEL“LABCYTE EPI-MODEL 24”
Ver. 8.3

LabCyte EPI-MODEL24 SKIN IRRITATION TEST-42 HOURS

S.O.P.

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TABLE OF CONTENTS
1. RATIONAL AND BACKGROUND................................................................................................... 4
1.1
1.2
1.3

LabCyte EPI-MODEL 24 SKIN IRRITATION TEST (SIT using LabCyte EPI-MODEL24)........ 4
BACKGROUND OF SIT using LabCyte EPI-MODEL24 .......................................................... 4
BASIS OF THE METOD........................................................................................................... 4

1.3.1
1.3.2

1.4
1.5
1.6

TEST SYSTEM DESCRIPTION...................................................................................................4
ASSAY QUALITY CONTROL.......................................................................................................5

LIMITATION OF THE METHOD............................................................................................... 6
BRIEF BASIC PROCEDURE ................................................................................................... 6
DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)......................................... 6

2. MATERIALS .................................................................................................................................... 7
2.1

LabCyte EPI-MODEL 24 .......................................................................................................... 7

2.1.1
2.1.2
2.1.3

2.2
2.3
2.4

LabCyte EPI-MODEL 24 KIT COMPONENTS .............................................................................7
SHIPMENT OF LabCyte EPI-MODEL24......................................................................................7
INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24............................................................7

TEST CHEMICALS .................................................................................................................. 7
CONSUMABLES...................................................................................................................... 7
OTHERS................................................................................................................................... 8

2.4.1
2.4.2

EQUIPMENT / INSTRUMENTS ...................................................................................................8
CONSUMMABLE ITEMS .............................................................................................................8

3. TEST METHOD ............................................................................................................................... 9
3.1

PREPARATIONS...................................................................................................................... 9

3.1.1
3.1.2
3.1.3
3.1.4

3.2

TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT............ 10

3.2.1
3.2.2

3.3

MTT SOLUTION ..........................................................................................................................9
POSITIVE CONTROL SUBSTANCE............................................................................................9
NEGATIVE CONTROL SUBSTANCE ..........................................................................................9
POLY WASH BOTTLE FOR PBS.................................................................................................9
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE ...............................................10
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT.............................................. 11

EXECUTION OF THE TEST .................................................................................................. 13

3.3.1
3.3.2
3.3.3
3.3.4
3.3.5

PREPARATION OF LabCyte EPI-MODEL 24 (DAY -1)..............................................................13
APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0) ................................................13
POST TREATMENT INCUBATION (DAY 0~2)...........................................................................16
MTT ASSAY (DAY 2) ..................................................................................................................16
FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2~3) ................................................17

4. ASSESSMENT .............................................................................................................................. 19
4.1
4.2

CONDITIONS FOR A SUCCESSFUL STUDY....................................................................... 19
ASSAY CRITERIA .................................................................................................................. 19

MDS 1:RECEIPT OF LABCYTE EPI-MODEL 24 ............................................................................. 21

MDS 2:

PRE-INCUBATION OF LABCYTE EPI-MODEL 24 (Section 3.3.1) .................................... 22

MDS 3-1: APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section
3.1.2, 3.3.2 ~ 3.3.3) ............................................................................................................................... 23
MDS 3-2: APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section
3.3.2~3.3.3) ........................................................................................................................................... 24
MDS 4:MTT ASSAY (Section 3.3.4) ................................................................................................. 25

MDS 5:FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.5) .................................. 26

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REVISION HISTORY............................................................................................................................. 27

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1. RATIONAL AND BACKGROUND
1.1

SKIN IRRITATION TEST using LabCyte EPI-MODEL 24 (SIT using LabCyte EPI-MODEL24)
The SIT using LabCyte EPI-MODEL24 is designed for the prediction of acute skin irritation ofchemicals by measurement of its cytotoxic effect, as reflected in the MTT assay, on the
Reconstructed Human Epidermis (RHE) model. The SIT using LabCyte EPI-MODEL24 is not a kit;
LabCyte EPI-MODEL24 tissues are commercially available per tissues item (with a minimum of 24
LabCyte EPI-MODEL24 tissues per order).

1.2 BACKGROUND OF SIT using LabCyte EPI-MODEL24
Performance standards for applying human skin models to in vitro skin irritation testing werealso defined based on the validated test EpiSkinTM test method (ECVAM SIVS, 2007). These
performance standards can be then used to evaluate the accuracy and reliability of otheranalogous test methods (also referred to as “me-too” tests) that are based on similar scientificprinciples and measure or predict the same biological or toxic effect.
Based on the GHS-EU classification, 12 irritants and 13 non-irritants in the draft performancestandards (ECVAM 2007) and the statement by ESAC (ESAC2009) were performed the validation
study through the 7 labs SIT using LabCyte EPI-MODEL24. Results were summarized at JSAAE
1st report and 2nd report on this validation study.

1.3 BASIS OF THE METHOD
Chemical-induced skin irritation, manifested by erythema and oedema, is the results of acascade of events beginning with penetration of the stratum corneum and damage to theunderlying layers of keratinocytes. The dying keratinocytes release mediators that begin the
inflammatory cascade which acts on the cells in the dermis, particularly the stromal andendothelial cells. It is the dilation and increased permeability of the endothelial cells that produce
the observed erythema and oedema. The RhE-based test methods measure the initiating eventsin the cascade.
The relative viability of the treated tissues was measured at the end of the treatment exposure(15 minutes) followed by a post-exposure period (42 hours) using MTT [(3-4,5-dimethyl thiazole2-yl) 2,5-diphenyltetrazoliumbromide] assay. A cutoff value of 50% viability of the negative control
value was considered and used to classify test substances as irritant (I) or non irritant (NI). The
culture environment might allow the detection of very small quantities of cytokines secreted by the
epidermis in response to topical application of test substances.

1.3.1
TEST SYSTEM DESCRIPTION
LabCyte EPI-MODEL24 is a new, commercially available RhE model produced by Japan Tissue
Engineering Co. Ltd. It consists of normal human epidermal keratinocytes whose biological origin
is neonate foreskin. In order to expand human keratinocytes while maintaining their phenotype,they were cultured with 3T3-J2 cells as a feeder layer (Rheinwald and Green, 1975; Green, 1978).
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Page 5 of 29Reconstruction of human cultured epidermis is achieved by cultivating and proliferating
keratinocytes on an inert filter substrate (surface 0.3 cm2) at the air-liquid interface for 13 days with
an optimized medium containing 5% fetal bovine serum. It constructs a multilayer structure
consisting of a fully differentiated epithelium with features of the normal human epidermis,
including a stratum corneum. LabCyte EPI-MODEL24 is embedded in an agarose gel containing
nutrient solution and shipped in 24-well plates at around 18°C.
1.3.1.1 Quality control of the test system
The LabCyte EPI-MODEL24 is manufactured according to defined quality assurance
procedures. Each batch production was provided with quality controls such as storage conditions,
RHE instructions for use, lot number and origin, histology (demonstration of human epidermis-likestructure with multilayered stratum corneum), cell viability, barrier function integrity (0.14  IC50 
0.4).

1.3.1.2 Precautions
The epidermal cells are taken from healthy donor negative to HIV, and Hepatitis. Nevertheless,handling procedures for biological materials should be followed:a) It is recommended to wear gloves during handling with the skin and kit components.
b) After use, the epidermis, the material and all media in contact with it should be
decontaminated prior to disposal (e.g. using special containers or autoclaving).

1.3.2

ASSAY QUALITY CONTROL

1.3.2.1 Assay Acceptance Criterion 1: Negative Control
The absolute OD of the negative control (NC) tissues (treated with sterile DPBS) in the MTT
assay is an indicator of tissue viability obtained in the testing laboratory after shipping and storingprocedures and under specific conditions of use.
0.7  Mean OD (A570/650) measured value  2.5
1.3.2.2 Assay Acceptance Criterion 2: Positive Control
A 5% SDS (in H2O) solution (see 7.6.3) is used as positive control (PC) and tested concurrently
with the test chemicals. Concurrent means here the PC has to be tested in each assay, but not
more than one PC is required per testing day. Viability of positive control should be within 95±1 %
confidence interval of the historical data.
Mean tissue viability  40%
1.3.2.3 Assay Acceptance Criterion 3: Standard Deviation (SD)
Since in each test skin irritancy potential is predicted from the mean viability determined on 3
single tissues, the variability of tissue replicates should be acceptably low.
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Page 6 of 29Standard Deviation (SD) of tissue viability of 3 identically treated replicates for negative
control and positive control  18 %1.4 LIMITATION OF THE METHOD
One limitation of this assay method is a possible interference of the test substance with the MTT
endpoint. A colored test substance or one that directly reduces MTT (and thereby mimics
dehydrogenase activity of the cellular mitochondria) may interfere with the MTT endpoint. However,these test substance are a problem only if at the time of the MTT test (i.e. 42 hours after test
substance exposure) sufficient amounts of the test substance are still present on (or in) the tissues.
In case of this unlikely event, the (true) metabolic MTT reduction and the contribution by a colored
test material or (false) direct MTT reduction by the test material can be quantified by a proceduredescribed in Section 3.2.
The method is not designed for testing of highly volatile test substances, gases and aerosols.

1.5 BRIEF BASIC PROCEDURE
On the day of receipt, LabCyte EPI-MODEL24 tissues are conditioned by incubation to releasetransportstress related compounds and debris overnight. After pre-incubation, tissues are topically
exposed to the test chemicals for 15 minutes. Preferably, three tissues are used per test chemical(TC) and for the positive control (PC) and negative control (NC). Tissues are then thoroughly
rinsed, blotted to remove the test substances, and transferred to fresh medium. After 42 hr
incubation period, the MTT assay is performed by transferring the tissues to the well containing
MTT medium (0.5 mg/ml). After 3 hr MTT incubation, the blue formazan salt formed by cellular
mitochondria is extracted with 0.3 mL/tissue of isopropanol and the optical density of the extracted
formazan is determined using a spectrophotometer at 570 nm and 650 nm as reference. Relativecell viability is calculated for each tissue as % of the mean of the negative control tissues. Skin
irritation potential of the test material is predicted if the remaining relative cell viability is below
50%.

1.6 DATA INTERPRETATION PROCEDURE (PREDICTION MODEL)
According to the GHS classification (Category 2 or no label), an irritant is predicted if the mean
relative tissue viability of three individual tissues exposed to the test substance is reduced below
50% of the mean viability of the negative controls.
In vitro results

In vivo prediction

Tissue viability is  50%

Irritant

Tissue viability is > 50%

Non Irritant

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

2. MATERIALS
2.1

LabCyte EPI-MODEL 24

2.1.1
LabCyte EPI-MODEL 24 KIT COMPONENTS
LabCyte EPI-MODEL 24 kit components are shown in Table 1.
Table 1 - LabCyte EPI-MODEL24 Kit Components
Component

Qty

LabCyte EPI-MODEL 24 plate

Description
Contains 24 culture inserts with tissues fixed in

1 platenutritive agar medium for transport (usable area:2

0.3cm ).
Assay Medium
24-well plate

2.1.2

1 bottle
1 plateBasic medium for incubation (30mL). Store at
refrigeration temperature.
Blank

plate

for

use

in

assay.

Storeatroom-temperature.

SHIPMENT OF LabCyte EPI-MODEL24

LabCyte EPI-MODEL 24 is packed in a special container (Icompo/NIPPON EXPRESS CO.,LTD) and delivered by NIPPON EXPRESS CO., LTD. After the Icompo is delivered, examine thecontents and make sure that all kit components (LabCyte EPI-MODEL24 plate, assay medium,and 24-well assay plate) are included in the package. Confirm lot numbers and expiration dates
also. Record details in the Methods Documentation Sheet (MDS) 1.
NIPPON EXPRESS will pick up the Icompo at a later date (generally, the day after the date ofdelivery), and we ask that you return it with a slip documenting receipt, as well as the insulating
materials.

2.1.3

INSTRUCTIONS FOR USE OF LabCyte EPI-MODEL 24

Begin incubating all of the culture inserts after opening the package. Do not store the culture
inserts again after opening.
The human epidermis cells used in LabCyte EPI-MODEL 24 originate from a normal donor andare HIV-, HBV-, HCV-, and HPV-negative. However, handle them with enough care and inaccordance with the laboratory biosafety guidelines since they contain raw materials of humanorigin.

2.2 TEST CHEMICALS
Coded test chemicals are delivered to each laboratory.

2.3

CONSUMABLES
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
The following consumables are required.
* The described quantities are necessary so that 1 to 6 samples can be assayed once.
• Assay Medium, 100mL (J-TEC: 402250)
1 bottle• MTT, 25mg (J-TEC: 403026)
1 bottle• Wide orifice cell saver tips for micro-pipettes (sterile) 96 tips
1 box
• 24-well assay plate (Becton,Dickinson and Company: 353047)
7 plates• 96-well plate (Becton,Dickinson and Company: 353072)
1 plates• Phosphate buffered saline (PBS) 500mL (Invitrogen: 14190-144)
2 bottles• Isopropanol 500mL (Wako Pure Chemical Industries: 164-08335)
1 bottle• SLS 25g (SIGMA:L4390)
1 bottle• Sterile distilled water 20mL (Otsuka Pharmaceutical: 36A1X00001) 1 bottles• Sterile cotton buds (JAPAN COTTON BUDS: 10A754D)
1 box

2.4

OTHERS

2.4.1

EQUIPMENT / INSTRUMENTS
•
•
•
•
•
•
•
•
•
•
•
•
•

2.4.2

Safety cabinet (or clean bench)
Water bath (37 °C)
CO2 incubator (37 °C, 5%CO2, capable of maintaining high humidity)
Autoclave
96-well multi-plate reader (required filters: 450nm, 570nm, 650nm)
Precision balance (0.1mg)
Aspirator
Stop-watches
Adjustable micro-pipette (10-200ȝL, 200-1000ȝL)Sharp-edged forceps (sterile)
Micro spatula (sterile)
Beaker (1~2L: sterile)
Sterilizable poly wash bottle (500~1000mL: sterile)

CONSUMMABLE ITEMS•
Micro-pipette tips (sterile: 10~200ȝL, 200~1000ȝL)•
Microtubes (1.5mL)•
Scalpel (KEISEI MEDICAL INDUSTRIAL: Keisei Scalpel 11A)

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

3. TEST METHOD*Perform operations in Section 3.1.1~3.1.4 and Section 3.3.1~3.3.2 aseptically in a safetycabinet (or clean bench).
*Operations other than above do not need to be performed with an aseptic technique. For
these operations, refer to Section 2.1.3 INSTRUCTIONS FOR USE OF LabCyte
EPI-MODEL 24

3.1

PREPARATIONS

3.1.1
MTT SOLUTION(1) Dissolve MTT in the assay medium to prepare the MTT medium (final concentration:
0.5mg/mL)
Use ultrasonic cleaning equipment or a vortex mixer as necessary in order to completely
dissolve the MTT.
*Store in a dark, cold place and use it within 24 hours.
(2) Record details of step (1) above in the MDS 4.

3.1.2
POSITIVE CONTROL SUBSTANCE(1) Weigh 500mg of SLS precisely.
(2) To prepare a positive control solution, put the SLS into a graduated cylinder or measuring
flask and dilute to 10mL with distilled water (final concentration: 5% w/v)]* Store in a dark, cold place and use it within 24 hours.
(3) Record details of steps (1) and (2) above in the MDS 3.

3.1.3
NEGATIVE CONTROL SUBSTANCE(1) Use distilled water.

3.1.4
POLY WASH BOTTLE FOR PBS(1) Sterilize poly wash bottle using an autoclave.
(2) Fill the sterilized poly wash bottle aseptically with sterile PBS.

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
3.2
TEST FOR DETECTING CHEMICALS THAT INTERFERE WITH MTT ENDPOINT
There are two kinds of test chemicals that interfere with the MTT assay as follows.
(a) Chemical that stains epidermis tissues.
(b) Chemical that is able to directly reduce MTT.
Test chemical that stains the epidermis tissues has a possibility to transfer from the epidermis
tissues to the extraction solution and to affect the optical density (OD) measurements.
Test chemical that is able to directly reduce MTT can affect the optical density (OD)measurements, if the test chemical is present in the epidermis tissues when the MTT viability
test is performed. Detection procedure of these test chemicals is described below.

3.2.1
DETECTION OF THE CHEMICALS THAT STAIN THE TISSUE
3.2.1.1 STEP1 (PRELIMINARY TEST)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical into wells of 24-well assay plate
preliminarily filled with 0.5mL of distilled water. Untreated distilled water is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 15
minutes.
(3) After incubation, shake the mixture gently and evaluate the staining of the distilled water
macroscopically.
(4) When the color of the solution changes significantly, the test chemical is presumed to havethe potential to stain the tissue and a functional check on viable tissues (Step2) should be
performed. When the color of the solution does not change significantly, the test chemical isdetermined not to have a potential to stain the tissue.
3.2.1.2 STEP2 (FUNCTIONAL CHECK ON VIABLE TISSUE)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color ofthe distilled water (Step1), onto the surface of the epidermis tissues. Distilled water is usedas negative control.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST.
However, incubate the tissue for 3 hours in culture media without MTT instead of incubating
in media containing MTT to evaluate the staining of the epidermis tissues.
(3) Calculate ratio of staining by test chemical from the following formula.

Ratio of staining by test
chemical (%) =OD test chemical – OD negative control
OD negative control

×100(4) When the ratio of staining by test chemical is <5%, correction of the results is not necessary.
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When the ratio is between 5% and 30%, the corrected MTT OD is calculated using thefollowing formula.test chemical
Corrected MTT OD =OD stained tissue (MTT assay)-OD stained tissue (no MTT assay)

When the ratio of staining by test chemical is >30%, the test chemical must be considered
incompatible with the test. However, when the Cell viability (%), which is calculated
according to the procedures described in this SOP Section 3.3.5.2, is <50%, the test
chemical is determined as irritant. Therefore correction of the results or determination ofincompatibility of the test chemical is not necessary.

3.2.2
DETECTION OF CHEMICALS THAT DIRECTLY REDUCE MTT
3.2.2.1 STEP3 (PRELIMINAY TEST)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical into wells of 24-well assay plate
preliminarily filled with 0.5mL of MTT medium. Untreated MTT medium is used as control.
(2) Close the lid of 24-well assay plate and incubate the mixture in CO2 incubator for 1 hour.
(3) After incubation, shake the mixture gently and evaluate the staining of the MTT medium
macroscopically.
(4) When the MTT medium turns blue/purple significantly, the test chemical can reduce MTT
and additional functional check (Step4) must be performed.

5% SLS

1-bromo hexane

1,1,1-tetrachloroe

5-(1,1-dimethylet

thanehyl)-2-methyl

cinnamaldehyde

eugenol

Photo 1 – Example of test for direct MTT reduction ability (STEP 3). Test substances5-(1,1-dimethylethyl)-2-methyl, cinnamaldehyde and eugenol have directly reduced
MTT. In these cases, Step 4 must be performed.

3.2.2.2 STEP4 (FUNCTIONAL CHECK ON VIABLE TISSUE)(1) Add 25ȝL (Liquid) or 25mg (Solid) of the test chemical, which clearly changed the color of
the MTT medium into blue/purple (Step3), onto the surface of the epidermis tissues.
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Distilled water is used as negative control.
(2) Follow all procedures described in this SOP Section 3.3 EXECUTION OF THE TEST.
However, use the epidermis tissues that has been freeze-killed at -20 °C or lower for morethan 24 hours instead of viable epidermis tissues.
(3) Calculate ratio of staining by test chemical from the following formula.

Ratio of staining by test
chemical (%) =OD test chemical – OD negative control
OD negative control

×100(4) When the ratio of staining by test chemical is <30%, correct OD data using the following
formula.

Corrected OD =OD (viable tissue) test chemical – [OD (freeze-killed tissue) test chemical- OD (freeze-killed tissue) negative control]

When the ratio of staining by test chemical is >30%, the test chemical must be considered
incompatible with the test. However, When the Cell viability (%), which is calculated
according to the procedures described in this SOP Section 3.3.5.2, is <50%, the test
chemical is determined as irritant. Therefore correction of the results or determination ofincompatibility of the test chemical is not necessary.

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EXECUTION OF THE TEST

3.3.1 PREPARATION OF LabCyte EPI-MODEL 24 (DAY -1)(1) Pre-warm the assay medium for 30 minutes to
Figure 1
Assay Plate
37 °C using a water bath.
st(2) Fill 3 wells of the 1 row of each 24-well assay 1 row(substance application)plate with the pre-warmed assay medium
2 row(0.5mL/well).
3 row
ĺ Figure 1(post-incubation)(3) Open the LabCyte EPI-MODEL 24 aluminum
4 row(MTT assay)
package.
(4) Open the LabCyte EPI-MODEL 24 plate lid
and pick up the culture inserts using sterile forceps.
*Do not touch the epidermis surface of culture inserts.
*Use forceps to remove agar medium sticking to the outside of the culture inserts.
(5) Transfer the culture inserts into assay
Figure 2
medium filled wells of the 1st row using sterile
Assay Plate
forceps.
1 row
ĺ Figure 2(substance application)
2 row*Avoid air bubble formation under the tissue
inserts.
3 row(post-incubation)(6) Place the plate (lid on) in a CO2 incubator.
row(7) Incubate overnight (15~30 hours) until 4(MTTassay)
Section 3.3.2 “APPLICATION OF TESTCHEMICALS AND RINSING.”(8) Record details of steps (1) - (7) above in the MDS 2.
st

nd

rd

th

st

nd

rd

th

3.3.2

APPLICATION OF TEST CHEMICALS AND RINSING (DAY 0)

3.3.2.1 PREPARATION OF WELLS FOR POST-INCUBATION (3RD ROW)
(1) Pre-warm the assay medium for 30 minutes to 37 °C using a water bath.
(2) Remove the assay plate from the CO2
Figure 3
incubator.
(3) Open the lid of the assay plate, and fill 3 wellsof the 3rd row with the pre-warmed assay
medium (1.0mL/well) using a micropipette.
ĺ Figure 3(4) Place the plate (lid on) in a CO2 incubator.
(5) Incubate until application of test chemicals

Assay Plate
1st row(substance application)
2nd row

3rd row(post-incubation)
4th row(MTT assay)(0~12 hours).
(6) Record details of steps (1) – (5) above in the MDS 3.

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3.3.2.2

Page 14 of 29

APPLICATION OF TEST CHEMICALS(1) Remove the assay plate from the CO2 incubator.
(2) Apply test chemicals onto the surface of epidermis tissues in the 1st row of the assay plate.
Use 3 wells per test chemical (N=3).
FOR LIQUIDs:

Carefully apply 25ȝL of the test chemical onto the central part of each

epidermis using a micropipette. After applied, close the lid of the assay plate and tap the sideof the plate outside the safe cabinet (or clean bench) in order for the liquid to spread out overthe entire epidermis surface. If necessary, use a

Photo 2 - Pipette tips for viscous liquids

micro spatula to coat the unapplied surface withliquids.

Do not push the epidermis surface too

hard with the spatula.
*Use wide orifice cell saver tips for viscous liquids.
ĺ Photo 2
Use a pipette, etc. to familiarize yourself with thenature of the test chemicals in advance.



FOR SOLIDs: Weigh out 25mg (±1mg) of the solid
Photo 3 – Applying a solid substancechemical with a precision balance in advance.
Apply first 25ȝL of distilled water and then theweighed test chemical onto the epidermis surface.
Use a micro spatula if necessary to gently spreadthe test chemical.
ĺ Photo 3*One 24-well assay plate should be used to assay

only one test chemical.
ĺ Figure 4
Figure 4(1 samples x 3(n) = 3 (culture inserts))

Assay Plate
Test substance(3) Apply test chemical onto each well at


st

1 row



(substance application)

1~3-minute intervals.

2nd row(4) Incubate each well for 15 minutes in the
3rd rowcabinet (lid on between the intervals).
*Close the lid of the assay plate at all times
except when applying samples.

(post-incubation)
4th row(MTT assay)

It might

affect the amount of test sample if the lid is kept open, due to air circulation in the safe
cabinet (or clean bench).
(5) Record details of steps (1) - (4) above in the MDS 3.

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
3.3.2.3 REMOVAL OF THE TEST CHEMICALS(1) 15 minutes (±30 seconds) after applying a chemical, open the assay plate and pick up a
culture insert with sterile forceps. Discard test

Photo 4 - Rinse 1

chemicals on the tissue by tilting and then tapping
the insert on the beaker.
(2) Fill and overflow the culture insert with PBS using a
PBS filled poly wash bottle. Hit the PBS stream
from the washing bottle on the side-wall of theculture insert and wash on the tissue surface by the
PBS current.

ĺ Photo 4
Attention: Must not to hit the PBS stream on the

Photo 5 - Rinse 2

tissue surface directly. Be careful not todamage the tissue surface.
(3) Discard the PBS into a beaker by tilting the insert.
If necessary, remove the PBS inside the culture
insert by tapping it on the beaker only once.
ĺ Photo 5

(4) Repeat steps (2) and (3) at least 15 times or moreas much as possible, and remove all residual test

Photo 6 - Rinse 3

chemical on the tissue surface almost completely.
Must not do discarding by tapping at only the lastwashing operation.
(5) Gently remove the leftover PBS outside the culture
insert with a sterile cotton bud. But don’t touch
inside the culture insert by a cotton bud.
ĺ Photo 6

Attention: Even if residues of washing PBS remain on the tissue surface, don’t do at all
because it is not necessary to
remove them.

(6) If test material remains on the epidermis

3

row

of

the

same

columnst(substance application)(7) Transfer the blotted culture insert to a well intheTest substance

1 row

surface, repeat steps (2) ~ (5) again.
rd

Assay Plate

Figure 5(for

2nd row

3rd row(post-incubation)

post-incubation).
ĺ Figure 5

4th row(MTT assay)

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*Avoid air bubble formation under the culture inserts.
(8) Repeat steps (1) ~ (7) for all the culture inserts at 1~3-minute intervals.
(9) Record details of steps (1) – (8) above in the MDS 3.

3.3.3

POST TREATMENT INCUBATION (DAY 0~2)

(1) Close the lid of the assay plate and place it in a CO2 incubator.
(2) Incubate for 42 hours.

3.3.4

MTT ASSAY (DAY 2)

3.3.4.1

PREPARATION OF WELLS FOR MTT ASSAY(1) Pre-warm MTT medium for 30 minutes to 37°C using a water bath.
(2) Remove the assay plate from the CO2 incubator.
Assay Plate

Figure 6(3) Open the lid of the assay plate, and fill each well

Test substance

of the 4th row with the pre-warmed MTT medium

st

1 row

(substance application)(0.5mL/well) using a micropipette.

2nd rowĺ Figure 6

3rd row(post-incubation)(4) Close the lid of the assay plate and place it in the

4th row

CO2 incubator.

(MTT assay)(5) Incubate until starting MTT assay (about 0 ~ 12hours).
(6) Record details of steps (1) – (5) above in the MDS 4.

3.3.4.2

MTT ASSAY(1) Remove the assay plate from the CO2 incubator after 42 hours (±1 hour) of post-incubation.
(2) Transfer each culture insert from the 3rd row to
the 4th row of the corresponding column.
ĺ Figure 7*Avoid dripping from the base end surface ofthe culture insert into other wells.

Figure 7

Assay Plate
Test substance


st

1 row

(substance application)

2nd row(post-incubation)

3rd row*Avoid air bubble formation under the culture
inserts.

4th row(MTT assay)(3) Close the lid of the assay plate and place it in
the CO2 incubator.
(4) Incubate for 3 hours.
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(5) Record details of steps (1) – (4) above in the MDS 4.

3.3.5

FORMAZAN EXTRACTION AND MEASUREMENT (DAY 2~3)

3.3.5.1

FORMAZAN EXTRACTION(1) Remove the assay plate(s) from the CO2 incubator 3 hours (±5 minutes) after the MTT
assay.
(2) Open the lid of the assay plate and pinch the cultured epidermis from each culture insert of
the 4th row with forceps.
Photo 7 - Detachment of epidermisĺ Photo 7*Use a micro spatula to scratch up the epidermis
or a scalpel to cut the membrane filter on the baseof the culture insert if the cultured epidermis
cannot be pinched due to damage from a test
chemical.
(3) Transfer the epidermis tissue into a 1.5mL micro



tube.
(4) Add 300ȝL of isopropanol to the micro tubes andsoak the entire epidermis tissue in the isopropanol.

(5) Incubate the micro tubes in a dark cold place (or refrigerator) overnight (more than 15hours) in order to completely extract pigments.
*Tighten the micro tube seal.
*Periodically shaking the micro tubes will contribute to a more
efficient extraction.
(6) Shake the micro tubes to mix the solution.
*If split epidermis tissues are suspended, wait until they sink or
gently centrifuge them (if a centrifuge is available).
(7) Transfer 200ȝL of the solution in each micro tube into each well on a 96-well plate.
*One well of 200ȝL of isopropanol should be set as a blank.
*Figure 8 shows an example of allocation in a 96-well plate.
Figure 8 – Allocation for a 96-well plate
A

1
Blank

B

DW-1

C

DW-2

D

DW-3

E
F
G

5%
SLS-1
5%
SLS-2
5%
SLS-3

2

3

4

5

6

7

8

9

10

11

Sample1-1
Sample1-2
Sample1-3
Sample2-1
Sample2-2
Sample2-3

Sample3-1
Sample3-2
Sample3-3Sample4-1
Sample4-2
Sample4-3

Sample5-1
Sample5-2
Sample5-3
Sample6-1
Sample6-2
Sample6-3

Sample7-1
Sample7-2
Sample7-3
Sample8-1
Sample8-2
Sample8-3

Sample9-1
Sample9-2
Sample9-3
Sample10-1
Sample10-2
Sample10-3

Sample11-1
Sample11-2
Sample11-3
Sample12-1
Sample12-2
Sample12-3

Sample13-1
Sample13-2
Sample13-3
Sample14-1
Sample14-2
Sample14-3

Sample15-1
Sample15-2
Sample15-3
Sample16-1
Sample16-2
Sample16-3

Sample17-1
Sample17-2
Sample17-3
Sample18-1
Sample18-2
Sample18-3

Sample19-1
Sample19-2
Sample19-3
Sample20-1
Sample20-2
Sample
20-3

12

H

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(8) Record details of steps (1) – (7) above in the MDS 5.

3.3.5.2

OPTICAL DENSITY MEASUREMENTS OF THE EXTRACTS(1) Using a 96-well plate reader, measure optical densities (OD) at 570nm and 650nm anddetermine the measured OD by subtracting the 570nm OD from the 650nm OD.
The equation is shown below:
Measured OD = [570nm ODsample – 570nm ODblank] – [650nm ODsample – 650nm ODblank]*Set the plate reader-calculated value as the measured OD if the 96-well plate reader performs
automatic calculations.

(2) Calculate the cell viability of a sample using the equation below. Furthermore, calculate thevariability (SD) of tissue replicates.
(3) Record details of steps (1) and (2) above in the MDS 5.

Cell Viability (%)㧩Measured ODsample
Mean Measured ODNC

×100

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

4. ASSESSMENT
4.1

CONDITIONS FOR A SUCCESSFUL STUDY

The skin irritation test should be considered successful if both of the following criteria have beenmet.
•

Tissue viability: 0.7  mean OD (A570/650) measured value for negative control  2.5.

•

Positive control: mean tissue viability for 5% SLS (positive control)  40%.

•

SD: SD (negative control and positive control) of tissue viability of 3 identically treatedreplicates  18 %4.2

ASSAY CRITERIA

The criteria for in vitro interpretation are shown below.
The test must be performed 3 times per a sample in total. Sort the tissue viabilities obtained
from the repeated tests in ascending order, and classify the irritancy based on the median of thosetissue viabilities.
Tissue Viability (primary)Classification

Tissue viability is  50%

Irritant

Tissue viability is > 50%

Non Irritant[FLOWCHART] ASSESSMENT FLOWCHART(1) Tissue viability in negative control ĺ (either criterion is not met) ĺ Assay Failure
0.7  Mean OD measured value  2.5
Positive control (5% SLS) result should be “irritant”
Mean tissue viability  40%
SD”
SD (negative control and positive control) of tissue viability of 3 identically treatedreplicates  18 %
Ļ(All criteria are met)Ļ(2) Assessment of test samples (3-time repeated tests: all tests satisfy (1))
The median of the 3 tissue viabilities (%)  50% ĺ (Yes) ĺ Classified as irritant
Ļ
(No)
Ļ
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Classified as non irritant

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
MDS 1:RECEIPT OF LABCYTE EPI-MODEL 24
Laboratory name:1.

Test name:

Test No. :LabCyte EPI-MODEL 24
Date received㧦
Lot No.㧦
Exouration date㧦(MM/DD/YYYY)

Accessories㧦 Assay medium, 30mL ‫ غ‬㧔Lot No.㧦Expiration date㧦㧕(MM/DD/YYYY)

24 well assay plate ‫غ‬
Note

2.

Assay medium
Date received㧦
Lot No.㧦
Expiration date㧦(MM/DD/YYYY)

Note

Date:

Operator:(MM/DD/YYYY)

Secretariat

Check date:Study director:(MM/DD/YYYY)

Check date:

Name:(MM/DD/YYYY)

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
MDS 2:
PRE-INCUBATION OF LABCYTE EPI-MODEL 24 (Section 3.3.1)
Laboratory name:1.

Test name:

Test No. :Warm up the assay medium and add 0.5mL of the assay medium to the wells of the 1st row on the
24-well assay plate.
Assay medium㧦㧔Lot No.㧦    Expiration date㧦㧕(MM/DD/YYYY)

Warm for 30 min.

‫غ‬

Add 0.5mL of assay medium to each well
Number of plates㧦

‫غ‬

2.

Transfer culture inserts to wells in the 1st row on the 24-well assay plate.
LabCyte EPI-MODEL 24㧦㧔Lot No.㧦    Expiration date㧦㧕(MM/DD/YYYY)

Time/date executed㧦
(MM/DD/YYYY HH:MM)‫غ‬

Confirm that there are no bubbles under the cell culture insert.

3.

LabCyte EPI-MODEL 24 is cultured in CO2 incubator overnight.

.

Time/date of culture start㧦
(MM/DD/YYYY HH:MM)

Planned time/date of exposure to test chemical㧦
(MM/DD/YYYY HH:MM)

Note

Date:

Operator:(MM/DD/YYYY)

Secretariat

Check date:Study director:(MM/DD/YYYY)

Check date:

Name:(MM/DD/YYYY)

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
MDS 3-1:
APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION (Section 3.1.2, 3.3.2~ 3.3.3)
Laboratory name:1.

Test name:

Test No.:

Preparation of positive control.
Weight of SLS

mg

 mL

Preparation vol.

Operation date㧦(MM/DD/YYYY HH/MM)

2.

Warm up the assay medium and add 1.0mL of the assay medium to the wells of the 3rd row on
the 24-well assay plate.
Assay medium㧦㧔Lot No.㧦    㧕Expiration date㧦(MM/DD/YYYY)

Warm for 30 min. Ƒ

Add 1.0mL of assay medium. ‫غ‬Time/date executed㧦
(MM/DD/YYYY HH/MM)

3.

Apply test chemicals to the LabCyte EPI-MODEL 24.
Time/date execution started㧦Time/date completed㧦(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)

4.

After exposure to test chemical for 15 min., wash out the LabCyte EPI-MODEL 24 and transfer
the culture inserts to the 3rd row on the 24-well assay plate.
㧔Lot No.㧦    

PBS㧦

㧕

Expiration date㧦(MM/DD/YYYY)

Time/date execution started㧦Time/date completed㧦(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)‫غ‬

Confirm that there are no bubbles under the cell culture insert.

5.

Test chemical information
Test chemical code No.

Lot No.

Distilled Water (Negative
control)
5%SLS(Positive control)

Date:

Operator:(MM/DD/YYYY)

Secretariat

Physical state

Test chemical vol.(weight)(measured weight)

Time ofapplication

Exposuretime
㧔15min.㧕

Liquid

25ȝL

㧦

‫غ‬

Liquid

25ȝL

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Check date:Study director:(MM/DD/YYYY)

Check date:

Name:(MM/DD/YYYY)

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
MDS 3-2:
APPLICATION OF TEST CHEMICALS, RINSING AND POST-INCUBATION(Section 3.3.2~3.3.3)
Laboratory name:5.

Test name:

Test No. :Test chemical information (continued)
Test chemical code No.

6.

Physical state

Test chemical vol.(weight)(measured weight)

Time ofapplication

Exposuretime
㧔15min.㧕

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Liquid, viscous, solid

25ȝL, (  mg,  mg,  mg)

㧦

‫غ‬

Lot No.

Culture LabCyte EPI-MODEL 24 in CO2 incubator for 42 hrs.
Time/date post-incubation started㧦(MM/DD/YYYY HH:MM)

Time/date post-incubation completed㧦(MM/DD/YYYY HH:MM)

Note

Date:

Operator:(MM/DD/YYYY)

Secretariat

Check date:Study director:(MM/DD/YYYY)

Check date:

Name:(MM/DD/YYYY)

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
MDS 4:MTT ASSAY (Section 3.3.4)
Laboratory name:1.

Test name:

Test No. :Preparation of MTT medium
Preparation vol.    mL

Lot No.     Time/date executed㧦
(MM/DD/YYYY HH/MM)

2.

Warm up the MTT medium and add 0.5mL of the MTT medium to the wells in the 4th row on the
24-well assay plate.
MTT medium.㧦㧔Lot No.㧦   Expiration date㧦       㧕(MM/DD/YYYY)

Warm for 30 min.

‫غ‬

Add 0.5mL of the MTT medium. ‫غ‬Time/date executed㧦
(MM/DD/YYYY HH/MM)

3.

After post-incubation, the LabCyte EPI-MODEL24 transfer to wells of 4th row of 24-well assay
plate.
Time/date started㧦Time/date completed㧦(MM/DD/YYYY HH/MM)(MM/DD/YYYY HH/MM)‫غ‬

Confirm that there are no bubbles under the cell culture insert.
4.

Store LabCyte EPI-MODEL 24 culture overnight in CO2 incubator for 42 hrs.
Information on MTT reaction time
Test chemical code No.

Lot No.

Distilled Water(Negative control)
5%SLS (Positive control)

MTT
reactionstart time

Time when
MTT
reaction
ends

㧦

MTT
reactionstart time

Time when
MTT
reaction
ends

㧦

㧦

㧦

Test chemical code No..

Lot No.

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

㧦

Note

Date:

Operator:(MM/DD/YYYY)

Secretariat

Check date:Study director:(MM/DD/YYYY)

Check date:

Name:(MM/DD/YYYY)

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
MDS 5:FORMAZAN EXTRACTION AND MEASUREMENT (Section 3.3.5)
Laboratory name:1.

Test name:

Test No. :After MTT reaction, use forceps to pick up the cultured epidermis from the cell culture insert andput it in a 1.5mL microtube.
‫غ‬

Did you use a scalpel to cut out the cultured epidermis?
Date of execution㧦
(MM/DD/YYYY)

2.

Add isopropanol (300ȝL) to microtube so that the cultured epidermis is completely immersed in
isopropanol.
Isopropanol
Lot No.
Immersion of the cultured epidermis in isopropanol.
Date of execution㧦

‫غ‬
‫غ‬

To add isopropanol (300ȝL)(MM/DD/YYYY)

3.

For MTT formazan extraction, allow micro tube to stand in a cold and dark space.
‫غ‬Place micro tube in a cold and dark space.

4.

Extract solution (200mL) is transferred to each well on the 96-well plate.
‫غ‬Transfer to the 96-well plate.
Time/date executed㧦
(MM/DD/YYYY HH/MM)

Sample location on 96-well plate.
1

2

3

4

5

6

7

8

9

10

11

12

blank

A

Distilled
Water-1
Distilled
Water-2
Distilled
Water-3

BC
D

E

5% SLS-1

F

5% SLS-2

G

5% SLS-3

H

5.

Analyze extract OD at 570nm and 650nm, and calculate the OD(570nm-650nm).
‫غ‬
‫غ‬
‫غ‬
‫غ‬
‫غ‬
‫غ‬

Analyze OD at 570nm and 650nm.
Calculate the OD(570nm-650nm).
Calculate cell viability and SD.
Cell viability and SD are recorded on a separate data sheet.
The data sheet is attached to the back of this sheet.
Check for input errors.
Time/date executed㧦
(MM/DD/YYYY HH/MM)

Note

Date:

Operator:(MM/DD/YYYY)

Secretariat

Check date:Study director:(MM/DD/YYYY)

Check date:

Name:(MM/DD/YYYY)

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Version 8.3
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REVISION HISTORY
Rev.

Content

Date Revised

Ver.1

1) First version

27/02/2008

Ver.2

1) Revised clerical error.

28/02/2008

Ver.3

1) Revised the post-incubation time and assessment criteria in

17/03/2008

compliance with the EpiSkin method described in “PerformanceStandards for Applying Human Skin Models to in vitro Skin Irritation
Testing”
2) Added photos and figures for instruction.
Ver.41) Added MDS 1~6.

15/05/2008

2) Added instruction and operational steps regarding the IL-1Į ELISA
kit.
3) Added subsections “Delivery of LabCyte EPI-MODEL24” and“Instruction For Use of LabCyte EPI-MODEL24” to Section 2.
4) Added the description regarding test chemicals to Section 2.
5) To Section 2, added the description of materials provided by J-TEC
separately from other materials.
6) Stated the specific calculation procedures in Section 3.2.5.2“OPTICAL DENSITY MEASUREMENTS OF EXTRACTS”.
Ver.4.1

1) Moved scalpel from Section 2.4 “MATERIALS PROVIDED BY

21/05/2008

J-TEC” to Section 2.5 “MATERIALS NOT PROVIDED WITH THE
J-TEC KITS”.
2) Removed the description regarding how to execute procedures
alone.
3) Moved IL-1Į ELISA reagents from Section 3.1 “PREPARATIONS” to
Section 3.2 “TEST METHOD”.
4) Added a flowchart for the IL-1Į ELISA procedures.
5) Changed from “in a cold dark place” to “in a cold dark place (orrefrigerator)” regarding formazan extraction.
6) Added the description of “ultrasonic cleaning equipment or vortex
mixer” as an example of an MTT dissolution method.
7) Changed the exposure time column from entering actual time tocheckboxes on the MDS 3.
Ver.5.0

1) Corrected typing errors in the section number for IL-1Į ELISA

27/08/2008

reagents.

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2) Removed the space for SLS lot numbers on the MDS 3.
3) Removed the space for PBS lot numbers on the MDS 3.
4) Added the space for isopropanol lot numbers on the MDS 5.
5) Added a checkbox about using a scalpel when removing tissues in
the MDS 5.
6) Added the space for IL-1Į ELISA kit lot numbers on the MDS 6.
7) Changed the applicable parts of product codes and kit components in
Section 2.2, with the change of IL-1Į ELISA kit types to a 96 well test
only.
8) Decreased the volume by half to 10mL and changed the storage
condition from within 1 month to within 24 hours in Section 3.1.2“POSITIVE CONTROL SUBSTANCE”.
9) Added the manufacturers and product codes of the 24-well plate and
96-well plate in Section 2.4 “MATERIALS PROVIDED BY J-TEC”.
10) Added specific time frames for incubation or culturing.
11) Added the conditions for a successful study in Section 4“ASSESSMENT”
12) Changed the specific method of applying liquids in Section 3.2.2.2“APPLICATION OF TEST CHEMICALS”.
13) Added descriptions in English on the MDS Sheets.
14) Changed the application time interval from 1 minute to
1~3minute(s).
15) Numbered figures and flowcharts.
16) Increased the size of spaces for lot numbers on the MDS Sheets.
17) Changed spaces for dates from MM/DD to MM/DD/YYYY.
18) Added director check date, study director, secretariat check dateand name at the end of each MDS.
19) Changed the size of matrixes for sample allocation to a 96-well platein the MDS 5 & 6.
20) Changed the test chemical name to test chemical code in the MDS
3 & 4.
21) Divided the MDS 3 into MDS 3-1 and 3-2, and added spaces for
date, operator, check date, study director at the end of the MDS 3-1,
and spaces for laboratory name, test name and test no. at the
beginning of the MDS 3-2.
Ver. 6.0

1) Removed the descriptions regarding the measurement of IL-1Į

27/02/2009

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
production, since the validation committee decided to use cell
viabilities only as an index for the skin irritancy test at the meeting in2009.
2) Revised the expression “the materials provided by J-TEC” for thevalidation study to that for a standard skin irritancy test preparation.
3) Clearly stated the cell viability equation to use the mean of measured
values.
4) Clearly stated to use the median of cell viabilities from the three-time
repeated tests as assay criteria.
Ver.

1) In order to avoid the possible influence of volatile test chemicals on

6.01

the results of other test chemicals, the types of test chemicals per

23/03/2009

plate was changed from 2 chemicals to just 1 chemical.
Ver.
7.01

1)Test for detecting chemicals that interfere with MTT endpoint was

03/07/2009

added to Section 3.2.

Ver. 7.2

1)Revised clerical error.

30/09/2009

Ver. 8.1

1) Added the description about rational and background as following

30/06/2010

chapters in Section 1.
LabCyte EPI-MODEL 24 SKIN IRRITATION TEST (SIT using
LabCyte EPI-MODEL24)BACKGROUND OF SIT using LabCyte EPI-MODEL24
BASIS OF THE METOD
LIMITATION OF THE METHODBRIEF BASIC PROCEDUREDATA INTERPRETATION PROCEDURE (PREDICTION MODEL)
2) Added photo about chemicals that directly reduce MTT in Section 3.
3) Added the washing protocol more detail in Section 3.
4) Added assessment about SD.
Ver.8.2

1) Changed description about the washing protocol in Section 3.

17/08/2010

2) Changed unit of consumable reagents and vessels from per avalidation study to per a test.
Ver.8.3

1) Changed description more briefly about the washing protocol in

14/06/2011

Section 3.

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