᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ  ᖺ  ᭶  ᪥                                  1R 
⓶⭵ឤసᛶヨ㦂௦᭰ἲゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖
࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 27 ᖺ 4 ᭶ 23 ᪥࡟ᮾிࠊᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸JaCVAM ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢᥦ᱌ࡀ࡞ࡉࢀࡓࠋ  ᥦ᱌ෆᐜ㸸 ゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖࡛㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ໬Ꮫ≀㉁ࢆᙉ࠸
ឤసᛶ≀㉁࡛࠶ࡿ GHS(Globally Harmonized System of Classification and Labelling ofChemicals)༊ศ㸯࡟ศ㢮ࡍࡿࡇ࡜ࡣ⾜ᨻୖྍ⬟࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⛥࡟ഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ୍᪉ࠊᮏヨ㦂ἲ࡛㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀ
ࡓሙྜࠊࡑࡢ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡣഇ㝜ᛶࡢྍ⬟ᛶࡀ࠶ࡾࠊᮏヨ㦂ἲ༢⊂࡛ࡑࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿࡇ࡜ࡣ㞴ࡋ࠸ࠋᮏヨ㦂ἲࡣࠊࡑࡢ≉ᛶࢆ༑ศ࡟⌮ゎࡋࡓୖ࡛ࠊIATA(Integrated Approaches to Testing and Assessment) ࢆᵓᡂࡍࡿࡑࡢ௚ࡢ᝟ሗ※࡜⤌ࡳྜࢃࡏ࡚㐺ษ࡟ホ౯ࡍࡿࡇ࡜ࡀࠊ⾜ᨻⓗ࡞ཷࡅධࢀ࡟ᚲせ࡛࠶ࡿ࡜⪃࠼ࡿࠋࡇࡢᥦ᱌᭩ࡣࠊOECD TG 442D; In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method࠾ࡼࡧ
JRC Scientific and Policy report,

EURL ECVAM Recommendation on the KeratinoSensTM assay forskin sensitisation testingࢆࡶ࡜࡟ࠊ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚ࠊJaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚⓶⭵ឤసᛶヨ㦂௦᭰ἲ ࣌ࣉࢳࢻ⤖ྜᛶ
ヨ㦂ࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ ኱㔝Ὀ㞝                      すᕝ⛅ె
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i

-D&9$0 ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧㣤ሯᑦᩥ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷ⏣ ⥳

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧Ώ㒊୍ே㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥ii

-D&9$0 㐠Ⴀጤဨ఍すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧㸸ጤဨ㛗ᕝす ᚭ  㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧᳜ᮧᒎ⏕㸦ཌ⏕ປാ┬ ་⸆㣗ရᒁ ᑂᰝ⟶⌮ㄢ㸧ᑠᕝஂ⨾Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧Ⳣ㔝 ⣧

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧಴ᣢ᠇㊰㸦ཌ⏕ປാ┬ ་⸆㣗ရᒁ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧㛵㔝♸Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧㧗ᮌ⠜ஓ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊 ື≀⟶⌮ᐊ㸧᮰㔝ṇ᫂㸦ཌ⏕ປാ┬ ་⸆㣗ရᒁ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧୰ᮧ㧗ᩄ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧᪥⏣ ඘

㸦ཌ⏕ປാ┬ ་⸆㣗ရᒁ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᗈ℩᫂ᙪ  㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶண
ホ౯㒊㸧
ᮏ㛫ṇ඘

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧ᑠᓥ ⫕㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊 ➨஧ᐊ㸧㸸஦ົᒁ

iii

iv

JaCVAM statement on ARE-Nrf2 Luciferase Test Method assay for skinsensitisation testing
At a meeting held on 23 April 2015 at the National Institute of Health Sciences (NIHS) in
Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:  
Proposal: When using the ARE-Nrf2 Luciferase Test Method in a regulatory context, it isreasonable for substances that give positive results to be classified as a strong
sensitiser, i.e., a Category 1 substance under the Globally Harmonized System ofClassification and Labelling of Chemicals (GHS). It is also necessary, however,to bear in mind that this assay occasionally yields false positive results forcertain substances. Conversely, it is quite possible that the ARE-Nrf2 Luciferase
Test Method will yield false negative results for some chemicals, which meansthat it would be unreasonable to use it as a standalone test for assessing skinsensitisation potency. We therefore conclude that the use of the ARE-Nrf2
Luciferase Test Method in a regulatory context requires a thoroughunderstanding of the assay’s strengths and weaknesses as a prerequisite to its
application within an integrated approach to testing and assessment (IATA) thatwill also take into account information from other sources.
This statement was prepared following a review of OECD TG 442D “In Vitro Skin
Sensitisation: ARE-Nrf2 Luciferase Test Method” as well as a JRC Scientific and Policy
report “EURL ECVAM Recommendation on the KeratinoSensTM assay for skin sensitisation
testing”. The JaCVAM Regulatory Acceptance Board acknowledges that the results of thisreview as well as of a study of materials prepared by the JaCVAM Editorial Committee
indicate that this assay is useful in a regulatory context.
Based on the above, we propose that regulatory agencies performing safety assessment of skinsensitisation potency consider using the ARE-Nrf2 Luciferase Test Method as an alternative to
testing with laboratory animals.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

20 August 2015v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:

Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Kazuhiro Kaneko (Japan Chemical Industry Association)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Akiyoshi Nishikawa (NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Koko Tanigawa (Japanese Society for Alternatives to Animal Experiments)
Mr. Takashi Yamada (National Institute of Technology and Evaluation)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Ms. Midori Yoshida (NIHS)
Mr. Takemi Yoshida (Japanese Society of Toxicology)
Mr. Isao Yoshimura (nominee by Chairperson)
Mr. Kazuto Watanabe (Japan Pharmaceutical Manufacturers Association)

Term: From 1st April 2014 to 31st March 2016vi

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

Mr. Akiyoshi Nishikawa (BSRC, NIHS): Chairperson
Mr. Toru Kawanishi (NIHS)
Mr. Mitsuru Hida (Ministry of Health, Labour and Welfare)
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. Kenji Kuramochi (Ministry of Health, Labour and Welfare)
Mr. Takatoshi Nakamura (Pharmaceutical & Medical Devices Agency)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Mr. Atsuya Takagi (Animal Management Section of the Division of Cellular andMolecular Toxicology, BSRC, NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Nobuo Uemura (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Section for the Evaluation of Novel Methods, Division of Risk
Assessment, BSRC, NIHS): Secretary

vii

viii

⓶⭵ឤసᛶヨ㦂௦᭰ἲゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------------------ 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------------------ 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS Test Guideline 442D,

In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method ------------------------ 23
JRC SCIENTIFIC AND POLICY REPORTS,
EURL ECVAM Recommendation on the KeratinoSensTM assay for skinsensitisation testing----------------------------------------------------------------------------------- 43

ixxホ౯఍㆟ሗ࿌᭩
ARE-Nrf2 Luciferase Test Method㸦ゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖㸧

JaCVAM ホ౯఍㆟

ᖹᡂ 27 ᖺ㸦2015 ᖺ㸧4 ᭶ 30 ᪥

1
1

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧㔠Ꮚ࿴ᘯ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ  㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᖹ㈡⚽᫂  㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷ⏣ ⥳㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧Ώ㒊୍ே㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧                ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥

22ゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖㸸Antioxidant response element (ARE)-Nrf21 Luciferase Test
Method㸦௨ୗࠊᮏヨ㦂ἲ࡜グࡍ㸧ࡣࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆண

ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊᚑ

᮶ࡢࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂㸦OECD TG406㸧
ࠊ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦Local Lymph Node Assay (LLNA)ࠊOECD TG429㸧࠾ࡼࡧ LLNA ࡢኚἲ࡛࠶ࡿ LLNA:DA(OECD TG442A)࡞ࡽࡧ࡟ LLNA:BrdU-ELISA (OECD TG442B)࡜ẚ࡭ࠊື≀ࢆ⏝࠸࡞࠸࡜࠸
࠺≉ᚩࢆ᭷ࡍࡿࠋᮏヨ㦂ἲࡣࠊ⓶⭵ឤసᛶࢆ᭷ࡍࡿከࡃࡢ໬Ꮫ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀ
࡚࠸ࡿ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜࠿ࡽࠊࡇࡢㄏᑟάᛶࢆᇵ㣴⣽⬊ࢆ⏝࠸࡚࡟ࡼࡾࠊ⓶⭵ឤసᛶࡢ᭷↓ࢆணᐃࡍࡿࡇ࡜ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊEuropean Union Reference Laboratory for Alternatives to Animal Testing (EURLECVAM)࡟ࡼࡿ➨୕⪅ホ౯ࡀ⤊஢ࡋ࡚࠸ࡿ㸯㸧ࠋJaCVAM ホ౯఍㆟ࡣࠊ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅
⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓ⓶⭵ឤసᛶヨ㦂 ゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖ࡢホ౯ሗ࿌᭩ࢆᇶ࡟ᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 ゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖㸸ARE-Nrf2 Luciferase Test Method௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 ࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂㸦OECD TG406㸧࠾ࡼࡧ࣐࢘ࢫ ࢆ ⏝ ࠸ ࡿ ᒁ ᡤ ࣜ ࣥ ࣃ ⠇ ヨ 㦂 [LLNA (OECD TG429) ࠊ LLNA:DA (OECD TG442A) ࠊ
LLNA:BrdU-ELISA (OECD TG442B)]
ヨ㦂ἲࡢᴫ␎㸸  ᮏヨ㦂ἲࡣࠊNrf2-Keap12-ARE pathway ࢆ฼⏝ࡋࠊࢣࣛࢳࣀࢧ࢖ࢺ⏤᮶
ARE-Nrf2 ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮㑇ఏᏊࢆ⏝࠸ࡓ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖࡛࠶ࡿ 1)ࠋ
 Nrf2-Keap1-ARE pathway ࡣࠊ㌿෗ᅉᏊ Nrf2ࠊNrf2 ࡢᢚไᅉᏊ Keap1 ࠾ࡼࡧ ARE ࡀ㛵ಀࡍࡿ㑇ఏᏊⓎ⌧⤒㊰࡛࠶ࡿࠋNrf2 ࡣ Keap1 ࡜⤖ྜࡋࠊARE ࡟౫Ꮡࡋ࡚Ⓨ⌧ࡍࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ࢆไᚚࡋ࡚࠸ࡿࠋKeap1 ࡢࢩࢫࢸ࢖ࣥṧᇶ࡟ồ㟁Ꮚᛶࡢ໬Ꮫ≀㉁ࡀ⤖ྜࡍࡿ࡜ࠊNrf2
ࡣ Keap1 ࠿ࡽゎ㞳ࡋࠊ᰾ෆ࡬⛣⾜ࡋ࡚ DNA ୖࡢ ARE ࡟⤖ྜࡍࡿࠋࡑࡢ⤖ᯝࠊୗὶࡢ㑇ఏᏊ⩌ࡢⓎ⌧ࡀㄏᑟࡉࢀࠊ໬Ꮫ≀㉁࡟ࡼࡿ㞀ᐖ࠿ࡽ⣽⬊ࢆಖㆤࡍࡿࡓࡵ࡟ᶵ⬟ࡍࡿࠋ⓶⭵ឤసᛶࢆ᭷ࡍࡿከࡃࡢ໬Ꮫ≀㉁ࡀ Nrf2-Keap1-ARE pathway ࢆάᛶ໬ࡍࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ
ᮏヨ㦂ἲ࡛ࡣࠊAKR1C2 㑇ఏᏊ㸦ᶞ≧⣽⬊࡟࠾࠸࡚⓶⭵ឤసᛶ≀㉁࡟ࡼࡾⓎ⌧ㄏᑟࡉࢀࡿ㑇ఏᏊࡢ 1 ࡘ㸧ࡢ ARE ࢆ⼥ྜࡉࡏࡓ SV40 ࣉ࣮ࣟࣔࢱ࣮ࢆ᭷ࡍࡿࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࡢࣉࣛࢫ࣑ࢻࢆᏳᐃⓗ࡟ᑟධࡋࡓ HaCaT ⣽⬊㸦ࣄࢺࢣࣛࢳࣀࢧ࢖ࢺ⏤᮶ᇵ㣴⣽⬊ᰴ㸧ࢆ⏝࠸ࡿࠋ໬Ꮫ≀㉁࡟ࡼࡾ Nrf2-Keap1-ARE pathway ࡀάᛶ໬ࡉࢀࡿ࡜ࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࡀ
Ⓨ⌧ࡍࡿࠋᇶ㉁ࢆῧຍࡋ࡚ࣝࢩࣇ࢙࣮ࣛࢮࡀゐ፹ࡍࡿ཯ᛂࡢⓎගᙉᗘࢆᐃࡍࡿࡇ࡜࡟ࡼ

ࡾࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡀホ౯ࡉࢀࡿࠋ
1
2

Nrf2㸸Nuclear factor (erythroid-derived 2)-like 2
Keap1: Kelch-like ECH-associated protein 133

㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶᮏヨ㦂ἲࡣࠊKeratinoSensTM3 ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 2-5)࡜ࡑࢀ࡟⥆ࡃ
EURL ECVAM Scientific Advisory Committee ࡟ࡼࡾ⊂❧ࡋࡓ➨୕⪅ホ౯ࡀᐇ᪋ࡉࢀ 1)ࠊ⓶⭵ឤసᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚⛉Ꮫⓗ࡟ጇᙜ࡛࠶ࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋᮏ㑥࡟࠾࠸࡚ࡣࠊJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡀࠊᮏヨ㦂ἲࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊබ㛤ࡉࢀ࡚࠸ࡿ᝟ሗ 1-5)ࢆᇶ࡟ホ౯ࡋࡓࠋホ౯ࡢ⤖ᯝࠊᮏヨ㦂ἲࡣࠊ⓶⭵ឤసᛶࢆ᭷ࡍࡿከࡃࡢ໬Ꮫ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀ࡚࠸ࡿ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿ࡜
࠸࠺⓶⭵ឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ㔜せ࡞࢖࣋ࣥࢺࢆ᳨ฟࡋ࡚࠾ࡾࠊ⓶⭵ឤసᛶヨ㦂ࡢ௦᭰
ヨ㦂ἲ࡜ࡋ࡚ཎ⌮ⓗጇᙜᛶࡀ࠶ࡿ࡜⪃࠼ࡽࢀࡓ 6)ࠋ
࡞࠾ࠊ෌⌧ᛶࢆ᳨ウࡋࡓࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚⿕㦂≀㉁㑅ᢥ࡟೫ࡾࡀ࠶ࡾࠊ᪋タෆ࣭᪋タ㛫෌⌧ᛶ࡟ࡣ␃ពࡍ࡭ࡁ࡛࠶ࡿ࡜ホ౯఍㆟ࡣุ᩿ࡋࡓࠋ
㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣࠊࣄࢺࢣࣛࢳࣀࢧ࢖ࢺ⏤᮶ᇵ㣴⣽⬊ᰴࢆ⏝࠸ࡿ in vitro ヨ㦂ἲ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓࠊ㸯⿕㦂≀㉁ᙜࡓࡾࡢࣛࣥࢽࣥࢢࢥࢫࢺࡶࠊ࣐࢘ࢫࢆ⏝࠸ࡿ LLNA
࡜ẚ㍑ࡋ࡚ 1/7 ⛬ᗘ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊヨ㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛࠶ࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ⡆౽ᛶ࠾ࡼࡧ⤒῭ᛶࡢ㠃࠿ࡽ᭷⏝࡜࠸࠼ࡿࠋ᭦࡟ࠊ⓶⭵ឤసᛶࢆ᭷ࡍࡿከࡃࡢ໬Ꮫ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀ࡚࠸ࡿ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣࠊࡇࡢㄏ
ᑟάᛶࢆᇵ㣴⣽⬊ࢆ⏝࠸࡚ᐃࡍࡿࡇ࡜࡟ࡼࡾࠊ⓶⭵ឤసᛶࡢ᭷↓ࢆணࡍࡿࡶࡢ࡛࠶ࡾࠊ

໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼ࡿࠋ
ࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ࡇࡢヨ㦂࡟౑⏝࡛ࡁࡿ⣽⬊⣔ࡣ KeratinoSensTM ࡢࡳ࡛࠶ࡾࠊ౑⏝࡟ࡣ⣽⬊⣔ࢆᶞ❧ࡋࡓ Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆྲྀᚓࡍࡿࡇ࡜ࡀᚲせ࡛࠶ࡾࠊ㈝⏝ࡶྵࡵ࡚฼⏝ྍ⬟࡞᮲௳ࡢヲ⣽ࡣ୙࡛᫂࠶ࡿࠋ୍᪉ࠊKeratinoSensTM ࢆ౑⏝ࡋ࡞࠸ሙྜࡣࠊOECD
సᡂࡢᮏヨ㦂ἲ࡟㛵ࡍࡿ Performance Standard㸦᱌㸧7)࡟ᚑ࠸ࠊጇᙜᛶࢆ♧ࡉ࡞ࡅࢀࡤ࡞ࡽ࡞
࠸ࠋࡲࡓࠊLogP ࡀ 7 ௨ୖࡢᙉ␯Ỉᛶ≀㉁ࡣࠊDMSO ࡜Ỉ࡬ࡢ⁐ゎ≉ᛶ࠿ࡽヨ㦂࡟㐺⏝࡛ࡁ࡞࠸ࠋ㓟↓Ỉ≀ࡣࠊࣜࢪࣥṧᇶ࡜཯ᛂᛶࢆ♧ࡍ≉ᚩࡀ࠶ࡾࠊࢩࢫࢸ࢖ࣥᇶ࡜ࡢ཯ᛂࡀᚲせ࡞ Nrf2 pathway ࢆㄏᑟࡋ࡞࠸ࡓࡵࠊഇ㝜ᛶ࡜ุᐃࡉࢀࡿྍ⬟ᛶࡀ࠶ࡿࠋࣝࢩࣇ࢙࣮ࣛࢮ㓝⣲࡟ᖸ΅ࡍࡿ໬Ꮫ≀㉁࡛ࡣࠊࣝࢩࣇ࢙࣮ࣛࢮάᛶࢆṇࡋࡃᐃࡍࡿࡇ࡜ࡀ࡛ࡁ࡞࠸ࡓࡵὀពࡀᚲせ࡛࠶ࡿࠋ㓟໬཯ᛂࡸ㓟໬ⓗ⬺࢔࣑ࣀ཯ᛂࢆᚲせ࡜ࡍࡿከࡃࡢࣉࣞࣁࣉࢸࣥࡣṇࡋࡃุᐃࡉࢀࡿࡀࠊࢩࢺࢡ࣒ࣟ P450 ࡟ࡼࡿάᛶ໬ࢆᚲせ࡜ࡍࡿࣉࣟࣁࣉࢸࣥࡣ᳨ฟ࡛ࡁ࡞࠸ࠋ
ᇵ㣴⣽⬊ࢆ⏝࠸ࡿヨ㦂⣔ࡢࡓࡵࠊ⣽⬊ẘᛶࡢᙉ࠸≀㉁ࡣ㐺ษ࡞⃰ᗘ࡛ヨ㦂ࡀ࡛ࡁ࡞࠸ሙྜ
ࡀ࠶ࡿࠋ100 ≀㉁௨ୖࢆホ౯ࡋࡓሗ࿌ 4,5)࡟࠾ࡅࡿᮏヨ㦂ἲࡢឤᗘࡣࠊ76.7%࡜ 77%࡛࠶ࡿࡓࡵࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀ࡚ࡶഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡍࡿᚲせࡀ࠶ࡿࠋࡲࡓࠊ≉␗ᗘࡶࠊ82.1%
3

KeratinoSensTM㸸Givaudan ♫࡟ࡼࡗ࡚ᶞ❧ࡉࢀࡓࢣࣛࢳࣀࢧ࢖ࢺ⏤᮶ ARE-Nrf2 ࣝࢩࣇ࢙ࣛ

࣮ࢮ࣏࣮ࣞࢱ࣮㑇ఏᏊࡢࣉࣛࢫ࣑ࢻࢆᏳᐃⓗ࡟ᑟධࡋࡓࣄࢺࢣࣛࢳࣀࢧ࢖ࢺ⏤᮶ᇵ㣴⣽⬊ᰴ

44

࡜ 79%࡛࠶ࡿࡇ࡜࠿ࡽࠊ㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ࡟ࡶࠊഇ㝧ᛶࡢྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃
ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶ࠾ࡼࡧ
⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸 ᮏヨ㦂ἲࡣࠊ⣽⬊ᇵ㣴ᢏ⾡࠾ࡼࡧ 96 ✰ࣉ࣮ࣞࢺ⏝࣑ࣝࣀ࣓࣮ࢱ࣮᧯సᢏ⾡ࢆಟᚓࡋ࡚࠸
ࢀࡤᐜ᫆࡟ᐇ᪋࡛ࡁࡿヨ㦂࡛࠶ࡿࠋࡓࡔࠊ⌧᫬Ⅼ࡛౑⏝࡛ࡁࡿ⣽⬊⣔ࡣ KeratinoSensTM ࡢࡳ
࡛࠶ࡾࠊ㈍኎ඖ࡛࠶ࡿ Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆྲྀᚓࡍࡿᚲせࡀ࠶ࡿࠋᮏヨ㦂ἲࡢᐇ᪋࡜⤖ᯝࡢゎ㔘࡟ᙜࡓࡗ࡚ࡣࠊ໬Ꮫ≀㉁ࡢᛶ㉁࡜ࡑࡢ㐺⏝㝈⏺ࢆぢᴟࡵࡿᚲせࡀ࠶ࡿࠋࡋ࠿
ࡋࠊᮏヨ㦂ἲࡣࠊ⓶⭵ឤసᛶࢆ᭷ࡍࡿከࡃࡢ໬Ꮫ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀ࡚࠸ࡿ㑇ఏᏊ
ࡢⓎ⌧ࢆㄏᑟࡍࡿ࡜࠸࠺⓶⭵ឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ㔜せ࡞࢖࣋ࣥࢺࢆ᳨ฟࡋ࡚࠾ࡾࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆ⪃࠼ࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼ࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡢ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࡶࡢ࡜⪃࠼ࡿࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸 ᮏヨ㦂ἲ࡛㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ໬Ꮫ≀㉁ࢆᙉ࠸ឤసᛶ≀㉁࡛࠶ࡿ
GHS(Globally Harmonized System of Classification and Labelling of Chemicals)༊ศ㸯࡟ศ㢮ࡍࡿࡇ࡜ࡣ⾜ᨻୖྍ⬟࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⛥࡟ഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ
࡞ࡽ࡞࠸ࠋ୍᪉ࠊᮏヨ㦂ἲ࡛㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡣഇ
㝜ᛶࡢྍ⬟ᛶࡀ࠶ࡾࠊᮏヨ㦂ἲ༢⊂࡛ࡑࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿࡇ࡜ࡣ㞴ࡋ࠸ࠋᮏヨ㦂ἲࡣࠊࡑࡢ≉ᛶࢆ༑ศ࡟⌮ゎࡋࡓୖ࡛ࠊIATA (Integrated Approaches to Testing and Assessment) ࢆᵓᡂࡍࡿࡑࡢ௚ࡢ᝟ሗ※࡜⤌ࡳྜࢃࡏ࡚㐺ษ࡟ホ౯ࡍࡿࡇ࡜ࡀࠊ⾜ᨻⓗ࡞ཷࡅධࢀ࡟ᚲせ࡛࠶ࡿ࡜⪃࠼ࡿࠋ

5
5

ᘬ⏝ᩥ⊩
1) EURL ECVAM (2012). ESAC Working Group Peer Review Consensus Report on
Givaudan-coordinated study transferability and reliability of the KeratinoSens assay for skinsensitisation testing.
2) DB-ALM (INVITTOX) (2013). Protocol 155: KeratinoSensTM .
3) Natsch A., Ryan C.A., Foertsch L., Emter R., Jaworska J., Gerberick G.F., Kern P. (2013). A
dataset on 145 chemicals tested in alternative assays for skin sensitization undergoingprevalidation. Journal of Applied Toxicology, 33, 1337-1352.
4) Emter R., Ellis G., Natsch A. (2010). Performance of a novel keratinocyte-based reporter cellline to screen skin sensitizers in vitro. Toxicology and Applied Pharmacology 245, 281-290.
5) EURL-ECVAM (2014). Recommendation on the KeratinoSensTM assay for skin sensitisation
testing, 42 pp.
Available at:
http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/recommendation-keratinosens-skin-sensitisation
6) JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸸⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ ARE-Nrf2
Luciferase Test Method㸦ᖹᡂ 27 ᖺ 4 ᭶ 28 ᪥㸧7) OECD (2014, in preparation). Performance Standards for the assessment of proposed similar or
modified in vitro skin sensitisation ARE-Nrf2 luciferase test methods in TG xxx. OECD
Environment, Health and Safety publications, Series on Testing and Assessment N. xxx, OECD,
Paris.

66⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩

ARE-Nrf2 Luciferase Test Methodゅ໬⣽⬊ᰴ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖

ᖹᡂ 27 ᖺ 4 ᭶ 28 ᪥

JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

1
7

JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍
ጤဨ㛗 ⟄஭ᑦஂ㸦⏣㎶୕⳻〇⸆ᰴᘧ఍♫㸧ጤဨᏳ㐩⋹Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧  㔠⃝⏤ᇶᏊ㸦୍⯡㈈ᅋἲே 㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧  ᑠᓥᖾ୍㸦୍⯡㈈ᅋἲே 㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧  బ⸨୍༤㸦ᅜ❧኱Ꮫἲே ⚟஭኱Ꮫ㸧  Ṋྜྷṇ༤㸦୍⯡㈈ᅋἲே ໬Ꮫ≀㉁ホ౯◊✲ᶵᵓ㸧  ᳃ᮏ㝯ྐ㸦ఫ཭໬Ꮫᰴᘧ఍♫㸧

2
8

せ᪨
⓶⭵ឤసᛶࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡞ホ౯㡯┠࡛࠶ࡾࠊᚑ᮶ࠊࣔࣝࣔࢵࢺ
ࡸ࣐࢘ࢫࢆ⏝࠸ࡓື≀ᐇ㦂࡟ࡼࡗ࡚ホ౯ࡉࢀ࡚ࡁࡓࠋ㏆ᖺ EU ࡟࠾ࡅࡿḢᕞ໬Ꮫရつไ࡛ࡣࠊᏳ ඲ ᛶ ホ ౯ ࡣ ࢥ ࣥ ࣆ ࣗ ࣮ ࢱ ࣮ ࢆ ⏝ ࠸ ࡓ ᐃ 㔞 ⓗ ᵓ 㐀 ά ᛶ ┦ 㛵 (QSAR: Quantitative
Structure-Activity Relationship)ࣔࢹࣝࡸ in vitro ヨ㦂ࡢ௦᭰ἲࡀ᥎ዡࡉࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶホ౯ࡉࢀࡓᡂศࢆྵࡴ໬⢝ရࡢ㍺ධ㈍኎ࡀ⚗Ṇࡉࢀࡓࡇ࡜࠿ࡽ㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠊື≀ࢆ⏝࠸࡞࠸ in vitro ヨ㦂ἲࡢ㛤Ⓨࡀᙉࡃᮃࡲࢀ࡚࠸ࡿࠋARE (Antioxidant response
element )-Nrf21 Luciferase Test Method ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀࡿ
㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࡇࡢㄏᑟάᛶ࡟ࡘ࠸࡚ᇵ㣴⣽⬊ࢆ⏝࠸࡚࢔ࢵࢭ࢖
ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌᭩ࡣࠊࡇࡢ ARE-Nrf2 Luciferase Test Method ࡟ࡘ࠸࡚ࠊᮏヨ㦂ἲࢆ㛤Ⓨࡋࡓ Givaudan ♫ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢᡂ⦼୪ࡧ࡟ EURL ECVAM㸦European
Union Reference Laboratory for Alternatives to Animal Testing㸧⛉Ꮫㅎၥጤဨ఍㸦ESAC㸧࡟ࡼࡾᐇ᪋ࡉࢀࡓ➨୕⪅ホ౯ሗ࿌᭩࡞࡝ࢆᇶ࡟ࡑࡢᡭ㡰ࢆ⧳ࡵࠊ᭷⏝ᛶ࡜㝈⏺ࢆホ౯ࡋࡓࡶࡢ࡛࠶
ࡿࠋ
ARE-Nrf2 Luciferase Test Method㸦௨ୗࠊᮏヨ㦂ἲ࡜グࡍ㸧ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨஧ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕཯ᛂ࠾ࡼࡧ Nrf2-Keap12-ARE pathwayࢆ฼⏝ࡋࡓヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
࣐࢘ࢫࢆ⏝࠸ࡿヨ㦂ἲ࡛࠶ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦LLNA:Local Lymph Node Assay㸧ࡢ⣙ 1/7⛬ᗘࡢࣛࣥࢽࣥࢢࢥࢫࢺ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊin vitro ヨ㦂ἲ࡛࠶ࡿࡇ࡜࠿ࡽࠊ᭷⏝ᛶࡣ㧗࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ᮏヨ㦂ἲ࡟౑⏝࡛ࡁࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ⣽⬊⣔ࡣ KeratinoSensTM
ࡢࡳ࡛࠶ࡾࠊࡑࡢ౑⏝࡟ࡣᮏ⣽⬊⣔ࢆᶞ❧ࡋࡓ Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆཷࡅࡿࡇ࡜ࡀᚲせ࡞ࡓࡵࠊ᪂つᑟධࡢᐜ᫆࡞ヨ㦂⣔࡜ࡣゝ࠸㞴࠸ࠋᮏヨ㦂ἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶࡣࠊ5 ᪋タ୰ 1 ᪋タ࡟࠾࠸࡚ࠊ
GHS(Globally Harmonized System of Classification and Labelling of Chemicals)༊ศ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁㸦ᙅ࠸ឤసᛶ≀㉁㸧࠾ࡼࡧ㠀ឤసᛶ≀㉁࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠿ࡗࡓࡓࡵࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦85㸣㸧࡟㐩ࡋ࡚࠾ࡽࡎࠊᙉ࠸ឤసᛶ≀㉁௨እ࡛ࡣุᐃࡀࡪࢀࡿᠱᛕࡀ࠶ࡿࠋ
୍᪉ࠊ᪋タ㛫෌⌧ᛶࡣࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦80㸣㸧ࢆୖᅇࡗࡓࠋ
ᮏヨ㦂ἲࡢឤᗘࡣࠊ⣙ 80㸣࡛࠶ࡾࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂࡟ࡼࡗ࡚☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽࡎࠊᮏヨ㦂ἲࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡲࡓࠊ≉␗ᗘࡶࠊ⣙ 80㸣࡛࠶ࡿࡇ࡜࠿ࡽࠊ㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ࡟ࡶࠊഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
ᮏヨ㦂ἲ࡛ࡣࠊάᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁ࡣࠊṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟࡉࢀ
࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓࠊ⣽⬊⣔࡛࠶ࡿࡓࡵࠊ␯Ỉᛶࡢ㧗࠸≀㉁࡛ࡣつᐃࡉࢀ࡚࠸ࡿ᭱㧗1
2

Nrf2㸸Nuclear factor (erythroid-derived 2)-like 2
Keap1: Kelch-like ECH-associated protein 139⃰ᗘ㸦2000 μM㸧࡛ࡢホ౯ࡀ㞴ࡋࡃࠊ㝜ᛶุᐃࡀୗࡏ࡞࠸ሙྜࡀ࠶ࡿࠋ௨ ୖࢆ ㋃ࡲ࠼ ࠊᮏ ጤဨ఍ ࡣࠊ ᮏヨ㦂 ἲࡀ ⓶⭵ឤ సᛶ ホ౯࡟ ỗ⏝ ࡉࢀࡿ ࡓࡵ ࡟ ࡣ ࠊ
KeratinoSensTM ࡀᏳ౯࡟ධᡭ࡛ࡁࡿࡇ࡜ࡀ๓ᥦ࡜⪃࠼ࡿࠋࡲࡓࠊᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ⪃៖ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢホ౯ࡣ୙༑ศ࡛࠶ࡾࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ㸦LLNAࠊࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂࡞࡝㸧࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ
1. ⥴ゝ ⓶⭵ឤసᛶࢆホ౯ࡍࡿࡇ࡜ࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡛࠶ࡿࠋ໬Ꮫ≀㉁ࡢ⓶⭵࡛ࡢ᥋ゐឤసᛶࡢࣜࢫࢡࢆື≀࡛ணࡍࡿヨ㦂ἲ࡜ࡋ࡚ࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶ

ヨ㦂㸦OECD TG406㸧ࡸ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦LLNA: Local Lymph Node Assay,
OECD TG429㸧ࡀ࠶ࡿࠋࡇࡢ[3H-Methyl]-thymidine ྲྀ㎸㔞ࢆ఩ඖ⣲㸦RI㸧ࢆ⏝࠸ࡎ ATP 㔞ࢆ㔞ࢆᐃࡍࡿ LLNA ௨እ࡟ᨺᑕᛶྠ

ᐃࡍࡿ LLNA:DA㸦OECD TG442A㸧ࡸ Bromodeoxyuridine

ᐃࡍࡿ LLNA:BrdU-ELISA㸦OECD TG442B㸧ࡀ࠶ࡿࠋࡇࡢࡼ࠺࡟ࠊ⌧ᅾ OECD ࠿ࡽ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚බ⾲ࡉࢀ࡚࠸ࡿヨ㦂ἲࡣࠊື≀ࢆ⏝࠸ࡓ in vivo ࡢヨ㦂ἲࡢࡳ࡛࠶ࡿࠋ
 EU ࡟࠾ࡅࡿḢᕞ໬Ꮫရつ๎㸦REACH: Registration, Evaluation, Authorisation and Restrictionof Chemicals㸧࡛ࡣࠊᏳ඲ᛶホ౯ࡣࢥࣥࣆ࣮ࣗࢱ࣮ࢆ⏝࠸ࡓᐃ㔞ⓗᵓ㐀άᛶ┦㛵㸦QSAR:
Quantitative Structure-Activity Relationship)ࣔࢹࣝࡸ in vitro ヨ㦂➼࡟ࡼࡿ௦᭰ἲࡀ᥎ዡࡉࢀ࡚࠾

ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆྵࢇࡔ໬⢝ရࡢ㍺ධ࠾ࡼࡧ㈍኎ࡀ⚗Ṇࡉࢀࡓ㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠋࡑࡢࡓࡵࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆホ౯ࡍࡿ௦᭰ἲࡢ㛤Ⓨࡀᙉࡃồࡵࡽࢀ࡚࠸ࡿࠋ ⌧ᅾࠊ࣌ࣉࢳࢻ࡜ࡢ⤖ྜ཯ᛂࢆ฼⏝ࡋࡓ Direct Peptide Reactivity Assay㸦DPRA㸧ࠊ༢⌫⣔⣽⬊ࡢάᛶ໬ࢆ฼⏝ࡋࡓ human Cell Line Activation Test㸦h-CLAT㸧࠾ࡼࡧ Myeloid U937 Skin
Sensitization Test㸦 MUSST 㸧ࠊ ࢣ ࣛࢳࣀ ࢧ ࢖ ࢺ⣽⬊ ⣔ ࡢ ᶆⓗ㑇 ఏ Ꮚ ࢆ⏝࠸ ࡓ ARE-Nrf2
Luciferase Test Method ࡞࡝ࡢ⓶⭵ឤసᛶヨ㦂ࡢື≀ࢆ⏝࠸࡞࠸ື≀ᐇ㦂௦᭰ἲࡀᥦ᱌ࡉࢀ
࡚࠾ࡾࠊEURL ECVAM㸦European Union Reference Laboratory for Alternatives to Animal Testing㸧➼࡟࠾࠸࡚ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀ࡚࠸ࡿࠋ
 ARE(Antioxidant response element )-Nrf2 Luciferase Test Method ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀ ARE ࡟ࡼࡾไᚚࡉࢀࡿ㑇ఏᏊࡢⓎ⌧ࢆㄏᑟࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࡇࡢㄏᑟάᛶ࡟ࡘ࠸࡚ᇵ
㣴⣽⬊ࢆ⏝࠸࡚ホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋKeratinoSens™ἲࡣᮏヨ㦂ἲࡢࡓࡵ࡟☜❧ࡉࢀࡓ⣽⬊ࢆ౑⏝ࡍࡿホ౯⣔࡛࠶ࡿࠋKeratinoSens™ἲࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙ
ࣥ◊✲ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊEURL ECVAM ⛉Ꮫㅎၥጤဨ఍㸦ESAC㸧࡟ࡼࡿ➨୕⪅ホ౯ࡀ᏶஢ࡋ࡚࠸ࡿ 1)ࠋJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸦௨ୗࠊᮏጤဨ఍㸧ࡀ ARE-Nrf2 Luciferase Test
Method ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊKeratinoSens™ἲࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝ➼ࠊ⌧ᅾࡲ࡛࡟බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗࢆࡶ࡜࡟ホ౯ࡋࡓࡢ࡛ࠊࡑࡢ⤖ᯝࢆሗ࿌ࡍࡿࠋ
410

2㸬ヨ㦂ἲࡢཎ⌮⓶⭵ឤసᛶࡣࠊࣄࢺ࡛ࡣ᥋ゐ⓶⭵⅖ࠊື≀㸦㱎ṑ㢮㸧࡛ࡣ᥋ゐ㐣ᩄ⑕࡜ࡋ࡚▱ࡽࢀࡿ໬Ꮫ≀㉁ࡢẘᛶࡢ୍ࡘ࡛࠶ࡿࠋOECD ࡀࡲ࡜ࡵࡓ Adverse Outcome Pathway (AOP)࡛ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ⓶⭵ឤసᛶࡣḟࡢ 4 ࡘࡢ Key event ࠿ࡽᡂࡿ࡜ࡉࢀ࡚࠸ࡿ 1)ࠋ
1) ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢࢩࢫࢸ࢖ࣥṧᇶ࠶ࡿ࠸ࡣࣜࢪࣥṧᇶ࡜ࡢඹ᭷⤖ྜ2) ࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕ᛶᛂ⟅࠾ࡼࡧ ARE-dependent pathway ࡟ࡼࡿ㑇ఏᏊⓎ⌧3) ᶞ≧⣽⬊ࡢάᛶ໬㸦≉␗ⓗ⣽⬊⾲㠃࣐࣮࣮࢝ࡢⓎ⌧ࠊࢣࣔ࢝࢖ࣥࡸࢧ࢖ࢺ࢝࢖ࣥࡢ⏘⏕㸧4) ࣜࣥࣃ⠇࡟࠾ࡅࡿ T ⣽⬊ࡢቑṪ
ARE-Nrf23 Luciferase Test Method ࡣୖグࡢ➨ 2 ࡢ Key event ࡟ᑐᛂࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡑ
ࡢᇶᮏⓗཎ⌮ࡣ Nrf2-Keap14-ARE pathway㸦ᅗ 1㸧ࢆ฼⏝ࡋࡓ࣏࣮ࣞࢱ࣮࢔ࢵࢭ࢖࡛࠶ࡿ 1)ࠋ
ᅗ 1㸬Nrf2-Keap1-ARE pathway ࡢᶍᘧᅗNrf2-Keap1-ARE pathway ࡣࠊ㌿෗ᅉᏊ Nrf2ࠊNrf2 ࡢᢚไᅉᏊ࡛࠶ࡿ Keap1 ࠾ࡼࡧ AREࡀ㛵ಀࡍࡿ㑇ఏᏊⓎ⌧⤒㊰࡛࠶ࡿࠋNrf2 ࡣ Keap1 ࡜⤖ྜࡋࠊARE ࡟౫Ꮡࡋ࡚Ⓨ⌧ࡍࡿ㑇ఏᏊ⩌ࡢⓎ⌧㔞ࢆไᚚࡋ࡚࠸ࡿࠋKeap1 ࡢࢩࢫࢸ࢖ࣥṧᇶ࡟ồ㟁Ꮚᛶࡢ໬Ꮫ≀㉁ࡀ⤖ྜࡍࡿ࡜ࠊ
Nrf2 ࡣ Keap1 ࠿ࡽゎ㞳ࡋࠊ᰾ෆ࡬⛣⾜ࡋ࡚ DNA ୖࡢ ARE ࡟⤖ྜࡍࡿࠋࡑࡢ⤖ᯝࠊୗὶࡢ
㑇ఏᏊ⩌ࡢⓎ⌧ࡀㄏᑟࡉࢀࠊ໬Ꮫ≀㉁࡟ࡼࡿ㞀ᐖ࠿ࡽ⣽⬊ࢆಖㆤࡍࡿࡓࡵ࡟ᶵ⬟ࡍࡿࠋከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀ Nrf2-Keap1-ARE pathway ࢆάᛶ໬ࡍࡿࠋ
ARE-Nrf2 Luciferase Test Method ࡛ࡣࠊAKR1C2 㑇ఏᏊ㸦ᶞ≧⣽⬊࡟࠾࠸࡚⓶⭵ឤసᛶ≀㉁࡟ࡼࡾⓎ⌧ㄏᑟࡉࢀࡿ㑇ఏᏊࡢ 1 ࡘ㸧ࡢ ARE ࢆ⼥ྜࡉࡏࡓ SV40 ࣉ࣮ࣟࣔࢱ࣮ࢆ᭷ࡍࡿ
ࣝࢩࣇ࢙࣮ࣛࢮ㑇ఏᏊࡢࣉࣛࢫ࣑ࢻࢆᏳᐃⓗ࡟ᑟධࡋࡓ HaCaT ⣽⬊㸦ࣄࢺࢣࣛࢳࣀࢧ࢖ࢺ
3
4

Nrf2 :Nuclear factor-erythroid 2-related factor 2
Keap1 : Kelch-like ECH-associated protein 1511⣔ᇵ㣴⣽⬊ᰴ㸧ࢆ⏝࠸ࡿࠋ໬Ꮫ≀㉁࡟ࡼࡾ Nrf2-Keap1-ARE pathway ࡀάᛶ໬ࡉࢀࡿ࡜ࣝࢩ
ࣇ࢙࣮ࣛࢮ㑇ఏᏊࡀⓎ⌧ࡍࡿࠋᇶ㉁ࢆῧຍࡋࠊࣝࢩࣇ࢙࣮ࣛࢮࡀゐ፹ࡍࡿ཯ᛂࡢⓎගᙉᗘࢆᐃࡍࡿࡇ࡜࡟ࡼࡾࠊ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࢆホ౯ࡍࡿࠋ3㸬ヨ㦂ᡭ㡰㸭ุᐃヨ㦂ᡭ㡰ࡣ≉࡟♧ࡉ࡞࠸㝈ࡾ KeratinoSensTM ࢆ⏝࠸ࡓሙྜࡢグ㍕࡛࠶ࡿࠋࢣࣛࢳࣀࢧ࢖ࢺ
⏤᮶ ARE-Nrf2 ࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮㑇ఏᏊ㸦keratinocyte-based ARE-Nrf2 luciferase
reporter gene㸧ࢆ⏝࠸ࡓᮏヨ㦂ἲࡢ⣽⬊࡜ࡋ࡚ KeratinoSensTM ௨እࡢ⣽⬊ࢆ⏝࠸ࡿሙྜࡣࠊ
OECD సᡂࡢ ARE-Nrf2 LuciferaseTest Method ࡟㛵ࡍࡿ Performance Standard㸦᱌㸧2)࡟ᚑ࠸
KeratinoSensTM ࢆ⏝࠸ࡓሙྜ࡜ྠ➼࠿࠶ࡿ࠸ࡣඃࡿಙ㢗ᛶࠊṇ☜ᗘࠊឤᗘࠊ≉␗ᗘ࡞࡝ࢆ♧ࡍࡇ࡜ࢆ☜ㄆࡋࡓࡢࡕ࡟౑⏝ࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ࠸ࡎࢀࡢሙྜࡶ᪥ᖖⓗ࡟⏝࠸ࡿ๓࡟OECD ࢞࢖ࢻࣛ࢖ࣥ㸦᱌㸧3)ࡢ Annex 2 ࡟ᚑ࠸ᢏ⾡ⓗ⇍㐩ᗘࢆ☜ㄆࡍࡿࡇ࡜ࢆ᥎ዡࡍࡿࠋ
3-1. ⣽⬊ࡢㄪ〇
ARE ไᚚୗࡢࣝࢩࣇ࢙࣮ࣛࢮ࣏࣮ࣞࢱ࣮㑇ఏᏊࢆᏳᐃⓗ࡟ྲྀࡾ㎸ࢇࡔࢺࣛࣥࢫࢪ࢙ࢽࢵ
ࢡ⣽⬊⣔ࢆ⏝࠸ࡿࠋ㸦⌧ᅾࡣࠊKeratinoSensTM ࡢࡳ࡛࠶ࡿࡓࡵࠊヨ㦂ἲࡢ㛤Ⓨ⪅࡜౑⏝チㅙዎ⣙ࢆ⤖ࢇࡔ࠺࠼࡛ࡇࡢ⣽⬊⣔ࢆධᡭࡋ࡚౑⏝ࡍࡿࡇ࡜࡟࡞ࡿ㸧ࠋᣦᐃࡉࢀࡓ⥅௦ᩘࡢ⣽⬊㸦2 ࠿ࡽ 4 ௦㸧ࢆቑṪࡋࠊศ๭ࡋ࡚ಖ⟶ࡋࠊࡇࢀࢆ୺ࢫࢺࢵࢡ⣽⬊࡜ࡍࡿࠋ୺ࢫࢺࢵࢡ⣽⬊࠿ࡽቑṪࡉࡏࠊᣦᐃࡉࢀࡓ⥅௦ᩘ㸦25 ௦㸧௨ෆ࡛ヨ㦂࡟౑⏝ࡍࡿࠋヨ㦂๓᪥࡟ 80㹼90㸣ࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡗࡓᇵ㣴ࣇࣛࢫࢥ࠿ࡽㄪ〇ࡋࡓᆒ୍࡞⣽⬊ᾮࢆ 4 ᯛ㸦3 ᯛࡣࣝࢩࣇ࢙࣮ࣛࢮάᛶ

ᐃࠊ1 ᯛࡣ⏕⣽⬊ᩘᐃ㸧ࡢ 96 ࢚࢘ࣝࣉ࣮ࣞࢺ࡟᧛✀㸦10,000 cells / well㸧ࡍࡿࠋ

3-2. ⿕㦂≀㉁࠾ࡼࡧᑐ↷≀㉁ࡢㄪ〇ཎ๎࡜ࡋ࡚ࠊヨ㦂ᙜ᪥ࡢㄪ〇࡜ࡍࡿࠋ⿕㦂≀㉁ࡣ DMSO ࡟⁐ゎࡋ࡚ 200 mM ࡢ⁐ᾮࢆㄪ〇ࡍࡿࠋDMSO ࡟୙⁐ࡢሙྜࡣ⁛⳦Ỉ࠶ࡿ࠸ࡣᇵ㣴ᾮ࡟࡚ྠᵝ࡟ㄪ〇ࡋࠊࡑࡢ⁐ᾮࡣࠊ⁛⳦㸦౛࠼ࡤℐ㐣㸧ࡍࡿࠋศᏊ㔞ࡀ୙᫂ࡢ⿕㦂≀㉁ࡢሙྜࡣ 40 mg/mL ࠶ࡿ࠸ࡣ 4㸣㸦W/V㸧ࡢ⁐ᾮ࡜ࡍࡿࠋࡇࢀࡽࡢ⁐ᾮࢆ DMSO㸦୙⁐ࡢሙྜࡣ⁛⳦Ỉࡲࡓࡣ⁛⳦ᇵ㣴ᾮ㸧࡛ಸࠎᕼ㔘ࡋ࡚ 12 ẁ㝵ࡢ⃰ᗘ㸦0.098㹼200 mM㸧
⁐ᾮࢆㄪ〇ࡍࡿࠋ
㝜ᛶᑐ↷㸦፹య㸧ࡣ 1 ࣉ࣮ࣞࢺ࠶ࡓࡾ 6 ࢚࢘ࣝศࢆྠᵝ࡟ㄪ〇ࡍࡿࠋ㝧 ᛶ ᑐ ↷ ࡣ Cinnamic Aldehyde 㸦 CAS No: 14371-10-9, trans-3-Phenyl-2-propenal,trans-Cinnamaldehyde㸧ࢆ⏝࠸ࠊDMSO ࡟⁐ゎࡋ࡚ 200 mM ࡢ⁐ᾮࢆㄪ〇ࡋࠊࡉࡽ࡟ DMSO࡛ᕼ㔘ࡋ࡚ 6.4 mM ࡜ࡍࡿࠋࡇࡢ⁐ᾮ࠿ࡽ DMSO ࡛ಸࠎᕼ㔘ࢆࡋ࡚ 5 ẁ㝵ࡢ⃰ᗘ㸦0.4㹼6.4
mM㸧⁐ᾮࢆㄪ〇ࡍࡿࠋࡉࡽ࡟ࠊࡍ࡭࡚ࡢ⁐ᾮࢆ⾑Ύྵ᭷ᇵ㣴ᾮ࡛ 25 ಸᕼ㔘ࡍࡿࠋࡇࢀࡽࡢㄪ〇⁐ᾮࢆྛ࢚࢘ࣝ
࡟ຍ࠼ࡿ㸦ࠕ⿕㦂≀㉁➼ࡢ㐺⏝ࠖཧ↷㸧࡜ࠊ᭱⤊⃰ᗘࡣ⿕㦂≀㉁࡛ 0.98㹼2000 μMࠊ㝧ᛶᑐ
612

↷࡛ 4㹼64 μM ࡜࡞ࡿࠋ㝜ᛶᑐ↷ࡢ DMSO ࡢ᭱⤊⃰ᗘࡣ௚ࡢㄪ〇ᾮ࡜ྠࡌ 1%࡜࡞ࡿࠋ ᑡ࡞ࡃ࡜ࡶ 2 ᅇࡢ⧞ࡾ㏉ࡋࡋᐃࢆ⾜࠺ࡀࠊ3 ᅇ┠ࢆ⾜࠺ሙྜࡶྵࡵ࡚ࠊࡑࢀࡒࢀࡢ⧞ࡾ㏉ᐃࡣ᪥ࢆኚ࠼࡚⾜࠸ࠊ⿕㦂≀㉁ࡢ⁐ᾮㄪ〇ࠊ⣽⬊ࡢ๓ᇵ㣴㸦⥅௦ᩘࡣྠࡌࡶࡢ㸧ࡶ⧞ࡾ㏉ࡋ

ᐃࡈ࡜࡟⾜࠺ࠋ

3-3. ⿕㦂≀㉁➼ࡢ㐺⏝24 ᫬㛫ᇵ㣴ᚋࡢ 4 ᯛࡢࣉ࣮ࣞࢺࡢᇵ㣴ᾮࢆᤞ࡚ࠊ1 ࢚࢘ࣝ࠶ࡓࡾ 150 μL ࡢ⾑Ύྵ᭷ᇵᆅ
㸦ᢠ⏕≀㉁୙ྵ㸧࡛⨨ࡁ᥮࠼ࡿࠋㄪ〇ࡋࡓ⿕㦂≀㉁⁐ᾮ➼ࢆ 50 μL ࡎࡘྛ࢚࢘ࣝ࡟ຍ࠼ࠊ48
᫬㛫ࠊ37±1Υࠊ5㸣CO2 ࢖࣮ࣥ࢟ࣗ࣋ࢱෆ࡛ᇵ㣴ࡍࡿࠋࡓࡔࡋࠊ1 ࢚࢘ࣝࡣ↓ฎ⨨㸦↓⣽⬊ࠊ✵࢚࢘ࣝ㸧࡜ࡍࡿࠋ࢚࢘ࣝ࠿ࡽࡢ⵨Ⓨࡸ஺ᕪởᰁࢆ㑊ࡅࡿࡓࡵ࡟ࣉ࣮ࣞࢺࡈ࡜࡟㐽ⶸࡍࡿࠋ
3-4. ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ

ᐃ㐺ษ࡞ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ

ᐃ࡟ࡣࠊ1) ឤᗘࡢⰋ࠸࣑ࣝࣀ࣓࣮ࢱࠊ2) ගࡢ஺ᕪ࡟ࡼࡿ

ᐃࡢጉᐖࢆ㜵ࡄ࡟༑ศ࡞࢚࢘ࣝࡢ㧗ࡉࢆᣢࡗࡓࣉ࣮ࣞࢺࠊ3) ༑ศ࡞ឤᗘ࡜ࣂࣛࡘࡁࡢప
࠸ᐃ್ࢆᚓࡿࡓࡵࡢࣝࢩࣇ࢙࣮ࣛࢮᇶ㉁ࡢ㑅ᢥࡀ㔜せ࡛࠶ࡿࠋࡇࢀࡽࢆ☜ㄆࡍࡿࡓࡵ࡟

Annex 3 3)࡟♧ࡉࢀࡓࢭࢵࢺ࢔ࢵࣉ᪉ἲࢆヨ㦂๓࡟☜ㄆࡍࡿࡇ࡜ࢆ່ࡵࡿࠋ
ᇵ㣴⤊஢ᚋࠊୖΎࢆᤞ࡚ࠊࣜࣥ㓟⦆⾪໬⏕⌮㣗ሷỈ୍࡛ᗘὙ࠺ࠋ⣽⬊⁐ゎ⏝⦆⾪ᾮࢆྛ࢚࢘ࣝ࡟ຍ࠼ࠊᐊ

࡛ 20 ศ㛫ฎ⌮ࡍࡿࠋ⣽⬊⁐ゎ≀ࢆྵࡴࣉ࣮ࣞࢺࢆ࣑ࣝࣀ࣓࣮ࢱ࡛

ᐃࡍࡿࡓࡵࠊྛ࢚࢘ࣝ࡟ࣝࢩࣇ࢙࣮ࣛࢮࡢᇶ㉁ᾮ 50 μL ࢆຍ࠼ࠊ1 ⛊ᚅࡕࠊ2 ⛊㛫ࡢⓎග㔞ࢆ✚⟬ࡍࡿࠋ
3-5. ⣽⬊⏕Ꮡ⋡ࡢᐃ⣽ ⬊ ࡢ ⏕ Ꮡ ⋡ ࢆᐃ ࡍ ࡿ ࣉ ࣞ ࣮ ࢺ ࡣ ࠊ ᇵ 㣴 ⤊ ஢ ᚋ ࡟ ᇵ ᆅ ࢆ MTT

㸦 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium
bromide; CAS No. 298-93-1㸧ྵ᭷᪂㩭ᇵᆅ࡟஺᥮ࡍࡿࠋ4 ᫬㛫ࠊ37Υࠊ5㸣CO2 ࢖࣮ࣥ࢟ࣗ࣋
ࢱෆ࡛ᇵ㣴ࡍࡿࠋᇵ㣴ᚋࠊMTT ྵ᭷ᇵᆅࢆᤞ࡚ࠊ10%SDS(Sodium Dodecyl Sulphate)⁐ᾮ࡞࡝୍࡛ኪ⣽⬊⁐ゎᚋࠊ600 nm ࡢ྾ගᗘࢆ
3-6.

ᐃࡍࡿࠋ

ᐃ್࠿ࡽ௨ୗࡢࣃ࣓࣮ࣛࢱ࣮ࢆồࡵࡿࠋ ࣭⿕㦂≀㉁࠾ࡼࡧ㝧ᛶᑐ↷࡛ほᐹࡉࢀࡓࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢ᭱኱ㄏᑟಸ⋡㸸Imax ࣭ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࡀ⁐፹ᑐ↷ࡢ 1.5 ಸࡢ㜈್㸦ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡀ 50%ቑຍ㸧ࢆ㉸࠼ࡓ⃰ᗘ㸸EC1.5
 ࣭⣽⬊⏕Ꮡ⋡ࡀ 50㸣࠾ࡼࡧ 30%ῶ࡜࡞ࡿ⃰ᗘ㸸IC50 ࠾ࡼࡧ IC30
ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟಸ⋡㸦Fold induction㸧ࡣᘧ 1 ࠿ࡽồࡵࡿࠋ඲యࡢ᭱኱ㄏᑟಸ
⋡ࡣࠊಶࠎࡢ⧞ࡾ㏉ࡋᐃࡢᖹᆒ࡜ࡋ࡚ồࡵࡿࠋᘧ㸯  Fold induction 㸻 (Lsample㸫Lblank) / (Lsolvent㸫Lblank)713   Lsample 㸸⿕㦂≀㉁ࡢⓎගᙉᗘ   Lblank  㸸↓⣽⬊ࠊ↓ฎ⨨ࡢᑐ↷࢚࢘ࣝࡢⓎගᙉᗘ   Lsolvent 㸹⣽⬊࡜⁐፹࠿ࡽ࡞ࡿ࢚࢘ࣝࡢᖹᆒⓎගᙉᗘ
EC1.5 ࡣᘧ 2 ࡟ࡼࡾ⥺ᙧ⿵㛫࡟ᇶ࡙࠸࡚ồࡵࡿࠋ඲యࡢ EC1.5 ࡣࠊಶࠎࡢ⧞ࡾ㏉ࡋᐃࡢᗄఱᏛⓗᖹᆒ࡛ồࡵࡿࠋ
ᘧ 2  EC1.5 = (Cb㸫Ca) × {(1.5㸫Ia) / (Ib㸫Ia)} 㸩Ca   Ca㸸1.5-fold induction ࢆ㉸࠼ࡓ᭱ప⃰ᗘ㸦μM㸧   Cb㸸1.5-fold induction ᮍ‶ࡢ᭱㧗⃰ᗘ㸦μM㸧   Ia㸸1.5-fold induction ࢆ㉸࠼ࡓ᭱ప⃰ᗘ࡛ࡢ fold induction㸦3 ᅇ   Ib㸸1.5-fold induction ᮍ‶ࡢ᭱㧗⃰ᗘ࡛ࡢ fold induction㸦3 ᅇ

ᐃࡢᖹᆒ㸧

ᐃࡢᖹᆒ㸧

⏕Ꮡ⋡㸦Viability㸧ࡣᘧ 3 ࠿ࡽồࡵࡿࠋ
ᘧ 3  Viability = (Vsample㸫Vblank) / (Vsolvent㸫Vblank) × 100   Vsample㸸⿕㦂≀㉁ࡢ࢚࢘ࣝࡢ MTT ྾ගᗘ   Vblank㸸↓⣽⬊ࠊ↓ฎ⨨ࡢᑐ↷࢚࢘ࣝࡢ MTT ྾ගᗘ   Vsolvent㸸⣽⬊࡜⁐፹࠿ࡽ࡞ࡿ࢚࢘ࣝࡢᖹᆒ MTT ྾ගᗘ
IC50 ࡜ IC30 ࡣᘧ 4 ࡟ࡼࡾ⥺ᙧ⿵㛫࡟ᇶ࡙࠸࡚ồࡵࡿࠋ඲యࡢ IC50 ࡜ IC30 ࡣࠊಶࠎࡢ⧞ࡾ
㏉ࡋ

ᐃࡢᗄఱᏛⓗᖹᆒ࡛ồࡵࡿࠋ

ᘧ 4  ICx 㸻 (Cb㸫Ca)×[{(100㸫x)㸫Va} / (Vb㸫Va)]㸩Ca   㹶㸸⃰ᗘࢆồࡵࡿῶᑡ⋡   Ca㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶࢆ㉸࠼ࡓ᭱ప⃰ᗘ㸦μM㸧   Cb㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶᮍ‶ࡢ᭱㧗⃰ᗘ㸦μM㸧   Va㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶࢆ㉸࠼ࡓ᭱ప⃰ᗘ࡟࠾ࡅࡿ⏕Ꮡ⋡   Vb㸸⣽⬊⏕Ꮡ⋡㹶㸣ῶᮍ‶ࡢ᭱㧗⃰ᗘ࡟࠾ࡅࡿ⏕Ꮡ⋡
1.5-fold induction ࢆ㉸࠼ࡓྛ⃰ᗘ࡟ࡘ࠸࡚ࠊࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࡀ⤫ィᏛⓗ࡟㝜ᛶᑐ↷࡟ᑐࡋ࡚᭷ព㸦p㸺0.05㸧࡛࠶ࡿ࠿ࢆ᳨ドࡍࡿࠋࢢࣛࣇࢆసᡂࡋ࡚どぬⓗ࡟☜ㄆࡍࡿࡇ࡜ࡶ᥎ዡࡍࡿࠋ᫂ࡽ࠿࡞⃰ᗘ౫Ꮡᛶࡀㄆࡵࡽࢀ࡞࠸ሙྜࠊ⃰ᗘ཯ᛂ᭤⥺ࡀ஧┦ᛶࢆ♧ࡍሙྜ࡟ࡣࠊᐃࢆ⧞ࡾ㏉ࡋࠊ⿕㦂≀㉁≉␗ⓗ࠿ࠊᐃ࢚࣮ࣛ࡞ࡢ࠿࡞࡝ࢆ☜ㄆࡍࡿࠋ஧┦ᛶ࡛࠶ࡿࡇ࡜ࡀ☜ㄆ࡛ࡁࡓሙྜࡣࠊࡼࡾప࠸ EC1.5 ್ࢆ㑅ᢥࡍࡿࠋ
3-7.

ᐃᡂ❧᮲௳

௨ୗࡢ 3 ᮲௳ࢆࡍ࡭࡚‶ࡓࡍሙྜ࡟ᡂ❧ࡍࡿࠋ1) 㝧ᛶᑐ↷ࡢ Cinnamic Aldehyde ࡣ㝧ᛶ࡛࡞ࡃ࡚ࡣ࡞ࡽ࡞࠸ࠋࡍ࡞ࢃࡕࠊ㝧ᛶᑐ↷ࡢㄏ8
14ᑟࡣᑡ࡞ࡃ࡜ࡶ 1 ⃰ᗘ࡛ 1.5 ࡢ㜈್௨ୖ࡛⤫ィᏛⓗ࡟᭷ព࡛࡞ࡃ࡚ࡣ࡞ࡽ࡞࠸ࠋ
2㸧Cinnamic Aldehyde ࡢ EC1.5 ್ࡣࣄࢫࢺࣜ࢝ࣝࢹ࣮ࢱ㸦7 μM ࡜ 30 μM ࡢ㛫ࡢࣂࣜࢹ࣮ࢩ
ࣙࣥࢹ࣮ࢱ࡟ᇶ࡙ࡁᐃᮇⓗ࡟᭦᪂ࡍࡿࡇ࡜㸧ࡢᖹᆒࡢ 2 ᶆ‽೫ᕪ್௨ෆ࡛࠶ࡿࡇ࡜࡜ࠊ
64 μM ࡢ Cinnamic Aldehyde ࡢ 3 ࣉ࣮ࣞࢺࡢᖹᆒࡢ Fold induction ࡣ 2 ࠿ࡽ 8 ࡢ㛫࡟࠶ࡿࡇ࡜ࢆ☜ㄆࡍࡿࠋᚋ⪅ࡀ‶ࡓࡉࢀ࡞࠸ሙྜࡣࠊCinnamic Aldehyde ࡟ࡼࡿࣝࢩࣇ࢙ࣛ
࣮ࢮάᛶࡢㄏᑟ࡜⃰ᗘ౫Ꮡᛶࡢ㛵㐃ࢆៅ㔜࡟☜ㄆࡋࠊ⃰ᗘ౫Ꮡᛶࡀ᫂ࡽ࠿࡞ሙྜ࡟ཷ
ࡅධࢀࡽࢀࡿࠋ
3) 3 ࣉ࣮ࣞࢺࡢ 6 ࢚࢘ࣝࡢ⁐፹ᑐ↷㸦ྜィ 18 ࢚࢘ࣝ㸧ࡢᖹᆒኚືಀᩘࡀ 20㸣௨ୗ࡛࠶ࡿࡇ࡜ࡀᚲせ࡛ࠊࡇࢀࡼࡾࡶ㧗࠸ሙྜࡣ↓ຠ࡜ࡍࡿࠋ
3-8. 㝧ᛶࡢุᐃ
2 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࡢ 2 ᅇ࠶ࡿ࠸ࡣ 3 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࡢ 2 ᅇ࡛ࠊ௨ୗࡢࡍ࡭࡚ࡢ᮲௳࡟
ྜ⮴ࡋࡓሙྜ࡟⿕㦂≀㉁ࡣឤసᛶ≀㉁࡜ุ᩿ࡍࡿࠋ
1) Imax ್ࡀ 1.5 ಸㄏᑟࡼࡾࡶ኱ࡁࡃ⁐፹ᑐ↷࡟ẚ㍑ࡋ࡚⤫ィᏛⓗ࡟᭷ព࡛࠶ࡿࡇ࡜ࠋ
2) 1.5 ಸ௨ୖࡢࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࢆ㉳ࡇࡋࡓ᭱ప⃰ᗘ࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡣ
70㸣௨ୖ࡛࠶ࡿࡇ࡜ࠋ
3) EC1.5 ್ࡀ 1000 μM ᮍ‶㸦ศᏊ㔞ᮍ▱ࡢሙྜࡣ 200 mg/mL ᮍ‶㸧࡛࠶ࡿࡇ࡜ࠋ
4) ࣝࢩࣇ࢙࣮ࣛࢮࡢㄏᑟ࡟᫂ࡽ࠿࡞඲యⓗ⃰ᗘ౫Ꮡᛶࡀ࠶ࡿࡇ࡜ࠋ
1㸧࠿ࡽ 3㸧ࡢ࠸ࡎࢀࡶࡀ‶ࡓࡉࢀࡓࡀࠊࣝࢩࣇ࢙࣮ࣛࢮࡢㄏᑟ࡟᫂ࡽ࠿࡞⃰ᗘ౫Ꮡᛶࡀ
ㄆࡵࡽࢀ࡞࠸࡜ࡁࠊ⤖ㄽࡣୗࡏࡎࠊ ࡉࡽ࡟⧞ࡾ㏉ࡋࡢᐇ㦂ࡀᚲせ࡜࡞ࡿࠋ1000 μM ᮍ‶㸦ศᏊ㔞ᮍ▱ࡢሙྜࡣ 200 mg/mL ᮍ‶㸧࡛㝜ᛶࡢሙྜࡶ⤖ㄽࡣୗࡏ࡞࠸ࠋ⣽⬊ẘᛶࢆ♧ࡍ⃰ᗘ㡿ᇦ࡛ࣝࢩࣇ࢙࣮ࣛࢮάᛶࡢㄏᑟࢆ♧ࡍ≀㉁ࡣࠊࢃࡎ࠿࡞⃰ᗘኚ໬࡛㝧ᛶࡢุᐃࡀそࡿ౛ࡀࡲࢀ࡟࠶ࡿࠋࡇࡢࡼ࠺࡞≀㉁ࡣࠊࡼࡾ⊃࠸⃰ᗘ⠊ᅖ࡛ࡼࡾᑠࡉ࠸ᕼ㔘⣔ิ㸦౛࠼ࡤ 1.33
࠶ࡿ࠸ࡣ 1.41㸧ࢆ⏝࠸࡚ࠊㄏᑟࡀ⣽⬊ẘᛶ⃰ᗘ࡛㉳ࡇࡿࡢ࠿ྰ࠿ࢆỴࡵࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ4㸬⢭ᗘGivaudan ♫ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 4)࡟࠾࠸࡚ࠊᢏ⾡⛣㌿ᛶࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀ᳨ウࡉࢀ࡚࠸ࡿࠋ
4-1. ᢏ⾡⛣㌿ᛶ㸦⾲ 1㸧
 7 ≀㉁ࢆ⏝࠸࡚୺ᑟ᪋タࡢ Givaudan ♫࠿ࡽ Procter&Gamble ♫ࠊBeiersdorf ♫ࠊInstitute forin vitro science㸦IIVS㸧࠾ࡼࡧ BASF ࡢ 4 ᪋タ࡬ࡢᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ホ౯ࡀ⾜ࢃࢀࡓࠋࡑ
ࡢ⤖ᯝࠊ┤᥋ࡢᢏ⾡ᣦᑟࢆࡏࡎ࡟ SOP ࡢᥦ౪ࡔࡅ࡛ᢏ⾡⛣㌿ࡣྍ⬟࡛࠶ࡗࡓࠋ
GHS(Globally Harmonized System of Classification and Labelling of Chemicals)࡛༊ศ 1A ࡟ศ㢮ࡉࢀࡿ 2,4-Dinitrochlorobenzene ࠾ࡼࡧ CitralࠊGHS ࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ Ethylene glycol
915dimethacrylate ࡣࠊ඲ 5 ᪋タ 3 ᅇ⧞ࡾ㏉ࡋ⾜ࡗࡓヨ㦂ࡢ⤖ᯝࡣࡍ࡭࡚㝧ᛶ࡛࠶ࡗࡓࠋ୍᪉ࠊ
GHS ࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ Hexyl cinnamic aldehyde ࡣ 2 ᪋タ࡛ 3 ᅇ୰ 3 ᅇ㝧ᛶ࡛࠶ࡗࡓࡀࠊ୺ᑟ᪋タࢆྵࡴ 3 ᪋タ࡛ࡣࡍ࡭࡚㝜ᛶ࠶ࡿ࠸ࡣࠊ㝧ᛶ 1 ᅇࠊ㝜ᛶ 2 ᅇࡢΰᅾࡋࡓุᐃ⤖ᯝࢆ♧ࡋࡓࠋ㠀ឤసᛶ≀㉁࡛࠶ࡿ ChlorobenzeneࠊMethyl salicylate ࠾ࡼࡧ Sulfanilamide ࡣࡍ࡭࡚ࡢ᪋タ࡛㝜ᛶ࡛࠶ࡗࡓࡀࠊ3 ᅇ⧞ࡾ㏉ࡋ୰ 1 ᅇ㝧ᛶࡢุᐃࢆ♧ࡍ᪋タࡀ࠶ࡗࡓࠋࡇࢀࡽࡢ
⤖ᯝࡣࠊ୺ᑟ᪋タࡀ᝿ᐃࡋࡓ⠊ᅖෆࡢ⤖ᯝ࡛࠶ࡿ࡜᥎

ࡉࢀࡿࡓࡵࠊᢏ⾡⛣㌿ᛶ࡟ၥ㢟ࡣ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ

⾲ 1ᢏ⾡⛣㌿ᛶࡢホ౯ᡂ⦼
CAS

Positve with EC 1.5 up to 1000 PM

GHS
potency
category

Lead Lab.
Histolocal
Data

No.

Chemical Name

1

2,4-Dinitrochlorobenzene

97-00-7

3 of 33 of 33 of 35392-40-5

2 of 22 of 23 of 3

Citral1A
1A

3 of 32

3 of 33 of 33 of 33 of 33 of 33456Ethylne glycol dimethacrylate
Hexyl cinnamic aldehyde
Chlorobenzene
M ethyl salicylate

97-90-5
101-86-0
108-90-7
119-36-8

1B
1B
No
No

2 of 22 of 20 of 20 of 23 of 3
1 of 30 of 30 of 33 of 30 of 30 of 3
1 of 33 of 3
1 of 30 of 30 of 33 of 33 of 30 of 3
1 of 33 of 33 of 3
1 of 3
1 of 37

Sulfanilamide

64-74-1No

0 of 20 of 30 of 30 of 3

1 of 30 of 3

Lead䚷lab.

Lab.1

Lab.2

Lab.3

Lab.4

4-2. ᪋タෆ෌⌧ᛶ㸦⾲ 2㸧 ࣈࣛ࢖ࣥࢻୗ࡛ホ౯ࡉࢀࡓ 21 ≀㉁࡟࠾࠸࡚ࠊ5 ᪋タࡢ᪋タෆ෌⌧ᛶ㸦3 ᅇࡢ⧞ࡾ㏉ࡋࡢ
ヨ㦂࡛ྠࡌ⤖ᯝ㸧ࡣࠊ୺ᑟ᪋タ㸸90.5%ࠊヨ㦂᪋タ 1㸸90.5%ࠊヨ㦂᪋タ 2㸸95.2%ࠊヨ㦂᪋
タ 3㸸95.2%ࠊヨ㦂᪋タ 4㸸81.0%࡛࠶ࡗࡓࠋᮏヨ㦂࡛ࡣࠊ㐩ᡂᇶ‽ࡣタᐃࡉࢀ࡚࠸࡞࠸ࡓࡵࠊDPRA ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛᥇⏝ࡉࢀࡓ 85㸣ࢆ┠Ᏻ࡟⪃࠼ࡓሙྜࠊ5 ᪋タ୰ 4 ᪋タࡀࡇࡢᇶ‽ࢆୖᅇࡗࡓࠋᇶ‽࡟㐩ࡋ࡞࠿ࡗࡓヨ㦂᪋タ 4 ࡟࠾࠸࡚෌⌧ᛶࡢᚓࡽࢀ࡞࠸≀㉁ࡣࠊGHS
࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ EugenolࠊPhenyl benzoateࠊ࠾ࡼࡧ㠀ឤసᛶ≀㉁࡛࠶ࡿ Diethyl phthalate
࠾ࡼࡧ Sodium lauryl sulfate ࡛࠶ࡗࡓࠋ ࡓࡔࡋࠊᮏホ౯࡟౑⏝ࡉࢀࡓ⓶⭵ឤసᛶ㝧ᛶ≀㉁ࡢෆヂࡣࠊGHS ࡛ 1A ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 11 ≀㉁࡟ᑐࡋࠊ᪋タෆ࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠸ሙྜࡢ࠶ࡿ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 4 ≀㉁࡜೫ࡾࡀ࠶ࡿࡇ࡜ࡣ␃ពࡍ࡭ࡁⅬ࡜⪃࠼ࡿࠋ
4-3. ᪋タ㛫෌⌧ᛶ㸦⾲ 2㸧 ࣈࣛ࢖ࣥࢻୗ࡛ホ౯ࡉࢀࡓ 21 ≀㉁ࡢ 5 ᪋タࡢ᪋タ㛫෌⌧ᛶ㸦5 ᪋タ࡛ྠࡌ⤖ᯝ㸧ࡣ 85.7%
࡛࠶ࡗࡓࠋᮏඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛ࡣࠊ㐩ᡂᇶ‽ࡣタᐃࡉࢀ࡚࠸࡞࠸ࡓࡵࠊDPRA ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛᥇⏝ࡉࢀࡓ 80㸣ࢆ┠Ᏻ࡟⪃࠼ࡓሙྜࠊࡇࡢᇶ‽ࢆୖᅇࡗࡓࠋ᪋タෆ෌⌧ᛶ࡜ྠᵝ࡟ࠊᮏホ౯࡟౑⏝ࡉࢀࡓ⓶⭵ឤసᛶ㝧ᛶ≀㉁ࡢෆヂ࡟೫ࡾࡀ࠶ࡿࡇ
࡜ࡣ␃ពࡍ࡭ࡁⅬ࡜⪃࠼ࡿࠋ
10
16

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

᪋タ㛫ኚື࠾ࡼࡧ᪋タෆኚືࡢホ౯ᡂ⦼
Chemical Name

CAS

2-M ercaptobenzothiazole
149-30-4
4-M ethylaminophenol sulphate 55-55-0
4-Nitrobenzylbromide
100-11-8
4-Phenylenediamine
106-50-3(5-Chloro)26172-55-4
methylisothiazolinone
Cinnamic aldehyde
104-55-2
Isoeugenol
97-54-1
Glyoxal107-22-2
M ethyldibromo glutaronitrile 35691-65-7
Oxazolone
15646-46-5
Tetramethylthiuramdisulfide
137-26-8
Cinnamyl alcohol
104-54-1
Eugenol
97-53-0
Imidazolidinyl urea
39236-46-9
Phenyl benzoate
93-99-2
Diethyl phthalate
84-66-2Isopropanol
67-63-0Glycerol
56-81-5Lactic acid50-21-5
Salicylic acid
69-72-7

21 Sodium lauryl sulfate

151-21-3

GHS
potency
category
1A
1A
1A
1A
1A
1A
1A
1A
1A
1A
1A
1B
1B
1B
1B
No
No
No
No
No
No

Within Laboratory Reproducibility

Positve with EC 1.5 up to 1000 PM
Lead䚷lab.

Lab.1

Lab.2

Lab.3

Lab.4

BetweenLaboratory
Reproducibility

3 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 3YYY
Y

3 of 33 of 33 of 33 of 33 of 3Y

3 of 33 of 33 of 33 of 33 of 33 of 33 of 3
1 of 32 of 30 of 30 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 32 of 3
1 of 33 of 30 of 30 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 30 of 3
1 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 30 of 30 of 30 of 30 of 30 of 30 of 33 of 33 of 33 of 33 of 33 of 33 of 33 of 3
1 of 33 of 3
1 of 32 of 30 of 30 of 30 of 30 of 3

Y
Y
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
Y

3 of 3

N

3 at
1 at
1 at
1 at
cytotox,conc. cytotox,conc. cytotox,conc. cytotox,conc.

90.5%(19/21) 90.5%(19/21) 95.2%(20/21) 100%(21/21) 85.7%(18/21)

85.7%(18/21)

5㸬ṇ☜ᗘ㸦ឤᗘ࠾ࡼࡧ≉␗ᗘ㸧
Givaudan ♫ࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 4)࡟࠾࠸࡚ࣈࣛ࢖ࣥࢻୗ࡛ホ౯ࡉࢀࡓ 21 ≀㉁ࢆᇶ
࡟ GHS ศ㢮࡜ࡢ୍⮴ᗘࠊឤᗘ࠾ࡼࡧ≉␗ᗘ࡟㛵ࡍࡿホ౯ࢆ⾜ࡗࡓࠋ ᐇ᪋ 5 ᪋タࡢᡂ⦼ࡢ✚⟬࡟ࡼࡿឤᗘ㸦Sensitivity㸧ࡣ 89.3%ࠊ≉␗ᗘ㸦Specificity㸧ࡣ 93.3%ࠊ
ṇ☜ᗘ㸦Accuracy㸧ࡣ 90.5%࡛࠶ࡗࡓࠋヨ㦂᪋タẖࡢᡂ⦼ࡣࠊ୺ᑟ᪋タ࠾ࡼࡧヨ㦂᪋タ 1㸸
ឤᗘ 86.7%ࠊ≉␗ᗘ 100%ࠊṇ☜ᗘ 90.5㸣ࠊヨ㦂᪋タ 2 ࠾ࡼࡧ 3㸸ឤᗘ 93.3%ࠊ≉␗ᗘ 100%ࠊṇ☜ᗘ 95.2㸣ࠊヨ㦂᪋タ 4㸸ឤᗘ 86.7%ࠊ≉␗ᗘ 66.7%ࠊṇ☜ᗘ 81.0㸣࡛࠶ࡗࡓࠋ
ᮏヨ㦂ἲࡢṇ☜ᗘ࡟㛵ࡋ࡚㛤Ⓨ᪋タ࡛࠶ࡿ Givaudan ♫࠿ࡽᥦฟࡉࢀࡓࣄࢫࢺࣜ࢝ࣝࢹ࣮
ࢱ࡛ࡣ 92.9㸣࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊึᮇࡢホ౯≀㉁ᩘࡀ୙༑ศ࡛࠶ࡿ࡜ࡢ ESAC ࡢࢥ࣓ࣥࢺࢆཷࡅࠊGivaudan ♫ࡀᐇ᪋ࡋࡓ 67 ≀㉁ 㸦ឤసᛶ≀㉁ 44ࠊ㠀ឤసᛶ≀㉁ 23㸧࡜ࠊࡉ
ࡽ࡟㏣ຍ࡛ᐇ᪋ࡋࡓ≀㉁ࢆྵࡴ 114 ≀㉁㸦ឤసᛶ≀㉁ 86ࠊ㠀ឤసᛶ≀㉁ 28㸧࡛ࡢホ౯⤖ᯝࡣ 4)ࠊ67 ≀㉁㸸ឤᗘ 86.4㸣ࠊ≉␗ᗘ 82.6㸣ࠊṇ☜ᗘ 85.1%ࠊ114 ≀㉁㸸ឤᗘ 76.7㸣ࠊ≉␗ᗘ
82.1㸣ࠊṇ☜ᗘ 78.1㸣࡛࠶ࡗࡓࠋࡉࡽ࡟ࠊNatsch ࡽ 5)ࡢ 145 ≀㉁ࢆ⏝࠸ࡓヨ㦂࡛ࡣࠊឤᗘ 77%ࠊ≉␗ᗘ 79%ࠊṇ☜ᗘ 72%࡛࠶ࡗࡓࠋࡲࡓࠊ⾲ 3 ࡟♧ࡍ㏻ࡾࠊGHS ࡛ 1A ࡟ศ㢮ࡉࢀࡿ≀㉁ࢆㄗࡗ࡚㝜ᛶ࡜ุ᩿ࡍࡿࡇ࡜ࡣ࡞࠿ࡗࡓࠋ

1117

⾲ 3 GHS ༊ศ 1A ≀㉁ࡢᡂ⦼
Imax

GHS
ChemicalCas no.
ศ㢮20086)

20107)

20118)INVITTOX4)

Citral5392-40-5

1A

9.8

96.4

22.3-104.4

96.4

Tetramethylthiuram disulfide

137-26-8

1A

ND

6.8

8.1-67.9

6.8

Cinnamic aldehyde

104-55-2

1A

31.6

16.2

9.8-44.7

16.2

2-Mercaptobenzothiazole

149-30-4

1A

10.9

8.8

4.9-64.1

8.8

Glyoxal107-22-2

1A

ND

28.2

14.5-195.0

28.2

Isoeugenol

97-54-1

1A

60.2

6.4

9.5-56.8

6.4

1,2-Dibromo-2,4-dicyanobutane

35691-65-7

1A

ND

4.0

2.1-7.8

4.0

4-(Methylamino)phenol sulfate

55-55-0

1A

32.4

5.9

4.5-12.2

10.3

1,4-Phenylenediamine

106-50-3

1A

12.7

26.8

19.4-45.2

26.8

4-Nitrobenzyl bromide

100-11-8

1A

ND

6.9

4.7-14.0

6.9

1-Chloro-2,4-dinitrobenzene

97-00-7

1A

12.3

14.8

4.3-19.5

14.8

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

26172-55-4

1A

7.2

7.2

4.8-13.5

7.2

Oxazolone

15646-46-5

1A

2.3

2.4

6.4-46.3

2.4

ND: Not determined

6. ホ౯ྍ⬟࡞≀㉁ࡢ⠊ᅖ
Emter ࡽࡀሗ࿌ࡋ࡚࠸ࡿࡼ࠺࡟ 6)ࠊᵝࠎ࡞ᵓ㐀ࢆ᭷ࡍࡿ໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡢண

ࡀྍ⬟࡛࠶ࡿ㸦⾲ 4㸧ࠋࡓࡔࡋࠊ⁐ゎᛶࡸ⁐፹୰࡛ࡢᏳᐃᛶ㸦౛࠼ࡤࠊ⁐፹୰࡛ࡢຍỈศゎ㸧ࡀၥ㢟࡛ホ౯୙ྍ⬟࡜࡞ࡿ≀㉁ࡀᏑᅾࡍࡿࠋලయⓗ࡟ࡣࠊLogP ࡀ 7 ௨ୖࡢᙉ␯ỈᛶศᏊࡣDMSO ࡜Ỉ࡬ࡢ⁐ゎ≉ᛶ࠿ࡽヨ㦂ࡀ࡛ࡁ࡞࠸ࠋ୍᪉ࠊLogP ࡀ 5 ࡲ࡛ࡢ໬Ꮫ≀㉁ࡣࠊỈ࠶ࡿ࠸ࡣ DMSO ࡟ྍ⁐࡞ࡓࡵࠊᐜ᫆࡟ヨ㦂ࡀྍ⬟࡛࠶ࡿࠋ㓟↓Ỉ≀ࡣࠊࢩࢫࢸ࢖ࣥṧᇶ࡛ࡣ࡞ࡃࣜࢪࣥṧᇶ࡜཯ᛂ≉ᚩࡀ࠶ࡾࠊࢩࢫࢸ࢖ࣥṧᇶ࡜ࡢ཯ᛂࡀᚲせ࡞ Nrf2 pathway ࢆㄏᑟࡋ࡞࠸ࡇ࡜ࡀ᥎ᐹࡉࢀࠊഇ㝜ᛶ࡜ุᐃࡉࢀࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋࡉࡽ࡟ࠊ㓟໬཯ᛂࡸ㓟໬ⓗ⬺࢔࣑ࣀ཯ᛂࢆᚲせ࡜ࡍࡿከࡃࡢࣉࣞࣁࣉࢸࣥࢆṇࡋࡃุᐃ࡛ࡁࡿࡀࠊP450 ࡟ࡼࡿάᛶ໬
ࡀᚲせ࡜᥎ᐃࡉࢀࡿࣉࣟࣁࣉࢸࣥࡣ᳨ฟ࡛ࡁ࡞࠸㸦KeratinoSensTM ࡢ⏤᮶⣽⬊࡛࠶ࡿ HaCaT⣽⬊ࡣ⸆≀௦ㅰ㓝⣲άᛶࢆ᭷ࡍࡿࡀ㝈ᐃⓗ࡛࠶ࡿ㸧ࠋࣝࢩࣇ࢙࣮ࣛࢮ㓝⣲࡟ᖸ΅ࡍࡿ໬Ꮫ≀㉁ࡶホ౯࡟ᙳ㡪ࡍࡿ 3)ࠋ

12
18

⾲ 4

Emter ࡽࡢሗ࿌Chemical Name

Sensitizers
Oxazolone
Benzoquinone(5-Chloro)-M ethylisothiazolinone
2,4-Dinitrochlorobenzene
4-nitrobenzylbromide
4-phenylenediamine
Glutaraldehyde
Benzoyl peroxide
Glyoxal4-M ethylaminophenol sulphate
Formaldehyde
M ethyldibromo glutaronitrile
Cinnamic aldehyde
2-Hydroxyethyl acrylate
Isoeugenol
Ethylenediamine
Benzylidene Acetone
M ethyl-2-nonynoate
2-M ercaptobenzothiazole
Benzyl salicylate
Tetramethylthiuramdisulfide
Diethylenetriamine
Thioglycerol
Phenylacetaldehyde
Resorcinol
Dihydroeugenol
Benzoisothiazolione
CitralHexyl cinnamic aldehyde
Eugenol
Abietic acidPhenyl benzoate
Lyral HM PCC
Benzocaine
Benzyl cinnamate
2,4-Dichloronitrobenzene
Cinnamyl alcohol
Hydroxycitronellal
Imidazolidinyl urea
Butyl glycidyl ether
Ethylene glycol dimethacrylate
Cobalt chloride
Nickel sulfate

LLNA EC3
䚷

KeratinoS ens results
ARE I max

ARE EC1.5

0.003
0.01
0.01
0.05
0.05
0.11
0.12
0.22
0.75
0.80
0.84
0.90
1.3
1.4
1.5
2.2
2.2
2.5
2.5
2.9
3.1
3.3
3.5
4.5

䚷
2.4
15.2
7.2
14.8
6.9
26.8
80.7
1.4
28.2
5.9
16.9
4.0
16.2
54.9
6.4
13.2
503.9
33.1
8.8
5.5
6.8
1.7
1.5
11.3

175.5
6.5
8.7
2.5
1.3
5.0
24.3
n.i.
89.1
9.4
63.2
7.8
16.1
32.3
16.1
99.9
9.7
1.8
48.1
8.4
0.8
1259.4
n.i.
28.5

5.9
6.8
7.8
9.8
9.9
10.1
11.6
13.6
17.1
17.1
18.4
20.0
21.0
23.0
24.0
30.9
32.9
Pos.
var.

1.0
1.5
24.0
96.4
2.7
1.3
11.4
1.3
16.1
3.0
8.7
2.9
1.7
137.1
2.9
340.7
188.4
23.3
4.2

n.i.
462.0
3.2
23.2
17.3
n.i.
16.6
n.i.
79.6
18.2
11.0
68.3
123.6
79.4
45.4
218.5
57.4
298.6
329.0

ARE IC50

Chemical Name

LLNA EC3

KeratinoS ens results
ARE I max

ARE EC1.5 ARE IC50
Non-sensitizers
1370.9 Sodium lauryl sulfate
var.
1.2
n.i.
44.7
104.5 Salicylic acid
var.
1.1
n.i.
>2000
7.1 M ethyl salicylate
var.
1.2
n.i.
>2000
8.2 Sulfanilamide
NC
1.4
n.i.
>2000
9.1 Diethyl phthalate
>100%
1.1
n.i.
>2000
438.9 Glycerol
>100%
1.2
n.i.
>2000
242.6 Propylene glycol
>100%
1.2
n.i.
>2000
567.6 Benzoic acid
>20%
1.1
n.i.
>2000
677.9 1-Butanol
>20%
1.1
n.i.
>2000
11.7 4-Hydroxybenzoic acid
>25%
1.1
n.i.
>2000
201.6 Sulfanilic acid
>25%
1.3
n.i.
>1000
25.6 Tartaric acid
>25%
1.2
n.i.
>2000
194.4 Propylparaben
>25%
9.7
14.5
813.1
207.2 Ethyl vanillin
>50%
5.4
161.7
>2000
731.4 Isopropanol
>50%
1.2
n.i.
>2000
>2000 Benzyl alcohol
>50%
1.2
n.i.
>2000
174.5 Dimethylisophtalate
NC
2.1
694.9
>2000
121.9 Dextran
NC
1.5
n.i.
>2000
1003.1 Tween 80
NC
2.7
19.3
399.8
111.0 Chlorobenzene
Neg.
1.2
n.i.
>2000
39.1 Lactic acid
Neg.
1.3
n.i.
>2000
>2000 Phenol
Neg.
1.3
n.i.
>2000
>2000 Benzaldehyde
>25
2.3
443.1
>2000
116.2 Octanoic acid
>50
1.1
n.i.
>2000
n.i.: no significant induction above threshold
>2000
759.2
ver.: Variable results
50.9
NC: Level not specified
Neg: Negative reference according to D. Basketter. Food Chem. Toxicol. 37, 1167-1174
182.8
26.3
1505.7
104.6
191.6
355.4
>2000
>2000
816.0
774.6
>2000
90.4
>2000
1655.81330.2998.7

7. ᭷⏝ᛶ࡜㝈⏺
ᮏヨ㦂ἲࡣࠊ⣽⬊ᇵ㣴ࡢᢏ⾡࡜ 96 ࢚࢘ࣝᑐᛂࡢ࣑ࣝࣀ࣓࣮ࢱ࣮ࡢ౑⏝ᢏ⾡ࡀ࠶ࢀࡤᐜ᫆
࡟ᐇ᪋ྍ⬟࡛࠶ࡿࠋKeratinoSensTM ἲ࠾ࡼࡧ LLNA ࡟㛵ࡍࡿ㈝⏝ࡢヨ⟬࡛ࡣࠊLLNA ࡛ࡣ 1≀㉁ᙜࡓࡾࡢࣛࣥࢽࣥࢢࢥࢫࢺࡀ⣙ 10 ୓෇࡛࠶ࡿࡢ࡟ᑐࡋࠊKeratinoSensTM ἲ࡛ࡣ 1 ≀㉁࡟ࡘ࠸࡚ 12 ⃰ᗘ࡛୕㔜ᐃࠊ࠿ࡘ 3 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࡢሙྜࡢࣛࣥࢽࣥࢢࢥࢫࢺࡣ⣙ 1.3

࠿ࡽ 1.5 ୓෇࡛࠶ࡿࠋᐇ㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛ᐇ᪋ྍ⬟࡛࠶ࡿࡇ࡜࠿ࡽࠊ⤒῭ᛶ࣭⡆౽ᛶࡢ㠃࠿ࡽ᭷⏝࡜⪃࠼ࡽࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ࡇࡢ
TMࡽࢀ࡚࠸ࡿ⣽⬊⣔ࡣ KeratinoSensᐃ࡟౑⏝࡛ࡁࡿࡇ࡜ࡀ▱ࡢࡳ࡛࠶ࡿࠋࡑࡢ౑⏝࡟ࡣࠊᮏ⣽⬊⣔ࢆᶞ❧ࡋࡓ

Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆཷࡅࡿࡇ࡜ࡀᚲせ࡜⪃࠼ࡽࢀࡿࡀࠊ㈝⏝ࡶྵࡵࡑࡢ᮲௳ࡢヲ⣽ࡣࢃ࠿ࡗ࡚࠸࡞࠸ࠋ୍᪉ࠊKeratinoSensTM ࢆ౑⏝ࡋ࡞࠸ሙྜ࡟ࡣࠊྠ➼ࡢ⣽⬊⣔ࢆᶞ❧ࡋࠊ
OECD సᡂࡢᮏヨ㦂ἲ࡟㛵ࡍࡿ performance standard㸦᱌㸧2)࡟ᚑ࠸ࠊጇᙜᛶࢆホ౯ࡋࡓୖ࡛
౑⏝ࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊ┦ᛂࡢ㈝⏝࡜᫬㛫ࡀᚲせ࡞ࡇ࡜ࡀᐜ᫆࡟᝿ീࡉࢀࡿࠋ
⣽⬊ᇵ㣴ᢏ⾡࡟⇍⦎ࡋࡓ◊✲⪅࡛࠶ࢀࡤࠊ⮬ື໬ࡋࡓᶵჾࢆ౑⏝ࡋ࡞ࡃ࡚ࡶࠊᑡ࡞ࡃ࡜
ࡶ 1 ≀㉁ 12 ẁ㝵ࡢ⃰ᗘ࡛ 42 ≀㉁ࡢ୕㔜ᐃࡢᐇ㦂ࢆ 1 㐌㛫࡛ᐇ᪋࡛ࡁࠊࡉࡽ࡟ࡇࢀࢆ 313
19ᅇࡢ⧞ࡾ㏉ࡋ࡛ᐇ᪋ࡍࡿ࡜ࠊ42 ≀㉁࡟ࡘ࠸࡚ 3 㐌㛫࡛᏶඲࡞⤖ᯝࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡿࡓࡵࠊ
ࣁ࢖ࢫ࣮ࣝࣉࢵࢺࡢヨ㦂ἲ࡜⪃࠼ࡽࢀࡿࠋ⮬ື໬ࡉࢀࡓᶵჾࢆ⏝࠸ࢀࡤ᭦࡞ࡿຠ⋡໬ࡶྍ⬟࡛࠶ࡿࠋ
EURL ECVAM ࡀᐇ᪋ࡋࡓ LLNA ࡜ࡢឤసᛶࡢ᭷↓ࡢẚ㍑㸵)࡛ࡣࠊṇ☜ᗘࡀ 77㸣㸦155/201㸧ࠊ
ឤᗘ 78㸣㸦71/91㸧ࠊ≉␗ᗘ 76㸣㸦84/110㸧࡛࠶ࡾࠊNatsch ࡽ 6)ࡢ 145 ≀㉁ࢆ⏝࠸ࡓヨ㦂࡛ࡶྠᵝࡢ⤖ᯝ㸦77%, 79%, 72%㸧ࡀᚓࡽࢀ࡚࠸ࡿࠋᮏヨ㦂ἲࡣឤసᛶ≀㉁࡜㠀ឤసᛶ≀㉁ࢆศ
ูࡍࡿࡢ࡟ຍ࠼࡚ࠊ⏝㔞-཯ᛂ᝟ሗࢆᚓࡿࡢ࡟ࡶᐤ୚ࡍࡿྍ⬟ᛶࡀ࠶ࡿ࡜ࡢሗ࿌ࡀ࠶ࡿࡀࠊ
⌧᫬Ⅼ࡛ࡑࡢ᰿ᣐࡣ༑ศ࡜ࡣゝ࠼࡞࠸ࠋᮏヨ㦂ἲࡣከᡂศ࠶ࡿ࠸ࡣΰྜ≀࡛ࡶᢏ⾡ⓗ࡟ࡣ㐺⏝ྍ⬟࡛࠶ࡿࡀࠊᐇ᪋౛ࡀ࡞࠸ࡓࡵࠊ
ៅ㔜࡞ุ᩿ࡀᚲせ࡛࠶ࡿࠋࡲࡓࠊ2000 μM ࢆ᭱㧗⃰ᗘ࡜ࡋ࡚ヨ㦂ࢆᐇ᪋ࡍࡿࡀࠊ㈋⁐ゎᛶࢆ♧ࡍ≀㉁࡟࠾࠸࡚ࠊࡇࡢ᭱㧗⃰ᗘ࡛ࡢホ౯ࡀᅔ㞴࡛ 1000 μM ᮍ‶࡛ᚓࡽࢀࡓ㝜ᛶࡢ⤖ᯝࢆᇶ࡟ࠊឤసᛶࡀ㝜ᛶ࡜ࡣุᐃ࡛ࡁ࡞࠸ࠋ
8. ⤖ㄽ
ARE-Nrf2 Luciferase Test Method ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨஧ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿ
ࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕཯ᛂ࠾ࡼࡧ Nrf2-Keap1-ARE pathway ࢆ฼⏝ࡋࡓ࣏࣮ࣞࢱ࣮࢔
ࢵࢭ࢖࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
࣐࢘ࢫࢆ⏝࠸ࡿ LLNA ࡢ 1/7 ⛬ᗘࡢࣛࣥࢽࣥࢢࢥࢫࢺ࡛ᐇ᪋ྍ⬟࡛࠶ࡾࠊin vitro ヨ㦂ἲ࡛࠶ࡿࡇ࡜࠿ࡽ᭷⏝ᛶࡣ㧗࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⌧᫬Ⅼ࡛ᮏヨ㦂ἲ࡟౑⏝࡛ࡁࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ⣽⬊⣔ࡣ KeratinoSensTM ࡢࡳ࡛࠶ࡾࠊࡑࡢ౑⏝࡟ࡣ⣽⬊⣔ࢆᶞ❧ࡋࡓ Givaudan ♫࠿ࡽࣛ࢖ࢭࣥࢫࢆཷࡅࡿࡇ࡜ࡀᚲせ࡞ࡓࡵࠊᑟධࡢᐜ᫆࡞ヨ㦂⣔࡜ࡣゝ࠸㞴࠸ࠋᮏヨ㦂ἲࡢඛ⾜ࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟࠾ࡅࡿ᪋タෆ෌⌧ᛶࡣࠊ5 ᪋タ୰ 1 ᪋タ࡟࠾࠸࡚ࠊ
GHS ༊ศ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁㸦ᙅ࠸ឤసᛶ≀㉁㸧࠾ࡼࡧ㠀ឤసᛶ≀㉁࡛෌⌧ᛶࡀᚓࡽࢀ࡞
࠿ࡗࡓࡓࡵࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦85㸣㸧࡟㐩ࡋ࡚࠾ࡽࡎࠊᙉ࠸ឤసᛶ≀㉁௨እ࡛ࡣุᐃ
ࡀࡪࢀࡿᠱᛕࡀ࠶ࡿࠋ୍᪉ࠊ᪋タ㛫෌⌧ᛶࡣࠊ┠Ᏻ࡜ࡋࡓ㐩ᡂᇶ‽㸦80㸣㸧ࢆୖᅇࡗࡓࡶࡢࡢࠊᮏホ౯࡟౑⏝ࡉࢀࡓ⓶⭵ឤసᛶ㝧ᛶ≀㉁ࡢෆヂࡣࠊGHS ࡛༊ศ 1A ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 11 ≀㉁࡟ᑐࡋࠊ᪋タෆ࡛෌⌧ᛶࡀᚓࡽࢀ࡞࠸ሙྜࡢ࠶ࡿ GHS ࡛༊ศ 1B ࡟ศ㢮ࡉࢀࡿ≀㉁ࡀ 4 ≀㉁࡜೫ࡾࡀ࠶ࡿࡇ࡜ࡣ␃ពⅬ࡜⪃࠼ࡿࠋ
100 ≀㉁௨ୖࢆホ౯ࡋࡓ஧ࡘࡢሗ࿌࡟࠾ࡅࡿᮏヨ㦂ἲࡢឤᗘࡣࠊ76.7㸣࡜ 77㸣࡛࠶ࡾࠊ㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊᮏヨ㦂ἲࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡲࡓࠊ≉␗ᗘࡶࠊ82.1%࡜ 79%࡛࠶ࡿࡇ࡜࠿ࡽࠊ㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜ࡟ࡶࠊഇ㝧ᛶࡢ⤖ᯝࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ
ᮏヨ㦂ἲ࡛ࡣࠊάᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁ࡣࠊṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟࡉࢀ
࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋࡲࡓࠊ⣽⬊ࢆ⏝࠸ࡓホ౯⣔࡛࠶ࡿࡓࡵࠊ⣽⬊ẘᛶࡸ␯Ỉᛶࡢ㧗࠸≀㉁࡛ࡣつᐃࡉࢀ࡚࠸ࡿ᭱㧗⃰ᗘ㸦2000 μM㸧࡛ࡢホ౯ࡀ㞴ࡋࡃࠊ㝜ᛶุᐃࡀୗࡏ࡞࠸ሙྜࡀ࠶
14
20

ࡿࠋ
௨ ୖࢆ ㋃ࡲ࠼ ࠊᮏ ጤဨ఍ ࡣࠊ ᮏヨ㦂 ἲࡀ ⓶⭵ឤ సᛶ ホ౯࡟ ỗ⏝ ࡉࢀࡿ ࡓࡵ ࡟ ࡣ ࠊ
KeratinoSensTM ࡀᏳ౯࡟ධᡭ࡛ࡁࡿࡇ࡜ࡀ๓ᥦ࡜⪃࠼ࡿࠋࡲࡓࠊᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ⪃៖ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢホ౯ࡣ୙༑ศ࡛࠶ࡾࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ㸦LLNAࠊࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂࡞࡝㸧࡜⤌ࡳྜࢃࡏ࡛ࡢホ౯ࢆ᥎ዡࡍࡿࠋ

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1) EURL ECVAM (2012) ESAC Working Group Peer Review Consensus Report on
Givaudan-coordinated study transferability and reliability of the KeratinoSens assay for skinsensitisation testing2) OECD (2014, in preparation) Performance Standards for the assessment of proposed similar or
modified in vitro skin sensitization ARE-Nrf2 luciferase test methods in TG xxx. OECD
Environment, Health and Safety publications, Series on Testing and Assessment N.XXX, OECD,
Paris.
3) OECD (2015) In Vitro Skin Sensitization: ARE-Nrf2 Luciferase Test Method, OECD Guidelines
for the Testing of Chemicals No. 442D.
4) DB-ALM (INVITTOX) (2013) Protocol 155: KeratinoSens™
5) Natsch A., Ryan C.A., Foertsch L., Emter R., Jaworska J., Gerberick F. and Kern P. (2013) A
dataset on 145 chemicals tested in alternative assays for skin sensitization undergoingprevalidation. Journal of Applied Toxicology, 33, 1337-1352.
6) Natsch A. and Emter R. (2008) Skin sensitizers induce antioxidant response element dependent
genes: application to the in vitro testing of the sensitization potential of chemicals. Toxicological
Science, 102, 110-119.
7) Emter R., Ellis G. and Natsch A. (2010) Performance of a novel keratinocyte-based reporter
cell line to screen skin sensitizers in vitro. Toxicology and Applied Pharmacology, 245,
281-290.
8) Natsch A., Bauch C., Foerttsch L., Gerberick F., Norman K., Hilberer A., Inglis H.,
Landsiedel R., Onken S., Reuter H., Schepky A. and Emter R. (2011) The intra- andinter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skinsensitizers in vitro: results of a ring-study in five laboratories. Toxicology in Vitro, 25,
733-744.
9) EURL ECVAM (2014). Recommendation on the KerationSensTM assay for skin sensitization
testing, 42 pp.
http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations/recommendationkeratinosens-skin-sensitisation.

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OECD GUIDELINE FOR THE TESTING OF CHEMICALSIn Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method

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25

TG 442D

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26

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OECD/OCDETG 442D

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27

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33

TG 442D

OECD/OCDE

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Results
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Discussion of the results 'LVFXVVLRQRIWKHUHVXOWVREWDLQHGZLWKWKH.HUDWLQR6HQV70WHVWPHWKRG &RQVLGHUDWLRQ RI WKH WHVW PHWKRG UHVXOWV ZLWKLQ WKH FRQWH[W RI DQ ,$7$ LI RWKHU UHOHYDQWLQIRUPDWLRQLVDYDLODEOH
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34

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OECD/OCDETG 442D

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© OECD, (2015)
35

TG 442D

OECD/OCDE 1DWVFK $ 5\DQ &$ )RHUWVFK / (PWHU 5 -DZRUVND - *HUEHULFN *) .HUQ 3   $
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*UDKDP & +DX[ & .ROOH 61 .UHLOLQJ 5 1DWVFK $ 0HKOLQJ $   (YDOXDWLQJ WKHVHQVLWL]DWLRQSRWHQWLDORIVXUIDFWDQWVLQWHJUDWLQJGDWDIURPWKHORFDOO\PSKQRGHDVVD\JXLQHDSLJ
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 %DXFK&.ROOH615DPLUH]7(OW]H7)DELDQ(0HKOLQJ$7HXEQHU:YDQ5DYHQ]ZDD\%/DQGVLHGHO5  3XWWLQJWKHSDUWVWRJHWKHUFRPELQLQJLQYLWURPHWKRGVWRWHVWIRUVNLQVHQVLWL]LQJSRWHQWLDOV5HJXO7R[LFRO3KDUPDFRO
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36

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OECD/OCDETG 442D

 5\DQ&$*LOGHD/$+XOHWWH%&'HDUPDQ5-.LPEHU,$QG*HUEHULFN*)  *HQH
H[SUHVVLQJ FKDQJHV LQ SHULSKHUDO EORRGGHULYHG GHQGULWLF FHOOV IROORZLQJ H[SRVXUH WR D FRQWDFWDOOHUJHQ7R[LFRO/HWW
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3HVWLFLGHV ± 4XDQWLWDWLYH  6WUXFWXUH $FWLYLW\ 5HODWLRQVKLS 4 6$5  *XLGDQFH 'RFXPHQWSS'LVSRQLEOHjO DGUHVVHVXLYDQWHKWWSZZZHSDJRYRSSIHDGLQWHUQDWLRQDOQDIWDWZJJXLGDQFHTVDUJXLGDQFHSGI© OECD, (2015)
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37

TG 442D

OECD/OCDEANNEX 1DEFINITIONS
Accuracy:7KHFORVHQHVVRIDJUHHPHQWEHWZHHQWHVWPHWKRGUHVXOWVDQGDFFHSWHGUHIHUHQFHYDOXHV,WLVD
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AOP (Adverse Outcome Pathway): VHTXHQFHRIHYHQWVIURPWKHFKHPLFDOVWUXFWXUHRIDWDUJHWFKHPLFDO
RUJURXSRIVLPLODUFKHPLFDOVWKURXJKWKHPROHFXODULQLWLDWLQJHYHQWWRDQin vivoRXWFRPHRILQWHUHVW  ARE: $QWLR[LGDQW UHVSRQVH HOHPHQW DOVR FDOOHG (S5( HOHFWURSKLOH UHVSRQVH HOHPHQW  LV D UHVSRQVH
HOHPHQWIRXQGLQWKHXSVWUHDPSURPRWHUUHJLRQRIPDQ\F\WRSURWHFWLYHDQGSKDVH,,JHQHV:KHQDFWLYDWHG
E\1IULWPHGLDWHVWKHWUDQVFULSWLRQDOLQGXFWLRQRIWKHVHJHQHV
Coefficient of variation: D PHDVXUH RI YDULDELOLW\ WKDW LV FDOFXODWHG IRU D JURXS RI UHSOLFDWH GDWD E\GLYLGLQJWKHVWDQGDUGGHYLDWLRQE\WKHPHDQ,WFDQEHPXOWLSOLHGE\IRUH[SUHVVLRQDVDSHUFHQWDJH
EC1.5: ,QWHUSRODWHGFRQFHQWUDWLRQIRUDIROGOXFLIHUDVHLQGXFWLRQIC30: &RQFHQWUDWLRQHIIHFWLQJDUHGXFWLRQRIFHOOXODUYLDELOLW\E\
IC50: &RQFHQWUDWLRQHIIHFWLQJDUHGXFWLRQRIFHOOXODUYLDELOLW\E\
Hazard: ,QKHUHQWSURSHUW\RIDQDJHQWRUVLWXDWLRQKDYLQJWKHSRWHQWLDOWRFDXVHDGYHUVHHIIHFWVZKHQDQ
RUJDQLVPV\VWHPRU VXE SRSXODWLRQLVH[SRVHGWRWKDWDJHQW
IATA (Integrated Approach to Testing and Assessment): $ VWUXFWXUHG DSSURDFK XVHG IRU KD]DUG
LGHQWLILFDWLRQ SRWHQWLDO KD]DUGFKDUDFWHULVDWLRQ SRWHQF\ DQGRUVDIHW\DVVHVVPHQW SRWHQWLDOSRWHQF\DQG
H[SRVXUH RIDFKHPLFDORUJURXSRIFKHPLFDOVZKLFKVWUDWHJLFDOO\LQWHJUDWHVDQGZHLJKWVDOOUHOHYDQWGDWD
WRLQIRUPUHJXODWRU\GHFLVLRQUHJDUGLQJSRWHQWLDOKD]DUGDQGRUULVNDQGRUWKHQHHGIRUIXUWKHUWDUJHWHGDQG
WKHUHIRUHPLQLPDOWHVWLQJ
Imax: 0D[LPDO LQGXFWLRQ IDFWRU RI OXFLIHUDVH DFWLYLW\ FRPSDUHG WR WKH VROYHQW QHJDWLYH  FRQWUROPHDVXUHGDWDQ\WHVWFKHPLFDOFRQFHQWUDWLRQKeap1: .HOFKOLNH(&+DVVRFLDWHGSURWHLQLVDVHQVRUSURWHLQWKDWFDQUHJXODWHWKH1UIDFWLYLW\8QGHU
XQLQGXFHG FRQGLWLRQV WKH .HDS VHQVRU SURWHLQ WDUJHWV WKH 1UI WUDQVFULSWLRQ IDFWRU IRU XELTXLWLQ\ODWLRQ
DQGSURWHRO\WLFGHJUDGDWLRQLQWKHSURWHDVRPH&RYDOHQWPRGLILFDWLRQRIWKHUHDFWLYHF\VWHLQHUHVLGXHVRI.HDSE\VPDOOPROHFXOHVFDQOHDGWRGLVVRFLDWLRQRI1UIIURP.HDS      
Mixture: $PL[WXUHRUDVROXWLRQFRPSRVHGRIWZRRUPRUHVXEVWDQFHVLQZKLFKWKH\GRQRWUHDFW  © OECD, (2015)

38



OECD/OCDETG 442D

Mono-constituent substance: $ VXEVWDQFH GHILQHG E\ LWV TXDQWLWDWLYH FRPSRVLWLRQ LQ ZKLFK RQH PDLQFRQVWLWXHQWLVSUHVHQWWRDWOHDVW ZZ 
Multi-constituent substance: $ VXEVWDQFH GHILQHG E\ LWV TXDQWLWDWLYH FRPSRVLWLRQ LQ ZKLFK PRUH WKDQ
RQH PDLQ FRQVWLWXHQW LV SUHVHQW LQ D FRQFHQWUDWLRQ   ZZ  DQG   ZZ  $ PXOWLFRQVWLWXHQWVXEVWDQFHLVWKHUHVXOWRIDPDQXIDFWXULQJSURFHVV7KHGLIIHUHQFHEHWZHHQPL[WXUHDQGPXOWLFRQVWLWXHQWVXEVWDQFHLVWKDWDPL[WXUHLVREWDLQHGE\EOHQGLQJRIWZRRUPRUHVXEVWDQFHVZLWKRXWFKHPLFDOUHDFWLRQ$PXOWLFRQVWLWXHQWVXEVWDQFHLVWKHUHVXOWRIDFKHPLFDOUHDFWLRQ
Nrf2: QXFOHDU IDFWRU HU\WKURLGGHULYHG  OLNH  LV D WUDQVFULSWLRQ IDFWRU LQYROYHG LQ WKH DQWLR[LGDQWUHVSRQVHSDWKZD\:KHQ1UILVQRWXELTXLWLQ\ODWHGLWEXLOGVXSLQWKHF\WRSODVPDQGWUDQVORFDWHVLQWRWKH
QXFOHXV ZKHUH LW FRPELQHV WR WKH $5( LQ WKH XSVWUHDP SURPRWHU UHJLRQ RI PDQ\ F\WRSURWHFWLYH JHQHVLQLWLDWLQJWKHLUWUDQVFULSWLRQ      
Positive control: $ UHSOLFDWH FRQWDLQLQJ DOO FRPSRQHQWV RI D WHVW V\VWHP DQG WUHDWHG ZLWK D VXEVWDQFH
NQRZQWRLQGXFHDSRVLWLYHUHVSRQVH7RHQVXUHWKDWYDULDELOLW\LQWKHSRVLWLYHFRQWUROUHVSRQVHDFURVVWLPH
FDQEHDVVHVVHGWKHPDJQLWXGHRIWKHSRVLWLYHUHVSRQVHVKRXOGQRWEHH[FHVVLYH
Relevance: 'HVFULSWLRQRIUHODWLRQVKLSRIWKHWHVWWRWKHHIIHFWRILQWHUHVWDQGZKHWKHULWLVPHDQLQJIXODQG
XVHIXO IRU D SDUWLFXODU SXUSRVH ,W LV WKH H[WHQW WR ZKLFK WKH WHVW FRUUHFWO\ PHDVXUHV RU SUHGLFWV WKHELRORJLFDOHIIHFWRILQWHUHVW5HOHYDQFHLQFRUSRUDWHVFRQVLGHUDWLRQRIWKHDFFXUDF\ FRQFRUGDQFH RIDWHVWPHWKRG  
Reliability: 0HDVXUHVRIWKHH[WHQWWKDWDWHVWPHWKRGFDQEHSHUIRUPHGUHSURGXFLEO\ZLWKLQDQGEHWZHHQODERUDWRULHV RYHU WLPH ZKHQ SHUIRUPHG XVLQJ WKH VDPH SURWRFRO ,W LV DVVHVVHG E\ FDOFXODWLQJ LQWUD DQGLQWHUODERUDWRU\UHSURGXFLELOLW\DQGLQWUDODERUDWRU\UHSHDWDELOLW\  
Reproducibility: 7KHDJUHHPHQWDPRQJUHVXOWVREWDLQHGIURPWHVWLQJWKHVDPHVXEVWDQFHXVLQJWKHVDPH
WHVWSURWRFRO VHHUHOLDELOLW\   
Sensitivity: 7KH SURSRUWLRQ RI DOO SRVLWLYH  DFWLYH FKHPLFDOV WKDW DUH FRUUHFWO\ FODVVLILHG E\ WKH WHVWPHWKRG,WLVDPHDVXUHRIDFFXUDF\IRUDWHVWPHWKRGWKDWSURGXFHVFDWHJRULFDOUHVXOWVDQGLVDQLPSRUWDQWFRQVLGHUDWLRQLQDVVHVVLQJWKHUHOHYDQFHRIDWHVWPHWKRG  Solvent/vehicle control: $ UHSOLFDWH FRQWDLQLQJ DOO FRPSRQHQWV RI D WHVW V\VWHP H[FHSW RI WKH WHVWFKHPLFDOEXWLQFOXGLQJWKHVROYHQWWKDWLVXVHG,WLVXVHGWRHVWDEOLVKWKHEDVHOLQHUHVSRQVHIRUWKHVDPSOHVWUHDWHGZLWKWKHWHVWFKHPLFDOGLVVROYHGLQWKHVDPHVROYHQWSpecificity: 7KH SURSRUWLRQ RI DOO QHJDWLYH  LQDFWLYH FKHPLFDOV WKDW DUH FRUUHFWO\ FODVVLILHG E\ WKH WHVWPHWKRG,WLVDPHDVXUHRIDFFXUDF\IRUDWHVWPHWKRGWKDWSURGXFHVFDWHJRULFDOUHVXOWVDQGLVDQLPSRUWDQWFRQVLGHUDWLRQLQDVVHVVLQJWKHUHOHYDQFHRIDWHVWPHWKRG  
Substance: &KHPLFDO HOHPHQWV DQG WKHLU FRPSRXQGV LQ WKH QDWXUDO VWDWH RU REWDLQHG E\ DQ\ SURGXFWLRQSURFHVV LQFOXGLQJ DQ\ DGGLWLYH QHFHVVDU\ WR SUHVHUYH WKH VWDELOLW\ RI WKH SURGXFW DQG DQ\ LPSXULWLHV© OECD, (2015)




39

TG 442D

OECD/OCDE
GHULYLQJIURPWKHSURFHVVXVHGEXWH[FOXGLQJDQ\VROYHQWZKLFKPD\EHVHSDUDWHGZLWKRXWDIIHFWLQJWKH
VWDELOLW\RIWKHVXEVWDQFHRUFKDQJLQJLWVFRPSRVLWLRQ  
Test chemical: 7KHWHUPWHVWFKHPLFDOLVXVHGWRUHIHUWRZKDWLVEHLQJWHVWHG
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS): $ V\VWHP SURSRVLQJ WKH FODVVLILFDWLRQ RI FKHPLFDOV VXEVWDQFHV DQG PL[WXUHV  DFFRUGLQJ WR
VWDQGDUGLVHGW\SHVDQGOHYHOVRISK\VLFDOKHDOWKDQGHQYLURQPHQWDOKD]DUGVDQGDGGUHVVLQJFRUUHVSRQGLQJFRPPXQLFDWLRQ HOHPHQWV VXFK DV SLFWRJUDPV VLJQDO ZRUGV KD]DUG VWDWHPHQWV SUHFDXWLRQDU\ VWDWHPHQWV
DQGVDIHW\GDWDVKHHWVVRWKDWWRFRQYH\LQIRUPDWLRQRQWKHLUDGYHUVHHIIHFWVZLWKDYLHZWRSURWHFWSHRSOH
LQFOXGLQJ HPSOR\HUV ZRUNHUV WUDQVSRUWHUV FRQVXPHUV DQG HPHUJHQF\ UHVSRQGHUV  DQG WKH HQYLURQPHQW UVCB: VXEVWDQFHV RI XQNQRZQ RU YDULDEOH FRPSRVLWLRQ FRPSOH[ UHDFWLRQ SURGXFWV RU ELRORJLFDOPDWHULDOVValid test method: $ WHVW PHWKRG FRQVLGHUHG WR KDYH VXIILFLHQW UHOHYDQFH DQG UHOLDELOLW\ IRU D VSHFLILF
SXUSRVHDQGZKLFKLVEDVHGRQVFLHQWLILFDOO\VRXQGSULQFLSOHV$WHVWPHWKRGLVQHYHUYDOLGLQDQDEVROXWH
VHQVHEXWRQO\LQUHODWLRQWRDGHILQHGSXUSRVH  © OECD, (2015)

40



OECD/OCDETG 442D

ANNEX 2PROFICIENCY SUBSTANCESIn Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method
3ULRUWRURXWLQHXVHRIDWHVWPHWKRGWKDWDGKHUHVWRWKLV7HVW*XLGHOLQHODERUDWRULHVVKRXOGGHPRQVWUDWH
WHFKQLFDOSURILFLHQF\E\FRUUHFWO\REWDLQLQJWKHH[SHFWHG.HUDWLQR6HQVSUHGLFWLRQIRUWKH3URILFLHQF\
6XEVWDQFHV UHFRPPHQGHG LQ 7DEOH  DQG E\ REWDLQLQJ WKH (& DQG ,& YDOXHV WKDW IDOO ZLWKLQ WKHUHVSHFWLYHUHIHUHQFHUDQJHIRUDWOHDVWRXWRIWKHSURILFLHQF\VXEVWDQFHV7KHVH3URILFLHQF\6XEVWDQFHV
ZHUH VHOHFWHG WR UHSUHVHQW WKH UDQJH RI UHVSRQVHV IRU VNLQ VHQVLWLVDWLRQ KD]DUGV 2WKHU VHOHFWLRQ FULWHULD
ZHUHFRPPHUFLDODYDLODELOLW\DYDLODELOLW\RIKLJKTXDOLW\in vivoUHIHUHQFHDQGDYDLODELOLW\RIKLJKTXDOLW\in vitroGDWDIURPWKH.HUDWLQR6HQV70WHVWPHWKRG

7DEOH  5HFRPPHQGHG VXEVWDQFHV IRU GHPRQVWUDWLQJ WHFKQLFDO SURILFLHQF\ ZLWK WKH .HUDWLQR6HQV 70 WHVWPHWKRG
EC1.5 (μM ) IC50 (μM )Reference Reference
Range (3)Range (3)Proficiency Substances

CASRN

PhysicalForm

In Vivo
Prediction (1)KeratinoSensTM
Prediction (2),VRSURSDQRO

/LTXLG

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1HJDWLYH!

!

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!

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!

&LQQDP\ODOFRKRO

6ROLG

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3RVLWLYH

!



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3RVLWLYH

!

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

(WK\OHQH
GLPHWKDFU\ODWHJO\FRO 7KHin vivoKD]DUG DQGSRWHQF\ SUHGLFWLRQVDUHEDVHGRQ//1$GDWD  7KH in vivoSRWHQF\LVGHULYHGXVLQJWKHFULWHULDSURSRVHGE\(&(72&   $ .HUDWLQR6HQV70 SUHGLFWLRQ VKRXOG EH FRQVLGHUHG LQ WKH IUDPHZRUN RI DQ ,$7$ DQG LQ DFFRUGDQFH ZLWK WKH SURYLVLRQV RI
SDUDJUDSKVDQGRIWKH7HVW*XLGHOLQH %DVHGRQWKHKLVWRULFDOREVHUYHGYDOXHV  

© OECD, (2015)




41

TG 442D

OECD/OCDEANNEX 3QUALITY CONTROL OF LUMINESCENCE MEASUREMENTS
Basic experiment for ensuring optimal luminescence measurements in the KeratinoSensTM assay
7KHIROORZLQJWKUHHSDUDPHWHUVDUHFULWLFDOWRHQVXUHREWDLQLQJUHOLDEOHUHVXOWVZLWKWKHOXPLQRPHWHU KDYLQJDVXIILFLHQWVHQVLWLYLW\JLYLQJDVWDEOHEDFNJURXQGLQFRQWUROZHOOV KDYLQJQRJUDGLHQWRYHUWKHSODWHGXHWRORQJUHDGLQJWLPHVDQG
 KDYLQJQROLJKWFRQWDPLQDWLRQLQDGMDFHQWZHOOVIURPVWURQJO\DFWLYHZHOOV
3ULRUWRWHVWLQJLWLVUHFRPPHQGHGWRHQVXUHKDYLQJDSSURSULDWHOXPLQHVFHQFHPHDVXUHPHQWVE\WHVWLQJDFRQWUROSODWHVHWXSDVGHVFULEHGEHORZ WULSOLFDWHDQDO\VLV 
Plate setup of first training experiment
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D

EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA EGDMA
0.98
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(*'0$ (WK\OHQHJO\FROGLPHWKDFU\ODWH &$61R DVWURQJO\LQGXFLQJFRPSRXQG&$ &LQQDPLFDOGHK\GHSRVLWLYHUHIHUHQFH &$61R The quality control analysis should demonstrate: DFOHDUGRVHUHVSRQVHLQURZ'ZLWKWKH,PD[!IROGDERYHEDFNJURXQG LQPRVWFDVHV,PD[YDOXHVEHWZHHQDQGDUHUHDFKHG  QR GRVHUHVSRQVH LQ URZ & DQG ( QR LQGXFWLRQ YDOXH DERYH  LGHDOO\ QRW DERYH   GXH WR
SRVVLEOHOLJKWFRQWDPLQDWLRQHVSHFLDOO\QH[WWRVWURQJO\DFWLYHZHOOVLQWKH(*'0$URZ
 QRVWDWLVWLFDOO\VLJQLILFDQWGLIIHUHQFHEHWZHHQWKHURZV$%&()DQG* LHQRJUDGLHQWRYHU
SODWH DQG
 YDULDELOLW\ LQ DQ\ RI WKH URZV $ % & ( ) DQG * DQG LQ WKH '062 ZHOOV LQURZ + VKRXOG EH
EHORZ LHVWDEOHEDFNJURXQG © OECD, (2015)

42



EURL ECVAM Recommendation on the
KeratinoSensTM assay for skinsensitisation testing

January 2014

October 2013

Report EUR 25946 EN

43European Commission
Joint Research Centre
Institute for Health and Consumer Protection (IHCP)
Contact information
European Reference Laboratory for Alternatives to Animal Testing
Address: Joint Research Centre, Via Enrico Fermi 2749, 21027 Ispra (VA), Italy
E-mail: JRC-ECVAM-CONTACT@ec.europa.eu
http://ihcp.jrc.ec.europa.eu/
http://www.jrc.ec.europa.eu/
This publication is a Reference Report by the Joint Research Centre of the European Commission.
Legal Notice
Neither the European Commission nor any person acting on behalf of the Commission
is responsible for the use which might be made of this publication.
Europe Direct is a service to help you find answers to your questions about the European Union
Freephone number (*): 00 800 6 7 8 9 10 11(*) Certain mobile telephone operators do not allow access to 00 800 numbers or these calls may be billed.

A great deal of additional information on the European Union is available on the Internet.
It can be accessed through the Europa server http://europa.eu/.
JRC87551
EUR 26427 EN
ISBN 978-92-79-34795-5
ISSN 1831-9424
Doi 10.2788/52914
Luxembourg: Publications Office of the European Union, 2013
© European Union, 2013
Reproduction is authorized provided the source is acknowledged.
Printed in Ispra

44

EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)EURL ECVAM RECOMMENDATION
on the KeratinoSensTM assay
for skin sensitisation testing

January 2014

145

ACKNOWLEDGEMENTS
This Recommendation was prepared by the EU Reference Laboratory for Alternatives to Animal Testing(EURL ECVAM), part of the Institute for Health and Consumer Protection (IHCP), Directorate-General
Joint Research Centre (DG JRC) of the European Commission.
The Recommendation was drafted on the basis of the ESAC Opinion and ESAC Working Group Reportoutlining the detailed scientific peer review of the Givaudan-coordinated study on the KeratinoSensTM
assay. The Recommendation further benefitted from comments and suggestions received frommembers of PARERE (EURL ECVAM's advisory body for Preliminary Assessment of Regulatory Relevancethat brings together representatives of Member State regulatory bodies as well as EU agencies including
ECHA, EFSA and EMA), and ESTAF (EURL ECVAM's Stakeholder Forum). Input was also provided bypartner organisations of EURL ECVAM in the framework of the International Collaboration on
Alternative Test Methods (ICATM), and by the general public.
Coordinator of the evaluation of the test submission was Silvia Casati. Coordinator of the ESAC Peer
Review and EURL ECVAM Recommendation was Claudius Griesinger.

Comments or enquiries related to this EURL ECVAM Recommendation should be sent to:
EU Reference Laboratory for Alternatives to Animal Testing
European Commission DG Joint Research Centre,
Institute for Health and Consumer Protection,
Via E. Fermi 2749,
I-21027 Ispra (VA), Italy.
e-mail: JRC-ECVAM-CONTACT@ec.europa.eu

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TABLE OF CONTENTSBACKGROUND TO EURL ECVAM RECOMMENDATIONS ............................................................. 3
EXECUTIVE SUMMARY ............................................................................................................. 4
1. Introduction ......................................................................................................................... 6
2. Test Method definition ........................................................................................................ 7
3. Overall Performance of the KeratinoSensTM test method ..................................................... 8
4. Limitations ........................................................................................................................... 9
5. Suggested regulatory use ................................................................................................... 11
6. Follow-up activities recommended by EURL ECVAM .......................................................... 11
7. Proprietary aspects ............................................................................................................ 12
References ............................................................................................................................. 13
Annex 1 ESAC OPINION ...................................................................................................... 17
Annex 2 EURL ECVAM request for ESAC advice ................................................................... 26

BACKGROUND TO EURL ECVAM RECOMMENDATIONS
The aim of a EURL ECVAM Recommendation is to provide EURL ECVAM views on the validity of the test
method in question, to advise on possible regulatory applicability, limitations and proper scientific useof the test method, and to suggest possible follow-up activities in view of addressing knowledge gaps.
During the development of its Recommendations, EURL ECVAM consults with its consultation body for
Preliminary Assessment of Regulatory Relevance (PARERE) and its EURL ECVAM Stakeholder Forum(ESTAF). Moreover, EURL ECVAM consults with other Commission services and its international
validation partner organisations of the International Cooperation on Alternative Test Methods (ICATM).
Before finalising its recommendations, EURL ECVAM also invites comments from the general public and,if applicable, from the test method submitter.

3
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EXECUTIVE SUMMARYThe KeratinoSensTM in vitro test method for skin sensitisation testing has been developed by Givaudan, aproducer of fragrances and flavours. From 2009 to 2010 Givaudan coordinated a validation study on the
KeratinoSensTM test method, focusing on its transferability and reproducibility. Following submission to
EURL ECVAM of the study data as well as supplementary information, EURL ECVAM charged ESAC toreview the KeratinoSensTM validation study which it finalised in December 2012. EURL ECVAM endorses
the conclusions of the ESAC opinion (Annex I) on the Givaudan-coordinated study and makes thefollowing recommendations.
(1) The Keap1-Nrf2-ARE pathway is considered a major regulator of cyto-protective responses toelectrophile and oxidative stress by controlling the expression of detoxification, antioxidant andstress response enzymes and proteins. Since the majority of chemical skin sensitisers areelectrophiles reacting with nucleophilic centres in skin proteins, the pathway is one relevant readout
for skin sensitisation (OECD, 2012).
(2) Since activation of the Keap1-Nrf2-ARE pathway addresses only one single biological mechanism, it
is likely that information from test methods based on this or similar pathways will not be sufficient
to conclude on the skin sensitisation potential of chemicals. Therefore the KeratinoSensTM assay
should not be considered a stand-alone full replacement method and data generated with the test
method should always be considered in the context of integrated approaches, e.g. Weight-ofEvidence (WoE) or Integrated Testing Strategies (ITS), combining them with complementary
information derived from in vitro assays addressing other key events of skin sensitisation (e.g. inchemico reactivity assays such as the Direct Peptide Reactivity Assay) as well as non-testing methods
including read-across from chemical analogues.
(3) Based on the data generated in the study, the KeratinoSensTM test method proved to be transferable
to laboratories experienced in cell culture and reproducible within- and between-laboratories (86%concordance in both cases).
(4) The Givaudan-coordinated validation study generated preliminary information on the test method's
predictive capacity and it was found that the accuracy of the KeratinoSensTM to discriminate skinsensitisers from non-sensitisers was 90% (sensitivity 87%, specificity 100%; n=21)1. The accuracy
calculated for an additional set of chemicals (77 sensitisers and 104 non-sensitisers) tested in-house
by Givaudan was 75%. These figures are similar to those recently published by Natsch et al. (2013)
based on in-house testing of about 145 chemicals (77% accuracy, 79% sensitivity, 72% specificity).
Taken together, this information indicates the usefulness of the KeratinoSensTM assay to contributeto the identification of sensitisers and non-sensitisers.
(5) The KeratinoSensTM assay also provides concentration-response information that may contribute to
the assessment of sensitising potency as recently proposed by Jaworska et al. (2013). Further work
is required to determine to which extent KeratinoSensTM results relate to potency categories basedon, preferentially, human data.

1

N.B. The values presented here differ from those presented in the ESAC WG report. The Givaudan submission to EURL ECVAM contained dataof the Givaudan-coordinated validation study plus data from in-house testing produced under non-validation conditions (e.g. no blind
testing). While the ESAC WG calculated the predictive capacity on the basis of all data points irrespective of how they had been
generated, the values presented above have been calculated a) on the basis of the validation study and b) on the basis of the additional
non-validation data. This ensures a consistent approach with regard to the presentation of the predictive capacity of other skinsensitisation test methods summarised in EURL ECVAM Recommendations (e.g. DPRA), where the preliminary predictive capacity of theassays has been calculated on the basis of a small validation set and compared to the predictive capacity from additional information
generated in-house by test submitters under non-validation conditions.

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(6) To support the development of integrated approaches employing information from cyto-protective
signalling pathways such as Keap1-Nrf2-ARE, the applicability of the KeratinoSensTM and its
limitations should be further characterised. Based on the available data from the validation study
and in-house testing of the submitter, the KeratinoSensTM assay seems applicable to a wide range ofchemicals. Nevertheless, negative results should be interpreted with some caution due to (a) the
cysteine-dependent mechanism of activation of the signalling pathway; (b) although some prohaptens are reported to be correctly predicted, those requiring biotransformation by P450 enzymes
are not detected; (c) while a variety of pre-haptens are reported to be detected, pre-haptens with aslow oxidation rate may go undetected unless oxidised before the actual experiment (Givaudan,2011).
(7) The KeratinoSensTM test method can be considered as a valuable component of integrated
approaches for skin sensitisation testing although further work is required to fully understand its
limitations and to be specific about what complementary data would be desirable depending on theuse case. Furthermore, its capacity to contribute to subcategorisation of sensitisers according to UN
GHS (UN, 2007) and to potency assessment needs to be defined, the latter preferentially on thebasis of human reference data.
(8) Respecting the provisions of Directive 2010/63/EU (EU, 2010) on the protection of animals used forscientific purposes, before embarking on animal experiments to identify substances with skinsensitisation potential, data from the KeratinoSensTM test method should be considered in
combination with complementary information in order to reduce and possibly avoid animal testing.
As provided for in Annex XI (point 1.2) of the REACH Regulation (EC, 2006), data from non-standard
testing methods, such as the KeratinoSensTM, may be used to adapt the standard information
requirement in the context of Weight-of-Evidence (WoE) judgments.

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1. Introduction
1) The assessment of skin sensitisation potential is an important component in the safety evaluation ofsubstances and represents a standard information requirement of legislation on chemicals in the
EU. These include: the Classification Labelling and Packaging of substances and mixtures (CLP)
Regulation (EC, 2008a), the REACH Regulation, the Plant Protection Products (PPP) Regulation (EC,2009a), the Biocides Directive (EC, 2012) and the Cosmetics Regulation (EC, 2009b). Determining
skin sensitisation hazard in terms of GHS classification is actually sufficient to satisfy the majority of
regulatory needs (EURL ECVAM, 2013). However, a more complete characterisation of the potencyof a skin sensitiser with regard to both induction as well as elicitation of contact dermatitis is often
required for a full risk assessment and the definition of appropriate risk management measures(e.g. setting of appropriate thresholds).
2) Currently only in vivo test methods are accepted by regulatory bodies for the generation of datasatisfying regulatory requirements on skin sensitisation. For instance, in the frameworks of theOrganisation for Economic Cooperation and Development (OECD) and the EU Test Methods
Regulation (EC, 2008b), there are four accepted guidelines, describing: the Buehler Test andGuinea-pig Maximisation Test, TG406 (OECD, 1992; EU test method B.6), the Local Lymph Node
Assay, TG429 (OECD, 2010a; EU test method B.42) and its non-radio-isotopic variants, the Local
Lymph Node Assay: DA (TG 442A; OECD, 2010b) and the Local Lymph Node Assay: BrdU Elisa (TG
442B; OECD, 2010c).
3) The key mechanistic events underpinning the skin sensitisation process that leads to Allergic Contact
Dermatitis (ACD) in humans have been identified and recently summarised in the OECD report on“The Adverse Outcome Pathway (AOP) for Skin Sensitisation Initiated by Covalent Binding toProteins“(OECD, 2012). These key events include 1) the covalent binding of the chemical to the skinprotein (haptenation), 2) the release of pro-inflammatory cytokines and the induction of cytoprotective pathways in keratinocytes 3) the maturation and mobilisation of dendritic cells (DC),immuno-competent cells in the skin, and 4) the antigen presentation to naïve T-cells andproliferation of memory T-cells. Considerable progress has been made in recent years towards thedevelopment of alternative non-animal methods that address these key mechanisms.
4) There is general agreement that it is unlikely that a single alternative method will be able to provide
sufficient information to fully replace the use of animals for this endpoint (Adler et al., 2011).
Instead it is held that information from different alternative testing and non-testing methods usedin combination will need to be integrated to address this health endpoint (Jowsey et al., 2006;
Adler et al., 2011). These methods should address different key events involved in skin sensitisation
thus covering the mechanistic complexity of this endpoint. Against this background, activities arebeing pursued by academia, industry and the European Commission to evaluate mechanisticallybased test methods that can contribute to skin sensitisation hazard identification andcharacterisation.
5) In May 2010, EURL ECVAM received a full submission reporting the experimental results generated
by five laboratories participating in a Givaudan-coordinated study for the evaluation of the protocol
transferability and the within- and between-laboratory reproducibility of the KeratinoSensTM in
vitro assay. Following the evaluation of the submitted information, EURL ECVAM judged that thewithin-laboratory reproducibility (WLR) was not sufficiently addressed to progress the study into
peer-review and requested Givaudan to generate additional experimental data on the WLR witheight coded chemicals provided by EURL ECVAM. In December 2010 EURL ECVAM received arevised full submission reporting the requested data plus results generated with an additional six
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chemicals, not previously tested with the KeratinoSensTM method. The revised full submission wascomplemented with supplementary information which included an updated analysis of the
KeratinoSensTM predictive capacity (PC) based on data from 47 chemicals in addition to the 67
chemicals originally considered for this purpose. Additional information on the PC of the method
for 80 non-sensitising chemicals with LLNA reference data was provided by Givaudan in the phaseof peer review. Besides the experimental data obtained with 21 coded chemicals in the Givaudancoordinated study, most of the information on the PC of the KeratinoSensTM has been generated inhouse by the test developer.
6) On the basis of the revised submission EURL ECVAM requested the ECVAM Scientific Advisory
Committee (ESAC) to provide an ESAC Opinion on the study and supportive information. The ESAC
Working Group (WG) "Skin Sensitisation", charged with reviewing validation studies on skinsensitisation test methods, was requested to prepare a detailed WG report (EURL ECVAM, 2012a)
on which basis ESAC adopted its Opinion (EURL ECVAM, 2012b; see Annex 1), endorsed on 17. 12.
2012.

2. Test Method definition
The important role of the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2) inpromoting the expression of genes coding for cyto-protective proteins (mainly phase 2 enzymes)
following electrophilic or oxidative stress is extensively described in the literature (e.g. Baird &Dinkova-Kistova, 2011; Kensler et al., 2007). The activity of Nrf2 is considered to be primarily
regulated by the cysteine-rich Keap1 sensor protein (Kelch-like ECH associated protein 1) althoughother signalling pathways are reported to be involved in its regulation (Baird & Dinkova-Kistova,2011). Under un-induced conditions the Keap1 protein targets the Nrf2 transcription factor for
ubiquitin-dependent proteasomal degradation (Itoh et al., 1999). It is proposed that covalentmodification of the cysteine residues in the Keap-1 protein by electrophiles/oxidants leads to thedissociation of the Keap-1 protein from the Nrf2 transcription factor and induces the translocationof the Nrf2 from the cytoplasm to the nucleus where it promotes the activation of cyto-protective
genes which have an antioxidant or electrophile response elements (AREs/EpREs) in their promoter
sequence (Itoh et al., 1997; Suzuki et al., 2013).
7) Although direct covalent binding to certain Keap1 cysteine residues is considered to be one of theplausible mechanisms through which the Keap1-Nrf2-ARE pathway is activated, other types ofmodifications of the protein, like oxidation or glutathionylation, are reported to be responsible for
its activation. In addition it is proposed that all electrophiles/oxidants may shift the redox balance ofthe cell through reaction with glutathione (GSH) which may in turn generate an oxidative burst able
to modify Keap1 cysteines (Holland & Fishbein, 2010).
8) As reviewed by Natsch (2010) there is increasing evidence that ARE-regulated genes are induced indifferent cell types after challenge with skin sensitisers. The relevance of the Keap1-Nrf2-ARE
regulatory pathway in the in vivo reaction to sensitisers was shown in studies with Nrf2 knockout
mice (Kim et al., 2008; El Ali et al., 2013; van der Veen et al., 2013 ).
9) The KeratinoSensTM test method is a reporter gene assay which uses an immortalised adherent cellline derived from an expanded clone of HaCaT human keratinocytes transfected with a selectable
plasmid. The plasmid contains the luciferase gene under the transcriptional control of the SV40
promoter fused with the ARE from the AKR1C2 gene which was identified as one of the genes upregulated by contact sensitisers in dendritic cells (Gildea et al., 2006; Ryan et al., 2004). This allows
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to quantitatively measure (by luminescence detection) luciferase gene induction, using wellestablished light producing luciferase substrates, as an indicator of the activity of the Nrf2
transcription factor in cells following exposure to electrophilic chemicals.
10) At present, the KeratinoSensTM test method is designed for the identification of sensitisers/nonsensitisers. Chemicals are classified as sensitisers if they induce a statistically significant induction of
the luciferase gene above a given threshold in two out of three experiments performed on different
days. This is established in parallel to cytotoxicity measurements to assess gene induction levels at
sub-cytotoxic concentrations. Since cells are exposed to 12 concentrations of the test chemicals, theconcentration needed for a statistically significant luciferase gene induction above the threshold(EC1.5 value) can be extrapolated from the dose response curve. In addition, the maximal foldinduction of the luciferase gene over solvent control (Imax) is determined.
11) As a result of the Givaudan-coordinated study (Natsch et al., 2011) and additional information
provided in the submission to EURL ECVAM, the standardised protocol was found to betransferable (to laboratories with cell culture technique experience) and reproducible within andbetween laboratories.
12) EURL ECVAM will disseminate a comprehensive description of the KeratinoSensTM method throughits database on alternative methods (DB-ALM, at http://ecvam-dbalm.jrc.ec.europa.eu; protocol
No. 155), together with all the necessary technical details (e.g. electronic data reporting formats)needed by an end-user laboratory to implement it in a reliable and self-sufficient manner.

3. Overall Performance of the KeratinoSensTM test method
Reference data
13) Reference classifications associated with the test chemicals were selected on the basis of a weightof evidence approach considering different data, i.e. the murine Local Lymph Node Assay (LLNA),
the Guinea Pig Maximisation Test (GPMT) and, where available, human data. Reference chemicals
from the LLNA performance standards (OECD, 2010a) were included in the chemical set. Additional
details can be found in the submission (Givaudan, 2011).
When interpreting the data of alternative methods, such as the KeratinoSensTM that have been
largely developed and validated using animal reference data such as LLNA or GPMT, it should be
kept in mind that the predictive relevance of these animal tests may not fully reflect the situationin the species of interest, i.e. humans. Notably, an evaluation of the LLNA in comparison to humandata has shown an accuracy of about 72% (Anderson et al., 2011), i.e. there is a risk of false
negative and false positive results. Moreover there is indication that the LLNA is deficient in
detecting low to moderate sensitisers as well as metals and organometal compounds (EC, 2000).
Transferability14) EURL ECVAM concludes that the KeratinoSensTM test method is transferable to laboratories
sufficiently experienced in cell culture techniques. Since stable background levels of the luciferase
gene are critical for the generation of reliable results, EURL ECVAM recommends that a number oftraining experiments, as described in the Standard Operating Procedure (SOP), be performed by
852new laboratories to ensure optimal luminescence measurements before the test method is used forroutine testing.
Reproducibility15) For the set of coded chemicals tested during the validation study, the KeratinoSensTM protocol
yielded concordant predictions within the Givaudan laboratory (86%; N=14) and between the fivelaboratories participating in the ring trial (86%, N=21).
Predictive Capacity
16) The accuracy of the test method in predicting the in vivo classification (sensitiser/non-sensitiser)
determined on the basis of existing evidence from LLNA, GPMT, Buehler Test and human data forthe 21 (coded) chemicals evaluated in the validation study was 90% (sensitivity 87%, specificity100%). However, since the chemicals selected by Givaudan to be used in the validation study havealready been used to develop and optimise the KeratinoSensTM prediction model, it is likely thatthese values reflect a best-case scenario. When calculating the predictive capacity on the basis of alarger set of data generated in-house by Givaudan, sensitivity and specificity are about 75% (n=77
sensitisers and 104 non-sensitisers). A recently published study correlating KeratinoSensTM data withclassifications in the LLNA reported an accuracy of 77% (sensitivity 79% and specificity 72%) for a setof 145 chemicals (Natsch et al., 2013). Thus, it is plausible that these figures might reflect the actual
performance of the test in discriminating between sensitisers and non-sensitisers.

4. Limitations
4.1 Technical limitations17) Solubility of test substances: Chemicals which are not soluble in either water or DMSO, being these
the two solvents prescribed by the SOP, cannot be tested in the KeratinoSensTM. Chemicals with acalculated octanol/water partition coefficient (cLogP) up to 5 were reported by the test developer tobe successfully tested with the method.
18) Solvent effects: As with many in vitro/in chemico assays, chemicals which are not stable in theprescribed solvents because of hydrolysis or other chemical reactions cannot be reliably tested.
4.2 Limitations with regard to applicability – negative results19) As the key mechanism leading to the activation of the Keap1-Nrf2-ARE pathway appears to be theelectrophilic reaction of stressors with nucleophilic thiols (cysteine sulfhydryl groups) of Keap-1 it ispossible that skin sensitising chemicals with selective reactivity towards other nucleophiles may not
be reliably identified by the KeratinoSensTM (e.g. amine reactive chemicals preferentially reactingwith lysine residues), thereby leading to false negative results. However, there is scientific evidence
that the pathway can be activated by other types of modification of Keap-1 cysteine residues, suchas oxidation or conjugation with glutathione, and that, moreover, the Nrf2 transcription factor may
be controlled by other signaling pathways. It is therefore plausible that sensitising chemicals notcovalently modifying Keap-1 cysteine residues (e.g. amine-reactive chemicals) can neverthelessactivate the Nrf2 pathway, leading to true positive responses in the KeratinoSensTM assay.
953

Complementary information from peptide reactivity assays may help addressing this uncertainty, inparticular assays able to distinguish between cysteine and lysine reactivity.
20) While a number of pro-haptens requiring enzymatic oxidation or deamination are reported to becorrectly classified by the KeratinoSensTM, pro-haptens requiring P450 activation are reported not tobe identified by the assay. According to the test developer, attempts to incorporate a metabolic
system in the KeratinoSensTM assay have recently been published (Natsch & Haupt, 2013).
21) A variety of pre-haptens have been reported as correctly predicted by the assay (e.g. 1,4phenylenediamine, hydroquinone and isoeugenol). However, some pre-haptens reported to have aslower rate of spontaneous oxidation (e.g. limonene) may require an oxidation step before theactual experiment.
22) Most of the misclassifications generated by the KeratinoSensTM concerns chemicals that aremoderate and weak sensitisers in vivo (see ESAC WG report, page 31,), while the false negative rate
for strong sensitisers is lower. This should be kept in mind when interpreting negative results.
4.3 Limitations with regard to applicability – positive results23) Considering the pathway monitored (i.e. electrophilic / oxidative stress), chemicals that do not act
as sensitisers but are nevertheless chemical stressors may lead to false positive results in the
KeratinoSensTM test method. This could for example include reactive chemicals that cause dermal
corrosion / irritation without, however, being skin sensitisers. Nevertheless, it was shown that
irritating surfactants, which often are predicted positive in the LLNA, are negative in the
KeratinoSens (Ball et al., 2011, Emter et al., 2010).

1054

5. Suggested regulatory use24) Due to the complexity of the mechanisms underlying skin sensitisation, it is likely that informationfrom different methods (in silico, in chemico, in vitro) is needed to reduce or replace the need foranimal testing, both for hazard identification and potency characterisation purposes.
25) Based on the validation study results and other available information, the KeratinoSensTM appears to
be a reliable test method that provides information on the ability of a chemical to activate the Nrf2
electrophilic and oxidative-stress response signalling pathway which has been shown to be arelevant pathway in the induction of skin sensitisation as demonstrated by studies in Nrf2-knockout
mice (Kim et al., 2008; El Ali et al., 2013; van der Veen et al., 2013). Therefore, Nrf2–dependent
luciferase induction measurements in the KeratinoSensTM assay when combined with informationfrom other non-animal methods in the context of a Weight-of-Evidence (WoE) approach or
Integrated Testing Strategy (ITS) may provide useful information about the sensitisation potential ofchemicals. Taking into consideration the dose-response information generated by the assay, it isplausible that KeratinoSensTM data may also contribute to characterisation of skin sensitisation
potency within integrated approaches. The extent of information needed to complement a
KeratinoSensTM result will depend on the intended application (e.g. hazard identification,
classification or potency assessment) and context (availability and quality of other information). An
example of the use of KeratinoSensTM data in a WoE approach for hazard assessment is published in
the scientific literature (Ball et al., 2011).
26) Notably, due to the nature of the pathway monitored (i.e. general electrophilic and oxidative stress),
KeratinoSensTM provides information on reactivity of chemicals that elicit protective stressresponses in exposed cells. Such data may be relevant for other health endpoints such as, forexample, dermal irritation and cancer (Reuter et al., 2010, Kansanen et al., 2013).
27) As outlined in more detail in section 4.2, negative KeratinoSensTM results should be interpreted withcare, taking into due consideration the possibility of false negatives due to (1) possible selective
reactivity of the chemical with amino acids other than cysteine, (2) the limited metabolic capacity of
the assay leading to possible misclassification of pro-haptens (especially those requiring
biotransformation by P450 enzymes), (3) the uncertain capacity to identify pre-haptens, (4) the
uncertain capacity to correctly identify moderate and especially weak sensitisers.
28) Chemicals able to activate the Keap1-Nrf2-ARE pathway by other mechanisms than covalent binding
to the Keap-1 cysteine residues may give false positive results in the KeratinoSensTM (see section
4.3).
29) Employed within an integrated approach, the KeratinoSensTM may be useful to satisfy information
requirements for Cosmetics (Regulation EC/1223/2009), Chemicals (Regulation EC/1907/2006),
Biocides (Regulation EC/528/2012) and Plant Protection Products (Regulation EC/1107/2009).

6. Follow-up activities recommended by EURL ECVAM(1) In view of further prospective testing with the KeratinoSensTM method, EURL ECVAM
recommends that the revised protocol available at EURL ECVAM's DB-ALM service(http://ecvam-dbalm.jrc.ec.europa.eu) be used: [DB-ALM protocol on KeratinoSensTM No. 155].
(2) Further testing should investigate possible limitations of the assay that relate to the cellularpathway chosen and the need for abiotic or biotic activation of some sensitisers (i.e. pre- and1155

pro-haptens). Moreover, since there is at present limited information on the applicability of the
KeratinoSensTM to chemical mixtures including plant extracts (Andres et al., 2013), additional
data may be helpful.
(3) Predictive capacity, applicability and limitations of the assay should be further evaluated in thecontext of its use as part of integrated approaches to testing and assessment. When doing so,
the limitations of available reference data e.g. from LLNA (EC, 2000) with regard to
reproducibility and relevance to the human situation should be however kept in mind. In
particular, the capability of the method to detect accurately weak and moderate skin sensitisers
should be further investigated.
(4) Further attention should be given to: (a) an evaluation of the possible contribution of
KeratinoSensTM data to sub-categorisation of sensitisers according to GHS (i.e. sub category 1A
and 1B); (b) an evaluation of whether and how the dose-response information generated by theassay could contribute to potency assessment allowing quantitative risk assessment. For such
evaluation, the use of human reference data will be particularly useful.
(5) Considering the limitations of the assay, integrated approaches using Nrf2–dependent luciferase
induction measurements should also make use of other information sources, in particularpeptide reactivity assays able to distinguish between cysteine and lysine reactivity. In addition,in silico methods (expert systems and QSAR models) may prove useful. In silico methods that
explicitly incorporate metabolic considerations (e.g. TIMES-SS: Patlewicz et al., 2007) may help
to identify pre- and pro-haptens. Analogues which have a similarly predicted mechanism ofaction, based on protein binding, can be found using the OECD QSAR Toolbox(www.qsartoolbox.org). The Toolbox also includes a specific profiler for the KeratinoSens TM
assay. A variety of proposals concerning the use of KeratinoSensTM data in combination withother information sources have been published and may support further work (Natsch et al.,2009; Bauch et al., 2012; Jaworska et al., 2013).
(6) EURL ECVAM supports the development of an OECD Test Guideline for the KeratinoSens TM. As
this test may be best employed in combination with complementary methods, it should beconsidered in the current initiative being undertaken at OECD to develop a guidance document
on Integrated Approaches for Testing and Assessment (IATA) for skin sensitization.
(7) Since the assay is amenable for automation, the development of an automated version of the
protocol is recommended.
(8) As the assay addresses a key signaling pathway of cyto-protective responses following
electrophilic and oxidative stress, the relevance of the test system for assessing othertoxicological endpoints should be considered.

7. PROPRIETARY ASPECTS
The 'KeratinoSens' name is a trade mark of the test method developer (Givaudan SA, Switzerland). EURLECVAM has received confirmation from Givaudan that the Keratinosens™ test method will be madeavailable to third parties subject to specific conditions including a one-time transfer fee.

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EC, 2006. Regulation (EC) No 1907/2006 of the European Parliament and of the Council of 18 December
2006 concerning the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH),
establishing a European Chemicals Agency, amending Directive 1999/45/EC and repealing Council
Regulation (EEC) No 793/93 and Commission Regulation (EC) No 1488/94 as well as Council Directive
76/769/EEC and Commission Directives 91/155/EEC, 93/67/EEC, 93/105/EC and 2000/21/EC. Official
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Journal No L 353, p. 0001-1355.
EC, 2008b. Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant toRegulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH). Official Journal L 142 , 31/05/2008 P.
0001 – 0739.

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EC, 2009a. Regulation (EC) No 1107/2009 of the European Parliament and of the Council of 21 October
2009 concerning the placing of plant protection products on the market and repealing Council Directives
79/117/EEC and 91/414/EEC Official Journal No L 309, p. 01-50.
EC, 2009b. Regulation (EC) No 1223/2009 of the European Parliament and of the Council of 30
November 2009 on cosmetic products. Official Journal No L 342, p. 01-59.
El Ali Z, Gerbeix C, Hemon P, Esser PR, Martin SF, Pallardy M, Kerdine-Römer S (2013) Allergic skininflammation induced by chemical sensitizers is controlled by the transcription factor Nrf2. Toxicol. Sci.,
134(1):39-48.
Emter R, Ellis G, Natsch A (2010) Performance of a novel keratinocyte-based reporter cell line to screen
skin sensitizers in vitro. Toxicol. Appl. Pharm., 245:281-290.
EU, 2010. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on
the protection of animals used for scientific purposes. Official Journal L 276, 20/10/2010 P. 0033 – 0079.
EU, 2012. Regulation (EU) No 528/2012 of the European Parliament and of the Council of 22 May 2012
concerning the making available on the market and use of biocidal products. Official Journal No L167,
p.01-123.
EURL ECVAM (2012a) ESAC Working Group Peer Review Consensus Report on a Givaudan-coordinatedstudy transferability and reliability of the KeratinoSens assay for skin sensitisation testing. Online(XXXX):http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations
EURL ECVAM (2012b) ESAC Opinion on a Givaudan-coordinated study concerning transferability andreliability of the KeratinoSens assay for skin sensitisation testing. Online(XXXX):http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/eurl-ecvam-recommendations
EURL ECVAM (2013). EURL ECVAM Strategy for Replacement of Animal Testing for Skin Sensitisation
Hazard Identification and Classification. JRC Scientific and Policy Reports. Publication Office of theEuropean Union. http://publications.jrc.ec.europa.eu/repository/handle/111111111/27708
Gildea LA, Ryan CA, Foertsch LM, Kennedy JM, Dearman RJ, Kimber I, Gerberick GF (2006) Identificationof gene expression changes induced by chemical allergens in dendritic cells: opportunities for skinsensitization testing. J. Invest. Dermatol., 126:1813-1822.
Givaudan (2011). Test method submission to EURL ECVAM: KeratinoSensTM assay for the testing of skinsensitizers.
Holland R, Fishbein JC (2010) Chemistry of the cysteine sensors in Kelch-like ECH-associated protein 1.
Antioxid. Redox Signal., 17:1749-1761.
Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y, Oyake T, Hayashi N, Satoh K, Hatayama I,
Yamamoto M, Nabeshima Y (1997) An Nrf2/small Maf heterodimer mediates the induction of phase II
detoxifying enzyme genes through antioxidant response elements. Biochem. Biophys. Res. Commun.,
236(2):313-322.
Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD, Yamamoto M (1999) Keap1 represses
nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal
Neh2 domain. Genes Dev., 13:76-86.
Jaworska J, Dancik Y, Kern P, Gerberick F, Natsch A (2013) Bayesian integrated testing strategy to assess
skin sensitization potency: from theory to practice. J. Appl. Toxicol. Epub ahead of print.
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Jowsey IR, Basketter DA, Westmoreland C, Kimber I (2006) A future approach to measuring relative skinsensitising potency: a proposal. J. Appl. Toxicol., 26:341-350.
Kansanen E, Kuosmanen SM, Leinonen H, Levonen AL (2013) The Keap1-Nrf2 pathway: Mechanisms ofactivation and dysregulation in cancer. Redox Biol., 1(1):45-49.
Kensler TW, Wakabayashi N, Biswal S (2007) Cell survival responses to environmental stresses via theKeap1-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol., 47:89-116.
Kim HJ, Barajas B, Wang M, Nel AE (2008) Nrf2 activation by sulforaphane restores the age-related
decrease of T(H)1 immunity: role of dendritic cells. J. Allergy Clin. Immunol., 121:1255-1261.
Natsch A, Emter R, Ellis G (2009) Filling the concept with data: integrating data from different in vitro
and in silico assays on skin sensitizers to explore the battery approach for animal-free skin sensitization
testing. Toxicol. Sci., 107:106-121.
Natsch A (2010) The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers-functional
relevance and a hypothesis on innate reactions to skin sensitizers. Toxicol. Sci., 113:284-292.
Natsch A, Bauch C, Foertsch L, Gerberick F, Norman K, Hilberer A, Inglis H, Landsiedel R, Onken S, Reuter
H, Shepky A, Emter R (2011) The intra- and inter-laboratory reproducibility and predictivity of the
KeratinoSensTM assay to predict skin sensitizers in vitro: results of a ring-study in five laboratories.
Toxicol. In Vitro., 25:733-44.
Natsch A, Ryan CA, Foertsch L, Emter R, Jaworska J, Gerberick F, Kern P (2013) A dataset on 145
chemicals tested in alternative assays for skin sensitisation undergoing prevalidation. J. Appl. Toxicol.
Epub, ahead of print.
Natsch A, Haupt T (2013). Utility of rat liver S9 fractions to study skin-sensitizing prohaptens in a
modified KeratinoSens assay. Toxicol. Sci., 135(2):356-368.
OECD (1992) OECD Guideline for the Testing of Chemicals No. 406: Skin Sensitisation. Paris, France:
Organisation for Economic Cooperation and Development. Online at: http://www.oecdilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4-healtheffects_20745788
OECD (2010a) OECD Guideline for the Testing of Chemicals No. 429: Skin Sensitisation: Local Lymph
Node Assay. Paris, France: Organisation for Economic Cooperation and Development. Online
at:http://www.oecd-ilibrary.org/environment/oecd-guidelines-for-the-testing-of-chemicals-section-4
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OECD (2010b) OECD Guideline for the Testing of Chemicals No. 442a Skin Sensitisation: Local Lymph
Node Assay DA. Paris, France: Organisation for Economic Cooperation and Development. Online at:
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health-effects_20745788
OECD (2010c) OECD Guideline for the Testing of Chemicals No. 442b: Skin Sensitisation: Local Lymph
Node Assay BrdU-ELISA. Paris, France: Organisation for Economic Cooperation and Development. Online
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OECD (2012) The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding toProteins. Part 1: Scientific Evidence. Series on Testing and Assessment No. 168. Online at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/PA
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Patlewicz G, Dimitrov SD, Low LK, Kern PS, Dimitrova GD, Comber MIH, Aptula AO, Phillips RD, Niemela J,
Madsen C, Wedebye EB, Roberts DW, Bailey PT, Mekenyan OG (2007) TIMES-SS – A promising tool forthe assessment of skin sensitization hazard. A characterisation with respect to the OECD Validation
Principles for (Q)SARs. Reg. Toxicol. Pharmacol., 48:225–239.
Ryan CA, Gildea LA, Hulette BC, Dearman RJ, Kimber I, Gerberick GF (2004) Gene expression changes inperipheral blood-derived dendritic cells following exposure to a contact allergen. Toxicol. Lett., 150(3):301-316.
Reuter S, Gupta SC, Chaturvedi MM, Aggarwal BB (2010) Oxidative stress, inflammation, and cancer:
how are they linked? Free Radic. Biol. Med., 49(11):1603-1616.
Suzuki T, Motohashi H, Yamamoto M (2013) Toward clinical application of the Keap1-Nrf2 pathway.
Trends Pharmacol. Sci., 34:340-346.
UN, United Nations N (2011). Globally Harmonised System of Classification and Labelling of Chemicals(GHS). Part 3: Health and Environmental Hazards. New York, NY, USA, and Geneva, Switzerland. United
Nations Economic Commission for Europe. Online
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van der Veen JW, Gremmer ER, Vermeulen JP, van Loveren H, Ezendam J (2013) Induction of skinsensitization is augmented in Nrf2-deficient mice. Arch. Toxicol., 87(4):763-766.

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ESAC OPINION

Annex 1ESAC OPINION

Opinion of the EURL ECVAM Scientific Advisory Committee (ESAC) on a
Givaudan-coordinated study on the transferability and reliability of the
KeratinoSens assay for skin sensitisation testing.
Ispra, 07.11.2012

Summary of the ESAC Opinion
The ESAC was asked to provide an opinion on a Givaudan-led study assessing the transferability
and reproducibility (within- and between-laboratories) of the KeratinoSens (primary objective of
the study) in view of its possible future use as part of a non-animal testing strategy for skinsensitization. The ESAC was also asked to provide an opinion on the predictive capacity of thetest method.
A wealth of information about the test chemicals, and the assessment of with laboratory
reproducibility (WLR), transferability, between laboratory reproducibility (BLR) and predictive
capacity of the test were presented. Also the applicability domain of the test was addressed indetail. The evaluation by the ESAC WG was complicated by the lack of detail in the body of thereport and the excessive reliance on annexes.
On the basis of the submitted and additionally requested information, the ESAC came to thefollowing conclusions:
Test chemicals:
The 114 selected chemicals represented a sufficient number of materials, reasonable structural
diversity and a variety of sensitising potency classes. Pre- and pro-haptens were included.
Therefore, the selection of chemicals was considered sufficient to gain information on theapplicability domain and limitations of the test method.
The small number of non-sensitizers (N=4) in the list of additional chemicals (N=47) considered
eligible for assessing the predictive capacity of the test was supplemented with 80 chemicals
with negative LLNA data.
WLR (14 chemicals/1 laboratory):
The ESAC considers the level of concordance acceptable and in agreement with target values(85%) for WLR performance.
Transferability (7 chemicals/4 laboratories):
Concordant predictions between the lead laboratory and the 4 naive laboratories wereobtained, demonstrating that the test method can be transferred to naive laboratories that areexperienced with cell culture techniques.
BLR (21 chemicals/5 laboratories):
The predictions were concordant for the large majority of chemicals, demonstrating an
acceptable level of between laboratory reproducibility.
Predictive capacity:
The conclusions regarding the predictivity are sound. A positive point is that reference
information from several in vivo tests were used for comparison as opposed to a single assay
outcome. Since approximately 1 in 5 sensitizers are likely to be missed, the test method should
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be considered in the future as part of an integrated testing strategy and not as a standalone
assay.
Applicability domain:
In principle, the applicability domain of this method is limited to cysteine reactive chemicals.
However, the evidence indicates that the applicability domain is wider, so it would be advisable
to assess this further by testing additional chemicals. Specific amine reactivity and metabolic
activation are among the key issues that need to be addressed.

1.

Mandate of the ESAC

The opinion of ESAC should support ECVAM with respect to the evaluation of the validity statusof the test method and possible necessary further work required to characterize the test
method's performance (predictive capacity, applicability and limitations of the test method).
Moreover, based on the evaluation of the data submitted, the ESAC should provide advice on
the potential usefulness of the KeratinoSens test method within a testing strategy for skinsensitization testing.

2.

Detailed opinion of the ESAC

Following a request from ECVAM to ESAC for peer review of and scientific advice on an ECVAMcoordinated prevalidation study concerning the KeratinoSens assay, an ESAC Working Group(ESAC WG) was set up by ESAC. The ESAC WG was charged with conducting a detailed scientificpeer review the ECVAM study concerning the transferability and reliability of the KeratinoSens
assay.
The ESAC WG had been set up by the ESAC during its meeting on March 2011 (ESAC 34). Basisfor the scientific review was the ECVAM request to ESAC concerning a scientific review (ESAC
request ER2011-04).
The date for the opinion was set to be 4-5 October 2011 (ESAC 35). However, unclarities andinconsistencies in the report required clarification by the test submitter. Two WG requests weresent: 16.12.2011 and 08.02.2012. These extra steps resulted in substantial additional
information that had to be reviewed and caused a 1 year delay.
The ESAC WG conducted the peer review from December 2011 to April 2012. Two face-to-facemeetings were organized (December 2011, and February 2012), followed by two telephone
conferences (February and April 2012) and finalized by written procedure.
The WG was a presented a wealth of information about the test chemicals, and the assessment
of WLR, transferability, BLR and predictive capacity of the test. Also the applicability domain of
the test was addressed in detail.
The data and the flow of events would have been more transparent if the report had followed
the EURL-ECVAM guidance and reporting template more closely. It would have been very
helpful if the test submitters had formulated their own conclusions/opinions when referring to
any of the numerous attachments that had followed the report. By plane referral to theattachments, the WG had to figure out itself what was meant and how data had to beinterpreted.
The WG identified a number of unclarities and inconsistencies which added hurtles to theevaluation of the report, without explanations being provided.
Issues that needed clarification:x It was not clear why the applied statistical approach was chosen for the evaluation of
the test results.
x The test design was not clear.
Inconsistencies:
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ESAC OPINIONx

Data analysis apparently moved from a test result oriented (e.g. Imax, EC1.5) to a
prediction (S/NS) oriented approach.
x Test acceptance criteria changed over time without explanation as to why this wasintroduced.
x Acceptance criteria were not consistently applied.
x Chemicals that were used for test development and refinement were inappropriately
included in the assessment of the BLR and the predicitive capacity.
x The WG addressed these issues by requesting additional information and re-analysis of
the data from the test submitter (See Annexes).
------------------------------------------------------------------------------------------------------The provided information did not provide any clarity about the statistical approach applied in
the study. The WG decide not to go into further discussion, and to focus on the outcome of theprediction model (S/NS).
The test design was sufficiently clarified, and the data were re-analysed on the basis of thevarious identified test acceptance criteria. This allowed the WG to properly assess
reproducibility, transferability and predictive capacity.
The WG attempted to recalculate the predictive capacity of the KeratinoSens based upon thechemicals that had not been included in test development and refinement. Since the number ofwell-characterized non-sensitizers (i.e. chemicals with negative LLNA outcome) among theeligible chemicals was considered too low, the WG requested data on more negative
compounds.
-------------------------------------------------------------------------------------------------------

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On the basis of the submitted and upon request acquired information the WG came to thefollowing conclusion:
Test chemicals:
The 114 selected chemicals were representing a sufficient number of materials, reasonablestructural diversity and a variety of sensitising potency classes. Pre- and pro-haptens were
included. Therefore, the selection of chemicals was considered sufficient to gain information on
the applicability domain and limitation of the test method.
The number of test items was considered sufficient to draw conclusions about thetransferability (N=7) and reproducibility (N=21) of the test.
The small number of non-sensitizers (N=4) in the extended list of chemicals (N=47) for assessing
the predictive capacity of the test was considered too low. The 67 chemicals used fordevelopment, refinement and evaluation of the test were not taken into consideration forassessing the predictive capacity by the WG.
WLR (14 chemicals, 1 laboratory):
Including all available data concordant results were obtained for 12/14 chemicals (85.7%). The
WG endorsed the conclusion of the VMG that the test is reproducible with laboratories. WG
considered this concordance in agreement with target values (85%) for WLR performancestandards as published in international accepted guidelines (e.g. Performance standards of
TG439 in vitro skin irritation).
The ESAC WG agreed that the re-analysis that was resubmitted upon request (see section 6.1)
was satisfying with regard to answering the question to which extent non-qualified test results
might have influenced the WLR analysis. The impact on WLR was felt to be negligible as evenunder the most stringent criteria (set 2 in Annex 4, p60) only 3 individual laboratory predictions
had not qualified.
Transferability (7 chemicals, 4 laboratories):
The conclusion on transferability was justified on the basis of concordant predictions (S/NS)
between the lead laboratory and the naive laboratories. The WG endorses the conclusion thatthe test method can be transferred to naive laboratories that are experienced with cell culture
techniques.
Concerns were raised about the reliability of luciferase measurements for transferability.
Differences in brand of luminometer or substrate were demonstrated by the test submitters not
to affect the liability of luminescence measurement. Based on this fact, it seems obvious to the
WG that the observed variation in luminescence measurements between laboratories is due tolack of experience, stressing the necessity of operating a number of training experiments in thenaïve laboratory before the test method can be used to identify skin sensitizers.
Regarding dose-response curve or EC1.5 (Attachment 8a & 8b), certain variability among thelaboratories was observed to cinnamic aldehyde and ethylene glycol dimethacrylate. But, nofurther explanation was given whether these variabilities originated from the chemicals’ own
physico-chemical characteristics or luminescence measurement issues.

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BLR (21 chemicals, 5 laboratories):
The S/NS prediction gave congruent results for the majority of chemicals (85.7 – 90.5%), taking
into consideration the explanations give for the outliers, also between laboratories. (See section
6.1).
The test acceptance criteria provided to the participating laboratories during the ring trial had
not been applied consequently when analysing the data. The reason for this inconsistency wasthat the criteria were found too stringent. In contrast to WLR and transferability assessment,these nonqualified data had an effect on the concordance of predictions (Annex 4, p62 (C. 2)).
There were no provisions made for re-testing in case of nonqualified predictions.
Predictive capacity:
The conclusions regarding the predictivity are sound given the overall value of 76.6%, the key
here is that weight of evidence data were used for comparison as opposed to a single assay
outcome.
The WG was impressed by the wealth of information that was provided by the test submitter on
the 114 chemicals assessed in this study. Based upon the 114 chemicals included in the study,
the predicitive capacity of the KeratinoSens was 78%. However, the 114 chemicals included the
67 chemicals of the Silver list. Including chemicals that were used for development, refinement
and evaluation of a test system might induce a bias in the assessment of the predictive capacity
and was therefore considered by the WG as inappropriate.
Considering only the new chemicals (43 sensitizers and 4 non-sensitizers), the calculations
showed that the predictive capacity (69%) was considerably lower than the 78% presented by
the submitter. It was noted that the number of new qualified non-sensitizers used in this study
was considered insufficient (N = 4).
The submitters were requested to submit additional data on chemicals with negative LLNA
reference data. Such data were provided for an additional 80 chemicals. Compiling all the data
provided by the submitters, the KeratinoSens revealed a sensitivity, specificity and accuracy of
79.3%, 79.8% and 79.5%. Omission of the seven reactive, peptide alkylating chemicals, for which
the LLNA data were not trusted despite absence of human data, the remaining chemicals
resulted in a sensitivity, specificity and accuracy of 79.3%, 84.5% and 81.7% (Annex 4, p64 (C8)).
The WG observed a poor performance of the test on weak sensitizers. Based on the predictions
using the 114 chemicals, 41% of the weak and 86% of the very weak sensitizers were missed(Table 4). Furthermore, the frequency false negative results were found to increase withdecreasing potency of the test chemical. This limitation is not clearly indicated in thesubmission.
Applicability domain:
The applicability domain was well described in the section 1.6 of KeratinoSens report. The
authors stated a variety of chemical classes which were expected to be successfully tested in
the KeratinoSens assay. These limitations were mainly limited to the issues of solubility or
stability in vehicle (e.g. interactions with vehicle, such as hydrolysis).
The WG discussed this issue (See section 2.2) and came to the conclusion that there is indirect
evidence that the applicability domain of test may extent to chemicals that not (only) react withthe cysteine residues of Keap1. Alternative mechanisms may lead to Nrf2 activation.
Study design allowed testing of some of the limitations of the applicability domain.
Readiness for standardized use:
The WG considered the test method sufficiently mature for classification and labelling ofchemicals (relevant to Regulation EC N° 1271/2008).
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Negative results have to be considered with care as weak sensitizers (and possibly also
moderate sensitizers at the lower end of the scala) will be probably missed (see section 9).
Unless this issue gets solved, the KeratinoSens has to be seen as a brick in an integrated testing
strategy of weight-of-evidence approach. The consideration of the chemistry /reactivity must be
included either by combination with a peptide reactivity test or predictive chemistryassessment. This reactivity assessment should include consideration concerning activating
mechanism(s). The KeratinoSens was considered useful for screening purposes, to identify
molecular initiators and to gain mechanistic information on the role of e.g. oxidative stress insensitization.
Identified gaps:
Weak and low-moderate sensitizers, as well as pro-haptens were performing poorly.
When considering cytotoxicity, more emphasis could have been paid to GSH status of the cells
and their GSH regenerating capacity. This system may have an impact the inherent chemical
reactivity whether directly conjugating to GSH or oxidising it (ref.).
The data do not support the expectation that this test can be used as a stand-alone(preliminary, waiting for PC and reproducibility assessment).
It appears that the correlation between in vivo and in vitro data needs further improvement as
there was a relative high variation among the in vitro scores of chemicals belonging to the samepotency class (Natsch et al., 2009).
Recommendations:
The test method can be used for S/NS identification of chemicals. Therefore, the test wasconsidered ready for the next steps in the ECVAM process. A Validation study should however
include more well-defined non-sensitizing compounds. Furthermore, a consistent use of
acceptance criteria nr 3 should be assured.
Since the test revealed issues around weak and low moderate sensitizers, negative resultscannot rule out a sensitization potential. This problem should be clearly flagged and/or
addressed to be solved.
At SOP level, the test submitters were recommended to modify the 96-well plate design, whichcurrently is prone to bias.
Integration of this assay with other predictive tests as they emerge needs to be based on the
better defined applicability domain.
Eventual combination of the KeratinoSens assay with a reactivity based approach needs toinclude unambiguous identification of reactivity and any specificity associated with it.
Training should be considered.

3.

Informative background to the Mandate and Opinion

Skin sensitization is the toxicological endpoint associated with substances that have the intrinsic
ability to cause Allergic Contact Dermatitis, ACD in humans. ACD represents the most common
manifestation of immunotoxicity in humans, i.e. adverse effects of xenobiotics involving theimmune system. The identification of the skin sensitization potential represents an importantcomponent of
the safety assessment of any new substance and especially those intended for topical use (e.g.
cosmetics). Current regulatory predictive tests for skin sensitization rely on the use of animals,
theseinclude:a) the traditional guinea pig tests: Buehler Test and Guinea-pig Maximization Test (OECD TG
406, Ref.1),
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ESAC OPINIONb) the Local Lymph Node Assay (LLNA, OECD TG 429, Ref.2) and its recently OECD adopted nonradioactive variants (OECD TG 422A, Ref.3 and OECD TG 422B, Ref.4).
Despite the progress that has been made in the development of alternative methods for skinsensitization hazard identification, there are currently no validated methods available. In
addition none of the tests under development/evaluation is able to fully characterize therelative potency of sensitizing substances and therefore, none of these assays is considered astand-alone method, capable of fully replacing current animal procedures.
The current view therefore is to combine different test methods in order to address different
key mechanisms of skin sensitization, these includes: skin bioavailability, haptenation (theprotein binding of chemicals which triggers immunological responses), epidermal inflammation,
dendritic cell activation and migration, T cell proliferation. Before these test methods can beroutinely used, their capacity to produce reproducible results needs to be demonstrated as afirst step.
There is evidence in the literature showing that the Nrf2-Keap1-ARE regulatory pathway is
induced by electrophilic chemicals. Since a considerable proportion of chemicals that lead toskin sensitization have these properties, the Nrf2-Keap1-ARE regulatory pathway is considered
one of the most relevant pathways for the identification of potential skin sensitizers (recently
reviewed by Natsch A, Ref.5). This knowledge was exploited by Givaudan to develop the
KeratinoSens assay which uses an adherent cell line derived from an expanded clone of HaCaT
keratinocytes transfected with a selectable plasmid. The plasmid contains the luciferase gene
under the transcription control of the SV40 promoter fused with the ARE (antioxidant response
element) from the AKR1C2 gene.
Using well-established light-producing luciferase substrates, the activity of ARE-bindingtranscription factors in the cells in response to exposure with soluble chemicals can be easily
measured. Luciferase induction is the read-out of the KeratinoSens test method and theconcentration at which the induction is 50% above the background level (EC1.5) is established inparallel to the IC50 value to classify chemicals as having skin sensitization potential.
The test method submitter proposes this method to be used in future as part of an integrated
approach for the full replacement of the animal tests or as a stand-alone method for skinsensitisation hazard identification. In relation to the ability of the test method to differentiate
between sensitizing and non-sensitizing chemicals, the test method submitter reported an
accuracy of 85.1% (sensitivity 86.4%, specificity 82.6%) with respect to in vivo data for a set of
67 chemicals tested in-house.
The KeratinoSens test method has been evaluated in a ring study involving 5 laboratories
including Givaudan who acted as the study coordinator. The transfer of the protocol was
evaluated with a set of 7 chemicals. 21 additional chemicals (15 sensitizers and 6 nonsensitizers) have been tested coded to generate information on the test method reliability andpredictive capacity. The laboratories consistently classified 18 of the 21 coded chemicals. The
accuracy of the in vitro classification with respect to the in vivo classification is reported to vary
between 85.4% and 96.7% for the different laboratories. Following the formal submission of the
KeratinoSens assay to ECVAM, Givaudan was asked to generate additional information on thewithin-laboratory reproducibility. In order to achieve this, ECVAM supplied Givaudan with 8
coded chemicals which have not been tested before with the KeratinoSens test method. Datafor these additional chemicals were generated at the Givaudan laboratories and were submitted
to ECVAM middle of March 2011.
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ESAC OPINION

With respect to the modular approach of validation (Hartung et al., 2004, Ref.6) the study
provides information on module 1) test definition, module 2) within laboratory reproducibility,
module 3) transferability and module 4) between laboratory reproducibility. Information formodule 5), predictive capacity, is only partially fulfilled.

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4.

ESAC OPINION

References

1. OECD, Organization for Economic Cooperation and Development (1992) Skin Sensitization
Guidelines for Testing of Chemicals No. 406, Paris.
2. OECD, Organization for Economic Cooperation and Development (2002) The Local Lymph
Node Assay. Guidelines for Testing of Chemicals No. 429, Paris.
3. OECD, Organization for Economic Cooperation and Development (2010a) Skin Sensitization:
Local Lymph Node Assay: DA, Guidelines for Testing of Chemicals No. 442A, Paris.
4. OECD, Organization for Economic Cooperation and Development (2010b) Skin Sensitization:
Local Lymph Node Assay: BrdU-ELISA, Guidelines for Testing of Chemicals No. 442B, Paris.
5. Natsch A. The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizersfunctional relevance and a hypothesis on innate reactions to skin sensitizers. Toxicological
Sciences 2010 113(2):284-92.
6. 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

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEANNEX 2 EURL ECVAM request for ESAC advice
EURL ECVAM request to ESAC for scientific advice on the Givaudancoordinated study on the transferability and reliability of the KeratinoSens
assay for skin sensitisation testing

Title page information
Abbreviated title of ESAC
requestESAC REQUEST Nr.
Template used for preparing
requestDate of finalising request
Date of submitting request to
ESAC
Request discussed throughOpinion expected at (date)
File name of this request

70

ESAC peer review of and ESAC opinion on the Givaudan-led study
on the KeratinoSens test method.
2011-04
EP 2.01
2011-03-07
2011-03-09
ESAC 34. 22-23 March 2011 and
ESAC 35. 4-5 October 2011 (mandate adopted:
objective/questions of review and ESAC WG)
ESAC 36. 20-21 March 2012
ER2011-04_KeratinoSens_ESACadopted.doc

26

ANNEX 2

1.

ECVAM REQUEST FOR ESAC ADVICETYPE OF REQUEST

Request Type

Identify request ("YES")

R1 ESAC Peer Review

YESof a Prevalidation Study or Validation Study
If R1)applies please specify further:►Prevalidation Study►Prospective Validation Study►Retrospective Validation Study►Validation Study based on PerformanceStandards

YES
The KeratinoSens assay for skin sensitisation testinghas been evaluated in a ring study involving fivelaboratories and led by Givaudan, a producer offragrances and flavours. The study has been
designed to generate information on the test
method's transferability and reproducibility to allow
recommendations to be made on these two aspects
in view of the future use of this test method in an
integrated approach for the full replacement of thecurrently used regulatory animal tests. In addition
the data generated in this study will inform possible
future evaluations of the test method's predictive
capacity.

No
No
No

R2 Scientific Advice on a test method submitted to
ECVAM for validation

No(e.g. the test method's biological relevance etc.)

No

R3 Other Scientific Advice(e.g. on test methods, their use; on technical issues such as cell
culturing, stem cells etc.)

2771ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

2.
TITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE
ESAC IS REQUESTED
Givaudan study on the KeratinoSens assay for skin sensitisation testing.

3.

BRIEF DESCRIPTION OF THE STUDY OR PROJECT

1) Background to skin sensitization and current predictive tests
Skin sensitisation is the toxicological endpoint associated with substances that have the intrinsic
ability to cause Allergic Contact Dermatitis, ACD in humans. ACD represents the most common
manifestation of immunotoxicity in humans, i.e. adverse effects of xenobiotics involving the immune
system. The identification of the skin sensitization potential represents an important component of
the safety assessment of any new substance and especially those intended for topical use (e.g.
cosmetics). Current regulatory predictive tests for skin sensitization rely on the use of animals, theseinclude:a) the traditional guinea pig tests: Buehler Test and Guinea-pig Maximisation Test (OECD TG 406,
Ref.1),b) the Local Lymph Node Assay (LLNA, OECD TG 429, Ref.2) and its recently OECD adopted nonradioactive variants (OECD TG 422A, Ref.3 and OECD TG 422B, Ref.4).
Despite the progress that has been made in the development of alternative methods for skinsensitisation hazard identification, there are currently no validated methods available. In addition
none of the tests under development/evaluation is able to fully characterise the relative potency ofsensitising substances and therefore, none of these assays is considered a stand-alone method,
capable of fully replacing current animal procedures.
The current view therefore is to combine different test methods in order to address different key
mechanisms of skin sensitisation, these includes: skin bioavailability, haptenation (the protein
binding of chemicals which triggers immunological responses), epidermal inflammation, dendritic
cell activation and migration, T cell proliferation. Before these test methods can be routinely used,their capacity to produce reproducible results needs to be demonstrated as a first step.
2) Background to the KeratinoSens
There is evidence in the literature showing that the Nrf2-Keap1-ARE regulatory pathway is induced
by electrophilic chemicals. Since a considerable proportion of chemicals that lead to skinsensitisation have these properties, the Nrf2-Keap1-ARE regulatory pathway is considered one of themost relevant pathways for the identification of potential skin sensitisers (recently reviewed by
Natsch A, Ref.5). This knowledge was exploited by Givaudan to develop the KeratinoSens assay
which uses an adherent cell line derived from an expanded clone of HaCaT keratinocytes transfectedwith a selectable plasmid. The plasmid contains the luciferase gene under the transcription controlof the SV40 promoter fused with the ARE (antioxidant response element) from the AKR1C2 gene.
Using well established light-producing luciferase substrates, the activity of ARE-binding transcription
factors in the cells in response to exposure with soluble chemicals can be easily measured.
Luciferase induction is the read-out of the KeratinoSens test method and the concentration at which
the induction is 50% above the background level (EC1.5) is established in parallel to the IC50 value toclassify chemicals as having skin sensitisation potential.
The test method submitter proposes this method to be used in future as part of an integrated
approach for the full replacement of the animal tests or as a stand alone method for skinsensitisation hazard identification.
In relation to the ability of the test method to differentiate between sensitising and non-sensitisingchemicals, the test method submitter reported an accuracy of 85.1% (sensitivity 86.4%, specificity
82.6%) with respect to in vivo data for a set of 67 chemicals tested in house.

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

3) Background to the KeratinoSens ring study
The KeratinoSens test method has been evaluated in a ring study involving 5 laboratories including
Givaudan who acted as the study coordinator. The transfer of the protocol was evaluated with a setof 7 chemicals. 21 additional chemicals (15 sensitisers and 6 non sensitisers) have been tested coded
to generate information on the test method reliability and predictive capacity. The laboratories
consistently classified 18 of the 21 coded chemicals. The accuracy of the in vitro classification withrespect to the in vivo classification is reported to vary between 85.4% and 96.7% for the different
laboratories.
Following the formal submission of the KeratinoSens assay to ECVAM, Givaudan was asked togenerate additional information on the within-laboratory reproducibility. In order to achieve this,
ECVAM supplied Givaudan with 8 coded chemicals which have not been tested before with the
KeratinoSens test method. Data for these additional chemicals are being generated at the Givaudan
laboratories and are expected to be submitted to ECVAM middle of March 2011.
With respect to the modular approach of validation (Hartung et al., 2004, Ref.6) the study provides
information on module 1) test definition, module 2) within laboratory reproducibility, module 3)transferability and module 4) between laboratory reproducibility. Information for module 5),predictive capacity, is only partially fulfilled.
References
OECD, Organisation for Economic Cooperation and Development (1992) Skin Sensitisation Guidelines
for Testing of Chemicals No. 406, Paris.
OECD, Organisation for Economic Cooperation and Development (2002) The Local Lymph Node
Assay. Guidelines for Testing of Chemicals No. 429, Paris.
OECD, Organisation for Economic Cooperation and Development (2010a) Skin Sensitization: Local
Lymph Node Assay: DA, Guidelines for Testing of Chemicals No. 442A, Paris.
OECD, Organisation for Economic Cooperation and Development (2010b) Skin Sensitization: Local
Lymph Node Assay: BrdU-ELISA, Guidelines for Testing of Chemicals No. 442B, Paris.
Natsch A. The Nrf2-Keap1-ARE toxicity pathway as a cellular sensor for skin sensitizers--functional
relevance and a hypothesis on innate reactions to skin sensitizers. Toxicological Sciences 2010
113(2):284-92.
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.

4.

OBJECTIVES, QUESTIONS, TIMELINES

4.1
OBJECTIVE
The opinion of ESAC on the present Prevalidation study of the KeratinoSens test
Objective
Why does ECVAM
require advice on
the current issue?

method should support ECVAM with respect to the evaluation of the validity
status of the test method at present and with regard to possible necessary further
work required to fully characterise the test method's performance (reproducibility,
predictive capacity, applicability, limitations of the test method).
Moreover, based on the evaluation of the data submitted, the ESAC should
provide advice on the potential usefulness of the KeratinoSens test method withina testing strategy for skin sensitisation testing and the proper scientific use of thetest method within such a testing strategy (e.g. with respect to its specificapplicability and limitations). It is explicitly noted that the ESAC is not requested tosuggest the precise placing of the submitted method in a hypothetical ITS, butrather to provide advice on the characteristics of the method relevant for its
subsequent integration into an ITS at a later point in time (i.e. when otherbuildings blocks of such an ITS are known).

29

73

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEQUESTION(S) TO BE ADDRESSED
4.2
Questions
1) DESIGN & CONDUCT OF STUDY: The ESAC is requested to review whether the
What are thequestions andissues that should
be addressed inview of achieving
the objective of
the advice?

submitted prevalidation study was conducted appropriately in view of theobjective of the study (see attachment 17e of the submission). The study objective
was to assess(1) the reproducibility of the KeratinoSens method in one (the lead) laboratory(n=14 substances plus further 28 substances which were however not tested in a
sufficient number of runs),(2) its transferability to other laboratories (n=7),(3) its reproducibility in other laboratories (BLR) when test items were tested
repeatedly, but in deviation from the complete procedure as conducted by thelead laboratory in the intra-laboratory study (n=21).
(4) Furthermore, the study aimed at assessing, in a preliminary manner, thepredictive capacity of the test method based on the testing of published reference
chemicals in the lead laboratory (n=114; this included 67 chemicals used for testdevelopment/optimisation and development of the prediction model) and during
the ring trial to assess transferability (n=7) and BLR (n=21).
When reviewing the design and conduct of the study, the following issues should
be addressed in particular:xxx

Clarity of the test definition (module 1)
Clarity of the definition of the study objective (see attachment 17e of thePrevalidation study report).
Appropriateness of the study design in view of study objective, inter alia:- Is the number of chemicals sufficient for the purposes of the study?
- Are the reference data used for assessing in particular the predictive
capacity appropriate and of good quality?

- Was the identification of chemicals conducted in an appropriate manner(i.e. presence or absence of selection criteria, justification etc.)?
- Is the adverse effect range of the selected chemicals appropriate for the
purpose of the study- In case of gaps (chemical class etc.) – are these justified?
- Is the number of laboratories sufficient?
Appropriateness of the study execution (e.g. were there pre-defined acceptancecriteria, were these respected? How were exceptions / deviations handled? Wereprovisions specified for retesting? Were the number of repetitions sufficient? etc.)
Appropriateness of the statistical analysis used for analysing WLR, transferabilitiy,
BLR and (preliminary) predictive capacity.

2) CONCLUSIONS OF STUDY: The ESAC is requested to assess whether theconclusions, as presented in the Test Submission Template (TST), Annex 17e, aresubstantiated by the information generated during prevalidation and are plausible
with respect to existing information and current views (e.g. literature).

74

30

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

In particular:x

Do the data on the basis of these chemicals provide sufficient information
on applicability and possible limitations of the test method, in particularin view of its potential use within an ITS for sensitisation?

x

Are the conclusions on reproducibility (WLR and BLR) as well astransferability justified and plausible?

x

Are the conclusions on predictive capacity justified and plausible withrespect to existing informationx

Are there possible gaps between study design and study conclusions
which remain to be addressed in view of the suggested conclusions / use(see also point 3)?

3) SUGGESTED USE OF THE TEST METHOD: The ESAC is requested (a) to evaluate,on the basis of the data submitted in the Prevalidation study, the possible use of
the validated method (also within a strategy) to identify skin sensitisers, (b) tomake additional recommendations (as required) on the proper scientific use of thetest method within such a strategy taking specific aspects of this method into
account (e.g. applicability, limitations etc.) and (c) to identify possible further
information required (i.e. are there gaps) to be able to conclude on the plausibilityof the suggested use (including within an ITS).

3175

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

4.3
TIMELINES
Timelines
Timeline
Finalised ESAC Opinion required by:concerning thisRequest to be presented to ESAC byrequestWhen does
ECVAM require
the advice?

5.

written procedure (e.g. due tourgency) prior to the next ESAC
Request to be presented to ESAC at
ESAC plenary meeting

Indication
ESAC 36, 20-21 March 2012
NO

ESAC 34, 22-23 March 2011 and ESAC
35, 4-5 October 2011 (mandate)

ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN ESAC

5.1
ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIREDSpecific
Structure(s) required
Required according to ECVAM? (YES/NO)
structures
required within S1 ESAC Rapporteur
NO
ESAC to address S2 ESAC Working GroupYES
the requestNO
S3 Invited Experts
Does the advice
require an ESAC
working group, an
ESAC rapporteuretc.?

Ad S3: If yes – list names andaffiliations of suggested
experts to be invited andspecify whether these aremember of the EEP
If other than above (S1-S3):

NO

5.2
DELIVERABLES AS PROPOSED BY ECVAM
Deliverables
Title of deliverable otherRequired? (YES/NO)
What deliverables than ESAC opinion(other than theNO
D1 ESAC Rapporteur Report
ESAC opinion) are
required for
addressing therequest?

76

and draft opinion
D2 ESAC Peer Review Reportand draft opinion
If other than above (D1-D2):

YES

6.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESACCount

Description of document

Available(YES/NO)

File name1

Validation study report (external
validation) of Givaudancoordinated KeratinoSens study
based on the ECVAM Test
Submission Template following

YES

ER201103_Ring_study_KeratinoSens.pdf

32

ANNEX 2

2

ECVAM REQUEST FOR ESAC ADVICEECVAM's Modular approach.
Review paper on biologicalrelevance of Nrf2-Keap1-ARE
toxicity pathway for sensitisation
testing

YES

ER2011-03_Toxicol. Sci.-2010Natsch.pdf

7.

TERMS OF REFERENCE OF THE ESAC WORKING GROUP

7.1

ESTABLISHMENT OF THE ESAC WORKING GROUP

During its 34th meeting on March 22-23 the ESAC plenary unanimously decided to establish an ESAC
Working Group Sensitisation charged with the detailed scientific review of four test methods for skinsensitisation.

7.2

TITLE OF THE ESAC WORKING GROUP

Full title:
ESAC Working Group on Skin Sensitisation Test Methods
Abbreviated title:
ESAC WG Sensitisation

7.3

MANDATE OF THE ESAC WG

The EWG is requested to conduct a scientific review of the relevant studies concerning four skinsensitisation test methods (DPRA, MUSST, h-CLAT, KeratinoSens). The review needs to address thequestions put forward to ESAC by ECVAM.
The review should focus on the appropriateness of design and conduct of the study in view of thestudy objective and should provide an appraisal to which extent the conclusions of the Validation
Management Team (VMT) / test method submitter are substantiated by the information generated
during the study and how the information generated relates to the scientific background available.

7.4

DELIVERABLE OF THE ESAC WG

The ESAC WG is requested to deliver to the chair of the ESAC and the ESAC Secretariat a detailed
ESAC Working Group Report outlining its analyses and conclusions. A reporting template has been
appended (Appendix 1) intended to facilitate the drafting of the report.
The conclusions drawn in the report should be based preferably on consensus. If no consensus canbe achieved, the report should clearly outline the differences in the appraisals and provide
appropriate scientific justifications.

7.5

PROPOSED TIMELINES OF THE ESAC WG

The ESAC Coordinator has proposed timelines* which should be agreed upon during the first
Teleconference (Item 1 in the table):
Item Proposed date/time
ActionDeliverable
1
7. September 2011 (Wednesday) Kick-off teleconference
Feedback on the
13:30 CET
Discussion of
mandate.
a) the submissionb) the mandate put forward by
the Secretariatc) the working procedure (ESAC
WG template)2
14. October 2011 (Friday)
Deadline for submitting first
Draft observations ofcomments within ESAC WG
each ESAC WG member
templatein the ESAC WG

33

77

ANNEX 2

3

24-26 October 2011 (Monday to
Wednesday)4

Further teleconferences andwork progress to be agreed
during meeting (Nr. 3).
15. February 2012 (Wednesday)

578

ECVAM REQUEST FOR ESAC ADVICEESAC WG meeting in Ispra.
Discussion of contentious items.
Drafting of the report.
Progressing of draft report

Final report to be delivered to
ESAC Coordinator/Secretariat.

34

template (to becompiled by ECVAM)
Draft report

Draft report

Final report

ANNEX 2

7.6

ECVAM REQUEST FOR ESAC ADVICEQUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WG

The ESAC WG is requested to address the three questions posed to the ESAC which have beenbroken down further in more specific questions by Secretariat (see section 4.2) and were discussedwith the ESAC WG and approved by the ESAC.
When preparing the final ESAC WG report to address these questions, the ESAC WG is requested touse a pre-defined reporting template. This template (see appendix 1) follows ECVAM's modular
approach and addresses to which extent the standard information requirements have beenaddressed by the study. In addition, the template allows for addressing the specific questions
outlined in section 4.2. The Secretariat will provide guidance if necessary.

35

79

ANNEX 2

APPENDIX 1

ECVAM REQUEST FOR ESAC ADVICEREPORTING STRUCTURE FOR THE ESAC WG REPORTThe following suggested structure follows the ECVAM information requirements ("modules") for
scientific review following validation and allows at the same time for the description of the analysis
and conclusions concerning more specific questions. A template has been created on the basis of thestructure below and this template will be made available to the ESAC.
The template can be used for various types of validation studies (e.g. prospective full studies,retrospective studies, performance-based studies and prevalidation studies). Depending on the
study type and the objective of the study, not all sections may be applicable. However, for reasonsof consistency and to clearly identify which information requirements have not been sufficiently
addressed by a specific study, this template is uniformly used for the evaluation of validation
studies.
1. Data collection
1.1 Information / data sources used
1.2 Search strategy
1.3 Selection criteria applied to the available information
2. Study objective and design
2.1 Clarity of the definition of the study objective
2.2 Analysis of the scientific rationale provided
2.3 Analysis of the regulatory rationale provided
2.4 Appropriateness of the study design
2.5 Appropriateness of the statistical evaluation
3. Test definition (Module 1)
3.1 Quality and completeness of the overall test definition
3.2 Quality of the background provided concerning the purpose of the test method
3.3 Quality of the documentation and completeness of (a) standardised protocols (SOPs) and (b)prediction models
4. Data quality
4.1 Overall quality of the evaluated data
4.2 Sufficiency of the evaluated data in view of the study objective
4.3 Quality of the reference data for evaluating reliability and relevance2
5. Test materials
5.1 Sufficiency of the number of evaluated test items in view of the study objective
5.2 Representativeness of the test items with respect to applicability
6. Within-laboratory reproducibility (Module 2)
6.1 Assessment of repeatability and reproducibility in the same laboratory
6.2 Conclusion on within-laboratory reproducibility as assessed by the study
7. Transferability (Module 3)
7.1 Quality of design and analysis of the transfer phase
7.2 Conclusion on transferability to a second laboratory/other laboratories as assessed by the study
8. Between-laboratory reproducibility (Module 4)
8.1 Assessment of reproducibility in different laboratories
8.2 Conclusion on reproducibility as assessed by the study
9. Predictive capacity (Module 5)
9.1 Adequacy of the assessment of the predictive capacity in view of the purpose
9.2 Overall relevance (biological relevance and accuracy) of the test method in view of the purpose
2

80OECD guidance document Nr. 34 on validation defines relevance as follows: "Description of relationship of the test to the effect ofinterest and whether it is meaningful and useful for a particular purpose. It sis the extent to which the test correctly measures or
predicts the biological effect of interest. Relevance incorporates consideration of accuracy (concordance) of a test method."36

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE

10. Applicability domain (Module 6)
10.1 Appropriateness of study design to conclude on applicability domain, limitations and exclusions
10.2 Quality of the description of applicability domain, limitations, exclusions
11. Performance standards (Module 7)
11.1 Adequacy of the proposed Essential Test Method Components
11.2 Adequacy of the Reference Chemicals
11.3. Adequacy of the defined Accuracy Values
12. Readiness for standardised use
12.1 Assessment of the readiness for regulatory purposes
12.2. Assessment of the readiness for other uses (in house screening etc.)12.3 Critical aspects impacting on standardized use
12.4 Gap analysis
13. Other considerations
14. Conclusions on the study
14.1 Summary of the results and conclusions of the study
14.2 Extent to which conclusions are justified by the study results alone
14.3 Extent to which conclusions are plausible in the context of existing information
15. Recommendations
15.1 General recommendations concerning the study
15.2 Recommendations concerning the test method (test system, protocol, prediction model)
16. References
17. Annexes

END OF EURL ECVAM RECOMMENDATION

37

81

European Commission
EUR 26427 – Joint Research Centre – Institute for Health and Consumer Protection
Title: EUR 26427 - EURL ECVAM Recommendation on the KeratinoSensTM assay for skin sensitisation testing
Luxembourg: Publications Office of the European Union2013 – 39 pp. – 21.0 x 29.7 cm
EUR – Scientific and Technical Research series – ISSN 1018-5593 (print), ISSN 1831-9424 (online)
ISBN 978-92-79-34795-5
doi: 10.2788/52914

Abstract
Identification of the skin sensitisation hazard of chemicals has traditionally relied on the use of animals. Progress in thedevelopment of alternative methods has been prompted by the increasing knowledge of the key biological mechanisms
underlying this human health effect, as documented by the OECD's recent report summarising the key biological events
leading to skin sensitisation ("Adverse Outcome Pathway" (AOP) for skin sensitisation). Within this AOP the activation ofcellular signalling pathways, such as the Keap1-Nrf2- antioxidant/electrophile response element (ARE)-dependent
pathway, known to play a relevant role in keratinocytes’ responses to skin sensitisers, is postulated to be a key event.
Therefore, test methods able to provide information on the ability of a chemical to activate this or other relevant
pathways in keratinocytes, may contribute to skin sensitisation hazard and safety assessment. The KeratinoSensTM test
method measures ARE-Nrf2 activation through a luciferase reporter gene. The test method has undergone a validation
study addressing mainly the test method’s transferability and within- and between-laboratory reproducibility. Following
independent scientific peer review by EURL ECVAM’s Scientific Advisory Committee (ESAC) and having considered the
input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded that the
KeratinoSensTM may prove a useful component of integrated approaches such as Weight of Evidence (WoE) or
Integrated Testing Strategies (ITS) for skin sensitisation hazard assessment. In addition to this, the KeratinoSensTM may
also be able to contribute to the assessment of sensitising potency, e.g. by supporting sub-categorisation of sensitisers
according to UN GHS. However it is recognised that further efforts are required to explore how KeratinoSensTM data
may contribute to potency assessment.

82

38

LB-NA-26427-EN-N

As the Commission’s in-house science service, the Joint Research Centre’s mission is to provide
EU policies with independent, evidence-based scientific and technical support throughout thewhole policy cycle.
Working in close cooperation with policy Directorates-General, the JRC addresses key societal
challenges while stimulating innovation through developing new standards, methods and tools,
and sharing and transferring its know-how to the Member States and international community.
Key policy areas include: environment and climate change; energy and transport; agriculture
and food security; health and consumer protection; information society and digital agenda;
safety and security including nuclear; all supported through a cross-cutting and multidisciplinary approach.

39

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