᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 29 ᖺ 3 ᭶ 21 ᪥
                                 No. 2016-03

⓶⭵ឤసᛶヨ㦂௦᭰ἲ human Cell Line Activation Test㸦h-CLAT㸧࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 29 ᖺ 2 ᭶ 24 ᪥࡟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 h-CLAT ࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊឤసᛶ≀㉁࡜ุᐃࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿ ࡀ ࠊ ឤ స ᛶ ᙉ ᗘ ศ 㢮 ࡸ United Nations (UN) Globally Harmonized System ofClassification and Labeling of Chemicals (GHS)ࡢࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮ࡣᅔ㞴࡛࠶ࡿࠋᮏ
ヨ㦂ἲ༢⊂࡛ࡢ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛࠶ࡾࠊ⿕㦂≀㉁ࡢ≉ᛶࢆ༑ศ࡟⌮ゎࡋࡓ
ୖ࡛ࠊIntegrated Approaches to Testing and Assessment (IATA) ࢆᵓᡂࡍࡿࡑࡢ௚ࡢ᝟ሗ
࡜⤌ࡳྜࢃࡏ࡚㐺ษ࡟ホ౯ࡍࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ࡞࠾ࠊᮏヨ㦂ἲࡢ฼⏝࡟࠶ࡓࡗ࡚
ࡣࠊ㐺⏝⠊ᅖࢆ༑ศ࡟㓄៖ࡋࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ
ࡇࡢᥦ᱌᭩ࡣࠊOrganisation for Economic Co-operation and Development (OECD) Test Guideline(TG) 442E; In Vitro Skin Sensitization: h-CLAT࠾ࡼࡧ h-CLAT Validation Study Report,

EURL

ECVAM Recommendation on the h-CLAT for skin sensitization 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 h-CLAT ࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ ኱㔝Ὀ㞝                      すᕝ⛅ె኱㔝Ὀ㞝JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i
JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥
                 
ii

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

iii

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JaCVAM statement on thehuman Cell Line Activation Test (h-CLAT) Skin Sensitization Test Method
At a meeting held on 24 February 2017 at the National Institute of Health Sciences (NIHS) in
Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:  
Proposal: Although it is possible to classify chemicals that yield positive results using theh-CLAT test method as sensitizers, it is not possible to assess accurately their
sensitization strength nor their subcategorization under the United Nations (UN)Globally Harmonized System of Classification and Labeling of Chemicals (GHS).
The h-CLAT test method is not suitable for predicting skin sensitization potential
on its own; in order to make a suitable assessment, the results of h-CLAT testing
must be used with a thorough understanding of the properties of each test
chemical in combination with other information as part of an integrated approachto testing and assessment (IATA). Furthermore, thorough consideration must be
given to the applicability domain when using this test.
This statement was prepared following a review of the Organisation for Economic
Co-operation and Development (OECD) Test Guideline (TG) 442E “In Vitro Skin
Sensitization: h-CLAT” as well as other documentation prepared by the Skin Sensitization
Testing JaCVAM Editorial Committee based on the “h-CLAT Validation Study Report” andthe “EURL ECVAM Recommendation on the h-CLAT for skin sensitization testing” toacknowledge that the results of a review and study by the JaCVAM Regulatory Acceptance
Board have confirmed the usefulness of this assay.
Based on the above, we propose the h-CLAT skin sensitization test method as a useful means
for safety assessment by regulatory agencies.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

March 21, 2017v

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

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

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

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

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⓶⭵ឤసᛶヨ㦂௦᭰ἲ
human Cell Line Activation Test㸦h-CLAT㸧┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------- 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------- 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 442E,
In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) ----------------31
JRC SCIENCE AND POLICY REPORT,EURL ECVAM Recommendation on the human Cell Line Activation Test
(h-CLAT) for skin sensitisation testing--------------------------------------------------53ix

xホ౯఍㆟ሗ࿌᭩

human Cell Line Activation Test㸦h-CLAT㸧

JaCVAM ホ౯఍㆟

ᖹᡂ 28 ᖺ㸦2016 ᖺ㸧12 ᭶ 20 ᪥

1

1

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ୖᬛᙲ㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒஭ᩍᏳ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰஭ಇྖ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟⃝ ⪽㸦᪥ᮏẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥

22

human Cell Line Activation Test㸦h-CLAT㸧ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀᶞ≧⣽⬊ࢆάᛶ໬ࡍࡿࡇ࡜ࢆ
฼⏝ࡋࠊᶞ≧⣽⬊ࡢࣔࢹࣝࡢࣄࢺ༢⌫⣔ᇵ㣴⣽⬊ THP-1 ⣽⬊࡟⿕㦂≀㉁ࢆ᭚㟢ࡋࡓ࡜ࡁࡢ⣽⬊⾲㠃ศᏊ
CD86 ࠾ࡼࡧ CD54 ࡢⓎ⌧㔞ࢆᐃࡍࡿࡇ࡜࡟ࡼࡾࠊ⓶⭵ឤసᛶࡢ᭷↓ࢆุᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ

h-CLAT ࡟ࡘ࠸࡚ࡣࠊEuropean Union Reference Laboratory for Alternatives to Animal Testing㸦EURL
ECVAM㸧࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀ 1)ࠊEURL ECVAM Scientific Advisory Committee㸦ESAC㸧࡟ࡼࡿ➨୕⪅ホ౯ࢆཷࡅࡓ 2) ࠋ2016 ᖺ 7 ᭶࡟ࡣࠊOrganisation for Economic Co-operation and Development

OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ Test Guideline: TG㸧442E ࡜ࡋ࡚ᢎㄆࡉࢀࡓ 3)ࠋJaCVAM ホ౯఍㆟ࡣࠊ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕ⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ human Cell Line Activation
Test㸦h-CLAT㸧ࠖ4)ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 human Cell Line Activation Test㸦h-CLAT㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 ࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂㸦OECD TG406㸧࠾ࡼࡧ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㹙Local Lymph Node Assay㸸LLNA㸦OECD TG429㸧ࠊLLNA㸸DA㸦OECD TG442A㸧ࠊ
LLNA㸸BrdU-ELISA㸦OECD TG442B㸧㹛
ヨ㦂ἲࡢᴫ␎㸸 ᮏヨ㦂ἲ࡛ࡣࠊᶞ≧⣽⬊ࡢࣔࢹࣝ࡜ࡋ࡚ࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊ⿕㦂≀㉁ࡀ THP-1 ⣽⬊ࢆάᛶ໬ࡍࡿ⬟ຊࢆホ౯ࡍࡿࠋTHP-1 ⣽⬊࡟⿕㦂≀㉁ࢆῧຍࡋ࡚ 24 ᫬㛫ᇵ㣴ࡋࡓᚋࠊfluorescein isothiocyanate㸦FITC㸧࡛⺯ගᶆ㆑ࡋࡓ CD86 ࠾ࡼࡧ CD54 ࡢ≉␗ᢠయࠊ࠾ࡼࡧ
propidium iodide㸦PI㸧ࢆ⏝࠸࡚⣽⬊ࢆᰁⰍࡋࠊࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜ࡟ࡼࡾ⣽⬊⾲㠃ศᏊ CD86 ཬࡧ
CD54 ࡢⓎ⌧㔞ࠊ୪ࡧ࡟⣽⬊⏕Ꮡ⋡ࢆᐃࡍࡿࠋ⿕㦂≀㉁࡛ฎ⌮ࡋࡓ࡜ࡁࡢ CD86 ࠾ࡼࡧ CD54 ࡑࢀࡒࢀࡢᖹᆒ⺯ගᙉᗘࢆࠊ⁐፹ࡢࡳࢆῧຍࡋࡓࢥࣥࢺ࣮ࣟࣝࡢ⺯ගᙉᗘ࡜ẚ㍑ࡋࠊ┦ᑐ⺯ගᙉᗘࢆ⟬ฟࡍࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶᮏヨ㦂ἲࡣࠊEURL ECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 1)࡜ࡑࢀ࡟⥆ࡃ ESAC ࡟ࡼࡿ➨୕⪅ホ౯࡟ࡼࡾࠊᐇ㦂ື≀ࢆ⏝࠸ࡓ⓶⭵ឤసᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚⛉Ꮫⓗ࡟ጇᙜ࡛࠶ࡿ࡜ሗ࿌ࡉࢀ࡚࠾ࡾ 2)ࠊ2016 ᖺ
7 ᭶࡟ OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ᢎㄆࡉࢀ࡚࠸ࡿ 3)ࠋJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡛
ࡣࠊ⌧ᅾࡲ࡛බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗ 2,3,5-8)ࢆᇶ࡟ᮏヨ㦂ἲࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ホ౯ࡋࡓࠋᮏヨ㦂ἲࡣࠊ⓶⭵ឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ㔜せ࡞ࢫࢸࢵࣉ࡛࠶ࡿᢠཎᥦ♧⣽⬊ࡢάᛶ໬཯ᛂࢆᣦᶆ࡟ࡋࡓࡶࡢ࡛ࠊάᛶ໬ࡢ㝿࡟ㄆࡵࡽࢀࡿ⣽⬊⾲㠃ศᏊࡢⓎ⌧ஹ㐍ࢆᐃࡍࡿ࡜࠸࠺Ⅼ࡛ཎ⌮ⓗ࡟ࡶጇᙜ࡛࠶ࡿࠋ

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㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣࠊࣄࢺ༢⌫⣔⏤᮶ᇵ㣴⣽⬊ᰴࢆ⏝࠸ࡿ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋ
ࡲࡓࠊ㸯ヨ㦂࠶ࡓࡾࡢ㈝⏝ࡣࠊ࣐࢘ࢫࢆ⏝࠸ࡿ LLNA ࡢ 1/5 ⛬ᗘ࡛࠶ࡾࠊヨ㦂ᮇ㛫ࡶ LLNA ࡟ẚ࡭࡚▷ᮇ㛫࡛࠶ࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ⤒῭ᛶ࠾ࡼࡧ㎿㏿ᛶࡢ㠃࠿ࡽ᭷⏝࡜࠸࠼ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊᮏヨ㦂ἲࡣࠊ⣽⬊ᐦᗘ࡜ᇵ㣴᫬㛫ࢆཝᏲࡍࡿࡇ࡜ࡸබẚ 1.2 ࡢ⃰ᗘタᐃࢆᚲせ࡜ࡍࡿࡓࡵࠊูࡢ⓶⭵ឤసⓎ⌧࢖࣋ࣥࢺࢆᣦᶆ࡜ࡍࡿ Direct Peptide Reactivity Assay㸦OECD TG442C㸧ࡸ ARE-Nrf2 Luciferase Test Method
㸦OECD TG442D㸧࡟ẚ࡭࡚ࠊ᧯సࡀ↹㞧࡛࠶ࡾࠊไ⣙ࡀከ࠸ࠋࡲࡓࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ
↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊ⫼ᬒࢹ࣮ࢱࢆసᡂࡍࡿࡇ࡜ࡀồࡵࡽࢀ࡚࠸ࡿࠋ

EURL ECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 1)࡟࠾࠸࡚ࠊ15 ≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タෆ෌⌧ᛶࡣࠊ
73.3㹼86.7%࡛࠶ࡗࡓࡀࠊヨ㦂᫬㛫ࡢཝᐦ࡞⟶⌮࡟ࡼࡾ෌⌧ᛶࡢྥୖࡀᅗࢀࡿ࡜ࡉࢀ࡚࠸ࡿࠋ୍᪉ࠊ24 ≀㉁ࢆ⏝࠸ 4 ᪋タ࡛ᐇ᪋ࡉࢀࡓ᪋タ㛫෌⌧ᛶࡣ 79.2㸣࡛࠶ࡗࡓࠋ୍⮴ࡋ࡞࠿ࡗࡓ≀㉁ࡣ᪋タෆ෌⌧ᛶࡶᝏ࠿ࡗࡓࠋ
EURL ECVAM1)ࠊTakenouchi ࡽ 7)ࠊ࠾ࡼࡧ Nukada ࡽ 8)ࡢ⤖ᯝࢆྜࢃࡏࡓ 142 ≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻101㸸41ࠊlog Kow ࡀ 3.5 ௨ୖ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ≀㉁࡜ΰྜ≀ࢆ㝖ࡃ㸧࡟ࡘ࠸࡚ࡢᡂ⦼ࡣࠊ
ṇ☜ᗘ 85㸣ࠊឤᗘ 93㸣ࠊ≉␗ᗘ 66㸣࡛࠶ࡾࠊᵝࠎ࡞໬Ꮫ≀㉁࡟㐺⏝ྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋࡋ࠿
ࡋࠊUnited Nations UN Globally Harmonized System of Classification and Labelling of Chemicals 㸦GHS㸧
ศ㢮࡟࠾ࡅࡿ㍍ᗘ㹼୰➼ᗘឤసᛶ≀㉁ࡣഇ㝜ᛶࡢุᐃࡀ⏕ࡌࡸࡍ࠸ഴྥࡀ࠶ࡿࠋࡲࡓࠊヨ㦂⁐፹࡟⁐ゎࡋ࡞࠸≀㉁࠾ࡼࡧヨ㦂⁐፹୰࡛୙Ᏻᐃ࡞≀㉁ࠊ᥹Ⓨᛶ≀㉁ࠊFITC ࡸ PI ࡜ྠࡌἼ㛗ᇦ࡟ᙉ࠸⺯ගࢆ᭷ࡍࡿ≀㉁ࠊࣉࣟࣁࣉࢸࣥ࠾ࡼࡧࣉࣞࣁࣉࢸࣥࡢࡼ࠺࡞άᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁࡛ࡣṇࡋࡃឤసᛶࡀ᳨ฟࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡾࠊヨ㦂⤖ᯝࡢゎ㔘࡟ࡣὀពࡀᚲせ࡛࠶ࡿࠋ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣࠊࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜ࡟⩦⇍ࡋࡓ᪋タ࡛࠶ࢀࡤᐇ᪋࡛ࡁࡿヨ㦂ἲ࡛࠶ࡾࠊ⺯ගᶆ㆑ࡉࢀ
ࡓ≉␗ᢠయࡶᕷ㈍ࡉࢀ࡚࠸ࡿࠋࡲࡓ THP-1 ⣽⬊ࡢධᡭࡣᐜ࡛᫆࠶ࡾࠊ▷ᮇ㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࡿࠋ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠾ࡾࠊ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲ࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊឤసᛶ≀㉁࡜ุᐃࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿࡀࠊឤసᛶᙉ
ᗘศ㢮ࡸ UN GSH ࡢࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮ࡣᅔ㞴࡛࠶ࡿࠋᮏヨ㦂ἲ༢⊂࡛ࡢ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛
࠶ࡾࠊ⿕㦂≀㉁ࡢ≉ᛶࢆ༑ศ࡟⌮ゎࡋࡓୖ࡛ࠊIntegrated Approaches to Testing and Assessment㸦IATA㸧ࢆᵓᡂࡍࡿࡑࡢ௚ࡢ᝟ሗ࡜⤌ࡳྜࢃࡏ࡚㐺ษ࡟ホ౯ࡍࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ࡞࠾ࠊᮏヨ㦂ἲࡢ฼⏝࡟࠶ࡓࡗ࡚ࡣࠊ㐺⏝⠊ᅖࢆ༑ศ࡟㓄៖ࡋࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ

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ཧ⪃ᩥ⊩
1)

EURL ECVAM (2013). human Cell Line Activation Teat (h-CLAT) Validation Study Report. Accessible at:https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/h-clat-validation-study-report.

2)

EURL ECVAM (2015). Recommendation on the human Cell Line Activation Test (h-CLAT) for skinsensitization testing. Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/eurl-ecvamrecommendation-on-the-human-cell-line-activation-test-h-clat-for-skin-sensitisation-testing3)OECD (2016). OECD Guideline for the Testing of Chemicals No. 442E: In Vitro Skin Sensitization: human
Cell Line Activation Test (h-CLAT). Available at: http://www.oecd-ilibrary.org/environment/test-no-442e-invitro-skin-sensitisation_9789264264359-en;jsessionid=3gdulzg8pwz1.x-oecd-live-02

4)

JaCVAM⓶⭵ឤసᛶ㈨ᩱ⦅⧩ጤဨ఍㸸⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ human Cell Line Activation Test㸦hCLAT㸧㸦2016ᖺ7᭶25᪥㸧㸬

5)

EURL ECVAM (2015). Protocol No. 158: human Cell Line Activation Test (h-CLAT). https://ecvamdbalm.jrc.ec.europa.eu/methods-and-protocols/search/h-clat

6)

Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N,
Itagaki H. (2010). A comparative evaluation of in vitro skin sensitization tests: the human cell-line activation
test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim., 38, 275-284.

7)

Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. (2013). Predictive performance of the human
Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-water partition coefficients. J.
Toxicol. Sci., 38, 599-609.

8)

Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skinsensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying
skin sensitization potency. Toxicol. In Vitro, 26, 1150-60.

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⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩human Cell Line Activation Test㸦h-CLAT㸧

JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍

ᖹᡂ 28 ᖺ 9 ᭶ 19 ᪥

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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

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せ᪨
⓶⭵ឤసᛶࡣ໬Ꮫ≀㉁ࡢᏳ඲ᛶホ౯࡟࠾࠸࡚㔜せ࡞ホ౯㡯┠࡛࠶ࡾࠊᚑ᮶ࠊࣔࣝࣔࢵࢺࡸ
࣐࢘ࢫࢆ⏝࠸ࡓື≀ᐇ㦂࡟ࡼࡗ࡚ホ౯ࡉࢀ࡚ࡁࡓࠋ㏆ᖺ EU ࡟࠾ࡅࡿḢᕞ໬Ꮫရつไ࡛ࡣࠊᏳ඲ᛶホ౯ࡣࢥࣥࣆ࣮ࣗࢱ࣮ࢆ⏝࠸ࡓᐃ㔞ⓗᵓ㐀άᛶ┦㛵㸦 Quantitative Structure-Activity
Relationship: QSAR㸧ࣔࢹࣝࡸ in vitro ࡢ௦᭰ἲࡀ᥎ዡࡉࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆྵࡴ໬⢝ရࡢ㈍኎ࡀ⚗Ṇࡉࢀࡓࡇ࡜࠿ࡽ㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠊື≀ࢆ⏝࠸࡞࠸ in vitro ヨ㦂ἲࡢ㛤Ⓨࡀᙉࡃᮃࡲࢀ࡚࠸ࡿࠋhuman Cell Line Activation Test
㸦h-CLAT㸧ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀᶞ≧⣽⬊ࢆάᛶ໬ࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶
ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊάᛶ໬࡟క࠸⣽⬊⾲㠃ୖࡢⓎ⌧㔞ࡀኚ໬ࡍࡿ CD86 ࡜ CD54 ࢆᐃࡍࡿࡇ࡜࡟ࡼࡾ⓶⭵ឤసᛶࡢ᭷↓ࢆุᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋᮏሗ࿌᭩ࡣࠊࡇࡢ h-CLAT ࡟ࡘ
࠸࡚ࠊEuropean Union Reference Laboratory for Alternatives to Animal Testing㸦EURL ECVAM㸧
࡟ࡼࡾᐇ᪋ࡉࢀࡓࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࠊホ౯㸦ࣆ࢔ࣞࣅ࣮ࣗ㸧ሗ࿌᭩࠾ࡼࡧヨ㦂ἲ㛤Ⓨ⪅ࡢᢞ✏ㄽᩥ࡞࡝ࢆᇶ࡟ヨ㦂ᡭ㡰ࢆࡲ࡜ࡵࠊ᭷⏝ᛶ࡜㝈⏺ࢆホ౯ࡋࡓࡶࡢ࡛࠶ࡿࠋ
h-CLAT ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨୕ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿᶞ≧⣽⬊ࡀάᛶ໬ࡍࡿ㝿ࡢ⣽⬊⾲㠃ศᏊࡢⓎ⌧ஹ㐍ࢆ฼⏝ࡋࡓ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
h-CLAT ࡛ࡣࠊ࣐࢘ࢫࢆ⏝࠸ࡿ Local Lymph Node Assay LLNA ࡢ 1/5 ⛬ᗘࡢ⤒㈝࡛⿕㦂≀㉁ࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿࡇ࡜ࡀྍ⬟࡜ヨ⟬ࡉࢀࠊヨ㦂ᮇ㛫ࡶ LLNA ࡟ẚ࡭▷ᮇ࡛࠶ࡿࡓࡵࠊ⤒῭ᛶ࣭㎿㏿ᛶࡢほⅬ࡛᭷⏝ᛶࡣ㧗࠸࡜ᛮࢃࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊDirect Peptide Reactivity
Assay DPRA ࡸ ARE-Nrf2 Luciferase Test Method ࡟ẚ࡭࡚ࠊ᧯సࡀ↹㞧࡛࠶ࡾࠊไ⣙ࡀከ࠸ࠋࡲࡓࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊࣄࢫࢺࣜ࢝ࣝ࡞ࢹ࣮ࢱ

࣮࣋ࢫࢆసᡂࡍࡿࡇ࡜ࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࠋ
ᮏヨ㦂ἲࡢ EURL ECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࠊ15 ≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タෆ෌⌧ᛶࡣ 73.3㹼86.7%࡛࠶ࡾࠊ඲⯡ⓗ࡟㧗ࡃ࡞࠿ࡗࡓࠋࡓࡔࡋࠊOrganisation for
Economic Co-operation and Development OECD ᑓ㛛ᐙ఍㆟࡟࠾࠸࡚ࠊヨ㦂࡟౑⏝ࡍࡿ THP1 ⣽⬊ࡢ๓ᇵ㣴᮲௳࠾ࡼࡧ⿕㦂≀㉁ࡢ᭚㟢᫬㛫ࢆࡼࡾཝᐦ࡟⟶⌮ࡍࡿࡇ࡜࡟ࡼࡾࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᅗࢀࡿࡇ࡜ࡀ♧ࡉࢀࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡢヨ㦂ᡭ㡰࡟ࡑࡢෆᐜࡀ཯
ᫎࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᮇᚅ࡛ࡁࡿ࡜⪃࠼ࡿࠋ୍᪉ࠊ24 ≀㉁ࢆ౑࠸ 4᪋タ࡛ᐇ᪋ࡉࢀࡓ᪋タ㛫෌⌧ᛶࡣ 79.2㸣࡛࠶ࡗࡓࠋ
OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖࡛ࣥࡣࠊ142 ≀㉁ࡢᡂ⦼࠿ࡽṇ☜ᗘ 85㸣ࠊឤᗘ 93㸣ࠊ≉␗ᗘ 66㸣࡜グ㍕ࡉࢀ࡚࠸ࡿࡀࠊlog Kow ࡀ 3.5 ௨ୖ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ≀㉁ࢆ㝖࠸ࡓୖ࡛ࡢᩘ್࡛࠶
ࡿࠋࡋࡓࡀࡗ࡚ࠊ≉࡟ log Kow ࡀ 3.5 ௨ୖࡢ≀㉁ࡢሙྜࡣഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋ
ᚓࡿ௚ࡢヨ㦂ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊᮏヨ㦂ἲࡢࡳ࡛⿕㦂≀㉁ࢆ⓶⭵ឤసᛶࡀ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸ࠋࡲࡓࠊ≉␗ᗘࡀ 66%࡛࠶ࡿࡓࡵഇ㝧ᛶ࡜ุᐃࡍࡿࡇ࡜ࡶ␃ពࡍࡿᚲせࡀ࠶ࡿࠋࡉࡽ࡟ࠊᮏヨ㦂ἲ࡟⏝࠸ࡿ⣽⬊ࡢ௦ㅰ⬟ࡣ㝈ᐃⓗ࡛࠶ࡿࡓࡵࠊάᛶ໬࡟௦
ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁࡛ࡣࠊṇࡋࡃࡑࡢ⓶⭵ឤసᛶࡀ᳨ฟࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
39ᮏጤဨ఍ࡣࠊୖグࡢᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ຺᱌ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛࠶ࡿ࡜⪃࠼ࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ࡜ࡢ⤌ྜࡏ࡛⏝࠸ࡿࡇ࡜ࢆ᥎
ዡࡍࡿࠋ

4
10

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Organisation for Economic Co-operation and Development: OECD Test Guideline: TG406㸧
ࡸ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㸦Local Lymph Node Assay(LLNA), OECD TG429㸧ࡀ࠶ࡿࠋࡇࡢ[3H-Methyl]-thymidine ྲྀ㎸㔞ࢆࡏࡎ࡟ ATP 㔞ࢆᐃࡍࡿ LLNA ௨እ࡟ᨺᑕᛶྠ఩ඖ⣲㸦RI㸧ࢆ౑⏝ᐃࡍࡿ LLNA: DA㸦OECD TG442A㸧ࡸ Bromodeoxyuridine 㔞ࢆᐃࡍࡿ

LLNA: BrdU-ELISA㸦OECD TG442B㸧ࡀ࠶ࡿࠋ EU ࡟ ࠾ ࡅ ࡿ Ḣ ᕞ ໬Ꮫ ရ つ ไ 㸦Registration, Evaluation, Authorization and Restriction ofChemicals: REACH㸧࡛ࡣࠊᏳ඲ᛶホ౯ࡣࢥࣥࣆ࣮ࣗࢱ࣮ࢆ⏝࠸ࡓᐃ㔞ⓗᵓ㐀άᛶ┦㛵㸦Quantitative Structure-Activity Relationship: QSAR㸧ࣔࢹࣝࡸ in vitro ➼࡟ࡼࡿ௦᭰ἲࡀ᥎ዡࡉ
ࢀ࡚࠾ࡾࠊື≀ᐇ㦂࡟ࡼࡾᏳ඲ᛶࡀホ౯ࡉࢀࡓᡂศࢆ౑⏝ࡋࡓ໬⢝ရࡢ㈍኎ࡀ⚗Ṇࡉࢀࡓ
㸦2013 ᖺ 3 ᭶඲㠃᪋⾜㸧ࠋࡑࡢࡓࡵࠊ໬Ꮫ〇ရࡢ⓶⭵ឤసᛶࢆุᐃࡍࡿ௦᭰ἲࡢ㛤Ⓨࡀᙉࡃồࡵࡽࢀ࡚࠸ࡿࠋ
 In chemico ヨ㦂ἲ࡜ࡋ࡚࣌ࣉࢳࢻ⤖ྜ཯ᛂࢆ฼⏝ࡋࡓ Direct Peptide Reactivity Assay㸦DPRA㸸OECD TG442C㸧࠾ࡼࡧ In vitro ヨ㦂ἲ࡜ࡋ࡚ࢣࣛࢳࣀࢧ࢖ࢺ⣽⬊⣔ࡢᶆⓗ㑇ఏᏊࢆ⏝࠸ࡓ ARE-Nrf2 Luciferase Test Method㸦OECD TG442D㸧ࡀ OECD ࠿ࡽヨ㦂ἲ࢞࢖ࢻࣛ࢖
ࣥ࡜ࡋ࡚බ⾲ࡉࢀ࡚࠸ࡿࠋࡲࡓࠊࡇࢀ௨እ࡟ࠊ༢⌫⣔⣽⬊ࡢάᛶ໬ࢆ฼⏝ࡋࡓ human Cell
Line Activation Test㸦h-CLAT㸧ࠊMyeloid U937 Skin Sensitization Test㸦U-SENS㸧࠾ࡼࡧ IL-8 Luc
assay ࡞࡝ࡢ⓶⭵ឤసᛶヨ㦂ࡢ in vitro ἲࡀᥦ᱌ࡉࢀ࡚࠾ࡾࠊEURL ECVAM㸦the European
Union Reference Laboratory for Alternatives to Animal Testing㸧➼࡟࠾࠸࡚ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀ࡚ࡁࡓࠋ
 h-CLAT ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀᶞ≧⣽⬊ࢆάᛶ໬ࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊάᛶ໬࡟క࠸⣽⬊⾲㠃࡛ࡢⓎ⌧㔞ࡀኚ໬ࡍࡿ CD86 ࡜
CD54 ࢆ

ᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋh-CLAT ࡢࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊEURL

ECVAM ࡟ࡼࡿホ౯㸦ࣆ࢔ࣞࣅ࣮ࣗ㸧ࡀ᏶஢ࡋ 1)ࠊ2016 ᖺ 7 ᭶࡟ OECD ࡢヨ㦂ἲ࢞࢖ࢻࣛ࢖
ࣥࣜࢫࢺ࡟㏣ຍࡉࢀࡓ㸦TG442E㸧7)ࠋ
JaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸦௨ୗࠊጤဨ఍㸧ࡣࠊh-CLAT ࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ࠊ⌧ᅾࡲ࡛࡟බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗࢆࡶ࡜࡟ホ౯ࡋࡓࡢ࡛ࠊࡑࡢ⤖ᯝࢆሗ࿌ࡍࡿࠋ
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㸦AOP㸧࡛ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ⓶⭵ឤసࡣḟࡢ 4 ࡘࡢ Key event ࠿ࡽᡂࡿ࡜ࡉࢀ࡚࠸ࡿࠋ
1) ໬Ꮫ≀㉁࡜ࢱࣥࣃࢡ㉁ࡢࢩࢫࢸ࢖ࣥṧᇶ࠶ࡿ࠸ࡣࣜࢪࣥṧᇶ࡜ࡢඹ᭷⤖ྜ5112) ࢣࣛࢳࣀࢧ࢖ࢺ࡟࠾ࡅࡿ⅖⑕ᛶᛂ⟅࠾ࡼࡧ Antioxidant/electrophile response element
㸦ARE㸧-dependent pathway ࡟ࡼࡿ㑇ఏᏊⓎ⌧3) ᶞ≧⣽⬊ࡢάᛶ໬㸦≉␗ⓗ⣽⬊⾲㠃࣐࣮࣮࢝ࡢⓎ⌧ࠊࢣࣔ࢝࢖ࣥࡸࢧ࢖ࢺ࢝࢖ࣥࡢ⏘⏕㸧4) ࣜࣥࣃ⠇࡟࠾ࡅࡿ T ⣽⬊ࡢቑṪh-CLAT ࡣୖグࡢ➨ 3 ࡢ Key event ࡟ᑐᛂࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡑࡢᇶᮏⓗཎ⌮ࡣࠊᶞ≧⣽⬊ࡢάᛶ໬᫬࡟Ⓨ⌧ࡀቑ኱ࡍࡿ⣽⬊⾲㠃ศᏊࡢᐃ࡛࠶ࡿࠋ ᶞ≧⣽⬊ࡢάᛶ໬᫬࡟ࡣࠊCD86ࠊCD54 ࡢࡼ࠺࡞⣽⬊⾲㠃ศᏊࡢⓎ⌧㔞ࡀቑ኱ࡍࡿࡇ࡜ࡀ
▱ࡽࢀ࡚࠸ࡿࠋCD86 ࡣࠊT ⣽⬊࡟ᢠཎᥦ♧ࡍࡿ㝿࡟ T ⣽⬊⾲㠃ࡢ CD28 ࡜┦஫స⏝ࡍࡿࡇ࡜࡟ࡼࡾ⿵ຓ่⃭ศᏊ࡜ࡋ࡚ᶵ⬟ࡍࡿࠋCD54 ࡣࠊูྡ Intercellular adhesion molecule-1
ICAM-1㸧࡜࠸࠺᥋╔ศᏊ࡛࠶ࡾࠊT ⣽⬊⾲㠃ࡢ Lymphocyte function-associated antigen 㸦
1 LFA1㸧࡜⤖ྜࡍࡿࠋ h-CLAT ࡛ࡣࠊᶞ≧⣽⬊ࡢࣔࢹࣝ࡜ࡋ࡚ࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊ໬Ꮫ≀㉁ࡀ THP-1 ⣽⬊ࢆάᛶ໬ࡍࡿ⬟ຊࢆホ౯ࡍࡿࠋTHP-1 ⣽⬊࡟᳨య࡛࠶ࡿ໬Ꮫ≀㉁ࢆῧ
ຍࡋ࡚ 24 ᫬㛫ᇵ㣴ࡋࡓᚋࠊCD86 ࠾ࡼࡧ CD54 ࡢⓎ⌧㔞ࢆࠊ⺯ගᶆ㆑ࡋࡓ≉␗ᢠయࢆ⏝࠸࡚ࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜ࡟ࡼࡾ

ᐃࡍࡿࠋ⁐፹ࡢࡳࢆῧຍࡋࡓࢥࣥࢺ࣮ࣟࣝ࡟ᑐࡍࡿ┦ᑐ⺯ගᙉᗘࢆ⟬ฟࡋࠊ໬Ꮫ≀㉁࡟ࡼࡿᶞ≧⣽⬊ࡢάᛶ໬ࡢᣦᶆ࡜ࡍࡿࠋ
3㸬ヨ㦂ᡭ㡰㸭ุᐃh-CLAT ࡢヨ㦂ᡭ㡰ࡣࠊ⏝㔞タᐃヨ㦂࡜ CD86/CD54 Ⓨ⌧

ᐃࡢ 2 ࡘ࡟ศࡅࡽࢀࡿࠋ

3-1㸬⣽⬊ㄪ〇࠾ࡼࡧヨ⸆ h-CLAT ࡛ࡣࠊTHP-1 ⣽⬊ࢆ౑⏝ࡍࡿࠋ⣽⬊ࡣ American Type Culture Collection㸦ATCC㸧
࠿ࡽධᡭ㸦TIB-202TM㸧ࡍࡿࡇ࡜ࡀᮃࡲࡋ࠸ࠋ
 THP-1 ⣽⬊ࡣࠊᇵ㣴⏝ᇵᆅ㸦RPMI-1620 ࡢᇶ♏ᇵᆅ࡟ 10% Fetal bovine serum, 0.05 mM 2Mercaptoethanol, 100 unit/mL Penicillin, 100 ȝg/mL Streptomycin ࢆ ຍ ࠼ ࡓ ࡶ ࡢ 㸧 ࡟ ࡚ ࠊ
0.1~1.0×106 cells/mL ࡢ⣽⬊⃰ᗘ࡛᧛✀ࡋࠊ2~3 ᪥ࡈ࡜࡟⥅௦ࢆ⾜࠸ࠊ⥔ᣢࡍࡿࠋ⣽⬊⃰ᗘࡣ
0.1~0.8×106 cells/mL ࢆ⥔ᣢࡋࠊ1.0×106 cells/mL ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡍࡿࠋࡲࡓࠊATCC ࠿ࡽධᡭᚋࡢึᅇ⼥ゎ 2 㐌㛫ᚋ࡟ࠊ2,4-Dinitrochlorobenzene㸦DNCB, CAS No.97-00-7ࠊ⣧ᗘӍ99%
௨ୖ㸧ࠊNickel sulfate㸦CAS NO. 10101-97-0ࠊ⣧ᗘӍ99%௨ୖ㸧࠾ࡼࡧ㝜ᛶᑐ↷࡜ࡋ࡚ Lactic
acid㸦CAS NO. 50-21-5ࠊ⣧ᗘӍ85%௨ୖ㸧ࢆ⏝࠸࡚ࠊ཯ᛂᛶࡢ☜ㄆࢆ⾜࠺ࠋDNCB ࡜ Nickel
sulfate ࡛ࡣࠊCD86 ࡜ CD54 ࡢ୧᪉࡛㝧ᛶ࡜࡞ࡾࠊLactic acid ࡛ࡣࠊCD86 ࡜ CD54 ࡢ࠸ࡎࢀ࡛ࡶ㝜ᛶࡢ⤖ᯝ࡜࡞ࡿࠋ཯ᛂᛶヨ㦂࡛ྜ᱁ࡋࡓ⣽⬊ࡢࡳࢆᮏヨ㦂ἲ࡛⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿࠋ
THP-1 ⣽⬊ࡣࠊ⼥ゎᚋ 2 ࣨ᭶ࡲ࡛⥅௦ࡉࡏࡿࡇ࡜ࡀ࡛ࡁࠊ⥅௦ᩘࡀ 30 ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡍ
࡭ࡁ࡛࠶ࡿࠋ
ヨ㦂ᐇ᪋๓࡟ࠊTHP-1 ⣽⬊ࢆ 0.1 ࡶࡋࡃࡣ 0.2×106 cells/mL ࡢ⣽⬊⃰ᗘ࡛ᇵ㣴⏝ࣇࣛࢫࢥ࡟᧛✀ࡋࠊࡑࢀࡒࢀ 72 ᫬㛫ࡶࡋࡃࡣ 48 ᫬㛫ᇵ㣴ࡍࡿࠋᐇ㦂᪥࡟ࠊ2×106 cells/mL ࡢ⃰ᗘ࡛612᪂ࡋ࠸ᇵ㣴⏝ᇵᆅ࡟෌ᠱ⃮ࡋࡓ THP-1 ⣽⬊ᠱ⃮ᾮࢆ 24 ✰ᖹᗏࣉ࣮ࣞࢺࡶࡋࡃࡣ 96 ✰ᖹᗏࣉ࣮ࣞࢺ࡟ࠊࡑࢀࡒࢀ 500 ȝL/well ࡶࡋࡃࡣ 80 ȝL/well ࡛ຍ࠼ࡿࠋ
3-2. ⏝㔞タᐃヨ㦂
⏝㔞タᐃヨ㦂ࡣࠊCV75㸦፹యᑐ↷࡜ẚ㍑ࡋ࡚ 75%ࡢ⣽⬊⏕Ꮡ⋡ࢆ♧ࡍ⿕㦂≀㉁⃰ᗘ㸧ࢆỴᐃࡍࡿࡓࡵ࡟ᐇ᪋ࡍࡿࠋCV75 ࡣ CD86 ࠾ࡼࡧ CD54 Ⓨ⌧㔞ᐃࡢ⃰ᗘタᐃ࡟฼⏝ࡉࢀࡿࠋ1) ⿕㦂≀㈨࠾ࡼࡧ㝜ᛶᑐ↷ࡢㄪ〇 ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡣࠊ⏝᫬ㄪ〇ࡍࡿࠋh-CLAT ࡛ࡣࠊ⿕㦂≀㉁ࡣ⁐ᾮࡶࡋࡃࡣᏳᐃ
࡞ᠱ⃮ᾮ࡟ㄪ〇ࡍࡿࠋ፹యࡣ⏕⌮㣗ሷỈ࠾ࡼࡧᇶ♏ᇵᆅࢆ➨୍ೃ⿵ࠊDimethyl sulfoxide㸦DMSO, ⣧ᗘӍ99%௨ୖ㸧ࢆ➨஧ೃ⿵࡜ࡋࠊ᭱⤊⃰ᗘࡣ⏕⌮㣗ሷỈ࠾ࡼࡧᇵᆅࡢሙྜࡣ 100
mg/mLࠊDMSO ࡢሙྜࡣ 500 mg/mL ࡜ࡍࡿࠋ100 mg/mL㸦⏕⌮㣗ሷỈ࠾ࡼࡧᇵᆅࡢሙྜ㸧࠾
ࡼࡧ 500 mg/mL㸦DMSO ࡢሙྜ㸧ࡢಖᏑ⁐ᾮࢆ⏝࠸࡚ࠊ௨ୗࡢᡭ㡰࡛ᕼ㔘᧯సࢆ⾜࠺ࠋ 㸫⏕⌮㣗ሷỈ࠾ࡼࡧᇶ♏ᇵᆅࢆ౑⏝ࡍࡿሙྜ㸸ࡑࢀࡒࢀࡢ፹యࢆ⏝࠸࡚බẚ 2 ࡛ᕼ㔘ࢆ⾜࠸ࠊ8 ⃰ᗘࡢಖᏑཎᾮࢆస〇ࡍࡿࠋࡇࢀࡽಖᏑཎᾮࡣࡑࢀࡒࢀᇵ㣴⏝ᇵᆅ࡛ࡉࡽ࡟ 50 ಸ࡟ᕼ㔘ࡍࡿ㸦౑⏝ᾮ㸧ࠋࡶࡋ᭱㧗⃰ᗘ࡛࠶ࡿ 1,000 ȝg/mL ࡛⣽⬊ẘᛶࡀ࡞࠸ሙྜࠊ᭱㧗⃰ᗘࢆୖࡆ࡚⣽⬊ẘᛶヨ㦂ࢆ෌ᐇ᪋ࡋ࡚Ỵᐃࡍࡿࡀࠊࣉ࣮ࣞࢺ࡟࠾ࡅࡿ᭱⤊⃰ᗘࡣࠊ5,000 ȝg/mLࢆ㉸࠼࡚ࡣ࠸ࡅ࡞࠸ࠋ
 㸫DMSO ࢆ౑⏝ࡍࡿሙྜ㸸DMSO ࢆ⏝࠸࡚බẚ 2 ࡛ᕼ㔘ࢆ⾜࠸ࠊ8 ⃰ᗘࡢಖᏑཎᾮࢆస
〇ࡍࡿࠋಖᏑཎᾮࡣࡑࡢᚋࠊᇵ㣴⏝ᇵᆅ࡛ࡉࡽ࡟ 250 ಸ࡟ᕼ㔘ࡍࡿ㸦౑⏝ᾮ㸧ࠋࡓ࡜࠼⣽⬊ẘᛶࡀ࡞ࡃ࡜ࡶࠊࣉ࣮ࣞࢺ୰ࡢ᭱⤊⃰ᗘࡣࠊ1,000 ȝg/mL ࢆ㉸࠼࡚ࡣ࠸ࡅ࡞࠸ࠋ ፹యᑐ↷࡟ࡣࠊ⿕㦂≀㉁ࡀ⏕⌮㣗ሷỈࡲࡓࡣᇶ♏ᇵᆅ࡛⁐ᾮࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮ᾮ࡜࡞ࡿሙྜ࡟ࡣᇵ㣴⏝ᇵᆅࢆ⏝࠸ࠊ⿕㦂≀㉁ࡀ DMSO ࡛⁐ᾮࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮ᾮ࡜࡞ࡿሙྜࡣ DMSO ࢆ⏝࠸࡚ࠊࡑࡢࣉ࣮ࣞࢺ࡟࠾ࡅࡿ᭱⤊⃰ᗘࡣ 0.2%࡜ࡍࡿࠋࡑࢀࡽࡣୖグࡢ౑⏝ᾮస〇ࡢᡭ㡰࡜ྠࡌᕼ㔘࡛⾜࠺ࠋ2) ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡢ᭚㟢 1) ࡛ㄪ〇ࡋࡓ⿕㦂≀㉁ࢆྵࡴ౑⏝ᾮ࠾ࡼࡧ፹యᑐ↷ࡣࠊ⣽⬊ᠱ⃮ᾮ࡜ 1:1 ࡛ΰྜࡋ㸦㏻ᖖࠊ᭱⤊⃰ᗘࡢ⠊ᅖࡣࠊ7.81~1,000 ȝg/mL㸧ࠊ24 ✰ᖹᗏࣉ࣮ࣞࢺࡶࡋࡃࡣ 96 ✰ᖹᗏࣉ࣮ࣞࢺ
࡟࡚ 37Υࠊ5%CO2 in air ࡛ 24 ᫬㛫㸦s0.5 ᫬㛫㸧ᇵ㣴ࡍࡿࠋ᥹Ⓨᛶࡢ㧗࠸⿕㦂≀㉁ࡢሙྜ
ࡣࠊ࢙࢘ࣝ㛫࡛⿕㦂≀㉁ࡢࢥࣥࢱ࣑ࡀ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࡢ࡛ࠊࣉ࣮ࣞࢺࢆࢩ࣮ࣝࡍࡿ࡞࡝
ࡢฎ⨨ࢆ᪋ࡍࡇ࡜ࡀᮃࡲࡋ࠸ࠋ
3)

Propidium iodide㸦PI㸧ᰁⰍ
24 ᫬㛫㸦s0.5 ᫬㛫㸧ࡢ᭚㟢ᚋࠊ⣽⬊ࡣࢧࣥࣉࣝࢳ࣮ࣗࣈ࡟⛣ࡋࠊ㐲ᚰ࡟ࡼࡾ⣽⬊ࢆ㞟ࡵࡿࠋୖ⃈ࡳࢆᤞ࡚ࡓᚋࠊ0.1%࢘ࢩ⾑Ύ࢔ࣝࣈ࣑ࣥ㸦Bovine Serum Albumin: BSA㸧ྵ᭷ࣜࣥ㓟⦆⾪ᾮ 200ȝL㸦96 ✰ࡢሙྜ㸧ࡶࡋࡃࡣ 600ȝL㸦24 ✰ࡢሙྜ㸧ࢆຍ࠼ࠊ෌ᠱ⃮ࡍࡿࠋࡇࡢ⣽⬊ᠱ⃮ᾮ 200ȝL ࡣࠊ96 ✰ࡢሙྜࡣ 96 ✰୸ᗏᆺࣉ࣮ࣞࢺ࡟ࠊ24 ✰ࡢሙྜࡣ࣐࢖ࢡࣟࢳ࣮ࣗ
713ࣈ࡟⛣ࡋࠊ96 ✰࡛ࡣ 200ȝL ࡢ 0.1% BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡛ࠊ24 ✰࡛ࡣ 600ȝL ࡢ 0.1%BSAྵ᭷ࣜࣥ㓟⦆⾪ᾮ ࡛ 2 ᅇ௨ୖὙίࡍࡿࠋ᭱⤊ⓗ࡟ࠊ⣽⬊ࡣ 0.1㸣BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ 㸦౛࠼ࡤࠊ400 ȝL㸧࡛෌ᠱ⃮ࡋࠊࡑࡇ࡟ 20 ȝL ࡢ PI ⁐ᾮ㸦PI ࡢ᭱⤊⃰ᗘࡣ 0.625 ȝg/mL㸧ࢆຍ࠼ࡿࠋ4) ⣽⬊ẘᛶࡢᐃ࡜ CV75 ࡢ⟬ฟPI ࡢྲྀࡾ㎸ࡳࡣࠊࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮࡛ศᯒࡍࡿࠋ⣽⬊ẘᛶࡣࠊ10,000 ࡢ⏕⣽⬊㸦PI ࢿ࢞ࢸ࢕ࣈ㸧ࡀ࢝࢘ࣥࢺ࡛ࡁࡿ᮲௳࡛ࠊ௨ୗࡢᘧ࡟ࡼࡾ⟬ฟࡍࡿࠋࡲࡓࠊ⣽⬊⏕Ꮡ⋡ࡀప࠸ሙྜ࡟ࡣࠊṚ⣽⬊ᩘࢆྵࡴ⣽⬊ᩘࢆ 30,000 ࡜ࡋࡓ᮲௳࡟࡚ࠊ⣽⬊⏕Ꮡ⋡㸦cell viability㸧ࢆ⟬ฟࡍࡿࠋࡲࡓࠊCV75㸦THP-1 ⣽⬊ࡢ 75%ࡀ⏕⣽⬊࡛࠶ࡿ⃰ᗘ㸧ࡣࠊḟᘧ࡟ࡼࡾ⟬ฟࡍࡿࠋ a㸸ヨ㦂ࡋࡓ⃰ᗘ࡟࠾࠸࡚⏕Ꮡ⋡ 75%ࢆ㉸࠼ࡿ᭱ᑠࡢ⏕Ꮡ⋡c㸸ヨ㦂ࡋࡓ⃰ᗘ࡟࠾࠸࡚⏕Ꮡ⋡ 75%ࢆ㉸࠼࡞࠸᭱኱ࡢ⏕Ꮡ⋡b㸸a ࡜࡞ࡿ⿕㦂≀㉁⃰ᗘ
d㸸c ࡜࡞ࡿ⿕㦂≀㉁⃰ᗘ
3-3㸬CD86 ࠾ࡼࡧ CD54 Ⓨ⌧ᐃ1) ⿕㦂≀㉁ࠊ㝜ᛶᑐ↷࠾ࡼࡧ㝧ᛶᑐ↷ࡢㄪ〇⿕㦂≀㉁ࢆ⁐ゎࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮ᾮ࡜ࡉࡏࡿ፹య࡟ࡣࠊ⏕⌮㣗ሷỈࠊᇶ♏ᇵᆅ࠾ࡼࡧDMSO ࠿ࡽ㐺ษ࡞ࡶࡢࢆ౑⏝ࡍࡿࠋ⿕㦂≀㉁ࡢ᭱㧗⃰ᗘࡣ⏝㔞タᐃヨ㦂࡟࡚⟬ฟࡉࢀࡓ
CV75 ࡢ 1.2 ಸ⃰ᗘ࡜ࡍࡿࠋࡶࡋࠊCV75 ࡀồࡵࡽࢀ࡞࠿ࡗࡓሙྜ㸦౛࠼ࡤࠊ⏝㔞タᐃヨ㦂࡛༑ศ࡞⣽⬊ẘᛶࡀㄆࡵࡽࢀ࡞࠿ࡗࡓሙྜ㸧࡟ࡣࠊ⿕㦂≀㉁ࡢ᭱኱⁐ゎࡶࡋࡃࡣᏳᐃ࡞ᠱ⃮⃰ᗘࢆ㛤ጞ⃰ᗘ࡜ࡍࡿࡀࠊࣉ࣮ࣞࢺࡢ᭱⤊⃰ᗘࡣࠊ⏕⌮㣗ሷỈࡶࡋࡃࡣᇶ♏ᇵᆅ࡛ࡣ 5,000 ȝg/mLࠊDMSO ࡢሙྜࡣ 1,000 ȝg /mL ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡍࡿࠋࡑࡢᚋࠊྠࡌ፹యࢆ⏝࠸࡚ࠊබẚ 1.2 ࡛ᕼ㔘ࡍࡿࡇ࡜࡛ 8 ⃰ᗘࡢಖᏑཎᾮ㸦⃰ᗘᖜ㸸0.335CV75㹼1.2×CV75㸸፹యࡀ⏕⌮㣗ሷᾮ࠾ࡼࡧᇶ♏ᇵᆅࡢሙྜࡣࡇࡢ 100 ಸࡢ⃰ᗘࠊDMSO ࡢሙྜࡣࡇࡢ 500 ಸࡢ⃰ᗘ㸧ࢆస〇ࡍࡿࠋಖᏑཎᾮࡣࡑࡢᚋࠊᇵ㣴⏝ᇵᆅࢆ⏝࠸࡚ࠊ፹యࡀ⏕⌮㣗ሷỈ࠾ࡼࡧᇶ♏ᇵᆅࡢሙྜࡣ 50 ಸࠊ፹యࡀ DMSO ࡢሙྜࡣ 250 ಸ࡟ᕼ㔘ࡋࠊ౑⏝ᾮࢆస〇ࡍࡿࠋࡇࢀࡽ౑⏝ᾮࡣࠊ᭱⤊ⓗ࡟⣽⬊ᠱ⃮ᾮ࡛ࡉࡽ࡟ 2 ಸᕼ㔘ࡉࢀࠊ᭚㟢ࡉࢀࡿࡇ࡜࡜࡞ࡿࠋࡶࡋ⤖ᯝࡀࠊᚋ㏙ࡍࡿヨ㦂ᡂ❧ࡢ᮲௳࡟ྜࢃ࡞࠸᫬ࡣࠊṇ☜࡞ CV75 ࢆᚓࡿࡓࡵ࡟⏝㔞タᐃヨ㦂ࢆ෌ᗘᐇ᪋ࡋ࡚ࡶ
ࡼ࠸ࠋCD86 ࠾ࡼࡧ CD54 Ⓨ⌧

ᐃ࡛ࡣࠊ24 well ࡢࡳࡀ౑ࢃࢀࡿࡇ࡜࡟ὀពࡍࡿᚲせࡀ࠶
8

14

ࡿࠋ
㝜ᛶᑐ↷/፹యᑐ↷ࡣࠊ⏝㔞タᐃヨ㦂࡜ྠᵝ࡟ㄪ〇ࡍࡿࠋ㝧ᛶᑐ↷࡟ࡣ DNCB ࢆ౑⏝ࡍࡿࠋ፹య࡟ DMSO ࢆ౑⏝ࡋࠊୖẁ࡟♧ࡍ࡜࠾ࡾࠊಖᏑཎᾮࢆస〇ࡍࡿࠋDNCB ࡣࠊCD86 ࠾ࡼࡧ
CD54 Ⓨ⌧ᐃࡢ㝧ᛶᑐ↷࡜ࡋ࡚ࠊ1 ⃰ᗘ㸦୍⯡ⓗ࡟ࡣࠊࣉ࣮ࣞࢺ୰ࡢ᭱⤊⃰ᗘࡀ 4 ȝg/mL㸧ࡀ౑ࢃࢀࡿࠋ4 ȝg/mL ࡜ࡍࡿࡓࡵ࡟ࠊDMSO ࢆ፹య࡟ࡋࡓ 2 mg/mL ࡢಖᏑཎᾮࢆᇵ㣴⏝ᇵ
ᆅ࡛ 250 ಸ࡟ᕼ㔘ࡋ࡚ࠊ8 ȝg/mL ࡢ౑⏝ᾮࢆస〇ࡍࡿࠋDNCB ⃰ᗘ࡟㛵ࡋ࡚ࡣࠊྛ᪋タ࡛Ỵᐃࡉࢀࡓ CV75 ࢆ㝧ᛶᑐ↷⃰ᗘ࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡶྍ⬟࡛࠶ࡿࠋ㝧ᛶᑐ↷࡟࠾࠸࡚ࡶࣉ࣮ࣞࢺࡢ᭱⤊⃰ᗘࡣࠊ⏕⌮㣗ሷỈࡶࡋࡃࡣᇶ♏ᇵᆅ࡛ࡣ 5,000
ȝg/mLࠊDMSO ࡢሙྜࡣ 1,000 ȝg/mL ࢆ㉸࠼࡞࠸ࡼ࠺࡟ࡋࠊ㝧ᛶᑐ↷ࡣ 1 ⃰ᗘ࡛࠶ࡿࡇ࡜࠿
ࡽᚋ㏙ࡍࡿ⿕㦂≀㉁ࡢヨ㦂ᡂ❧ࡢ᮲௳ࡢ࠺ࡕࠊ᭱⤊㡯┠ࡢࡳᑐ㇟እ࡜࡞ࡿࠋ
2) ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡢ᭚㟢⿕㦂≀㉁ࡢ⓶⭵ឤసᛶࡢண
ࡢ CD86 ࠾ࡼࡧ CD54 Ⓨ⌧⤖ᯝ㸦㝧ᛶࡶࡋࡃࡣ㝜ᛶ㸧ࢆᚓࡿࡓࡵ࡟ࡣࠊᑡ࡞ࡃ࡜ࡶ 2 ᅇᐃࢆ⧞ࡾ㏉ࡍᚲせࡀ࠶ࡿࠋ2 ᅇࡢྠ᪥ࡢ࡝ࡕࡽ࡛ࡶⰋ࠸ࡀࠊࡑࢀࡒࢀࡢ

ᐃࡣࠊ␗࡞ࡿ᪥ࡶࡋࡃࡣࠊ

ᐃ࡛ಖᏑཎᾮ, ⿕㦂≀㉁ࡢ౑⏝ᾮ࠾ࡼࡧᰁⰍ⏝ᢠయ⁐ᾮࡣูࠎ࡟ㄪ〇ࡉࢀࡿࡇ࡜ࠊ౑⏝ࡉࢀࡿ⣽⬊ሢࢆศࡅࡿ㸦౛࠼ࡤࠊ⣽⬊ࡣ␗࡞ࡿࣇࣛࢫࢥ
࠿ࡽ㞟ࡵࡿ࡞࡝㸧ࡇ࡜ࡀᚲせ࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⣽⬊ࡣྠࡌ⥅௦ᩘ⏤᮶࡛ࡶᵓࢃ࡞࠸ࠋ
౑⏝ᾮ࡜ࡋ࡚ㄪ〇ࡋࡓ⿕㦂≀㉁࠾ࡼࡧ㝜ᛶᑐ↷ࡣࠊࡑࢀࡒࢀ⣽⬊ᠱ⃮ᾮ࡜ 1:1 ࡢ๭ྜ࡛ΰྜ
ࡋࠊ37Υࠊ5%CO2 in airࠊ24 ᫬㛫s0.5 ᫬㛫ࠊᇵ㣴ࡍࡿࠋᑡ࡞ࡃ࡜ࡶ 2 ᅇࡢ࡛ࠊࡑࢀࡒࢀࡢ

ᐃࢆᐇ᪋ࡍࡿࡢ

ᐃ࡛ࡣࠊ ᐃෆࡢ⿕㦂≀㉁ࡢྛ⃰ᗘ࠾ࡼࡧ㝜ᛶᑐ↷ࡣࠊn=1 ࡛࠶ࡗ࡚ࡶၥ㢟࡞࠸ࠋ3) ⣽⬊ࡢᰁⰍ࠾ࡼࡧศᯒ
24 ᫬㛫ࡢ᭚㟢ᚋࠊ⣽⬊ᾮࢆࢧࣥࣉࣝࢳ࣮ࣗࣈ࡟⛣ࡋࠊ㐲ᚰ࡟ࡼࡾ⣽⬊ࢆ㞟ࡵࠊ1 mL ࡢ
0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ㸦pH 7.4㸧࡛ 2 ᅇὙίࡍࡿࠋὙίᚋࠊ600 ȝL ࡢ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡟ 0.01%㸦w/v㸧ࡢ⃰ᗘ࡛ࢢࣟࣈࣜࣥ㸦Cohn fraction II, III, Human; SIGMA, #238810G㸧ࢆຍ࠼ࡓࡶࡢࢆຍ࠼ࠊ4Υ࡛ 15 ศࠊ࢖࣮ࣥ࢟ࣗ࣋ࢺࡋ࡚ࠊࣈࣟࢵࢡࢆ⾜࠺ࠋࡑࡢᚋࠊ
⣽⬊ࡣࠊ180 ȝL ࡎࡘ 3 ࡘ࡟➼ศࡋࠊ୸ᗏ 96 ✰ࣉ࣮ࣞࢺࡶࡋࡃࡣ࣐࢖ࢡࣟࢳ࣮ࣗࣈ࡟⛣ࡍࠋ㐲ᚰᚋࠊ⣽⬊ࡣࠊ50 ȝL ࡢ Fluorescein isothiocyanate㸦FITC㸧ᶆ㆑ᢠ CD86 ᢠయࠊFITC ᶆ㆑ᢠ CD54 ᢠయࡶࡋࡃࡣ FITC ᶆ㆑࣐࢘ࢫ IgG1 ᢠయ㸦࢔࢖ࢯࢱ࢖ࣉ㸧࡟࡚ࠊ4Υ࡛ 30 ศ㛫ࠊᰁⰍࡍࡿࠋh-CLAT DB-ALM ࣉࣟࢺࢥ࣮ࣝ 2)࡛グ㍕ࡉࢀ࡚࠸ࡿᢠయ࡛ࡣࠊᢠ CD86 ᢠయ㸦BDPharMingen #555657; Clone:Fun-1㸧࡛ࡣ 3:25 ࡢ๭ྜ࡛ᢠయ࡜ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮࢆΰྜࡋࠊᢠ CD54 ᢠయ㸦DAKO, #F7143; Clone: 6.5B5㸧࠾ࡼࡧ IgG1㸦DAKOࠊ#X0927㸧࡛ࡣ
3:50 ࡢ๭ྜ࡛ᢠయ࡜ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮࢆΰྜࡍࡿࠋ
ᢠయᰁⰍᚋࡢ⣽⬊ࡣ 200ȝL ࡢ 0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡛ 3 ᅇὙίᚋࠊ400 ȝL ࡢ
0.1%BSA ྵ᭷ࣜࣥ㓟⦆⾪ᾮ࡛෌ᠱ⃮ࡋࠊࡑࡇ࡟ PI ⁐ᾮ㸦PI ⃰ᗘࡀ 0.625 ȝg/mL ࡛࠶ࢀࡤ20 ȝL㸧ࡶࡋࡃࡣࡑࡢ௚ࡢ⣽⬊ẘᛶ࣐࣮࣮࢝ᾮࢆῧຍࡍࡿࠋCD86 ࠾ࡼࡧ CD54 ࡢⓎ⌧ࠊ⣽⬊⏕Ꮡ⋡ࡣࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮ࢆ⏝࠸࡚

ᐃࡍࡿࠋ
9
15

3-4 ࢹ࣮ࢱ࠾ࡼࡧሗ࿌
1) ࢹ࣮ࢱࡢホ౯
CD86 ࡜ CD54 ࡢⓎ⌧ࡣࠊࢳࣕࣥࢿࣝ FL-1 ࢆ᭷ࡍࡿࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮࡛

ᐃࡍࡿࠋᖹᆒ⺯ගᙉᗘ㸦Mean Fluorescence Intensity: MFI㸧ࢆᇶ࡟ࠊ௨ୗࡢᘧ࠿ࡽࠊ㝧ᛶᑐ↷࠾ࡼࡧ⿕㦂≀㉁࡛ฎ⌮ࡋࡓ⣽⬊࡟࠾ࡅࡿ CD86 ࠾ࡼࡧ CD54 ࡢ┦ᑐ⺯ගᙉᗘ㸦Relative Fluorescence
Intensity: RFI㸧ࢆồࡵࡿࠋ࢔࢖ࢯࢱ࢖ࣉࢥࣥࢺ࣮ࣟࣝ㸦࣐࢘ࢫ IgG1 ᢠయ࡛ᰁⰍࡋࡓࡶࡢ㸧ࡢ⣽⬊⏕Ꮡ⋡ࡣࠊ3-2 4)ࡢ
ᘧࢆ⏝࠸࡚ィ⟬ࡍࡿࠋ
2) ண

᪉ἲ

CD86 ࠾ࡼࡧ CD54 Ⓨ⌧ᐃ࡛ࡣࠊࡑࢀࡒࢀࡢ⿕㦂≀㉁ࡢண 㸦㝧ᛶࡶࡋࡃࡣ㝜ᛶ㸧ࢆ⾜࠺ࡓࡵ࡟ࠊᑡ࡞ࡃ࡜ࡶ 2 ᅇࡢ⊂❧ࡋࡓࡢᐃࡢ࠺ࡕ 2 ᅇࠊࡶࡋࡃࡣ 3 ᅇࡢᐃࢆ⾜࠺ᚲせࡀ࠶ࡿࠋ௨ୗࡢ≧ἣࡢ࠺ࡕ㸯ࡘࡀ 2 ᅇᐃࡢ࠺ࡕ 2 ᅇ࡛ㄆࡵࡽࢀࡓሙྜࠊh-CLAT ࡟ࡼࡿணࡣࠊ㝧ᛶ࡜ุᐃࡍࡿࠋ
࣭⣽⬊⏕Ꮡ⋡ 50%௨ୖ࡜࡞ࡿ࠸ࡎࢀ࠿ࡢ⃰ᗘ࡟࠾࠸࡚ࠊCD86 ࡢ┦ᑐ⺯ගᙉᗘࡀ 150%௨
ୖ࡜࡞ࡿሙྜ
࣭⣽⬊⏕Ꮡ⋡ 50%௨ୖ࡜࡞ࡿ࠸ࡎࢀ࠿ࡢ⃰ᗘ࡟࠾࠸࡚ࠊCD54 ࡢ┦ᑐ⺯ගᙉᗘࡀ 200%௨
ୖ࡜࡞ࡿሙྜ ᭱ึࡢ 2 ᅇࡢணᐃ࡟࠾࠸࡚ࠊCD86 ࠾ࡼࡧ/ࡶࡋࡃࡣ CD54 ࡀ࡜ࡶ࡟㝧ᛶ࡛࠶ࢀࡤࠊh-CLATࡣ㝧ᛶ࡜ุᐃࡋࠊ3 ᅇ┠ࡢᐃࡣ୙せ࡛࠶ࡿࠋྠᵝ࡟᭱ึࡢ 2 ᅇࡢ

ࡧ CD54 ࡀඹ࡟㝜ᛶ࡛࠶ࢀࡤࠊ3 ᅇ┠ࡢ
2 ᅇࡢ

ᐃࡣ୙せ࡛ h-CLAT ண

ᐃ࡛ࠊCD86 ࠾ࡼࡣ㝜ᛶ࡜ุᐃࡍࡿࠋ୍᪉ࠊ

ᐃ࡛஧ࡘࡢ࣐࣮࣮࢝ࡢ࠺ࡕ࠸ࡎࢀࡶ㐃⥆ࡋ࡚㝧ᛶࢆ♧ࡉ࡞࠿ࡗࡓሙྜࠊ࠶ࡿ࠸ࡣ୧

⪅࡜ࡶ㐃⥆ࡋ࡚㝜ᛶ࡜࡞ࡽ࡞࠿ࡗࡓሙྜࠊ3 ᅇ┠ࡢᐃࡀᚲせ࡛ࠊ᭱⤊ࡢண

ࡣࠊ3 ᅇࡢ

ᐃࡢከᩘ⤖ᯝࢆࡶࡗ࡚Ỵᐃࡍࡿࠋ2 ᅇࡢ┠ࡢᐃࢆ⾜࠸ࠊ1 ᅇ┠ࡣ CD86 ࡀ㝧ᛶࠊ2 ᅇ┠

ᐃࡀᚲせ࡛࠶ࡿࠋࡑࡢ⤖ᯝࠊ3 ᅇ┠ุᐃࡍࡿࠋ୍᪉ࠊ3 ᅇ┠ࡢᐃ࡛ CD54 ࡀ㝧ᛶ࡜࡞ࡗࡓሙྜࠊ3 ᅇᐃࡀ㝜ᛶ࡜࡞ࡗࡓሙྜࠊh-CLAT ணࡣ㝜ᛶ࡜ᐃ࡛ࠊ࡝ࡕࡽ࠿ࡢ࣐࣮࣮࢝㸦CD86 ࡶࡋࡃࡣ CD54㸧ࡀ㝧ᛶࠊࡶࡋࡃࡣ୧࣐࣮࣮࢝ࡀ㝧ᛶ࡜࡞ࡗࡓሙྜࠊh-CLAT ணࡣ㝧ᛶ࡜ุᐃࡍࡿࠋ

ḟ࣮࣌ࢪ࡟ࣇ࣮ࣟᅗ㸦ᅗ 1㸧ࢆ♧ࡍࠋ
3-5㸬ヨ㦂ᡂ❧ࡢ᮲௳h-CLAT ࡢሙྜࠊ௨ୗࡢ᮲௳ࡀᡂ❧ࡍࡿᚲせࡀ࠶ࡿࠋ࣭ᇶ♏ᇵᆅᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢ⣽⬊⏕Ꮡ⋡ࡀࠊ90%௨ୖ࡛࠶ࡿࠋ
࣭፹యᑐ↷࡟࠾ࡅࡿ CD86 ࠾ࡼࡧ CD54 ࡢ୧᪉ࡢ┦ᑐ⺯ගᙉᗘ್ࡀ㝧ᛶᇶ‽್㸦CD86 ࡛
1016

ࡣ 150㸣ࠊCD54 ࡛ࡣ 200㸣㸧ࢆ㉸࠼࡞࠸ࠋ⁐፹ᑐ↷ࡢ┦ᑐ⺯ගᙉᗘ್ࡣࠊ3-4

1)࡟グ㍕ࡢ

ᘧ࡟ࡼࡾィ⟬ࡉࢀࡿ㸦ࡑࡢ㝿ࠊࠕMFI of chemicalࠖࡣࠕMFI of solvent/vehicleࠖ࡟ࠊࠕMFI ofsolvent/vehicleࠖࡣࠕMFI of (medium) controlࠖ࡟ㄞࡳ᥮࠼ࡿᚲせࡀ࠶ࡿࠋ㸧࣭ᇶ♏ᇵᆅᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢ୧᪉࡟࠾࠸࡚ࠊ࢔࢖ࢯࢱ࢖ࣉ࡟ᑐࡍࡿ CD86 ࡜ CD54 ࡢ୧᪉ࡢᖹᆒ⺯ගᙉᗘࡢ๭ྜࡀ 105%ࡼࡾ኱ࡁࡃ࡞ࡿࠋ
࣭㝧ᛶᑐ↷㸦DNCB㸧࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡀ 50%௨ୖࢆ♧ࡋࠊCD86 ࠾ࡼࡧ CD54 ࡢ┦ᑐ⺯ගᙉᗘࡀ࠸ࡎࢀࡶ㝧ᛶᇶ‽࡜࡞ࡿࠋ࣭⿕㦂≀㉁࡟࠾࠸࡚ࠊࡑࢀࡒࢀࡢ

ᐃ࡛ヨ㦂ࡉࢀࡓᑡ࡞ࡃ࡜ࡶ 4 ⃰ᗘ࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡀ 50㸣ࢆ㉸࠼ࡿ᭱㧗⃰ᗘ㸦1.2CV75㸧࡛ࡢ⣽⬊⏕Ꮡ⋡ࡀ 90㸣ࡼࡾᑠࡉ࠸ሙྜࠊ㝜ᛶ⤖ᯝࡣཷࡅධࢀࡽ
ࢀࡿࡀࠊ1.2CV75 ࡢ⣽⬊⏕Ꮡ⋡ࡀ 90㸣௨ୖ࡛࠶ࡗࡓሙྜ࡟ࡣࠊ㝜ᛶ⤖ᯝࡣཷࡅධࢀࡽࢀ
࡞࠸ࠋࡑࡢࡼ࠺࡞ሙྜ࡟ࡣࠊ෌ᗘ CV75 ࢆỴᐃࡍࡿࡇ࡜࡛ࠊ⃰ᗘタᐃࢆࡸࡾ┤ࡍࡇ࡜ࡀᮃ
ࡲࡋ࠸ࠋࡓࡔࡋࠊ፹య࡟⏕⌮㣗ሷỈࠊᇶ♏ᇵᆅ࠾ࡼࡧࡑࡢ௚ࡢᇵᆅࢆ౑⏝ࡋࡓ 5,000
ȝg/mLࠊDMSO ࢆ౑⏝ࡋࡓ 1,000 ȝg/mL ࠾ࡼࡧ᭱኱⁐ゎ⃰ᗘࡀヨ㦂ࡢ᭱㧗⃰ᗘ࡛࠶ࡿሙ
ྜࠊ⏕Ꮡ⋡ࡀ 90%௨ୖ࡛࠶ࡗ࡚ࡶࠊ㝜ᛶ⤖ᯝࡣチᐜࡉࢀࡿࠋ

1117

ᅗ 1 ண᪉ἲP1㸸CD86 ࡢࡳࡀ㝧ᛶࡢሙྜࠊP2㸸CD54 ࡢࡳࡀ㝧ᛶࡢሙྜࠊP12㸸CD86 ࡜ CD54 ࡀ࡜ࡶ࡟㝧ᛶࡢሙྜࠊN㸸CD86 ࡜ CD54 ࡀ࡜ࡶ࡟㝜ᛶࡢሙྜࠊ*㸸᭱ึࡢ 2 ᅇࡢࡳྜࢃࡏࠊ 㸸᭱ึࡢ 2 ᅇࡢ㸡ᐃ⤖ᯝ࡟ᇶ࡙࠸࡚ᐇ᪋ࡉࢀࡓ 3 ᅇ┠ࡢ

ࢃࡏ 

12
18

ᐃ⤖ᯝࡢ⤌

ᐃ⤖ᯝࡢ⤌ࡳྜ

4㸬⢭ᗘ
EURL ECVAM ࡟ࡼࡾᐇ᪋ࡉࢀࡓࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 3)࡟ࡣ 4 ᪋タ㸦Kao Corporation㸦୺ᑟ᪋タ㸧ࠊShiseido Quality Assessment Center㸦୺ᑟ᪋タ㸧ࠊEURL ECVAMࠊBioassay GmbH㸧ࡀཧຍࡋࠊᢏ⾡⛣㌿ᛶࠊ᪋タෆ෌⌧ᛶ࠾ࡼࡧ᪋タ㛫෌⌧ᛶࡀ᳨ウࡉࢀ࡚࠸ࡿࠋ
4-1. ᢏ⾡⛣㌿ᛶ5 ≀㉁㸦3 ឤసᛶ≀㉁㸸DNCBࠊNickel Sulfateࠊ Phenylacetaldehyde ࠾ࡼࡧ 2 㠀ឤసᛶ≀㉁㸸
Sodium lauryl sulfateࠊLactic acid㸧ࡢࢹ࣮ࢱྲྀᚓࢆ⾜࠸ࠊ୺ᑟ᪋タ࡛࠶ࡿ Kao ࡜ Shiseido ࠿ࡽ EURL ECVAM ࡜ Bioassay ࡢ 2 ᪋タ࡬ࡢᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ホ౯ࡀ⾜ࢃࢀࠊࣂࣜࢹ࣮ࢩ
ࣙࣥሗ࿌᭩࡛ࡣࠊᢏ⾡⛣㌿ᛶ࡟ၥ㢟࡞࠸࡜グ㍕ࡉࢀ࡚࠸ࡿࠋࡓࡔࡋࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)࡛ࡣࠊᮏἲࢆ౑⏝ࡍࡿ๓࡟ヨ㦂᪋タ࡟࠾࠸࡚ᢏ⬟⩦⇍⏝ 10 ≀㉁࠾ࡼࡧ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷ࠊ፹యᑐ↷ࢆ⏝࠸࡚཯ᛂᛶࢆ☜ㄆࡋࠊᢏ⾡ࡀ⩦⇍ࡋ࡚࠸ࡿࡇ࡜ࢆ♧ࡍࡇ࡜ࢆồࡵ࡚࠸ࡿࠋຍ࠼࡚ࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊࣄࢫࢺࣜ࢝ࣝ࡞ࢹ࣮ࢱ࣮࣋ࢫࡢసᡂࢆồࡵ࡚࠸ࡿࠋ
4-2. ᪋タෆ෌⌧ᛶ㸦⾲ 1㸧 ┣᳨ୗ࡛ホ౯ࡉࢀࡓ 15 ≀㉁࡟ࡘ࠸࡚ࠊ4 ᪋タࡢ᪋タෆ෌⌧ᛶ㸦3 ᅇࡢ⧞ࡾ㏉ࡋࡢヨ㦂࡛ྠࡌ⤖ᯝ㸧ࡣࠊKao㸸86.7%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸BenzylsalicylateࠊMethylsalicylate㸧ࠊShiseido㸸
80.0%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸Kathon CGࠊBeryllium sulfateࠊFormaldehyde㸧ࠊBioassay㸸73.3%
㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸FormaldehydeࠊBenzylsalicylateࠊMethylsalicylateࠊXylene㸧ࠊECVAM㸸
80.0%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸Chlorpromazine HClࠊBenzylcinnamateࠊDimethylisophthalate㸧
࡛࠶ࡗࡓࠋ
⾲ 1

᪋タෆ෌⌧ᛶࡢホ౯ᡂ⦼

ID

Chemical name

10 Kathon CG
11 Beryllium sulfate
12 Formaldehyde
13 Chloramine T
14 Chlorpromazine HCl
15 2-mercaptobenzothiazole
16 Benzylsalicylate
17 Benzylcinnamate
18 R(+) Limonene
19 M ethylsalicylate
20 Isopropanol
21 Dimethylisophtalate
22 4-aminobenzoic acid
23 Nickel chloride
24 Xylene

GHS
potency
category

LLNA potency

LLNA

Kao

Shiseido

Bioassay

ECVAM

26172-55-4

1A

extreme

P

3/3

2/3

3/3

3/3

7787-56-6

1A

extreme

P

0/3

2/3

0/3

3/3

50-00-0

1A

strong

P

3/3

2/3

2/3

3/3

CAS NoPositve with EC 150 or EC200

149358-73-6

1A

strong

P

3/3

3/3

3/3

3/3

69-09-0

1A

strong

P

3/3

3/3

3/3

2/3

149-30-4

1A

moderate

P

3/3

3/3

3/3

3/3

118-58-1

1B

moderate

P

1/3

0/3

1/3

3/3

103-41-3

1B

weak

P

0/3

3/3

0/3

1/3

5989-27-5

1B

weak

P

3/3

3/3

3/3

3/3

119-36-8

1B

weak

N

2/3

0/3

1/3

3/3

67-63-0

NC

NC

N

0/3

0/3

0/3

0/3
1/3

1459-93-4

NC

NC

N

0/3

0/3

0/3

150-13-0

NC

NC

N

0/3

0/3

0/3

0/3

7718-54-9

1B

NC (false neg)

N

3/3

3/3

3/3

3/3

1330-20-7NC

weak (false pos)P

Number of concordancy
Concordancy rate (%)

0/3

0/3

1/3

3/3

13/15
86.7

12/15
80.0

11/15
73.3

12/15
80.0

13
19

ᮏヨ㦂࡛ࡣࠊ㐩ᡂᇶ‽ࢆ 85㸣࡟タᐃࡋ࡚࠸ࡓࡀࠊࡇࡢᇶ‽ࢆୖᅇࡗࡓࡢࡣ 4 ᪋タ୰ 1 ᪋タࡢࡳ࡛࠶ࡗࡓࠋ෌⌧ᛶࡢᚓࡽࢀ࡞࠸≀㉁ࡣࠊUN GHS 1A ศ㢮ࡀ 4 ≀㉁ࠊ1B ศ㢮ࡀ 3 ≀㉁ࠊศ㢮୙ྍ≀㉁ࡀ 2 ≀㉁ࡢ 9 ≀㉁࡛࠶ࡗࡓࠋࡲࡓࠊ୍ࡘࡢ᪋タ࡛ࡶഇ㝜ᛶࢆ♧ࡋࡓ≀㉁
ࡣࠊUN GHS 1A ศ㢮ࡀ 1 ≀㉁㸦Beryllium sulfate㸧ࠊ1B ศ㢮ࡀ 3 ≀㉁㸦Benzylsalicylateࠊ
BenzylcinnamateࠊMethylsalicylate㸧ࡢ 4 ≀㉁࡛࠶ࡗࡓࠋࡓࡔࡋࠊOECD ᑓ㛛ᐙ఍㆟࡟࠾࠸࡚ࠊヨ㦂࡟౑⏝ࡍࡿ THP-1 ⣽⬊ࡢ๓ᇵ㣴᮲௳࠾ࡼࡧ⿕㦂≀㉁ࡢ᭚㟢᫬㛫ࢆࡼࡾཝᐦ࡟⟶⌮ࡍࡿࡇ࡜࡟ࡼࡾࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᅗࢀࡿࡇ࡜ࡀ
♧ࡉࢀ㸦ῧ௜㈨ᩱ㸧ࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)ࡢヨ㦂ᡭ㡰࡟ࡑࡢෆᐜࡀ཯ᫎࡉࢀ࡚࠸ࡿࠋ
4-3. ᪋タ㛫෌⌧ᛶ㸦⾲ 2㸧 ┣᳨ୗ࡛ホ౯ࡉࢀࡓ 24 ≀㉁ࡢ 4 ᪋タࡢ᪋タ㛫෌⌧ᛶ㸦4 ᪋タ࡛ྠࡌ⤖ᯝ㸧ࡣ 79.2%㸦୍⮴ࡋ࡞࠿ࡗࡓ≀㉁㸸 Beryllium sulfateࠊBenzylsalicylateࠊBenzylcinnamateࠊMethylsalicylateࠊ
Xylene㸧࡛࠶ࡾࠊ㐩ᡂᇶ‽ 80㸣ࢆୗᅇࡗࡓࠋ୍⮴ࡋ࡞࠿ࡗࡓ≀㉁ࡣ᪋タෆ෌⌧ᛶࡢヨ㦂࡟࠾࠸୍࡚⮴ࡋ࡞࠿ࡗࡓ 9 ≀㉁࡟ྵࡲࢀࡿࠋࡋࡓࡀࡗ࡚ࠊ᪋タෆ෌⌧ᛶ࡜ྠᵝ࡟ࠊᮏἲ࡛ࡣ GHS
1A ศ㢮ࡢ≀㉁ࡀ㐺ษ࡟ุᐃࡉࢀ࡞࠸ሙྜࡢ࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋ⾲ 2᪋タ㛫෌⌧ᛶࡢホ౯ᡂ⦼

ID

Chemical name

1 Benzoquinone
2 4-phenylenediamine
3 Dihydroeugenol
4 1-thioglycerol
5 Imidazolidinylurea
6 Methylmethacrylate
7 Glycerol
8 2,4-dichloronitrobenzene
9 Benzyl alcohol
10 Kathon CG
11 Beryllium sulfate
12 Formaldehyde
13 Chloramine T
14 Chlorpromazine HCl
15 2-mercaptobenzothiazole
16 Benzylsalicylate
17 Benzylcinnamate
18 R(+) Limonene
19 Methylsalicylate
20 Isopropanol
21 Dimethylisophtalate
22 4-aminobenzoic acid
23 Nickel chloride
24 Xylene

LLNA
potency

LLNA
Kao

Shiseido106-51-4

1A

extreme

P

1/1

1/1

1/1

1/1

Y

106-50-3

1A

strong

P

1/1

1/1

1/1

1/1

Y

2785-87-7

1B

moderate

P

1/1

1/1

1/1

1/1

Y

96-27-5

1B

moderate

P

1/1

1/1

1/1

1/1

Y

39236-46-9

1B

weak

P

1/1

1/1

1/1

1/1

Y

80-62-6

1B

weak

P

0/1

0/1

0/1

0/1

Y

56-81-5

NC

NC

N

0/1

0/1

0/1

0/1

Y

611-06-3

NC

NC

N

1/1

1/1

1/1

1/1

Y

100-51-6

NC

NC

N

1/1

1/1

1/1

1/1

Y

26172-55-4

1A

extremeP

7787-56-6

1A

extreme

P

3/3
0/3

2/3
2/3

3/3
0/3

3/3
3/3

Y
N

50-00-0

1A

strong

P

3/3

2/3

2/3

3/3

Y

149358-73-6

1A

strong

P

3/3

3/3

3/3

3/3

Y

69-09-0

1A

strong

P

3/3

3/3

3/3

2/3

Y

149-30-4

1A

moderateP

118-58-1

1B

moderate

P

3/3
1/3

3/3
0/3

3/3
1/3

3/3
3/3

Y
N

Positve with EC 150 or EC200
Bioassay

103-41-3

1B

weak

P

0/3

3/3

0/3

1/3

N

5989-27-5

1B

weak

P

119-36-8

1B

weak

N

3/3
2/3

3/3
0/3

3/3
1/3

3/3
3/3

Y
N

67-63-0

NC

NC

N

0/3

0/3

0/3

0/3

Y

1459-93-4

NC

NC

N

0/3

0/3

0/3

1/3

Y

150-13-0

NC

NC

N

0/3

0/3

0/3

0/3

Y

3/3
0/3

3/3
0/3

3/3
1/3

3/3
3/3

Y
N

7718-54-9

1B

NC (false neg)N1330-20-7NC

weak (false pos)

P

14
20

Between4 Laboratory
Reproducibili
ECVAM
ty

GHS
potency
category

CAS NoNumber of concordancy
Concordancy rate (%)

19/24
79.2

ᮏヨ㦂ࡣࠊ2 ẁ㝵࡛ᐇ᪋ࡉࢀ࡚࠸ࡿࠋ1 ẁ㝵┠ࡢ ID No. 1-9 ࡢ≀㉁࡟ࡼࡿヨ㦂ࡣ 1 ᅇࡢࡳࡢᐇ᪋࡛࠶ࡿࠋ2 ẁ㝵┠ࡢ ID No. 10-24 ࡢ≀㉁ࢆ⏝࠸ࡓヨ㦂ࡣ 3 ᅇᐇ᪋ࡋࠊ2 ᅇ࠶ࡿ࠸ࡣ 3ᅇྠࡌ࡛࠶ࡗࡓ⤖ᯝࢆ᥇⏝ࡋ࡚࠸ࡿࠋࡑࡢ⤖ᯝࠊn=3 ࡛ᐇ᪋ࡋࡓ 15 ≀㉁࡛ࡢ᪋タ㛫෌⌧ᛶࡣ 66.7㸣࡜࡞ࡾࠊn=1 ࡛ࡢホ౯ࢆྵࡵࡓ 79.2㸣ࢆୗᅇࡿ⤖ᯝ࡜࡞ࡗࡓࠋࡇࢀࡣ᪋タෆ෌⌧ᛶࡢヨ㦂࡟࠾࠸࡚ࠊn=3 ࡛ࡢ⧞ࡾ㏉ࡋࡢ㝿ࠊ3 ᅇ࡜ࡶྠࡌ⤖ᯝࢆ♧ࡉ࡞࠸ࢣ࣮ࢫࡀ 60 ᅇ୰ 12ᅇ㸦20.0㸣㸧ྵࡲࢀࡿࡇ࡜ࡀཎᅉ࡜⪃࠼ࡽࢀࡓࠋ
5㸬ṇ☜ᗘ㸦ឤᗘ࠾ࡼࡧ≉␗ᗘ㸧
EURL ECVAMࡀ24≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻16㸸8㸧࡛⾜ࡗࡓࣂࣜࢹ࣮ࢩࣙࣥ
ヨ㦂2)࡛ࡣࠊṇ☜ᗘ76㸣ࠊឤᗘ81㸣ࠊ≉␗ᗘ66㸣࡛࠶ࡗࡓࠋࡇࡢᡂ⦼ࡣࠊAshikagaࡽ4)࡟ࡼ
ࡿ100≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻72㸸28㸧ࡢᡂ⦼ṇ☜ᗘ84㸣ࠊឤᗘ88㸣ࠊ≉␗ᗘ
75㸣㸧࡜ẚ㍑ࡋ࡚ప࠸ࡶࡢ࡛࠶ࡗࡓࠋ
Takenouchiࡽ5)ࡀỈ࡬ࡢ⁐ゎᛶࡀప࠸≀㉁ࡀഇ㝜ᛶࢆ♧ࡍᙳ㡪ࢆㄪ࡭ࡿࡓࡵ࡟ᐇ᪋ࡋࡓ37≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻30㸸7㸧ࡢ⤖ᯝ࡛ࡣࠊṇ☜ᗘ68%ࠊឤᗘ70%ࠊ≉␗ᗘ
57%࡛࠶ࡾࠊ30ࡢឤసᛶ≀㉁ࡢ࠺ࡕ9≀㉁ࡀ㝜ᛶ࡜ุᐃࡉࢀࡓࠋࡇࡢ୰ࡢ2≀㉁ࡣᇵ㣴ᾮ୰࡟ỿẊࡀほᐹࡉࢀࠊ5≀㉁࡛ࡣἜ⁲ࡀほᐹࡉࢀࡓࠋࡇࡢ9≀㉁ࡢlog Kowࡣࠊ3.5௨ୖ࡛࠶ࡗ
ࡓࠋࡉࡽ࡟ࠊNukadaࡽ6)࡟ࡼࡿ106≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻75㸸31㸧ࡢᡂ⦼࡜ྜ
ࢃࡏྜィ143≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻105㸸38㸧᳨࡛ウࡋࡓ⤖ᯝࠊṇ☜ᗘࡣ
80%ࠊឤᗘࡣ83%ࠊ≉␗ᗘࡣ71%࡛࠶ࡾࠊ8≀㉁ࡀഇ㝜ᛶࢆ♧ࡋࡓࠋࡇࡢ࠺ࡕࡢ5≀㉁ࡣ3.5
ᮍ‶ࡢlog Kow࡛࠶ࡾࠊࡑࡢ࠺ࡕ2≀㉁ࡣࣉࣟࣁࣉࢸࣥ/ࣉࣞࣁࣉࢸ࡛ࣥ࠶ࡗࡓࠋࡲࡓࠊ11≀㉁ࡀഇ㝧ᛶࢆ♧ࡋࡓࠋ
OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)࡟ࡣࠊEURL ECVAM3)ࡢ 24 ≀㉁࡜ࠊTakenouchi ࡽ 5)ࡢ 143 ≀㉁ࡢ⤖ᯝࢆ⤫ྜࡋࠊ142 ≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻101㸸41㸧ࡢᡂ⦼ࢆࡶ࡜࡟ࠊṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࢆồࡵࠊࡑࢀࡒࢀࠊ85㸣ࠊ93㸣ࠊ66㸣࡜⾲グࡉࢀ࡚࠸ࡿࠋࡇࡢ୰࡛ࠊ୧ሗ
࿌࡛㔜」ࡍࡿ 10 ≀㉁࡟ࡘ࠸࡚ࡣ EURL ECVAM3)ࡢࢹ࣮ࢱࢆ᥇⏝ࡋࠊTakenouchi ࡽ 5)ࡢࢹ࣮
ࢱ࠿ࡽࡣࠊࡇࡢ 10 ≀㉁࡜ log Kow ࡀ 3.5 ௨ୖࡢ 14 ≀㉁࡜ΰྜ≀ 1 ✀ࢆ㝖ࡃ 118 ≀㉁ࡢࢹ࣮
ࢱࢆ᥇⏝ࡋ࡚࠸ࡿࠋ୍᪉ࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)ࡢ 142 ≀㉁࡟ Takenouchi ࡽ 5)ࡢㄽᩥ
ࡢ log Kow ࡀ 3.5 ௨ୖࡢ 14 ≀㉁ࢆຍ࠼ࡓ 156 ≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻113㸸43㸧࡛ࡢṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࡣࠊࡑࢀࡒࢀ 83㸣ࠊ68㸣ࠊ79㸣࡜࡞ࡾࠊឤᗘࡀపୗࡍࡿഴྥࡀࡳ
ࡽࢀࡓࠋ
6. ホ౯ྍ⬟࡞≀㉁ࡢ⠊ᅖOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ 7)࡟グ㏙ࡢ࠶ࡿ 142 ≀㉁࡟ࡼࡿホ౯࡛ࡣࠊ⾲ 3 ࡟♧ࡍ㏻ࡾࠊ
ᵝࠎ࡞໬Ꮫ≀㉁ࡢ⓶⭵ឤసᛶࡢண

ࡀྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠋ

15
21

⾲3
Chemical Name
Benzo(a)pyrene
Diphenylcyclopropenone
Oxazolone
Dinitrofluorobenzene
Tetrachlorosalicylanilide
Bandrowskis base
1-Benzoylacetone
4-Nitrobenzyl bromide
2,4-Dinitrochlorobenzene
Potassium dichromate
Beryllium sulfate
Kathon CG (1.2% CM I)
Benzoquinone
Glutaraldehyde
1,4-Dihydroquinone
Phthalic anhydride
M aleic anhydride
Hexyl salicylate
Benzyl bromide
Benzyl peroxide
Lauryl gallate
Propyl gallate
2-Aminophenol
2-Nitro-4-phenylenediamine
Cobalt chloride
Chloramine T (CAS No. 149358-73-6)
CD-3 (CAS No. 25646-71-3)
Iodopropynyl butylcarbamate
1,2-Dibromo-2,4-dicyanobutane
Chlorpromazine HCl
4-Phenylenediamine
Chloramine T (CAS No. 127-65-1)
Formaldehyde
Isoeugenol
1-Naphtol
1-Phenyl-1,2-propanedione
2-Hydroxyethyl acrylate
Glyoxal
Bisphenol A-diglycidyl ether
Vinyl pyridine
2-M ethyl-2H-isothiazolone
3-Dimethylaminopropylamine
Ethylene diamine
1,2-Benzisothiazoline-3-one
M ethyl-2-nonynoate
Phenylacetaldehyde
Cinamic aldehyde
3-Aminophenol
Diethyl sulphate
3-Propylidenephthalide
Benzylidene acetone
2,4-Heptadienal
5-M ethyl-2-phenyl-2-hexanal
Alpha-methyl cinnamic aldehyde
Nickel sulfate
Tetramethylthiuramdisulfide
Trans-2-hexenal
Resorcinol
3,4-Dihydrocoumarin
Diethyl maleate2-M ethoxy-4-methyl-phenol
Diethylenetriamine
2-Phenylpropionaldehyde
4-Chloroaniline-damascone
Perillaaldehyde
Trimellitic anhydride2-M ercaptobenzothiazole
Benzylsalicylate
1-Thioglycerol
Dihydroeugenol

LLNA

h-CLAT

Potency
EC3 (%) Results
CD86
Category
Extreme
0.00009P+
Extreme
0.003
P
Extreme
0.003
P+
Extreme
0.03
P+
Extreme
0.04
P
Extreme
0.04
P+
Extreme
0.04
P
Extreme
0.05
P+
Extreme
0.05
P+
Extreme
0.08
P+
Extreme0.001
N
Extreme
0.009
P+
Extreme0.0099P+
Strong0.1
P+Strong0.11
P+Strong0.16
NStrong0.16
PStrong0.18
PStrong0.2
PStrong0.22
NStrong0.3
P+Strong0.32
PStrong0.4
P+Strong0.5
PStrong0.6
PStrong0.6
P+Strong0.6
P+Strong0.9
PStrong0.9
P+Strong0.14
PStrong0.11
P+Strong0.4
P+Strong0.61
P+
M oderate
1.2
N
M oderate
1.3
P+
M oderate
1.3
P+
M oderate
1.4
P+
M oderate
1.4
P
M oderate
1.5
P+
M oderate
1.6
P+
M oderate
1.9
P+
M oderate
2.2
P+
M oderate
2.2
P+
M oderate
2.3
P
M oderate
2.5
P+
M oderate
3.0
P+
M oderate
3.0
P+
M oderate
3.2
P
M oderate
3.3
P+
M oderate
3.7
P+
M oderate
3.7
P+
M oderate
4.0
P+
M oderate
4.4
P
M oderate
4.5
P+
M oderate
4.8
P+
M oderate
5.2
P+
M oderate
5.5
P+
M oderate
5.5
P
M oderate
5.6
P
M oderate
5.8
P+
M oderate
5.8
P+
M oderate
5.8
N
M oderate
6.3
P+
M oderate
6.5
P+
M oderate
6.7
P+
M oderate
8.1
P+
M oderate
9.2
P+
M oderate
1.7
P
M oderate
2.9
N
M oderate
3.6
P
M oderate
6.8
P
2013
: Takenouchi: h-CLAT Validation Study Report 2012

CD54
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

Chemical Name
2-Ethylhexyl acrylate
Amyl cinnamic aldehyde
2,3-Butanedion
CitralEugenolOxalic acid
Lyral (CAS No. 31906-04-4)
4-Allylanisole
Lilial (CAS No. 80-54-6)
Pentachlorophenol
Phenyl benzoate
Cinnamic alcohol-iso-methylionone
Benzocaine
Geraniol
5-M ethyl-2,3-hexanedione
2,2'-dihydroxyazobenzene
Ethyleneglycol dimethacrylate
Penicillin G
Linalool
Butyl glycidyl ether
Hydroxycitronellal
Pyridine
AnilineNonanoic acid
Benzylcinnamate
Imidazolidinylurea
R(+) Limonene
M ethylmethacrylate
Furil (CAS No. 109-65-9)
1-Butanol
1-Iodohexane
2-Acetylcyclohexanone
2-Hydroxypropyl methacrylate
4-Hydroxybenzoic acid
6-M ethylcoumarin
Acetanisole
Benzalchonium chloride
Benzaldehyde
Benzoic acid
Chlorobenzene
Clofibrate
Coumarin
DextranDiethylphthalate
Dimethyl formamide
Ethyl benzoylacetate
Ethyl vanillin
Furil (CAS No. 492-94-4)
Kanamycin
Lactic acid
Octanoic acid
Propyl paraben
Propylene glycol
Saccharin
Salicylic acid
Streptomycin sulfate
Sulfanilamide
Tween 80
Vanillin
Zinc sulfate
Glycerol
2,4-Dichloronitrobenzene
Benzyl alcohol
M ethylsalicylate
Isopropanol
Dimethylisophthalate
4-Aminobenzoic acid
Sodium lauryl sulfate
Nickel chloride
Xylene

LLNA
Potency
EC3 (%) Results
Category
Weak
10
P
Weak
11
P
Weak
11
P
Weak
13
P
Weak
13
P
Weak
15
P
Weak
17
P
Weak
18
P
Weak
19
P
Weak
20
P
Weak
20
P
Weak
21
P
Weak
21.8
P
Weak
22
P
Weak
26
P
Weak
26
P
Weak
27.9
P
Weak
28
P
Weak
30
P
Weak
30
P
Weak
31
N
Weak
33
P
Weak
72
P
Weak
89
P
Weak
21
P
Weak
18.4
N
Weak
24
P
Weak
69
P
Weak
90
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
P
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Non-sensitizer
ND
N
Others
14
N
OthersND
P
Others
95.8
Nh-CLAT
CD86

CD54

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

-

-

7. ᭷⏝ᛶ࡜㝈⏺
ᮏἲࡣ⣽⬊ᐇ㦂࡜ࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜࢆ⤌ࡳྜࢃࡏࡓヨ㦂ἲ࡛࠶ࡾࠊ⣽⬊ᇵ㣴ᶵჾ࠾ࡼࡧࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮ࢆಖ᭷ࡋࠊࡑࡢᢏ⾡࡟⩦⇍ࡋࡓ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡜ᛮࢃࢀࡿ ࠋ ࡲ ࡓ ࠊ ᮏ ἲ ࡣ ື ≀ ࢆ ⏝ ࠸ ࡞ ࠸ in vitro ࡢ ᡭ ἲ ࡛ ࠶ ࡾ ࠊ ⛉ Ꮫ ⓗ ┠ ⓗ 1622

     ࡢࡓࡵ࡟ᐇ᪋ࡉࢀࡿື≀ᐇ㦂࡟㛵ࡋࠊࠕື≀ࡢឡㆤཬࡧ⟶⌮࡟㛵ࡍࡿἲᚊࠖ࠾ࡼࡧ 3R ࡢ⢭⚄࡜ྜ⮴ࡋ࡚࠸ࡿࠋࡉࡽ࡟ h-CLAT ࠾ࡼࡧ LLNA ᐇ᪋ࡢ㝿࡟ᚲせ࡜࡞ࡿᾘ⪖ရ㈝ࡣࠊLLNA
࡛ࡣ 1 ࢔ࢵࢭ࢖ᙜࡓࡾ⣙ 10 ୓෇࡛࠶ࡿࡢ࡟ᑐࡋࠊh-CLAT ࡛ࡣ⣙ 2 ୓෇࡛࠶ࡾ 1/5 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡜ヨ⟬ࡉࢀࡓࠋヨ㦂ᮇ㛫ࡶ LLNA ࡼࡾ▷ᮇ㛫࡛ᐇ᪋ྍ⬟࡛࠶ࡿࡇ࡜࠿ࡽࠊヨ㦂ἲ࡜ࡋ࡚㎿㏿ᛶ࣭⤒῭ᛶࡢ㠃࠿ࡽ᭷⏝࡜ᛮࢃࢀࡿࠋ
ᢏ⾡ⓗ㝈⏺࡜ࡋ࡚ࠊᾮ┦࡛ࡢ཯ᛂࢆᚲせ࡜ࡍࡿヨ㦂⣔࡛࠶ࡿࡓࡵࠊ⏕⌮㣗ሷᾮ࠶ࡿ࠸ࡣᇵ
ᆅ࡟ 100 mg/mLࠊࡇࢀࡽ࡟ᡤᐃࡢ⃰ᗘ࡛୙⁐ࡢሙྜࡣࠊDMSO ࡟ 500 mg/mL ࡢ⃰ᗘ࡛⁐ゎ࠶ࡿ࠸ࡣᏳᐃⓗ࡟ศᩓࡍࡿᚲせࡀ࠶ࡿ㸦ࡓࡔࡋࠊ⛉Ꮫⓗ᰿ᣐࡀ࠶ࢀࡤ௚ࡢ⁐፹ࡶ౑⏝ྍ⬟࡜ࡉࢀ࡚࠸ࡿ㸧
ࠋࡲࡓࠊᮏἲࡣࣇ࣮ࣟࢧ࢖ࢺ࣓࣮ࢱ࣮ࢆ⏝࠸ࡿᡭἲ࡛࠶ࡾࠊ⺯ගࢆ᭷ࡍࡿ≀㉁ࡶホ౯ࡣྍ⬟࡛࠶ࡿࡀࠊFITC ࡸ PI ࡜ྠ୍Ἴ㛗࡟ᙉ࠸⺯ගࢆ᭷ࡍࡿ≀㉁ࡣ

ᐃࢆጉᐖࡍࡿྍ⬟ᛶࡀ࠶ࡿࠋ

᭦࡟ࠊ㐣ᗘࡢ⣽⬊ẘᛶࢆ᭷ࡍࡿ≀㉁ࡣ⣽⬊ࡢᵓ㐀ኚ໬ࢆᘬࡁ㉳ࡇࡋṇࡋࡃホ౯ࡉࢀ࡞࠸ྍ
⬟ᛶࡀ࠶ࡾࠊ௚ࡢ⣽⬊ࢆ⏝࠸ࡿヨ㦂⣔࡜ྠᵝ࡟ࠊ᥹Ⓨᛶ≀㉁ࡣ㣕ᩓ࡟ࡼࡿ≀㉁ࡢࣟࢫࡸ㏆㞄ࡢ࢙࢘ࣝ࡬ࡢࢡࣟࢫࢥࣥࢱ࣑ࢆ㉳ࡇࡍࡓࡵࠊ㐺ษ࡟ホ౯ࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
≀ᛶ࡟㛵ࡍࡿ㐺⏝㝈⏺࡜ࡋ࡚ࡣ log Kow ࡀ 3.5 ࢆ㉸࠼ࡿ≀㉁ࡣഇ㝜ᛶࢆ⏕ࡌࡸࡍ࠸ഴྥ࡟
࠶ࡿࡀࠊヱᙜ≀㉁࡟ࡼࡿ㝧ᛶࡢ⤖ᯝࡣ࠶ࡿ⛬ᗘಙ㢗࡛ࡁࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ
ᮏ⣽⬊ࡢ௦ㅰ⬟ࡣ㝈ᐃⓗ࡛࠶ࡿࡓࡵࠊࣉࣟࣁࣉࢸࣥ㸦P450 ➼࡟ࡼࡿ௦ㅰάᛶ໬ࢆᚲせ࡜ࡍࡿ≀㉁㸧ࡸࣉࣞࣁࣉࢸࣥ㸦㓟໬࡟ࡼࡾάᛶ໬ࡉࢀࡿ≀㉁㸧ࡣഇ㝜ᛶࢆ⏕ࡌࡿྍ⬟ᛶࡀ࠶ࡿࠋ
ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡛ホ౯ࡋࡓ㔠ᒓሷ࡛ࡣ United Nations (UN) Globally Harmonized Systemof Classification and Labelling of Chemicals 㸦GHS㸧 1A ࡟ศ㢮ࡉࢀࡿ Beryllium sulfate ࡀࠊഇ㝜ᛶ࡜࡞ࡗࡓࡀࠊ᪤ሗ 4)࡟ࡼࡿ࡜ Potassium dichromateࠊCobalt chlorideࠊNickel sulfate ࡣṇࡋࡃホ౯ࡉࢀ࡚࠸ࡿࡓࡵࠊ㔠ᒓሷࢆ㐺⏝⠊ᅖእ࡜ࡍࡿ᰿ᣐࡣ࡞࠸ࠋ
ᮏἲ࡟ࡼࡿഇ㝜ᛶࡢุᐃࡣࠊᙉᗘឤసᛶ≀㉁㸦UN GHS 1A ศ㢮㸧ࡼࡾࡶ㍍ᗘ㹼୰➼ᗘឤసᛶ≀㉁㸦UN GHS 1B ศ㢮㸧࡛⏕ࡌࡸࡍ࠸ഴྥ࡟࠶ࡿࠋࡇࢀࡽࡢ⤖ᯝ࠿ࡽࠊ㝧ᛶ࡜ุᐃࡉ
ࢀࡓሙྜࡣឤసᛶ㝧ᛶ࡜ุ᩿ࡍࡿࡇ࡜ࡣྍ⬟࡜⪃࠼ࡿࡀࠊ1 ๭⛬ᗘࡣഇ㝧ᛶࡢྍ⬟ᛶࡀ࠶ࡿࡇ࡜࡟␃ពࡍࡿᚲせࡀ࠶ࡿࠋ ࡲࡓࠊᮏἲࡣᢏ⾡ⓗ࡟ࡣΰྜ≀࡟ࡶᛂ⏝ྍ⬟࡜ࡉࢀ࡚࠸ࡿࡀࠊΰྜ≀࡛ࡢᐇ㦂౛ࡣᴟࡵ࡚
㝈ࡽࢀ࡚࠸ࡿࠋ
ᚑࡗ࡚ࠊ⌧᫬Ⅼ࡛ࡣ௨ୗࡢ≀㉁⩌࡬ࡢ㐺⏝ᛶࡣ᫂☜࡛࡞࠸࡜⪃࠼ࡿࠋ㸫ヨ㦂⁐፹࡟⁐ゎࡋ࡞࠸≀㉁࠾ࡼࡧヨ㦂⁐፹୰࡛୙Ᏻᐃ࡞≀㉁㸫ࣉࣟࣁࣉࢸࣥࠊࣉࣞࣁࣉࢸࣥ
㸫᥹Ⓨᛶ≀㉁㸫FITC ࡸ PI ࡜ྠࡌἼ㛗ᇦ࡟ᙉ࠸⺯ගࢆ᭷ࡍࡿ≀㉁࡞࠾ࠊࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢᡂ⦼࠿ࡽࡣࠊᮏἲࡢឤసᛶᙉᗘศ㢮ࡸ UN GHS ࡢࢧࣈ࢝ࢸ
ࢦ࣮ࣜศ㢮࡬ࡢᛂ⏝࡟ࡣ㐺ࡉ࡞࠸࡜⪃࠼ࡽࢀࡿࠋ

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8. ⤖ㄽ
h-CLAT ࡣࠊឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ➨୕ẁ㝵ࡢ࢖࣋ࣥࢺ࡛࠶ࡿᶞ≧⣽⬊ࡀάᛶ໬ࡍࡿ㝿ࡢ⣽⬊⾲㠃ศᏊࡢⓎ⌧ஹ㐍ࢆ฼⏝ࡋࡓ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ໬Ꮫ≀㉁ࡢឤసᛶࢆุ᩿ࡍࡿୖ࡛㔜せ࡞᝟ሗࢆ୚࠼࡚ࡃࢀࡿࠋ
࣐࢘ࢫࢆ⏝࠸ࡿ LLNA ࡢ 1/5 ⛬ᗘࡢ⤒㈝࡛ᐇ᪋ྍ⬟࡜ヨ⟬ࡉࢀࠊヨ㦂ᮇ㛫ࡶ LLNA ࡟ẚ࡭▷ᮇ࡛࠶ࡿࡓࡵࠊ᭷⏝ᛶࡣ㧗࠸࡜ᛮࢃࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ⣽⬊ᐦᗘ࡜ᇵ㣴᫬㛫ࢆཝᏲࡍࡿࡇ࡜ࠊබẚ 1.2 ࡢ⃰ᗘタᐃࡀᚲせ࡞ࡓࡵࠊDPRA ࡸ ARE-Nrf2 Luciferase Test Method ࡟ẚ࡭࡚ࠊ᧯సࡀ↹㞧࡛࠶ࡾࠊไ⣙ࡀከ࠸ࠋࡲࡓࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊࣄࢫࢺࣜ࢝ࣝ࡞ࢹ࣮ࢱ࣮࣋ࢫࢆసᡂࡍࡿࡇ࡜ࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࠋ
ᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂࡟࠾࠸࡚ࠊ15 ≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タෆ෌⌧ᛶࡣࠊ
73.3㹼86.7%࡛࠶ࡾࠊ඲⯡ⓗ࡟㧗ࡃ࡞࠿ࡗࡓࠋࡓࡔࡋࠊOECD ᑓ㛛ᐙ఍㆟࡟࠾࠸࡚ࠊヨ㦂࡟౑⏝ࡍࡿ THP-1 ⣽⬊ࡢ๓ᇵ㣴᮲௳࠾ࡼࡧ⿕㦂≀㉁ࡢ᭚㟢᫬㛫ࢆࡼࡾཝᐦ࡟⟶⌮ࡍࡿࡇ࡜࡟
ࡼࡾࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᅗࢀࡿࡇ࡜ࡀ♧ࡉࢀࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡢヨ㦂ᡭ㡰࡟ࡑࡢෆᐜࡀ཯ᫎࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊ᪋タෆ෌⌧ᛶࡢྥୖࡀᮇᚅ࡛ࡁࡿ࡜⪃࠼ࡿࠋ୍᪉ࠊ24≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タ㛫෌⌧ᛶࡣࠊ79.2㸣࡛࠶ࡗࡓࠋ
OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖࡛ࣥࡣࠊ142 ≀㉁ࡢᡂ⦼࠿ࡽṇ☜ᗘ 85㸣ࠊឤᗘ 93㸣ࠊ≉␗ᗘ 66㸣࡜グ㍕ࡉࢀ࡚࠸ࡿࡀࠊlog Kow ࡀ 3.5 ௨ୖ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ≀㉁ࢆ㝖࠸ࡓୖ࡛ࡢᩘ್࡛࠶
ࡿࠋࡲࡓࠊOECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥࡢ 142 ≀㉁࡟ log Kow ࡀ 3.5 ௨ୖࡢ 14 ≀㉁ࢆຍ࠼ࡓ
156 ≀㉁ࡢṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࡣࠊࡑࢀࡒࢀ 83㸣ࠊ68㸣ࠊ79㸣࡜࡞ࡾࠊឤᗘࡀపୗࡍࡿഴ
ྥࡀࡳࡽࢀࡓࠋࡋࡓࡀࡗ࡚ࠊ≉࡟ log Kow ࡀ 3.5 ௨ୖࡢ≀㉁ࡢሙྜࡣഇ㝜ᛶࡢྍ⬟ᛶࢆ⪃៖ࡋࠊ⿵᏶ࡋᚓࡿ௚ࡢヨ㦂ࡼࡾ☜ㄆࡋ࡞ࡅࢀࡤ࡞ࡽ࡞ࡃࠊᮏヨ㦂ἲࡢࡳ࡛⓶⭵ឤసᛶࢆ㝜ᛶ࡜ุᐃࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠸࡜⪃࠼ࡿࠋࡲࡓࠊ≉␗ᗘࡀ 66%࡛࠶ࡿࡓࡵഇ㝧ᛶ࡜ุᐃࡍࡿࡇ࡜
ࡶ␃ពࡍࡿᚲせࡀ࠶ࡿࠋࡉࡽ࡟ࠊUN GHS ศ㢮ࡢ≀㉁࡛ࠊ㍍ᗘឤసᛶ≀㉁࡛ࡣഇ㝜ᛶࡀ⏕ࡌࡸࡍࡃࠊᙉᗘឤసᛶ≀㉁࡛ࡣࡑࡢẚ⋡ࡣప࠸ࠋࡇࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣࠊ≀㉁ࡢឤసᛶᙉᗘศ㢮ࡸ UN GHS ࡢࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮࡬ࡢᛂ⏝࡟ࡣ㐺ࡉ࡞࠸࡜⪃࠼ࡿࠋࡲࡓࠊᮏヨ㦂ἲ࡟⏝࠸ࡿ⣽⬊ࡢ௦ㅰ⬟ࡣ㝈ᐃⓗ࡛࠶ࡿࡓࡵࠊάᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁࡛ࡣࠊṇࡋࡃࡑࡢឤసᛶࡀ᳨ฟ
ࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ
ᮏጤဨ఍ࡣࠊୖグࡢᮏヨ㦂ἲࡢᵝࠎ࡞㝈⏺ࢆ຺᱌ࡍࡿ࡜ࠊᮏヨ㦂ἲ༢⊂࡛ࡣ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛᭷ࡾࠊドᣐࡢ㔜ࡳ௜ࡅࡸ௚ࡢヨ㦂ἲ࡜ࡢ⤌ྜࡏ࡛⏝࠸ࡿࡇ࡜ࢆ᥎ዡࡍ
ࡿࠋ

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ᘬ⏝ᩥ⊩
1㸬EURL ECVAM (2015) Recommendation on the human Cell Line Activation Test (h-CLAT) forskin sensitization testing. Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/eurlecvam-recommendation-on-the-human-cell-line-activation-test-h-clat-for-skin-sensitisation2㸬DB-ALM (INVITTOX) (2014) Protocol 158: human Cell Line Activation Test (h-CLAT), 23pp.
Accessible at: https://ecvam-dbalm.jrc.ec.europa.eu/methods-and-protocols/search/h-clat
3㸬EURL ECVAM (2013) human Cell Line Activation Teat (h-CLAT) Validation Study Report.
Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/h-clat-validation-study-report
4. Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y,Miyazawa M, Ito Y,
Nishiyama N, Itagaki H. (2010) A comparative evaluation of in vitro skin sensitization tests: thehuman cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab.
Anim., 38, 275-284.
5. Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. (2013) Predictive performance ofthe human Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-waterpartition coefficients. J. Toxicol. Sci., 38, 599-609.
6. Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012)
Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT)
and an attempt at classifying skin sensitization potency. Toxcol. In Vitro, 26, 1150-60.
7. OECD (2016) OECD Guideline for the Testing of Chemicals No. 442E: In Vitro Skin Sensitization:
human

Cell

Line

Activation

Test(h-CLAT).

Availableat:

http://www.oecd-

ilibrary.org/environment/test-no-442e-in-vitro-skin-sensitisation_9789264264359-en

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OECD/OCDE

442E
Adopted:29 July 2016

OECD GUIDELINE FOR THE TESTING OF CHEMICALSIn Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)INTRODUCTION
1. A skin sensitiser refers to a substance that will lead to an allergic response following skin contact as
defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals(UN GHS) (1). This Test Guideline (TG) describes the in vitro procedure called human Cell Line
Activation test (h-CLAT), to be used for supporting the discrimination between skin sensitisers and nonsensitisers in accordance with the UN GHS (1).
2. There is general agreement regarding the key biological events underlying skin sensitisation. The current
knowledge of the chemical and biological mechanisms associated with skin sensitisation has beensummarised in the form of an Adverse Outcome Pathway (AOP) (2), starting with the molecular initiating
event through intermediate events to the adverse effect, namely allergic contact dermatitis. In this instance,
the molecular initiating event (i.e. the first key event) is the covalent binding of electrophilic substances tonucleophilic centres in skin proteins. The second key event in this AOP takes place in the keratinocytes
and includes inflammatory responses as well as changes in gene expression associated with specific cellsignalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways. The
third key event is the activation of dendritic cells (DC), typically assessed by expression of specific cellsurface markers, chemokines and cytokines. The fourth key event is T-cell proliferation, which is indirectly
assessed in the murine Local Lymph Node Assay (LLNA) (3).
3. The assessment of skin sensitisation has typically involved the use of laboratory animals. The classicalmethods that use guinea-pigs, the Guinea Pig Maximisation Test (GPMT) of Magnusson and Kligman, andthe Buehler Test (TG 406) (4), assess both the induction and elicitation phases of skin sensitisation. The
murine tests, the LLNA (TG 429) (3) and its two non-radioactive modifications, LLNA: DA (TG 442 A)(5) and LLNA: BrdU-ELISA (TG 442 B) (6), all assess exclusively the induction response, and have also
gained acceptance, since they provide an advantage over the guinea pig tests in terms of animal welfare
together with an objective measurement of the induction phase of skin sensitisation.
4. More recently mechanistically-based in chemico (OECD TG 442C; Direct Peptide Reactivity Assay
addressing the first key event of the skin sensitisation AOP) (7) and in vitro (OECD TG 442D; ARE-Nrf2
Luciferase Test Method addressing the second key event of the skin sensitisation AOP) (8) test methods
have been adopted for contributing to the evaluation of the skin sensitisation hazard potential of chemicals.
However, a combination of non-animal methods (in silico, in chemico, in vitro) within Integrated
1© OECD, (2016)
You are free to use this material
http://www.oecd.org/termsandconditions/.

subject

to

the

terms

and

conditions

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

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OECD/OCDE

Approaches to Testing and Assessment (IATA) will be needed to be able to fully substitute for the animal
tests currently in use given the restricted AOP mechanistic coverage of each of the currently available nonanimal test methods (2)(9).
5. The h-CLAT method is proposed to address the third key event of the skin sensitisation AOP by
quantifying changes in the expression of cell surface markers associated with the process of activation of
monocytes and DC (i.e. CD86 and CD54), in the human monocytic leukaemia cell line THP-1, following
exposure to sensitisers (10). The measured expression levels of CD86 and CD54 cell surface markers arethen used for supporting the discrimination between skin sensitisers and non-sensitisers.
6. The h-CLAT method has been evaluated in a European Union Reference Laboratory for Alternatives toAnimal Testing (EURL ECVAM)-coordinated validation study and subsequent independent peer review
by the EURL ECVAM Scientific Advisory Committee (ESAC). Considering all available evidence andinput from regulators and stakeholders, the h-CLAT was recommended by EURL ECVAM (11) to be usedas part of an IATA to support the discrimination between sensitisers and non-sensitisers for the purpose ofhazard classification and labelling. Examples of the use of h-CLAT data in combination with otherinformation are reported in the literature (12) (13) (14) (15) (16) (17) (18) (19).
7. Definitions are provided in Annex I.

INITIAL CONSIDERATIONS AND LIMITATIONS
8. Skin sensitisers have been reported to induce the expression of cell membrane markers associated withDC activation (2). Test methods such as the h-CLAT which measure markers of monocyte activation andmay be related to DC activation (20) are therefore considered relevant for the assessment of the skinsensitisation potential of chemicals. However, since DC activation represents only one key event of theskin sensitisation AOP, information generated with test methods measuring markers of DC activation may
not be sufficient on its own to conclude on the absence of skin sensitisation potential of chemicals.
Therefore, data generated with the h-CLAT method should be considered in the context of integrated
approaches, such as IATA, and combined with other complementary information e.g. derived from in vitro
assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including
read-across from chemical analogues.
9. The test method described in this Test Guideline can be used to support the discrimination between skinsensitisers (i.e. UN GHS Category 1) and non-sensitisers in the context of IATA. This Test Guidelinecannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as
defined by UN GHS (1), for authorities implementing these two optional subcategories, nor to predict
potency for safety assessment decisions. However, depending on the regulatory framework, a positive
result with the h-CLAT may be used on its own to classify a chemical into UN GHS category 1.
10. The h-CLAT method proved to be transferable to laboratories experienced in cell culture techniques
and flow cytometry analysis. The level of reproducibility in predictions that can be expected from the test
method is in the order of 80% within and between laboratories (11) (21). Results generated in thevalidation study (22) and other published studies (23) overall indicate that, compared with LLNA results,
the accuracy in distinguishing skin sensitisers (i.e. UN GHS Cat.1) from non-sensitisers is 85% (N=142)
with a sensitivity of 93% (94/101) and a specificity of 66% (27/41) (based on a re-analysis by EURLECVAM (21) considering all existing data and not considering negative results for chemicals with a Log
Kow greater than 3.5 as described in paragraph 12). False negative predictions with the h-CLAT are more
likely to concern chemicals showing a low to moderate skin sensitisation potency (i.e. UN GHS

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subcategory 1B) than chemicals showing a high skin sensitisation potency (i.e. UN GHS subcategory 1A)(12) (22) (24). Taken together, this information indicates the usefulness of the h-CLAT method tocontribute to the identification of skin sensitisation hazards. However, the accuracy values given here forthe h-CLAT as a stand-alone test method are only indicative, since the test method should be considered in
combination with other sources of information in the context of an IATA and in accordance with theprovisions of paragraph 9 above. Furthermore, when evaluating non-animal methods for skin sensitisation, it
should be kept in mind that the LLNA test as well as other animal tests may not fully reflect the situation inhumans.
11. The term "test chemical" is used in this Test Guideline to refer to what is being tested 1 and is not
related to the applicability of the h-CLAT to the testing of mono-constituent substances, multi-constituent
substances and/or mixtures. On the basis of the data currently available, the h-CLAT method was shown tobe applicable to test chemicals covering a variety of organic functional groups, reaction mechanisms, skinsensitisation potency (as determined in in vivo studies) and physicochemical properties (11) (23) (24).
Limited information is currently available on the applicability of the h-CLAT method to multi-constituent
substances/mixtures (24). The test method is nevertheless technically applicable to the testing of multiconstituent substances and mixtures. However, before use of this Test Guideline on a mixture forgenerating data for an intended regulatory purpose, it should be considered whether, and if so why, it may
provide adequate results for that purpose2. Such considerations are not needed when there is a regulatory
requirement for the testing of the mixture. Moreover, when testing multi-constituent substances or
mixtures, consideration should be given to possible interference of cytotoxic constituents with the observed
responses.
12. The h-CLAT method is applicable to test chemicals soluble or that form a stable dispersion (i.e. a
colloid or suspension in which the test chemical does not settle or separate from the solvent/vehicle into
different phases) in an appropriate solvent/vehicle (see paragraph 21). Test chemicals with a Log Kow
greater than 3.5 tend to produce false negative results (23). Therefore negative results with test chemicals
with a Log Kow greater than 3.5 should not be considered. However, positive results obtained with test
chemicals with a Log Kow greater than 3.5 could still be used to support the identification of the test
chemical as a skin sensitiser. Furthermore, because of the limited metabolic capability of the cell line used(25) and because of the experimental conditions, pro-haptens (i.e. substances requiring enzymatic
activation for example via P450 enzymes) and pre-haptens (i.e. substances activated by oxidation) in
particular with a slow oxidation rate may also provide negative results in the h-CLAT (24). Fluorescent test
chemicals can be assessed with the h-CLAT (26), nevertheless, strong fluorescent test chemicals emittingat the same wavelength as fluorescein isothiocyanate (FITC) or as propidium iodide (PI), will interferewith the flow cytometric detection and thus cannot be correctly evaluated using FITC-conjugatedantibodies or PI. In such a case, other fluorochrome-tagged antibodies or other cytotoxicity markers,
respectively, can be used as long as it can be shown they provide similar results as the FITC-tagged
antibodies (see paragraph 31) or PI (see paragraph 25) e.g. by testing the proficiency substances in Annex
II. In the light of the above, negative results should be interpreted in the context of the stated limitations
and together with other information sources within the framework of IATA. In cases where there isevidence demonstrating the non-applicability of the h-CLAT method to other specific categories of test
chemicals, it should not be used for those specific categories.
13. As described above, the h-CLAT method supports the discrimination between skin sensitisers from
non-sensitisers. However, it may also potentially contribute to the assessment of sensitising potency (12)(13) (17) when used in integrated approaches such as IATA. Nevertheless, further work, preferably based
1

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

2

This sentence was proposed and agreed at the April 2014 WNT meeting.

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on human data, is required to determine how h-CLAT results may possibly inform potency assessment.

PRINCIPLE OF THE TEST
14. The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e.
CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to
the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may
mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker
expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies.
Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker
expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers
compared to solvent/vehicle control are calculated and used in the prediction model (see paragraph 33), to
support the discrimination between sensitisers and non-sensitisers

DEMONSTRATION OF PROFICIENCY
15. Prior to routine use of the test method described in this Test Guideline, laboratories should demonstratetechnical proficiency, using the 10 Proficiency Substances listed in Annex II. Moreover, test method users
should maintain an historical database of data generated with the reactivity checks (see paragraph 18) andwith the positive and solvent/vehicle controls (see paragraphs 27-29), and use these data to confirm thereproducibility of the test method in their laboratory is maintained over time.

PROCEDURE
16. This Test Guideline is based on the h-CLAT DataBase service on ALternative Methods to animal
experimentation (DB-ALM) protocol no. 158 (27) which represents the protocol used for the EURLECVAM-coordinated validation study. It is recommended that this protocol is used when implementing
and using the h-CLAT method in the laboratory. The following is a description of the main components
and procedures for the h-CLAT method, which comprises two steps: dose finding assay and CD86/CD54
expression measurement.
Preparation of cells
17. The human monocytic leukaemia cell line, THP-1, should be used for performing the h-CLAT method.
It is recommended that cells (TIB-202™) are obtained from a well-qualified cell bank, such as theAmerican Type Culture Collection.
18. THP-1 cells are cultured, at 37°C under 5% CO2 and humidified atmosphere, in RPMI-1640 medium
supplemented with 10% foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillinand 100 μg/mL streptomycin. The use of penicillin and streptomycin in the culture medium can be
avoided. However, in such a case users should verify that the absence of antibiotics in the culture medium
has no impact on the results, for example by testing the proficiency substances listed in Annex II. In any
case, in order to minimise the risk of contamination, good cell culture practices should be followed
independently of the presence or not of antibiotics in the cell culture medium. THP-1 cells are routinely
seeded every 2-3 days at the density of 0.1 to 0.2 × 106 cells/mL. They should be maintained at densities
from 0.1 to 1.0 × 106 cells/mL. Prior to using them for testing, the cells should be qualified by conducting a

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reactivity check. The reactivity check of the cells should be performed using the positive controls, 2,4dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity) and nickel sulfate (NiSO4) (CAS n. 1010197-0, ≥ 99% purity) and the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity), two weeks
after thawing. Both DNCB and NiSO4 should produce a positive response of both CD86 and CD54 cellsurface markers, and LA should produce a negative response of both CD86 and CD54 cell surface markers.
Only the cells which passed the reactivity check are to be used for the assay. Cells can be propagated up to
two months after thawing. Passage number should not exceed 30. The reactivity check should be
performed according to the procedures described in paragraphs 27-31.
19. For testing, THP-1 cells are seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, andpre-cultured in culture flasks for 72 hours or for 48 hours, respectively. It is important that the cell density
in the culture flask just after the pre-culture period be as consistent as possible in each experiment (byusing one of the two pre-culture conditions described above), because the cell density in the culture flask
just after pre-culture could affect the CD86/CD54 expression induced by allergens (28). On the day of
testing, cells harvested from culture flask are resuspended with fresh culture medium at 2 × 10 6 cells/mL.
Then, cells are distributed into a 24 well flat-bottom plate with 500 μL (1 × 106 cells/well) or a 96-wellflat-bottom plate with 80 μL (1.6 × 105 cells/well).
Dose finding assay
20. A dose finding assay is performed to determine the CV75, being the test chemical concentration that
results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value is used todetermine the concentration of test chemicals for the CD86/CD54 expression measurement (see paragraphs
27-31).
Preparation of test chemicals and control substances
21. The test chemicals and control substances are prepared on the day of testing. For the h-CLAT method,
test chemicals are dissolved or stably dispersed (see also paragraph 12) in saline or medium as firstsolvent/vehicle options or dimethyl sulfoxide (DMSO, t 99% purity) as a second solvent/vehicle option if
the test chemical is not soluble or does not form a stable dispersion in the previous two solvents/vehicles,
to final concentrations of 100 mg/mL (in saline or medium) or 500 mg/mL (in DMSO). Other
solvents/vehicles than those described above may be used if sufficient scientific rationale is provided.
Stability of the test chemical in the final solvent/vehicle should be taken into account.
22. Starting from the 100 mg/mL (in saline or medium) or 500 mg/mL (in DMSO) stock solutions of thetest chemicals, the following dilution steps should be taken:− For saline or medium as solvent/vehicle: Eight stock solutions (eight concentrations) are prepared,by two-fold serial dilutions using the corresponding solvent/vehicle. These stock solutions are thenfurther diluted 50-fold into culture medium (working solutions). If the top final concentration in theplate of 1000 μg/mL is non-toxic, the maximum concentration should be re-determined by
performing a new cytotoxicity test. The final concentration in the plate should not exceed 5000
μg/mL for test chemicals dissolved or stably dispersed in saline or medium.
− For DMSO as solvent/vehicle: Eight stock solutions (eight concentrations) are prepared, by two-foldserial dilutions using the corresponding solvent/vehicle. These stock solutions are then further
diluted 250-fold into culture medium (working solutions).The final concentration in plate should notexceed 1000 μg/mL even if this concentration is non-toxic.
The working solutions are finally used for exposure by adding an equal volume of working solution to thevolume of THP-1 cell suspension in the plate (see also paragraph 24) to achieve a further two-fold dilution(usually, the final range of concentrations in the plate is 7.81–1000 μg/mL).
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23. The solvent/vehicle control used in the h-CLAT method is culture medium (for test chemicals
solubilised or stably dispersed (see paragraph 12) either with medium or saline) or DMSO (for test
chemicals solubilised or stably dispersed in DMSO) tested at a single final concentration in the plate of
0.2%. It undergoes the same dilution as described for the working solutions in paragraph 22.
Application of test chemicals and control substances
24. The culture medium or working solutions described in paragraphs 22 and 23 are mixed 1:1 (v/v) withthe cell suspensions prepared in the 24-well or 96-well flat-bottom plate (see paragraph 19). The treatedplates are then incubated for 24±0.5 hours at 37°C under 5% CO2. Care should be taken to avoid
evaporation of volatile test chemicals and cross-contamination between wells by test chemicals, e.g. by
sealing the plate prior to the incubation with the test chemicals (29).
Propidium iodide (PI) staining
25. After 24±0.5 hours of exposure, cells are transferred into sample tubes and collected by centrifugation.
The supernatants are discarded and the remaining cells are resuspended with 600 μL (in case of 96-well) or
1mL (in case of 24-well) of a phosphate buffered saline containing 0.1% bovine serum albumin (staining
buffer). 200 μL of cell suspension is transferred into 96-well round-bottom plate (in case of 96-well) or
micro tube (in case of 24-well) and washed twice with 200 μL (in case of 96-well) or 1mL (in case of 24well) of staining buffer. Finally, cells are resuspended in staining buffer (e.g. 400 μL) and PI solution (e.g.
20 μL) is added (for example, final concentration of PI is 0.625 μg/mL). Other cytotoxicity markers, suchas 7-Aminoactinomycin D (7-AAD), Trypan blue or others may be used if the alternative stains can beshown to provide similar results as PI, for example by testing the proficiency substances in Annex II.
Cytotoxicity measurement by flow cytometry and estimation of CV75 value
26. The PI uptake is analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000
living cells (PI negative) are acquired. The cell viability can be calculated using the following equation by
the cytometer analysis program. When the cell viability is low, up to 30,000 cells including dead cells
should be acquired. Alternatively, data can be acquired for one minute after the initiation of the analysis.
Cell Viability =Number of living cells

× 100

Total Number of acquired cells

The CV75 value (see paragraph 20), i.e. a concentration showing 75% of THP-1 cell survival (25%cytotoxicity), is calculated by log-linear interpolation using the following equation:
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Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively

Cell viability (%)

100

a

50
c
0
1

b d
10
100
1000
Test dose (Pg/mL)

Other approaches to derive the CV75 can be used as long as it isdemonstrated that this has no impact on the results (e.g. by testing
the proficiency substances).
CD86/CD54 expression measurement

Preparation of the test chemicals and control substances
27. The appropriate solvent/vehicle (saline, medium or DMSO; see paragraph 21) is used to dissolve or
stably disperse the test chemicals. The test chemicals are first diluted to the concentration corresponding to
100-fold (for saline or medium) or 500-fold (for DMSO) of the 1.2 × CV75 determined in the dose finding
assay (see paragraph 26). If the CV75 cannot be determined (i.e. if sufficient cytotoxicity is not observedin the dose finding assay), the highest soluble or stably dispersed concentration of test chemical preparedwith each solvent/vehicle should be used as starting concentration. Please note that the final concentrationin the plate should not exceed 5000 μg/mL (in case of saline or medium) or 1000 μg/mL (in case of
DMSO). Then, 1.2-fold serial dilutions are made using the corresponding solvent/vehicle to obtain thestock solutions (eight concentrations ranging from 100×1.2 × CV75 to 100×0.335 × CV75 (for saline or
medium) or from 500×1.2 × CV75 to 500×0.335 × CV75 (for DMSO)) to be tested in the h-CLAT method(see DB-ALM protocol NO. 158 for an example of dosing scheme). The stock solutions are then further
diluted 50-fold (for saline or medium) or 250-fold (for DMSO) into the culture medium (working
solutions). These working solutions are finally used for exposure with a further final two-fold dilution
factor in the plate. If the results do not meet the acceptance criteria described in the paragraphs 35 and 36
regarding cell viability, the dose finding assay may be repeated to determine a more precise CV75. Please
note that only 24-well plates can be used for CD86/CD54 expression measurement.
28. The solvent/vehicle control is prepared as described in paragraph 23. The positive control used in the hCLAT method is DNCB (see paragraph 18), for which stock solutions are prepared in DMSO and diluted
as described for the stock solutions in paragraph 27. DNCB should be used as the positive control for
CD86/CD54 expression measurement at a final single concentration in the plate (typically 4.0 μg/mL). To
obtain a 4.0 μg/mL concentration of DNCB in the plate, a 2 mg/mL stock solution of DNCB in DMSO is
prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution. Alternatively,
the CV75 of DNCB, which is determined in each test facility, could be also used as the positive control
concentration. Other suitable positive controls may be used if historical data are available to derivecomparable run acceptance criteria. For positive controls, the final single concentration in the plate should
not exceed 5000 μg/mL (in case of saline or medium) or 1000 μg/mL (in case of DMSO). The run
acceptance criteria are the same as those described for the test chemical (see paragraph 35), except for thelast acceptance criterion since the positive control is tested at a single concentration.

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Application of test chemicals and control substances
29. For each test chemical and control substance, one experiment is needed to obtain a prediction. Eachexperiment consists of at least two independent runs for CD86/CD54 expression measurement (seeparagraphs 33 and 34). Each independent run is performed on a different day or on the same day provided
that for each run: a) independent fresh stock solutions and working solutions of the test chemical andantibody solutions are prepared and b) independently harvested cells are used (i.e. cells are collected fromdifferent culture flasks); however, cells may come from the same passage. Test chemicals and control
substances prepared as working solutions (500 μL) are mixed with 500 μL of suspended cells (1x106 cells)
at 1:1 ratio, and cells are incubated for 24±0.5 hours as described in paragraphs 27 and 28. In each run, asingle replicate for each concentration of the test chemical and control substance is sufficient because aprediction is obtained from at least two independent runs.
Cell staining and analysis
30. After 24±0.5 hours of exposure, cells are transferred from 24 well plate into sample tubes, collected bycentrifugation and then washed twice with 1mL of staining buffer (if necessary, additional washing steps
may be done). After washing, cells are blocked with 600 μL of blocking solution (staining buffer
containing 0.01% (w/v) globulin (Cohn fraction II, III, Human: SIGMA, #G2388-10G)) and incubated at
4°C for 15 min. After blocking, cells are split in three aliquots of 180 μL into a 96-well round-bottom plateor micro tube.
31. After centrifugation, cells are stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse
IgG1 (isotype) antibodies at 4°C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol
no. 158 (27) should be used by diluting 3:25 (v/v, for CD86 (BD-PharMingen, #555657; Clone: Fun-1)) or
3:50 (v/v, for CD54 (DAKO, #F7143; Clone: 6.5B5) and IgG1 (DAKO, #X0927)) with staining buffer.
These antibody dilution factors were defined by the test method developers as those providing the best
signal-to-noise ratio. Based on the experience of the test method developers, the fluorescence intensity of
the antibodies is usually consistent between different lots. However, users may consider titrating theantibodies in their own laboratory's conditions to define the best concentrations for use. Other
fluorochrome-tagged anti-CD86 and/or anti-CD54 antibodies may be used if they can be shown to provide
similar results as FITC-conjugated antibodies, for example by testing the proficiency substances in Annex
II. It should be noted that changing the clone or supplier of the antibodies as described in the h-CLAT DBALM protocol no. 158 (27) may affect the results. After washing with 200 μL of staining buffer threetimes, cells are resuspended in staining buffer (e.g. 400 μL), and the PI solution (e.g. 20 μL to obtain a
final concentration of 0.625 μg/mL) or another cytotoxicity marker's solution (see paragraph 25) is added.
The expression levels of CD86 and CD54, and cell viability are analysed using flow cytometry.

DATA AND REPORTING
Data evaluation
32. The expression of CD86 and CD54 is analysed with flow cytometry with the acquisition channel FL-1.
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of
CD86 and CD54 for positive control (ctrl) cells and chemical-treated cells are calculated according to thefollowing equation:
RFI =MFI of chemical-treated cells − MFI of chemical-treated isotype control
x100
cells
MFI of solvent/vehicle-treated ctrl cells − MFI of solvent/vehicle-treated isotype ctrl
cells
8

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The cell viability from the isotype control (ctrl) cells (which are stained with mouse IgG1 (isotype)antibodies) is also calculated according to the equation described in paragraph 26.
Prediction model
33. For CD86/CD54 expression measurement, each test chemical is tested in at least two independent runs
to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE
if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise
the h-CLAT prediction is considered NEGATIVE (Figure 1):− The RFI of CD86 is equal to or greater than 150% at any tested concentration (with cell viability ≥50%);− The RFI of CD54 is equal to or greater than 200% at any tested concentration (with cell viability ≥50%).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, theh-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if
the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE (withdue consideration of the provisions of paragraph 36) without the need for a third run. If however, the firsttwo runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and thefinal prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In thisrespect, it should be noted that if two independent runs are conducted and one is only positive for CD86
(hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third
run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT
prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P 1 or
P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE.

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Figure 1: Prediction model used in the h-CLAT test method. An h-CLAT prediction should beconsidered in the framework of an IATA and in accordance with the provision of paragraphs 9, 11 and 12.
P1: run with only CD86 positive; P2; run with only CD54 positive; P12: run with both CD86 and CD54
positive; N: run with neither CD86 nor CD54 positive. *The boxes show the relevant combinations ofresults from the first two runs, independently of the order in which they may be obtained. #The boxes show
the relevant combinations of results from the three runs on the basis of the results obtained in the first two
runs shown in the box above, but do not reflect the order in which they may be obtained.
34. For the test chemicals predicted as POSITIVE with the h-CLAT, optionally, two Effective
Concentrations (EC) values, the EC150 for CD86 and EC200 for CD54, i.e. the concentration at which thetest chemicals induced a RFI of 150 or 200, may be determined. These EC values potentially could
contribute to the assessment of sensitising potency (3) when used in integrated approaches such as IATA
(12) (13) (14) (15) (16). They can be calculated by the following equations:
EC150 (for CD86) = Bconcentration + [(150 - BRFI) / (ARFI - BRFI) × (Aconcentration - Bconcentration)]
EC200 (for CD54) = Bconcentration + [(200 - BRFI) / (ARFI - BRFI) × (Aconcentration - Bconcentration)]where
Aconcentrationis the lowest concentration in μg/mL with RFI > 150 (CD86) or 200 (CD54)
Bconcentration is the highest concentration in μg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)

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BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54)
For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs for
CD86/CD54 expression measurement may be required. The final EC150 and EC200 values are thendetermined as the median value of the ECs calculated from the three independent runs. When only two of
three independent runs meet the criteria for positivity (see paragraph 33), the higher EC150 or EC200 of
the two calculated values is adopted.
Acceptance criteria
35. The following acceptance criteria should be met when using the h-CLAT method (22) (27).
-The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.

-

In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive
criteria (CD86 RFI t 150% and CD54 RFI t 200%). RFI values of the solvent/vehicle control arecalculated by using the formula described in paragraph 32 ("MFI of chemical" should be replacedwith "MFI of solvent/vehicle", and "MFI of solvent/vehicle" should be replaced with "MFI of(medium) control").

-

For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype
control should be > 105%.

-

In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria(CD86 RFI t 150 and CD54 RFI t 200) and cell viability should be more than 50%.

-

For the test chemical, the cell viability should be more than 50% in at least four tested concentrations
in each run.

36. Negative results are acceptable only for test chemicals exhibiting a cell viability of less than 90% at thehighest concentration tested (i.e. 1.2 × CV75 according to the serial dilution scheme described in paragraph27). If the cell viability at 1.2 × CV75 is equal or above 90% the negative result should be discarded. Insuch a case it is recommended to try to refine the dose selection by repeating the CV75 determination. Itshould be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in
DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, anegative result is acceptable even if the cell viability is above 90%.
Test report
37. The test report should include the following information.
Test chemical- Mono-constituent substancex

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

Physical appearance, Log Kow, water solubility, DMSO solubility, molecular weight, andadditional relevant physicochemical properties, to the extent available;x

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

Storage conditions and stability to the extent available;x

Justification for choice of solvent/vehicle for each test chemical.

- Multi-constituent substance, UVCB and mixture:x

Characterisation as far as possible by e.g. chemical identity (see above), purity, quantitative
occurrence and relevant physicochemical properties (see above) of the constituents, to theextent available;x

Physical appearance, water solubility, DMSO solubility and additional relevant
physicochemical properties, to the extent available;x

Molecular weight or apparent molecular weight in case of mixtures/polymers of known
compositions or other information relevant for the conduct of the study;xTreatment prior to testing, if applicable (e.g. warming, grinding);xConcentration(s) tested;x

Storage conditions and stability to the extent available;x

Justification for choice of solvent/vehicle for each test chemical.

Controls- Positive controlx

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

Physical appearance, Log Kow, water solubility, DMSO solubility, molecular weight, andadditional relevant physicochemical properties, to the extent available and where applicable;x

Purity, chemical identity of impurities as appropriate and practically feasible, etc.;x

Treatment prior to testing, if applicable (e.g. warming, grinding);xConcentration(s) tested;x

Storage conditions and stability to the extent available;x

Reference to historical positive control results demonstrating suitable run acceptance criteria,
if applicable.

- Negative and solvent/vehicle controlx

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

Purity, chemical identity of impurities as appropriate and practically feasible, etc.;x

Physical appearance, molecular weight, and additional relevant physicochemical properties in
the case other control solvent/vehicle than those mentioned in the Test Guideline are used andto the extent available;

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Storage conditions and stability to the extent available;x

Justification for choice of solvent/vehicle for each test chemical.

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Test method conditions-

Name and address of the sponsor, test facility and study director;-

Description of test method used;-

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

Flow cytometry used (e.g. model), including instrument settings, globulin, antibodies and cytotoxicity
marker used;-

The procedure used to demonstrate proficiency of the laboratory in performing the test method bytesting of proficiency substances, and the procedure used to demonstrate reproducible performance ofthe test method over time, e.g. historical control data and/or historical reactivity checks’ data.

Test acceptance criteria-

Cell viability, MFI and RFI values obtained with the solvent/vehicle control in comparison to theacceptance ranges;- Cell viability and RFI values obtained with the positive control in comparison to the acceptance
ranges;-Cell viability of all tested concentrations of the tested chemical.

Test procedure-

Number of runs used;
Test chemical concentrations, application and exposure time used (if different than the one
recommended)
Duration of exposure (if different than the one recommended);
Description of evaluation and decision criteria used;
Description of any modifications of the test procedure.

-

Results--

Tabulation of the data, including CV75 (if applicable), individual geometric MFI, RFI, cell viability
values, EC150/EC200 values (if applicable) obtained for the test chemical and for the positive control
in each run, and an indication of the rating of the test chemical according to the prediction model;
Description of any other relevant observations, if applicable.

Discussion of the results-

Discussion of the results obtained with the h-CLAT method;

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Consideration of the test method results within the context of an IATA, if other relevant information
is available.

Conclusions

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LITERATURE
1. United Nations UN (2013), Globally Harmonized System of Classification and Labelling of Chemicals(GHS). Fifth revised edition. New York & Geneva: United Nations Publications. ISBN: 978-92-1117006-1. Available at: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev05/05files_e.html].
2. 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. Available at:
http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/P
ART1&docLanguage=En
3. OECD (2010), The Local Lymph Node Assay. OECD Guideline for the Testing of Chemicals No. 429,
Organisation for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/env/testguidelines
4. OECD (1992), Skin Sensitisation. OECD Guideline for the Testing of Chemicals No. 406, Organisation
for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/env/testguidelines
5. OECD (2010), Skin Sensitisation: Local Lymph Node Assay: DA. OECD Guideline for the Testing ofChemicals No. 442A, Organisation for Economic Cooperation and Development, Paris. Available at:
http://www.oecd.org/env/testguidelines
6. OECD (2010), Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA. OECD Guideline for the
Testing of Chemicals No. 442B, Organisation for Economic Cooperation and Development, Paris.
Available at: http://www.oecd.org/env/testguidelines
7. OECD (2015), OECD Guideline for the Testing of Chemicals No. 442C: In Chemico Skin Sensitisation:
Direct Peptide Reactivity Assay (DPRA). Paris, France: Organisation for Economic Cooperation andDevelopment. Available at: http://www.oecd.org/env/testguidelines
8. OECD (2015), OECD Guideline for the Testing of Chemicals No. 442D: In Vitro Skin Sensitisation:
ARE-Nrf2 Luciferase Test Method. Paris, France: Organisation for Economic Cooperation andDevelopment. Available at: http://www.oecd.org/env/testguidelines
9. Adler S, Basketter D, Creton S, Pelkonen O, van Benthem J, Zuang V, Andersen KE, Angers-Loustau
A, Aptula A, Bal-Price A, Benfenati E, Bernauer U, Bessems J, Bois FY, Boobis A, Brandon E, Bremer
S, Broschard T, Casati S, Coecke S, Corvi R, Cronin M, Daston G, Dekant W, Felter S, Grignard E,
Gundert-Remy U, Heinonen T, Kimber I, Kleinjans J, Komulainen H, Kreiling R, Kreysa J, Leite SB,
Loizou G, Maxwell G, Mazzatorta P, Munn S, Pfuhler S, Phrakonkham P, Piersma A, Poth A, Prieto P,
Repetto G, Rogiers V, Schoeters G, Schwarz M, Serafimova R, Tähti H, Testai E, van Delft J, vanLoveren H, Vinken M, Worth A, Zaldivar JM. (2011), Alternative (non-animal) methods for cosmetics
testing: current status and future prospects-2010. Archives of Toxicology 85, 367-485.
10. Ashikaga T, Yoshida Y, Hirota M, Yoneyama K, Itagaki H, Sakaguchi H, Miyazawa M, Ito Y, Suzuki
H, Toyoda H. (2006), Development of an in vitro skin sensitization test using human cell lines: The
human Cell Line Activation Test (h-CLAT) I. Optimization of the h-CLAT protocol. Toxicol. In Vitro
20, 767–773.
11. EC EURL-ECVAM (2013), Recommendation on the human Cell Line Activation Test (h-CLAT) forskinsensitisation
testing.
Accessibleat:https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvamrecommendations
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12. Takenouchi O, Fukui S, Okamoto K, Kurotani S, Imai N, Fujishiro M, Kyotani D, Kato Y, Kasahara T,
Fujita M, Toyoda A, Sekiya D, Watanabe S, Seto H, Hirota M, Ashikaga T, Miyazawa M. (2015), Test
battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a
139 chemical data set for predicting skin sensitizing potential and potency of chemicals. J Appl Toxicol.
35, 1318-1332.
13. Hirota M, Fukui S, Okamoto K, Kurotani S, Imai N, Fujishiro M, Kyotani D, Kato Y, Kasahara T,
Fujita M, Toyoda A, Sekiya D, Watanabe S, Seto H, Takenouchi O, Ashikaga T, Miyazawa M. (2015),
Evaluation of combinations of in vitro sensitization test descriptors for the artificial neural networkbased risk assessment model of skin sensitization. J Appl Toxicol. 35, 1333-1347.
14. Bauch C,kolle SN, Ramirez T, Fabian E, Mehling A, Teubner W, van Ravenzwaay B, Landsiedel R.
(2012), Putting the parts together: combining in vitro methods to test for skin sensitizing potencials.
Regul Toxicol Parmacol. 63, 489-504.
15. Van der Veen JW, Rorije E, Emter R, Natch A, van Loveren H, Ezendam J. (2014), Evaluating theperformance of integrated approaches for hazard identification of skin sensitizing chemicals. Regul
Toxicol Pharmacol. 69, 371-379.
16. Urbisch D, Mehling A, Guth K, Ramirez T, Honarvar N, Kolle S, Landsiedel R, Jaworska J, Kern PS,
Gerberick F, Natsch A, Emter R, Ashikaga T, Miyazawa M, Sakaguchi H. (2015), Assessing skinsensitization hazard in mice and men using non-animal test methods. Regul Toxicol Parmacol. 71, 337351.
17. Jaworska JS, Natsch A, Ryan C, Strickland J, Ashikaga T, Miyazawa M. (2015), Bayesian integrated
testing strategy (ITS) for skin sensitization potency assessment: a decision support system forquantitative weight of evidence and adaptive testing strategy. Arch Toxicol. 89, 2355-2383.
18. Strickland J, Zang Q, Kleinstreuer N, Paris M, Lehmann DM, Choksi N, Matheson J, Jacobs A, Lowit
A, Allen D, Casey W. (2016), Integrated decision strategies for skin sensitization hazard. J Appl
Toxicol. DOI 10.1002/jat.3281.
19. Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012),
Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT)
and an attempt at classifying skin sensitization potency. Toxicol. In Vitro 26, 1150-60.
20. Miyazawa M, Ito Y, Yoshida Y, Sakaguchi H, Suzuki H. (2007), Phenotypic alterations and cytokine
production in THP-1 cells in response to allergens. Toxicol. In Vitro 21, 428-437.
21. EC EURL ECVAM (2015), Re-analysis of the within and between laboratory reproducibility of thehuman Cell Line Activation Test (h-CLAT). Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/eurlecvam-recommendations/eurl-ecvam-recommendation-on-the-human-cell-line-activation-test-h-clatfor-skin-sensitisation-testing
22. EC EURL ECVAM (2012), human Cell Line Activation Test (h-CLAT) Validation Study Report
Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/eurl-ecvam-recommendations
23. Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. (2013), Predictive performance of thehuman Cell Line Activation Test (h-CLAT) for lipophilic with high octanol-water partition coefficients.
J. Toxicol. Sci. 38, 599-609.
24. Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y,

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NishiyamaN, Itagaki H. (2010), A comparative evaluation of in vitro skin sensitisation tests: thehuman cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab.
Anim. 38, 275-284.
25. Fabian E., Vogel D., Blatz V., Ramirez T., Kolle S., Eltze T., van Ravenzwaay B., Oesch F.,
Landsiedel R. (2013), Xenobiotic metabolizin enzyme activities in cells used for testing skinsensitization in vitro. Arch Toxicol 87, 1683-1969.
26. Okamoto K, Kato Y, Kosaka N, Mizuno M, Inaba H, Sono S, Ashikaga T, Nakamura T, Okamoto Y,
Sakaguchi H, Kishi M, Kuwahara H, Ohno Y. (2010), The Japanese ring study of a human Cell Line
Activation Test (h-CLAT) for predicting skin sensitization potential (6th report): A study forevaluating oxidative hair dye sensitization potential using h-CLAT. AATEX 15, 81-88.
27. DB-ALM (INVITTOX) (2014), Protocol 158: human Cell Line Activation Test (h-CLAT), 23pp.
Accessible at: http://ecvam-dbalm.jrc.ec.europa.eu/
28. Mizuno M, Yoshida M, Kodama T, Kosaka N, Okamato K, Sono S, Yamada T, Hasegawa S, Ashikaga
T, Kuwahara H, Sakaguchi H, Sato J, Ota N, Okamoto Y, Ohno Y. (2008), Effects of pre-culture
conditions on the human Cell Line Activation Test (h-CLAT) results; Results of the 4th Japanese interlaboratory study. AATEX 13, 70-82.
29. Sono S, Mizuno M, Kosaka N, Okamoto K, Kato Y, Inaba H, , Nakamura T, Kishi M, Kuwahara H,
Sakaguchi H, Okamoto Y, Ashikaga T, Ohno Y. (2010), The Japanese ring study of a human Cell Line
Activation Test (h-CLAT) for predicting skin sensitization potential (7th report): Evaluation of volatile,
poorly soluble fragrance materials. AATEX 15, 89-96.
30. OECD (2005), Guidance Document No.34 on “the Validation and International Acceptance of New or
Updated Test Methods for Hazard Assessment”. OECD Series on Testing and Assessment.
Organization for Economic Cooperation and Development, Paris, France, 2005, 96 pp.
31. ECETOC (2003). Contact sensitization: Classification according to potency. European Centre for
Ecotoxicology & Toxicology of Chemicals (Technical Report No. 87).
32. Ashikaga T, Sakaguchi H, Okamoto K, Mizuno M, Sato J, Yamada T, Yoshida M, Ota N, Hasegawa S,
Kodama T, Okamoto Y, Kuwahara H, Kosaka N, Sono S, Ohno Y. (2008), Assessment of the human
Cell Line Activation Test (h-CLAT) for Skin Sensitization; Results of the First Japanese Interlaboratory Study. AATEX 13, 27-35.

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OECD/OCDEANNEX I
DEFINITIONS

Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of relevance. The term is often used interchangeably
with concordance to mean the proportion of correct outcomes of a test method (30).
AOP (Adverse Outcome Pathway): sequence of events from the chemical structure of a target chemicalor group of similar chemicals through the molecular initiating event to an in vivo outcome of interest (2).
CV75: The estimated concentration showing 75% cell viability.
EC150: the concentrations showing the RFI values of 150 in CD86 expression
EC200: the concentrations showing the RFI values of 200 in CD54 expression
Flow cytometry: a cytometric technique in which cells suspended in a fluid flow one at a time through afocus of exciting light, which is scattered in patterns characteristic to the cells and their components; cells
are frequently labeled with fluorescent markers so that light is first absorbed and then emitted at alteredfrequencies.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
organism, system or (sub) population is exposed to that agent.
IATA (Integrated Approach to Testing and Assessment): A structured approach used for hazard
identification (potential), hazard characterisation (potency) and/or safety assessment (potential/potency andexposure) of a chemical or group of chemicals, which strategically integrates and weights all relevant data
to inform regulatory decision regarding potential hazard and/or risk and/or the need for further targeted andtherefore minimal testing.
Medium control: An untreated replicate containing all components of a test system. This sample isprocessed with test chemical-treated samples and other control samples to determine whether thesolvent/vehicle interacts with the test system.
Mixture: A mixture or a solution composed of two or more substances in which they do not react.
Mono-constituent substance: A substance, defined by its quantitative composition, in which one main
constituent is present to at least 80% (w/w).
Multi-constituent substance: A substance, defined by its quantitative composition, in which more than
one main constituent is present in a concentration ≥ 10% (w/w) and < 80% (w/w). A multi-constituent
substance is the result of a manufacturing process. The difference between mixture and multi-constituent
substance is that a mixture is obtained by blending of two or more substances without chemical reaction. A
multi-constituent substance is the result of a chemical reaction.

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Positive control: A replicate containing all components of a test system and treated with a substanceknown to induce a positive response. To ensure that variability in the positive control response across time
can be assessed, the magnitude of the positive response should not be excessive.
Pre-haptens: chemicals which become sensitisers through abiotic transformation
Pro-haptens: chemicals requiring enzymatic activation to exert skin sensitisation potential
Relative fluorescence intensity (RFI): Relative values of geometric mean fluorescence intensity (MFI) in
chemical-treated cells compared to MFI in solvent/vehicle-treated cells.
Relevance: Description of relationship of the test to the effect of interest and whether it is meaningful anduseful for a particular purpose. It is the extent to which the test correctly measures or predicts the
biological effect of interest. Relevance incorporates consideration of the accuracy (concordance) of a test
method (30).
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility and intra-laboratory repeatability (30).
Run: A run consists of one or more test chemicals tested concurrently with a solvent/vehicle control andwith a positive control.
Sensitivity: The proportion of all positive/active chemicals that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results, and is an important considerationin assessing the relevance of a test method (30).
Staining buffer: A phosphate buffered saline containing 0.1% bovine serum albumin.
Solvent/vehicle control: An untreated sample containing all components of a test system except of the test
chemical, but including the solvent/vehicle that is used. It is used to establish the baseline response for the
samples treated with the test chemical dissolved or stably dispersed in the same solvent/vehicle. Whentested with a concurrent medium control, this sample also demonstrates whether the solvent/vehicle
interacts with the test system.
Specificity: The proportion of all negative/inactive chemicals that are correctly classified by the test. It is ameasure of accuracy for a test method that produces categorical results and is an important consideration inassessing the relevance of a test method (30).
Substance: Chemical elements and their compounds in the natural state or obtained by any production
process, including any additive necessary to preserve the stability of the product and any impurities
deriving from the process used, but excluding any solvent which may be separated without affecting thestability of the substance or changing it composition.

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Test chemical: The term "test chemical" is used to refer to what is being tested.
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS): A system proposing the classification of chemicals (substances and mixtures) according tostandardised types and levels of physical, health and environmental hazards, and addressing corresponding
communication elements, such as pictograms, signal words, hazard statements, precautionary statements
and safety data sheets, so that to convey information on their adverse effects with a view to protect people(including employers, workers, transporters, consumers and emergency responders) and the environment(1).
UVCB: substances of unknown or variable composition, complex reaction products or biologicalmaterials.
Valid test method: A test method considered to have sufficient relevance and reliability for a specificpurpose and which is based on scientifically sound principles. A test method is never valid in an absolutesense, but only in relation to a defined purpose (30).

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ANNEX IIPROFICIENCY SUBSTANCES

Prior to routine use of a test method that adheres to this Test Guideline, laboratories should demonstratetechnical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 substances
recommended in Table 1 and by obtaining CV75, EC150 and EC200 values that fall within the respective
reference range for at least 8 out of the 10 proficiency substances. Proficiency substances were selected torepresent the range of responses for skin sensitisation hazards. Other selection criteria were that thesubstances are commercially available, and that high-quality in vivo reference data as well as high quality
in vitro data generated with the h-CLAT method are available. Also, published reference data are available
for the h-CLAT method (11) (23).
Table 1: Recommended substances for demonstrating technical proficiency with the h-CLAT method
In vivoprediction1

CV75
Reference
Range in
Pg/mL2h-CLAT resultsfor CD86(EC150 Reference
Range in μg/mL)2

h-CLAT resultsfor CD54(EC200 Reference
Range in μg/mL)2

Proficiency substances

CASRN

Physicalstate

2,4-Dinitrochlorobenzene

97-00-7

Solid

Sensitiser(extreme)

2-12

Positive(0.5-10)

Positive(0.5-15)

4-Phenylenediamine

106-50-3

Solid

Sensitiser(strong)

5-95

Positive(<40)

Negative(>1.5)3

Nickel sulfate

10101-97-0

SolidSensitiser(moderate)

30-500

Positive(<100)

Positive(10-100)

2-Mercaptbenzothiazole

149-30-4

Solid

Sensitiser(moderate)

30-400

Negative(>10)3

Positive(10-140)

R(+)-Limonene

5989-27-5

LiquidSensitiser(weak)>20

Negative(>5)3

Positive(<250)

Imidazolidinyl urea

39236-46-9

SolidSensitiser(weak)

25-100

Positive(20-90)

Positive(20-75)Isopropanol

67-63-0LiquidNon-sensitiser>5000

Negative(>5000)

Negative(>5000)Glycerol

56-81-5

LiquidNon-sensitiser>5000

Lactic acid50-21-5

LiquidNon-sensitiser

1500-5000

4-Aminobenzoic acid150-13-0

Solid

Non-sensitiser>1000

Negative(>5000)
Negative(>5000)
Negative(>1000)

Negative(>5000)
Negative(>5000)
Negative(>1000)Abbreviations: CAS RN = Chemical Abstracts Service Registry Number
1
The in vivo hazard and (potency) prediction is based on LLNA data (11) (23). The in vivo potency isderived using the criteria proposed by ECETOC (31).
2Based on historical observed values (22) (32).
3Historically, a majority of negative results have been obtained for this marker and therefore a negative
result is mostly expected. The range provided was defined on the basis of the few historical positive
results observed. In case a positive result is obtained, the EC value should be within the reported
reference range.

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EURL ECVAM Recommendationon the human Cell Line Activation Test
(h-CLAT) for skin sensitisation testing2015

Report EUR 27022 EN

53

European Commission
Joint Research Centre
Institute for Health and Consumer Protection
Contact information
EURL ECVAM
Address: Joint Research Centre, via Enrico Fermi 2749, 21027 Ispra (Va), Italy
E-mail: JRC-ECVAM-CONTACT@ec.europa.eu
https://ec.europa.eu/jrc/en/institutes/ihcp
https://ec.europa.eu/jrc
Legal NoticeThis publication is a Science and Policy Report by the Joint Research Centre, the European Commission’s in-house science
service. It aims to provide evidence-based scientific support to the European policy-making process. The scientific output
expressed does not imply a policy position of the European Commission. 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.
All images © European Union 2015
JRC93307
EUR 27022 EN
ISBN 978-92-79-44706-8 (PDF)
ISSN 1831-9424 (online)
doi:10.2788/29986
Luxembourg: Publications Office of the European Union, 2015
© European Union, 2015
Reproduction is authorised provided the source is acknowledged.

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 summarised in the OECD report on: "The Adverse Outcome Pathway (AOP) for Skin
Sensitisation Initiated by Covalent Binding to Proteins". Within this AOP the activation of dendritic cells (DC), typicallyassessed by expression of cell surface markers, chemokines and cytokines, is considered to be a key event. Therefore, test
methods able to provide information on the ability of a chemical to up-regulate markers of DC activation may contributeto skin sensitisation hazard assessment. The human Cell Line Activation Test (h-CLAT) measures the upregulation of theCD86 and CD54 markers of DC activation in THP-1 cells, a human monocytic leukemia cell line. The test method has
undergone a validation study addressing the test method's transferability and within- and between-laboratory
reproducibility. Following independent peer-review by the EURL ECVAM's Scientific Advisory Committee (ESAC) and having
considered input from regulators, stakeholders, international partners and the general public, EURL ECVAM concluded thatthe h-CLAT test method should prove valuable within Integrated Approaches to Testing and Assessment (IATA) for hazard
assessment. The h-CLAT may also be able to contribute to the assessment of sensitising potency, however it is recognisedthat further efforts are required to explore how h-CLAT data may contribute to potency assessment.

54

EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)EURL ECVAM RECOMMENDATION
on the human Cell Line Activation Test (h-CLAT)
for skin sensitisation testing

February 201555

Table of Contents
Executive Summary .......................................................................................................................... 3
1. Introduction.................................................................................................................................. 4
2. Test Method definition................................................................................................................. 5
3. Overall performance of the h-CLAT test method ......................................................................... 6
4. Limitations .................................................................................................................................... 8
5. Suggested regulatory use ............................................................................................................. 9
6. Follow-up activities recommended by EURL ECVAM ................................................................. 10
References ...................................................................................................................................... 12

Background to EURL ECVAM Recommendations
The aim of a EURL ECVAM Recommendation is to provide EURL ECVAM views on the validity ofthe test method in question, to advise on possible regulatory applicability, limitations andproper scientific use of the test method, and to suggest possible follow-up activities in view ofaddressing knowledge gaps.
During the development of its Recommendations, EURL ECVAM consults with its advisory bodyfor 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.

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.

256

Executive Summary
The human Cell Line Activation Test (h-CLAT) for skin sensitisation testing was developed by Kao
Corporation and Shiseido (Japan). With a view to facilitating its use as a component ofintegrated approaches to assessing the skin sensitisation potential of chemicals, EURL ECVAM
coordinated a validation study to assess the reliability of the h-CLAT method and to gain somepreliminary insight into its predictive capacity. On completion of the study, EURL ECVAM
requested ESAC to conduct a scientific peer review of the validation study report and theresulting ESAC opinion was delivered in May 2014. Following consideration of the ESAC opinion
EURL ECVAM makes the following observations and recommendations:1) The h-CLAT addresses one of the key events of the skin sensitisation Adverse Outcome
Pathway (AOP) by measuring markers of dendritic cell (DC) activation in THP-1 cells, ahuman monocytic leukemia cell line. Therefore information generated by the h-CLAT isconsidered relevant for the assessment of the skin sensitisation potential of chemicals.
2) The validation study demonstrated that the h-CLAT test method is transferable tolaboratories experienced in cell culture techniques and flow cytometry analysis. The withinlaboratory and between-laboratory reproducibly, as characterised on the basis ofconcordant classifications of the chemicals employed (n=15 and n=24 respectively), wereboth in the order of 80%.
3) The accuracy of h-CLAT to discriminate sensitisers from non-sensitisers was calculated to be
76% (sensitivity 81% and specificity 66%) with the chemicals tested (n=24). However thisresult is only an approximation since the validation study was clearly not designed to fully
assess the predictive capacity of the h-CLAT as a stand-alone method. Published information
actually reports a higher accuracy (80%) for a larger set of chemicals (n=143; Takenouchi etal., 2013).
4) Based on the outcome of the validation study and reports from the scientific literature, data
generated with the h-CLAT method should prove valuable as part of Integrated Approaches
to Testing and Assessment (IATA) together with complementary information (e.g. inchemico or other in vitro data, QSAR or read-across predictions).
5) Besides providing information that contributes to the assessment of the skin sensitisation
potential of chemicals, the h-CLAT assay also generates concentration-response informationthat may contribute to the assessment of potency. Nevertheless, additional work is stillrequired to determine to which extent h-CLAT results can contribute to potency prediction.
6) The h-CLAT method should be further assessed with respect to its response to chemicals
that need to be activated (e.g. through biotransformation or auto-oxidation) before eliciting
their sensitisation effect, and to its applicability to chemical mixtures and polymers.
7) EURL ECVAM fully supports the development of an OECD Test Guideline for the h-CLAT.
8) Respecting the provisions of Directive 2010/63/EU (EU, 2010) on the protection of animals
used for scientific purposes, h-CLAT data should be considered before embarking on animal
experiments for assessing skin sensitisation potential. As described in Annex XI of the
REACH Regulation (EC, 2006), h-CLAT data may be used to adapt the standard information
requirement in the context of Weight-of-Evidence (WoE) judgments (point 1.2) or by theuse of in vitro methods (point 1.4).

357

1. Introduction
1) The assessment of skin sensitisation potential is an important component in the safety
evaluation of substances 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 Biocidal Products Regulation (EU,2012) and the Cosmetics Regulation (EC, 2009b). Determining skin sensitisation hazard
according to the Globally Harmonised System to Classification and Labelling (GHS) is actually
sufficient to satisfy the majority of regulatory needs (EURL ECVAM, 2013a). However, amore complete characterisation of the potency of a skin sensitiser with regard to both
induction as well as elicitation of contact dermatitis is often required for classification of
mixtures, appropriate risk management measures (e.g. setting of appropriate exposurelevels) and eventually a full risk assessment.
2) Traditionally, skin sensitisation hazard assessment has involved the use of laboratory
animals. In the framework of the Organisation for Economic Cooperation and Development(OECD) and the EU Test Methods Regulation (EC, 2008b), there are four accepted
guidelines, describing: the Buehler Test and Guinea-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).
Following the ESAC peer review of the validation studies and the publication of the EURLECVAM Recommendations on the in chemico Direct Peptide Reactivity Assay (DPRA) (EURLECVAM, 2013b) and the in vitro KeratinoSens¥ test method (EURL ECVAM, 2014a), OECD
Test Guidelines on these two assays have been developed and are in the acceptanceprocess.
3) The key biological events underpinning the skin sensitisation process are well establishedand have been summarised in the OECD report on "The Adverse Outcome Pathway (AOP)for Skin Sensitisation Initiated by Covalent Binding to Proteins" (OECD, 2012a, 2012b). These
key events include 1) the covalent binding of the chemical to skin proteins (haptenation), 2)
the release of pro-inflammatory cytokines and the induction of cyto-protective pathways inkeratinocytes, 3) the maturation and mobilisation of dendritic cells (DC), immunocompetent 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 the development of alternative non-animal methods that address these key
mechanisms. Following the ESAC peer review of the validation studies and the publicationof the EURL ECVAM Recommendations on the in chemico Direct Peptide Reactivity Assay(DPRA) (EURL ECVAM 2013b), and the in vitro KeratinoSensTM test method (EURL ECVAM
2014a), OECD Test Guidelines on these two non-animal methods have been recently
adopted (TG 442C, OECD 2015a and TG 442D, OECD 2015b).
4) There is general agreement that it is unlikely that alternative (non-animal) methods
designed to address a single key event of the skin sensitisation pathway will be able toprovide sufficient information to fully replace the use of animals for this endpoint (Adler etal., 2011). Instead, what is likely needed is some combination of information fromcomplementary alternative methods (Jowsey et al., 2006; Adler et al., 2011). Against thisbackground, activities are being pursued by academia, industry and the European
Commission to evaluate mechanistically-based test methods that can contribute to skinsensitisation hazard identification and characterisation.

4585) In 2008, EURL ECVAM received a joint submission by Kao Corporation and Shiseido (Japan)
on the h-CLAT test method that described the extensive work performed to develop andoptimise the method. This included the results of multiple laboratories studies (Sakaguchi etal., 2006, 2010; Ashikaga et al., 2006, 2008; Kosaka et al., 2008; Sono et al., 2008; Mizuno etal., 2008) and h-CLAT data for 100 chemicals. As a consequence, in the period between
November 2009 and November 2012, EURL ECVAM coordinated a validation study on the hCLAT (EURL ECVAM 2013c). The study was designed to generate information according to
the modular approach to validation (Hartung et al., 2004) with the primary objective of fully
assessing the reliability of the h-CLAT (i.e. its transferability and within and between
laboratory reproducibility). Only as a secondary study objective, the experimental data
generated were used to perform a preliminary evaluation of the ability of the h-CLAT todiscriminate between skin sensitising and non-sensitising chemicals, as defined by theUnited Nations (UN) Globally Harmonised System (GHS) to classification and labelling (UNGHS, 2013). Assessment of the preliminary predictive capacity of the h-CLAT was performed
as a step towards determining the potential contribution of the method within integrated
approaches to skin sensitisation hazard assessment. In addition, where possible, theexperimental data were used to derive preliminary considerations on the ability of the test
method to sub-categorise sensitising chemicals, e.g. into sub-categories 1A and 1B as
defined by the UN GHS.
6) Following completion of the study and finalisation of the Validation Study Report (EURLECVAM, 2013c), EURL ECVAM requested the ECVAM Scientific Advisory Committee (ESAC)
to provide an ESAC Opinion on the study. The ESAC Working Group (WG) "Skin
Sensitisation" prepared a detailed WG report (EURL ECVAM, 2013d) which formed the basisof the ESAC Opinion (EURL ECVAM, 2014b; see Annex 1), endorsed by members at an ESAC
meeting in March 2014 and formally delivered to EURL ECVAM in May 2014.

2. Test Method definition
7) The important role played by Dendritic Cells (DC) in the initiation of adaptive immune
responses is well established, including the cutaneous immune response to chemicalallergens. DC can recognise and internalise antigens such as haptenated proteins, transportthem via the lymphatic system to the regional lymph nodes and present them via major
histocompatibility complex (MHC) molecules to naïve T lymphocytes to induce
differentiation and proliferation of specific memory T-cells. Recognition and processing of
the antigen by DC requires the local production of various danger signals including
inflammatory mediators resulting from the activation of the innate immunity of the skin
(Ainscough et al., 2013; Kaplan et al., 2012; Vocanson et al., 2009). Once activated, DC
migrate from the skin to the lymph nodes and undergo a process of maturation
characterised by phenotypic and functional changes resulting in the loss of their capability
to process antigens and in the acquisition of the functionality of antigen presentation. The
maturation process involves the decrease of phagocytic activity, increased expression of
MHC molecules on the cell surface, changes in cytokines and chemokines secretion and upregulation of several co-stimulatory (e.g. CD80, CD86, and CD40) and intercellular adhesion
molecules (e.g. CD11a, and ICAM-1/CD54) (Quah and O'Neill, 2005; Vocanson et al., 2009;
Kimber et al., 2011).
Some of these biomarkers have been considered in the development of cell-based assays
for assessing the skin sensitisation potential of chemicals (Aiba et al., 1997; Casati et al.,2005; dos Santos et al., 2009; Vandebriel et a.l., 2010). It is recognised that the mechanismsthat lead in vitro to the augmentation of these membrane markers by sensitising chemicals
559

may only partially reflect the complexity of the mechanisms inducing DC maturation in
integral biological models such as rodents and humans (Kimber et al., 2011; 2013).
However, assays based on human myeloid cell lines and measuring markers known to beover-expressed during DC maturation in vivo, are considered to provide mechanistic
information that can contribute to the in vitro assessment of the skin sensitisation potentialof chemicals (OECD 2012a; 2012b; Adler et al., 2011; Vanderbriel et al., 2010; van Helden etal., 2008) .
8) In the h-CLAT method the modulation of the CD86 and CD54 membrane markers in THP-1
cells (Tsuchiya et al., 1980), a human monocytic leukemia cell line used as a surrogate modelfor DC (van Helden et al., 2008), is measured by flow cytometry following 24 hours of
exposure to eight serial concentrations of test chemical selected on the basis of a predetermined CV75 (i.e. the concentration of test chemical that allows 75% of cell survival).
The h-CLAT test method is designed to discriminate between sensitising and non-sensitisingchemicals whereby chemicals are classified as sensitisers if the relative fluorescenceintensity (RFI) of either CD86 and/or CD54 exceeds a defined threshold (i.e. RFI CD86≥150
and RFI CD54≥200; Sakaguchi et al., 2009) compared to the vehicle control wells at any
tested concentration, in at least two out of three independent measurements (i.e.
repetitions). Cell viability is measured concurrently by Propidium Iodide (PI) staining and RFI
values are considered for the prediction only if cell viability is above 50%.
9) Since the THP-1 cells are exposed to 8 serial concentrations of test chemicals, for positive
chemicals it is generally possible to calculate from the concentration-response curve an
Estimated Concentration (EC) 1 value for the CD86 and the CD54 representing theconcentration of test chemical needed to induce an RFI equal to the respective thresholdvalues, i.e. CD86 EC150 and CD54 EC200. Proposals have been made on how to use thesevalues for potency prediction (Nukada et al., 2011, 2013), nevertheless additional work is
still required to determine how h-CLAT data may inform potency assessment.
10) As a result of the validation study a revised and more detailed Standard Operating
Procedure (SOP) was defined (EURL ECVAM, 2013c) which EURL ECVAM will disseminate,
together with a comprehensive description of the h-CLAT method through its database on
alternative methods (DB-ALM, see http://ecvam-dbalm.jrc.ec.europa.eu; protocol No. 158).
The SOP contains all the necessary technical details (including electronic data reporting
templates) needed by an end-user laboratory to implement it in a reliable and self-sufficientmanner. In addition, EURL ECVAM intends to make available an online video tutorial withpractical demonstration of how to perform the most critical steps of the h-CLAT SOP.

3. Overall performance of the h-CLAT test method
Reference data
11) A key criterion employed for selecting the validation test chemicals was availability of highquality in vivo data from the murine LLNA and GPMT or Buehler test, with concordantclassification from these assays. In addition, chemicals with available human data and/or
which are known to produce misleading responses in the animal tests (e.g. Nickel chloride
and Xylene which produce false negative and false positive responses in the LLNA test,
1

Estimated Concentrations are not to be confused with Effect Concentrations which are also usuallyabbreviated "EC".

660

respectively) were considered in the selection. The set of chemicals used in the study
comprised one third of non-sensitisers and two thirds of sensitisers, with a balanced
representation of potency classes (weak, moderate, strong and extreme) for the sensitisers.
Also included in the reference set were: chemicals from the LLNA performance standards(OECD 2010a), two well characterised pre-haptens (i.e. chemicals requiring abiotic
activation to exert their sensitisation potential), 4-Phenylendiamine and R(+)-Limonene, awell-known pro-hapten (i.e. a chemical requiring metabolic activation to act as sensitiser),
Dihydroeugenol, to challenge the potential of THP-1 cells to metabolically activate inert
substances, and two metal salts, Berillium sulphate and Nickel chloride. Additional details
on chemical selection can be found in the Validation Study Report (EURL ECVAM, 2013c).
12) When interpreting the data from alternative non-animal methods such as the h-CLAT thathave been largely developed and validated using animal reference data such as LLNA or
GPMT, it should be kept in mind that the animal tests are not fully reflective of the humansituation. Notably, an evaluation of the LLNA in comparison to human data has shown an
accuracy of about 72% (Anderson et al., 2011) indicating an appreciable risk of both false
negative and false positive predictions for humans. Moreover there is indication that the
LLNA is deficient in detecting low to moderate sensitisers as well as metals and organometalcompounds (EC, 2000).
Transferability13) EURL ECVAM concludes that the h-CLAT test method is transferable to laboratories
sufficiently experienced in cell culture techniques and flow cytometry analysis and that havereceived proper training. The h-CLAT procedure is composed of several tasks which need tobe performed sequentially, i.e., the qualification of the cell batch, the determination of an
accurate CV75 value, the cell staining and measurement of CD86 and CD54 expression byflow cytometry and the data analysis and interpretation. EURL ECVAM recommends
therefore that a step-wise approach similar to the one implemented in the transferability
phase of the validation study is used when implementing the method before the test is
performed for routine testing.
Reproducibility14) The between laboratory reproducibility, assessed in the validation study by testing a set of
24 coded chemicals and determining concordant predictions of sensitiser versus nonsensitiser, met the expected value of 80% set a priori by the Validation Management Team
(VMT). The overall within laboratory reproducibility (calculated from 15 of the 24 chemicals
tested) was found to be 80%, which was lower than the expected value of 85% set by the
VMT.
15) ESAC raised concerns in relation to within laboratory reproducibility since the VMT target
value was not met. EURL ECVAM acknowledges this but notes that the VMT targets were
derived from quite limited historical data on between laboratory reproducibility only,
generated under non-blinded conditions (Sakaguchi et al., 2010). EURL ECVAM believes thatthese VMT target values should not be interpreted as 'cut-off' validation criteria since what
can be considered as acceptable in terms of reproducibility typically depends on the context
of use, such as within an IATA. However, as with data from any other experimental method,
the reproducibility of h-CLAT needs to be taken into account when it is applied in any
decision-making context. In this respect, it is worth noting that the reproducibility of the hCLAT for discriminating between skin sensitisers and non-sensitisers appears to be761comparable to that of the LLNA (i.e. 70-80%, as calculated from the data available in the
NICEATM database, see: http://ntp.niehs.nih.gov).
16) The ESAC peer review of the h-CLAT study included valuable expert discussion of various
statistical approaches to assess within and between laboratory reproducibility. As a followup, EURL ECVAM proposes to re-analyse the data from the validation study with a view to
exploring the merits of various statistical methods for describing the reproducibility of a test
method that produces a classification-based prediction.
17) As indicated by the ESAC, further fine-tuning of the h-CLAT testing protocol and additional
characterisation of the test system (THP-1 cells) may lead to an improved performance ofthe test method including the level of reproducibility that can be achieved. Nevertheless,the validation of the h-CLAT did not highlight any specific feature of the test method thatwould require additional optimisation in the short term to substantially improve its
performance.
Predictive Capacity
18) Full evaluation of the predictive capacity of the h-CLAT was not within the scope of the EURLECVAM study since the test method is not proposed as a stand-alone full replacement
method. Nevertheless, the accuracy of the h-CLAT in predicting the in vivo classification(sensitiser/non-sensitiser) determined on the basis of concordant results in the LLNA,
guinea pig tests and where available human data (see paragraph 11), was determined as
76% (sensitivity 81% and specificity 66%) (EURL ECVAM, 2013c). A recently published study
that reported data on 143 chemicals (Takenouchi et al., 2013) suggested an accuracy of 80%
in predicting LLNA classifications indicating that the actual performance of the h-CLAT test
in discriminating between sensitisers and non-sensitisers may thus be actually higher. The
accuracy of the h-CLAT in predicting human skin sensitising potential is indicated in thescientific literature to be 83% (sensitivity 88%, specificity 67%) for a set of 66 chemicals forwhich human patch test data and case reports are available (Nukada et al., 2011) and for asmaller set of chemicals (n=23; sensitivity 81%, specificity 86%) (Bauch et al., 2011).

4. Limitations
4.1 Technical limitations
19) Solubility of test substances: The test chemicals should be dissolved in a solvent compatible
with the cell culture conditions. Therefore, chemicals which are not soluble in either
medium, saline or DMSO, these being the solvents prescribed by the SOP, cannot be tested
in the h-CLAT assay.
20) Test substance stability: As with many in vitro and in chemico assays, chemicals which are
not stable in the prescribed solvents because of hydrolysis or other chemical reactions
cannot be reliably tested.
21) Maximum testable concentration: In order to prevent osmotic stress of the cells, themaximum concentration of test substance should not exceed 5000 μg/mL.
22) Interference with flow cytometry analysis: Since the h-CLAT uses a fluorescein
isothiocyanate (FITC)-labelled antibody, strong fluorescent test chemicals emitting at thesame wavelength as FITC may interfere with the flow cytometry light-signal acquisition. To
circumvent the problem, antibodies labelled with alternative fluorescent dyes may be used8

62

provided that it can be shown that equivalent results to those obtained with the FITClabelled antibodies are obtained. Also, flow cytometry analysis cannot be conducted
correctly in the case of excessive cytotoxicity due to artefacts arising from diffuse labellingof cytoplasmic structures.
4.2 Limitations with regard to applicability23) A recently published analysis of h-CLAT data suggests that chemicals with an octanol-waterpartition coefficient (log Kow) value lower than 3.5 can be tested in the assay and provide
accurate predictions, whereas chemicals with a log Kow greater than 3.5 tend to produce
false negative results (Takenouchi et al., 2013). For this reason, it was suggested that
positive h-CLAT predictions obtained with chemicals with a log Kow greater than 3.5 arelikely to be trustworthy whereas a negative prediction should be considered inconclusive.
24) As for many other assays based on an individual cellular model, the metabolic capacity(biotransformation) of the h-CLAT only partially represents the skin metabolism in vivo
(Hennen et al, 2011; Chipinda et al., 2011; Fabian et al., 2013). Therefore, pro-haptens suchas Isoeugenol may not be correctly identified by the assay. Nevertheless, putative prohaptens such as 2-Aminophenol, Eugenol, 1-Naphtol and 2-Methoxy-4-methylphenol(Gerberick et al., 2009) have been reported in the h-CLAT submission to EURL ECVAM as
being correctly predicted by the assay. In addition, Dihydroeugenol, a well characterised
pro-hapten, was correctly classified as a sensitiser by all of the laboratories participating in
the validation study (EURL ECVAM, 2013c).
25) Some pre-haptens are reported to be false negative in the h-CLAT (e.g. Abietic Acid)
whereas others are reported as being correctly predicted by the assay (e.g. Geraniol and
Linalool) (Ashikaga et al., 2010). The two pre-haptens evaluated in the EURL ECVAM
validation study, 1,4-Phenylendiamine and R(+)Limonene, were correctly detected as
potential sensitisers by all of the laboratories.
26) Most of the misclassifications generated by the h-CLAT in the EURL ECVAM study (EURLECVAM 2013c), and in other published studies (Ashikaga et al., 2010) concerns chemicals
that are weak sensitisers in vivo while the false negative rate for strong sensitisers is much
lower. This should be kept in mind when interpreting negative results.

5. Suggested regulatory use27) Due to the complexity of the mechanisms underlying skin sensitisation, it is likely that
information from different methods (in silico, in chemico, in vitro) is needed to reduce orreplace the need for animal testing, both for hazard identification and potency
characterisation purposes.
28) Based on the validation study results and other available information, the h-CLAT test
method appears to be effective in providing information on the ability of a chemical toenhance the expression of the CD54 and/or CD86 cell membrane markers in THP-1 cells.
Such markers are considered useful readouts for the identification of skin sensitisingchemicals (OECD 2012a; 2012b). In addition, evidence in the literature clearly indicates thepredictive value of h-CLAT data when combined with complementary information (Bauch etal., 2012; Nukada et al., 2013; Hirota et al., 2013; Tsujita-Inoue et al., 2014; van der Veen etal., 2014). Therefore results from the h-CLAT assay can be used within an IATA to determine
the sensitisation potential of chemicals.
96329) Taking into consideration the concentration-response information generated by the assay, itis plausible that h-CLAT may potentially contribute within an IATA to the characterisation ofskin sensitisation potency. The extent of additional evidence needed to complement a hCLAT result will depend on the intended application (e.g. hazard identification or potency
assessment) and context (availability and quality of other information). Examples of the useof h-CLAT data in integrated non-animal approaches for hazard and potency assessment
have been published in scientific literature (Bauch et al., 2012; Nukada et al., 2013; Hirota etal., 2013; Tsujita-Inoue et al., 2014; van der Veen et al., 2014).
30) Negative h-CLAT results should be interpreted with care, taking into due consideration (1)the limited capacity of the assay to metabolise (biotransform) pro-haptens, (2) the fact thatsome pre-haptens may not be sufficiently oxidised under the h-CLAT experimental
conditions, and (3) the high rate of false negative predictions obtained with chemicals witha log Kow greater than 3.5.
31) Employed within an appropriate IATA, the h-CLAT assay 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
32) When applying the h-CLAT method, EURL ECVAM recommends that the revised protocol
available at EURL ECVAM's DB-ALM service (http://ecvam-dbalm.jrc.ec.europa.eu, protocol
No. 158) be used.
33) EURL ECVAM will undertake additional statistical analysis of the validation study results tobetter describe and understand aspects of reproducibility of this method.
34) Further testing to assess the performance of the h-CLAT method should include emphasis
on assessing pre-and pro-haptens. In addition, its applicability to chemical mixtures andpolymers (Jung YS et al., 2011) should be further investigated.
35) Predictive capacity of the assay for the discrimination between sensitisers and nonsensitisers should be further evaluated in the context of its inclusion within IATA. 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.
36) Integrated approaches using the h-CLAT method should also make use of other information
sources, in particular from testing and non-testing methods (e.g. chemoinformatics, readacross and QSAR models). In silico methods that incorporate metabolic considerations (e.g.
TIMES-SS: Patlewicz et al., 2007) may also help to identify pre- and pro-haptens. Analogues
which have a similarly predicted mechanism of action, e.g. based on protein binding, can befound using the OECD QSAR Toolbox (www.qsartoolbox.org). The Toolbox also includes a
specific profiler based on the h-CLAT assay. A variety of proposals concerning the use of hCLAT data in combination with other information sources to discriminate between
sensitising and non-sensitising chemicals have been published (Bauch et al., 2012; Nukada
et al., 2013; van der Veen et al., 2014) and may support further work.
37) The possible contribution of h-CLAT CD86 EC150 and CD54 EC200 values derived from theconcentration-response curve to support sub-categorisation of sensitisers according to GHS(i.e. sub category 1A and 1B) and to contribute to potency assessment should be evaluatedin the context of integrated approaches. Examples are published in the scientific literature
1064on how these values can contribute to both purposes (Ashikaga et al., 2010; Hirota et al.,2013; Nukada et al., 2013; Tsujita-Inoue et al., 2014). For such evaluation, the use of humanreference data (Basketter et al., 2014) will be particularly useful.
38) To reduce the cost and time needed for deriving a h-CLAT prediction for the purpose of skinsensitisation hazard identification, consideration should be given to adapting the h-CLAT
SOP to eliminate the need for a third run in case of consistent and unequivocal predictionsin the first two runs.
39) EURL ECVAM supports the development of an OECD Test Guideline for the h-CLAT. As thistest 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 IATA for skin sensitisation.

11

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EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

ANNEX 1ESAC OPINION

on the ECVAM-led study of the human Cell Line Activation
Test (h-CLAT)TABLE OF CONTENTS
SUMMARY OF THE ESAC OPINION ................................................................................................................. 17
1.

MANDATE OF THE ESAC ........................................................................................................................ 19

2.

DETAILED OPINION OF THE ESAC .......................................................................................................... 20
1) STUDY DESIGN – TRANSFERABILITY, RELIABILITY AND RELEVANCE. ................................................................................ 202) CONCLUSIONS OF THE STUDY ............................................................................................................................... 21

3.

INFORMATIVE BACKGROUND TO THE MANDATE AND OPINION........................................................... 23

4.

REFERENCES .......................................................................................................................................... 24

Ispra, 11 March 2014

Summary of the ESAC Opinion
The ESAC was requested to provide a scientific opinion on an EURL-ECVAM led validation study
assessing mainly the transferability and reproducibility (within- and between-laboratories) of the hCLAT test method (primary objective of the study) in view of its possible future use as part of a nonanimal testing strategy for skin sensitization. The study had also been designed to provide
preliminary information on a) the predictive capacity of the test method and b) its potential use forcontributing to sub categorisation of sensitizing chemicals.
Overall, the conclusions made by the ESAC based on the ESAC WG report correspond well with theconclusions drawn by the Validation Management Group overseeing the study and as described inthe Validation Study Report, indicating that, generally, the conclusions are supported by the results
shown in the report (see Section 15.1).
The ESAC disagreed, however, with the VMG conclusion concerning the Within Laboratory
Reproducibility (WLR).
- Acceptance criteria were determined at the start of the study by the VMG. WLR was assessedusing 15 chemicals in three independent experiments. The average reproducibility of 80%(KAO (86.7%), Shiseido (80.0%), EURL-ECVAM (80.0%) and Bioassay (73.3%)) did not meet the
85% reproducibility target set by the VMG. Actually, only one out of four participants met
this target. Despite missing the expected performance level, the VMG neverthelessconcluded that the h-CLAT is a reproducible method within laboratories. This conclusion was
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partly based on the premise that a subset of chemicals consistently drove the discrepancies
in reproducibility, and that some of these problem chemicals might fall outside theapplicability domain. While the chemical limitations of the test are appreciated, the ESAC isconcerned that there may be other inherent characteristics or critical aspects of the h-CLAT
test method, which could be important sources of variability (e.g. the time course forexpression of the cell surface markers; the state of cell differentiation be a source ofvariability). The other reason given by the VMG to support their conclusion is that the h-CLAT
assay is intended to be used as part of an ITS. Generally, the ESAC does not support thisreasoning: a low reproducibility, i.e. high variability will cause problems when using a test
method in practice and this is independent of whether it is used as a stand-alone test
method or within integrated approaches. Other information sources within an integrated
approach will not be able to remedy the intrinsic variability of one information source. With
regard to the h-CLAT assay, the ESAC Working Group is concerned that the poor
reproducibility of the assay may actually create difficulties with respect to the interpretationof data generated as part of an ITS, as results are likely to be conflicting.
The data were considered strong enough to support transferability of the test to properly
equipped, trained and staffed laboratories with the appropriate analytical capabilities.
Five of the 24 chemicals produced a discordant classification by the laboratories resulting inan average BLR reproducibility of 81.3%, meeting the target (80%).
For S/NS classification, values (accuracy: 76%; sensitivity: 81.3%; specificity: 65.6%) are,overall, lower than the values (84%, 87%, 75%, respectively) resulting from the historical dataon 100 chemicals (Ashikaga et al., 2010), which were provided to EURL-ECVAM as part of thetest submission. Due to this discrepancy the ESAC concludes that the number of substances
and the information available for these substances in the peer reviewed publication was
insufficient for allowing more than a purely preliminary indication on the predictive capacity
in terms of S/NS.
For sub-categorization, the data generated and statistically assigned cut-offs propose amaximum accuracy of 58% accuracy, which is in contrast to previously published data
(N=100) that reported an accuracy of 72% (Ashikaga et al. 2010). The ESAC does notunderstand why the VMT considers the values obtained in the validation study as promising.
Our conclusion is that the number of substances and the information available for thesesubstances was insufficient for allowing more than a purely preliminary indication on thepredictive capacity in terms of potency classification.
The number of chemicals did not allow us to draw conclusions about the applicability domainof the test (which, notably, was not one of the study objectives). Empirically the applicability
domain seems to exclude pro-haptens, auto-fluorescent compounds, chemicals with limited
water solubility/stability, metal salts and volatile compounds. However, pre-/pro-haptens
were reported as correctly identified.

The predictive capacity, applicability domain and limitations of the test are not, in our view, yet fully
defined. The submitted study does not provide strong evidence supporting the usefulness of the hCLAT for GHS sub-categorisation of sensitizers. However, recent studies substantiate the preliminary
data of the VSR (Nukada et al., 2012; Nukada et al., 2013).
-

The ESAC recommends that the sources of the unsatisfactory WLR (below the 85% target) be
identified and addressed;

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better defining (1) the predictive capacity and (2) the applicability domain of the h-CLAT (toeliminate the uncertainty currently associated with a negative result) either through further
testing (i.e. prospective validation) or through retrospective analysis of existing information(retrospective validation: data grouping / meta-analysis);to adapt the SOP to reduce resource costs by eliminating the need for a third evaluation run
in case where the first two runs are consistent;
to reassess the amended SOP version 7 using existing/historical results with the purpose to
re-evaluate the predictive capacity of this test method.
that further studies be conducted to determine the potential of the test method to properly
sub-categories chemicals with skin sensitisation potential.

Recently, Nukada et al. (2013) reported a data integration strategy including the h-CLAT, the Direct
Peptide Reactivity Assay (DPRA) and the knowledge-based expert system 'DEREK' for thedevelopment of a test battery to predict the skin sensitizing potential and potency of chemicals.
Using a tiered strategy of h-CLAT and DPRA an accuracy of 86% and 73% for the potential andpotency prediction, respectively, was obtained. Further studies are needed to identify the best
integrated testing strategy or strategies able to address the different regulatory goals and risk
assessments (hazard identification, classification, potency assessment, etc.) in reliable and relevant amanner.

1.

Mandate of the ESAC

The opinion of ESAC should support ECVAM with respect to the development of recommendations
regarding the reliability (transferability, within and between laboratory reproducibility) of the h-CLAT
and the potential regulatory use of the test method.
1. Study design – transferability, reliability and relevancex

x

The ESAC was requested to review whether the validation study was conducted
appropriately in view of the objective of the study:

Reproducibility of the h-CLAT method within laboratories (WLR);

Transferability;

Reproducibility between laboratories (BLR);

Predictive capacity of the test method.

With respect to the design and conduct of the study, the following issues were to beaddressed:
 Clarity of the test definition (module 1)
 Clarity of the definition of the study objective
 Appropriateness of the study design in view of study objective
 Appropriateness of the study execution:
 Appropriateness of the statistical analysis used for analysing WLR, transferability, BLR
and (preliminary) predictive capacity.

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2. Conclusions of the studyx

The ESAC was requested to assess the justification and plausibility of

Reproducibility (WLR and BLR) and transferability;Preliminary predictive capacity;
Possible gaps between study design and study conclusions which remain to beaddressed in view of the suggested conclusions/use;
Applicability and possible limitations of the test method, in particular in view of its
potential use within an ITS for sensitisation testing and assessment.



3. The ESAC is requested (a) to evaluate, on the basis of the data submitted in the validation study,
the possible use of the test method (also within a strategy) to identify skin sensitizers, (b) to makeadditional recommendations (as required) on the proper scientific use of the test method within sucha 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 theplausibility of the suggested use (including within an ITS).

2.

Detailed opinion of the ESAC

The ESAC was asked to provide an opinion on a EURL-ECVAM-coordinated study assessing thetransferability and reproducibility (within- and between-laboratories) of the h-CLAT (primaryobjective of the study) in view of its possible future use as part of a non-animal testing strategy forskin sensitization. The study had also been used to provide preliminary information on a) the
predictive capacity of the test method and b) its potential use for contributing to sub-categorisationof sensitizing chemicals.
1) Study design – transferability, reliability and relevance.
xx

The Test Definition of the h-CLAT assay would benefit from a more detailed rationale
behind the selection of the THP-1 cell line, and CD86 and CD54 membrane markers; inparticular as to why both of the markers are required. Furthermore, their biological andmechanistic relevance to the human situation is not sufficiently explained. There is
ample evidence showing that CD86 and CD54 are generally up-regulated in response tochallenges that cause cell damage, inflammation and cytotoxicity. There is a need to
explain what special features of the test or the prediction model are making the test
specific for sensitization.
The WLR was assessed at the level of concordance with a binary prediction (S/NS). An
average reproducibility of 80% did not meet the 85% reproducibility target set by the
VMG. Actually, only one out of the four participating laboratories met this target. The
definition of the reproducibility target (85%) set by the VMG was based on theperformance of methods previously evaluated at EURL-ECVAM. The expected
performance of the test (WLR) is derived from the BLR calculated from the test
submissions. The ESAC does not consider 85% to be an unreasonably high target.
Furthermore, the explanation offered according to which it was a small number ofcompounds with special properties that caused problems with reproducibility was not

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x

x

x

x

x

x

ESAC OPINION
further substantiated. Indeed, it was noted that the problem concerned 9 out of 15
chemicals and none of the problem chemicals gave issues in all 4 laboratories.
The ESAC is concerned, in the absence of evidence, that low WLR was caused by thecharacteristics of the chemicals tested and that there may be inherent characteristics ofthe h-CLAT, which could be important sources of WLR variability The low reproducibilityof the test, raised also the concern that the h-CLAT as potential ITS building block withpoor reproducibility might actually create more difficulties in interpreting data as part ofan ITS due to conflicting results.
The training and transfer phases of the validation study were well planned and executed.
All the stages appear well documented. Some key issues have been identified during theprocess of transfer to the naïve laboratories and effort has been put into identifying andsolving these issues. These changes were taken up in SOP version 5 (used for transfer)and resulted in SOP versions 6 and 7. It is clear from the transfer data that adopting thismethod in a laboratory requires sufficient experience in flow cytometry and cell culture.
The BLR was assessed in terms of concordance in predictions. Two BLR values weregenerated by testing 24 chemicals, one comparing the consistency of the two naïve labs
with the first lead lab and the second comparing them with the second lead lab. ESAC
agreed with the VMG's conclusions with respect to the acceptability of the BLR becauseof the marginal difference between the lowest BLR (79.2%) and expected performance of
80%. The chemicals that drove discrepancies in the BLR study were the same as thosedriving discrepancies in the WLR study. The ESAC notes, in the absence of a defined
applicability domain, that some of these test chemicals may have physicochemical
properties making them incompatible with this test method.
The ESAC recognizes the fact that this study was not designed to address the predictive
capacity of the h-CLAT due to the low number of chemicals. This also applies to the subcategorization. Three chemicals (methyl methacrylate, DCNB and benzyl alcohol) wereconsistently and reproducibly wrongly classified.
The project was described and designed in clearly recognizable and well describedphases including Test Definition (Module 1), Transferability (Module 3), Within
Laboratory Reproducibility (WLR) (Module 2), Between Laboratory Reproducibility (BLR)(Module 4). The data were also used for a preliminary evaluation of Predictive Capacity(Module 5).
Overall, the chosen statistical approach was considered appropriate. The ‘expected
proportion’ of concordant classifications (between laboratories) was calculated to be
90% on the basis of available data on between-laboratory reproducibility as submitted to
ECVAM (see Appendix 2 of VSR, page 5). However, it was not clear why a power of 75%
rather than the more conventional 80% or 90% power had been applied. This power
allows for detecting 25% changes in each direction and, as a consequence, leads to alower limit of the confidence interval of 65 % (90%-25%).

2) Conclusions of the studyxxOverall, the study design, including the chemicals and their associated reference data,
were considered appropriate for the purpose of addressing the first objective of thestudy: Assessing the WLR (N=15) and BLR (N=24) of the h-CLAT.
Overall, the conclusions made by the ESAC correspond well with the conclusions drawn
by the VMG as described in the VSR, tending for confirm that these conclusions aresupported by the results shown in the report.

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ANNEX 1x

x
x

ESAC OPINION
The ESAC disagreed, however, with the VMG conclusion concerning the WLR. An
average reproducibility with the validation study test chemicals of 80% (not meeting the
85% VMG reproducibility target) set by the VMG. Actually, only one out of four
participating laboratories met this target. The validation study did not fully establish thereasons for the WLR performance figures obtained.
The conclusion on the BLR is considered reasonable in light of the marginal difference
between the lowest BLR (79.2%) and expected performance of 80%. See above
The accuracy values for S/NS classification (76%) and sub-categorization (57%) are lower
than those reported earlier (84% (S/NS) and 72% (sub-categorization)) and based on
historical data on 100 chemicals (Ashikaga et al. 2010. ATLA 38; 275-284) which were
submitted to EURL-ECVAM as part of the test submission. The ESAC believes this may be
explained in part by the smaller number of chemicals used for the validation study,some of which were not part of the historical data set. A separate communication fromthe test developers suggests that the historical data may have contained
proportionately fewer difficult chemicals (e.g. the three chemicals consistently wrongly
classified in the validation study are not part of the 100 chemical set).
Assessment/description of the applicability domain was not the objective of this study.
Consequently, the small number of chemicals used in the validation study, which was set
to satisfy the primary goal of the study, is not sufficient on its own to draw robust
conclusions on the applicability domain.

3) Possible use of the test method, i.e. to identify (also within a strategy) skin sensitizers, andadditional recommendations (as required) on the proper scientific use of the test method within sucha strategy.
xx

As yet no applicability domain has been described for this method. Deciding whether or
not a chemical falls within the applicability domain of the test will be a challenge withregard to pro-haptens, metal (salts), chemicals with limited solubility/stability in water,
volatile compounds and auto-fluorescent compounds.
Regarding potency class, the data obtained did not support the use of the h-CLAT as astand-alone assay for potency classification. This is in agreement with the statement of
the VMG that the assay should be further evaluated for its capacity to "contribute" to apotency classification.

Recommendations:xx

The ESAC considered that the target value for WLR was a realistic and justified one andwere therefore concerned that three of the four laboratories failed to meet this target.
The ESAC recommends that the sources of variability be identified (e.g. the time course
for expression of the cell surface markers; the state of cell differentiation be a source ofvariability), and that solutions be provided. Poor reproducibility may create difficulties ininterpreting data as part of an ITS due to conflicting results. A review of the existing 100
chemical/24 chemical datasets might identify properties of chemicals for which this test
is not an appropriate method for investigating skin sensitisation potential.
The ESAC recommends better explaining, clarifying or defining (1) the predictive capacity,
the ability of the cell system and biomarkers to selectively identify skin sensitisation, andthe sources of variability; and (2) the applicability domain of the h-CLAT to reduce thefrequency to inconsistent results, either through further testing (i.e. prospective

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x

3.

ESAC OPINION
validation) or through retrospective analysis of existing information (retrospective
validation: data grouping / meta-analysis).
For greater efficiency, the SOP could be adapted by eliminating the need for a third
evaluation run in case where the first two runs are consistent as a third inconsistent run
does not change the outcome.
Based on the ESAC assessment of the validation study data, the available limited
evidence does not support the use of the test method for GHS sub-classification ofsensitizers: that was not, however, a primary objective of the validation study. Additional
information and evidence are required when further consideration is given to the use of
the test method for this purpose (see for example Nukada et al., 2012; 2013). Nukada etal. (2013) reported a data integration strategy including HCLAT, DPRA and DEREK for thedevelopment of a test battery to predict the skin sensitizing potential and potency ofchemicals. Using a tiered system of h-CLAT and DPRA an accuracy of 86% and 73% for the
potential and potency prediction was obtained. The tiered system showed a higher
sensitivity (from 88 to 96%) compared with h-CLAT alone. Further studies are needed to
identify the best integrated testing strategy or strategies necessary to cover the different
regulatory goals and risk assessments (hazard identification, classification, potency
assessment, etc.).

Informative background to the Mandate and Opinion

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 for those intended for topical application(e.g. cosmetics). Current regulatory predictive tests for skin sensitization rely on the use of animals,
these include: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
non-radioactive 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 currently under development/evaluation is able to fully characterise the relative
potency of sensitising substances and therefore, none of these assays is considered a stand-alone
method, capable of fully replacing current animal procedures, in particular as regards to cosmetics.
The current view therefore is to combine different test methods in order to address different key
mechanisms of skin sensitisation: skin bioavailability, haptenation (the protein binding of chemicals
which triggers immunological responses), epidermal inflammation, dendritic cell activation andmigration, T cell proliferation. Test methods are currently under development which have been
specifically designed to address these key mechanistic steps involved in skin sensitisation. Before
these test methods can be routinely used, e.g. in ITSs, their capacity to produce reproducible results
needs to be demonstrated as a first step. There is ample evidence showing that maturation markers
in general, and CD86 and CD54 in specific, are generally up-regulated in response to challenges thatcause inflammation and cytotoxicity. There is however a window in which only sensitizers (or the
ESAC Opinion on the EURL ECVAM-led study of the human Cell Line Activation Test (h-CLAT) for skinsensitisation testing

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ESAC OPINION

majority of them) activate dendritic cells (DCs). Cellular stress induced by allergens is different fromthe one triggered by irritants. Furthermore, hypersensitivity reactions are the result of normallybeneficial immune responses acting inappropriately against benign antigens, causing inflammatory
reactions and tissue damage. Just for clarification, DCs are recognized as important antigen
presenting cells in adaptive immunity because of their capacity to stimulate naïve lymphocytes(Banchereau et al., 2000). Langerhans cells (LC) are resident immature DCs in the skin capable to takeup and process contact allergens. During this process LC differentiate into mature
immunostimulatory cells up-regulating the expression of co-stimulatory molecules such as CD80,
CD86 and CD40 and adhesion molecules including CD2, CD11a, CD54, CD58 (Quah and O’Neill, 2005).
Activated LC move from the epidermis into the dermis, and into the regional lymphatic system. In thelymph node, LC differentiate into mature dendritic cells and present antigen to specific T lymphocyte
using MHC class II molecules to hold the processed antigen in place. Adhesion molecules on both theantigen-presenting cell (i.e. CD86) and the T-cell (i.e. CD28) ensure appropriate contact and costimulation. Following appropriate stimulus, a clone of T cells with the ability to react to the antigen,which caused their expansion, is produced. The h-CLAT it is a test method that allows for quantitative
analysis of a chemical’s potential to induce activation of THP-1 cells (used as a surrogate for human
myeloid dendritic cells). This method has been initially proposed by Ashikaga et al. (2002) to identify
sensitizers, and Yoshida et al. (2003) reported that naïve THP-1 could respond to sensitizers
specifically through augmented expression of co-stimulatory molecules, CD54 and CD86, andconsidered this as a possible tool to be used as an in vitro sensitization test.

4.

References

Ashikaga T, Hoya M, Itagaki H, Katsumula Y and Aiba S (2002). Evaluation of CD86 expression and
MHC calss II molecule internalization in THP-1 human monocytic cells as predictive endpoints forcontact sensitizers. Toxicol. In Vitro 16: 711-716.
Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M,Ito Y, Nishiyama N,
Itagaki H. (2010) A comparative evaluation of in vitro skin sensitisation tests: the human cell-line
activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. Aug;38(4):275-84.
Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B and Palucka K (2000).
Immunobiology of dendritic cells. Annu. Rev. Immunol. 18: 767-811.
Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP, (2004) Development of a
peptide reactivity assay for screening contact allergens. Toxicol Sci. 81; 332-43.
Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittevin JP, (2007). Quantification ofchemical peptide reactivity for screening contact allergens: a classification tree model approach.
Toxicol Sci. 97, 417-27.
Landsteiner K & Jacobs J (1936) Studies on the sensitisation of animals with simple chemicalcompounds. Journal of Experimental Medicine 64, 625-639
Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012) Prediction ofskin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt atclassifying skin sensitization potency. Toxicol In Vitro. Oct;26(7):1150-60.
ESAC Opinion on the EURL ECVAM-led study of the human Cell Line Activation Test (h-CLAT) for skinsensitisation testing

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Nukada Y, Miyazawa M, Kazutoshi S, Sakaguchi H, Nishiyama N. (2013) Data integration of nonanimal tests for the development of a test battery to predict the skin sensitizing potential andpotency of chemicals. Toxicol In Vitro. Mar;27(2):609-18.
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
Quah BJC, and O’Neill HC (2005). Maturation of function in dendritic cells for tolerance andimmunity. J. Cell. Mol. Med. 9: 643-54.
Takenouchi O, Miyazawa M, Saito K, Ashikaga T and Sakaguchi H (2013). Predictive performance ofthe human Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-waterpartition coefficients. J. Toxicol. Sci. 38: 599-609.
Yoshida Y, Skaguchi H, Ito Y, Okuda M and Suzuki H (2003). Evaluation of chemicals using expressionof co-stimulatory molecules, CD54 and CD86, on the naive THP-1 cell line. Toxicol. In Vitro 17: 221228

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ANNEX 1

ESAC OPINIONEURL ECVAM Scientific Advisory Committee
x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

Prof. Jürgen BORLAK
Dr. Neil CARMICHAEL
Dr. Edward CARNEY
Dr. Harvey CLEWELL
Prof. Lucio COSTA
Dr.Kristina KEJLOVÁ
Dr. David John KIRKLAND
Prof. Annette KOPP-SCHNEIDER
Dr. Renate KRÄTKE
Prof. Claus-Michael LEHR
Dr. José Maria NAVAS
Dr. Aldert PIERSMA
Dr. Jonathan David RICHMOND
Dr. Erwin L. ROGGEN
Dr. Dorothea SESARDIC

ESAC Working Group Skin Sensitisation
x
x
x
x
x
x
x
x

Dr. Erwin ROGGEN (ESAC member, Chair of ESAC WG and rapporteur)Dr. Ed CARNEY (ESAC member)Prof. A. Wallace HAYES (ESAC member)Dr. Maja ALECSIC (external expert)
Dr. Emanuela CORSINI (external expert)
Dr. David LOVELL (external expert)
Dr. Michael WOOLHISER (external expert)
Prof. Yong HEO (external expert, ICATM nomination)

ESAC / ESAC WG Coordination
Dr. Claudius GRIESINGER

ESAC Opinion on the EURL ECVAM-led study of the human Cell Line Activation Test (h-CLAT) for skinsensitisation testing

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEANNEX 2

ECVAM REQUEST FOR ESAC ADVICEonan ECVAM-coordinated study concerning the transferability andreliability of the human Cell Line Activation Test (h-CLAT) for skinsensitisation 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

h-CLAT test method for skin sensitisation testing
2013-02
EP 2.01
3/6/2013
3/6/2013 for discussion at ESAC38 18/19 June 2013
ESAC 38
Q4 of 2013: ESAC plenary meeting or through written procedure
ESAC REQUEST 2013-03 h-CLAT-final.doc

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICETABLE OF CONTENTS
1. TYPE OF REQUEST.............................................................................................. 29
2. TITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE ESAC IS
REQUESTED .............................................................................................................. 30
3. BRIEF DESCRIPTION OF THE STUDY OR PROJECT ................................................. 30
4. OBJECTIVES, QUESTIONS, TIMELINES ................................................................. 34
4.1 OBJECTIVE ......................................................................................................................................... 34
4.2 QUESTION(S) TO BE ADDRESSED ............................................................................................................ 34
4.3 TIMELINES ............................................................................................................................................... 35

5.

ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN ESAC ............. 36

5.1 ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIRED ..................................... 36
5.2 DELIVERABLES AS PROPOSED BY ECVAM............................................................................................... 37

6.
7.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESAC ............................... 38
TERMS OF REFERENCE OF THE ESAC WORKING GROUP ....................................... 39

7.1

ESTABLISHMENT OF THE ESAC WORKING GROUP ........................................................................ 39

7.2

TITLE OF THE ESAC WORKING GROUP ........................................................................................... 39

7.3

MANDATE OF THE ESAC WG .......................................................................................................... 39

7.4

DELIVERABLE OF THE ESAC WG ...................................................................................................... 39

7.5

PROPOSED TIMELINES OF THE ESAC WG ....................................................................................... 39

7.6

QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WG ................................................... 40

APPENDIX 1 REPORTING STRUCTURE FOR THE ESAC WG REPORT ................ Error! Bookmark not defined.

28

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ANNEX 2

1.

ECVAM REQUEST FOR ESAC ADVICETYPE OF REQUEST

Request Type

Identify request ("YES")

R1 ESAC Peer Review

YES: Validation study addressing mainly reliabilityof a Prevalidation Study or Validation Study
If R1)applies please specify further:►Prevalidation Study►Prospective Validation StudyIn the period between 2010 and 2012 EURLECVAM coordinated a validation study focusing
on an assessment of reliability of three test
methods for skin sensitisation testing: 1) the
Direct Peptide Reactivity Assay (DPRA), 2) the
human Cell Line Activation Test (h-CLAT), 3) the
Myeloid U937 Skin Sensitisation Test (MUSST).
This request focuses on the h-CLAT test method.

►Retrospective Validation Study►Validation Study based on PerformanceStandards

R2 Scientific Advice on a test method submitted to
ECVAM for validation(e.g. the test method's biological relevance etc.)

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

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ANNEX 2

2.

ECVAM REQUEST FOR ESAC ADVICETITLE OF STUDY OR PROJECT FOR WHICH SCIENTIFIC ADVICE OF THE
ESAC IS REQUESTED

Validation of the reliability of the Human Cell Line Activation Test (h-CLAT)

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 skin allergy, leading to the disease called allergic contact dermatitis (ACD) in humans.
The identification of the skin sensitisation potential represents an important component of thesafety assessment of new and existing substances including cosmetic ingredients. Current regulatory
predictive tests for skin sensitisation rely on the use of animals. These include: guinea-pig tests(Buehler Test and Guinea-pig Maximisation Test) (TG 406, OECD 1992; TM B06, EC 2008a), themurine Local Lymph Node Assay (LLNA) (TG 429, OECD 2010a; TM B42, EC 2008a) and its non-radioisotopic variants (TG 422a, OECD 2010b; TG 422b, OECD 2010c).
The key events underlying of the induction of skin sensitisation are well understood and have been
recently documented by the OECD in its report on: “The Adverse Outcome Pathway (AOP) for Skin
Sensitisation Initiated by Covalent Binding to Proteins” (OECD 2012a; 2012b). These include: 1) theability of the chemical to penetrate the skin and reach the site of haptenation (skin bioavailability),2) the covalent binding of the chemical to skin proteins (haptenation), 3) the release of proinflammatory signals and the induction of cyto-protective cellular pathways in keratinocytes 4) the
activation and maturation of Dendritic cells (DC) the skin immunocompetent cells, 5) the migrationof DC from skin to the regional lymph nodes, 6) the presentation by DC of the haptenated protein to
T cells and the clonal expansion of memory T cells (lymphocytes capable of being stimulated andactivated specifically by the haptenated protein).
Progress has been made in recent years in the development of mechanistically-based alternative
methods for hazard identification some of which might also be able to contribute to potency
prediction. However, none of these tests is currently regarded to have the potential to function as astand-alone method to fully replace the animal tests. Instead, it is proposed that a combination of inin silico, in chemico and in vitro tests, addressing the key biological events of skin sensitisation, will
be needed to achieve this goal.
Proposals on how to use these methods in Integrated Testing Strategies (ITS)/Integrated Approaches
to Testing and Assessment (IATA) for both hazard identification and potency prediction are
emerging.

2) The Human Cell Line Activation Test (h-CLAT).
30

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEThe Human Cell Line Activation Test addresses the role that Langerhans cell (LC) and dermal
dendritic cells (DC) play in the induction of skin sensitization. These cells are important mediators inthe skin sensitization process since they are capable of presenting the hapten-protein conjugate toresponsive T lymphocytes in the lymph nodes draining the site of exposure (Kimber andCumberbatch, 1992). The maturation process of LC and DC from antigen processing cells to antigen
presenting cells is considered a key event in the acquisition of skin sensitisation. This maturation
process involves the modulation of the expression of cell surface phenotypic markers, those mostcommonly reported being CD54, CD80, CD86 and major histocompatibility complex (MHC) class II(Galvao dos Santos et al., 2009). This knowledge has been exploited in the development of in vitro
tests based on the use of DC-like immortalized cell-lines to screen the skin sensitization potential ofchemicals.

The h-CLAT measures the modulation of CD86 and CD54 protein markers on the surface of THP-1
cells (human monocytic cell line) by flow cytometric analysis, following 24 hour cell exposure to 8
concentrations of a test substance. The concentrations used in the main experiment are selected on
the basis of the CV75 value, the estimated concentration of test substance yielding 75% cell viability,
previously determined with a propidium iodide viability assay. A chemical is classified as sensitiser if
the expression of either the CD86 and/or the CD54 is equal or exceeds a defined threshold in at least
2 of 3 independent evaluations.

The h-CLAT test method was jointly developed by Kao Corporation and Shiseido. Extensive
development/optimisation/evaluation work including assessment of the test method’s performancein multi-laboratory ring trials was conducted prior submission to ECVAM. The submission to ECVAM
reported results for 100 chemicals with an accuracy of 84% for distinguishing sensitisers from nonsensitisers compared to LLNA data.

3) Study objectives and design

The validation of the h-CLAT test method was part of larger validation study involving theassessment of two other test methods, the Direct Peptide Reactivity Assay (DPRA) and the Myeloid
U937 Skin Sensitisation Test (MUSST). The validation study was coordinated by ECVAM in the period
between 2010 and 2012 with the primary objective of assessing the test methods’ transferability
and within and between laboratory reproducibility in view of their potential future use in integrated
non-animal approaches intended to reduce and replace the currently used animal tests for skinsensitisation hazard identification.

As a secondary goal of the study, the experimental data were used to perform:a) A preliminary evaluation of the ability of the three tests to reliably discriminate skin sensitising (S)from non-sensitising (NS) chemicals as defined by the Globally Harmonised System (GHS) of
classification and labelling of substances (category 1; no category) (UN, 2011) and as implemented in
the European Commission Regulation on classification, labelling and packaging (CLP) of substancesand mixtures (EC, 2008b).

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEb) Where possible, a preliminary consideration of the ability of the three tests to contribute topotency categorisation e.g. GHS sub-category 1A (strong sensitisers) and 1B (other sensitisers) as
defined in the fourth revised edition of GHS (2011).

24 coded test items were tested by each of the four laboratories participating in the study for theevaluation of the h-CLAT (Kao and Shiseido as the lead laboratories, Biossay and EURL ECVAM as thenaïve laboratories) to generate information on the between-laboratory reproducibility. A subset of
15 chemicals was tested two additional times in each laboratory for the evaluation of the withinlaboratory reproducibility.

With respect to the ECVAM's modular approach to validation (Hartung et al., 2004) the study
generated information on modules 1) test definition, 2) within laboratory reproducibility, 3)transferability and 4) between laboratory reproducibility. In addition, the experimental data
contributed to modules 5) predictive capacity and 6) applicability domain. However, the number ofchemicals used in this validation study, which was based on statistical considerations related tothe evaluation of the reproducibility only, was not sufficient on its own to conclude on the last
two modules.

4) Study results

The main results for the study’s primary goal are summarised in the table below:

ModuleModule 2
WLR

Module 3
Transferability
Module 4
BLR

Results
Evaluation of the WLR for a subset (n=15) of the validation study
chemicals in each laboratory focused on the concordance of predictions(sensitizer versus non-sensitiser) as determined by the results of three
independent experiments.
Kao Laboratory

WLR=86.7%

Shiseido Laboratory

WLR=80%EURL ECVAM Laboratory

WLR=80%

Bioassay Laboratory

WLR=73.3%

Both naïve laboratories (EURL ECVAM and Bioassay) succeeded intransferring the protocol to their testing facilities.
Evaluation of the BLR for the 24 chemicals focused on the concordanceof the predictions (sensitiser versus non-sensitiser) and was calculated
by comparing the two naïve laboratories with each of the two leadlaboratories separately.
Naïve and Kao

BLR=83.3%

Naïve and Shiseido

BLR=79.2%

Overall

BLR=79.2%
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ECVAM REQUEST FOR ESAC ADVICE

5) Conclusions of the VMG
The VMG concluded that the information generated in this validation study demonstrates that the hCLAT is a robust test method that can be easily transferred to properly equipped laboratories
sufficiently experienced in cell culture and flow cytometry analysis. In addition the study results
support the fact that the h-CLAT is a reproducible test method that can contribute to thedetermination of the sensitization potential of substances.

References

EC 2008a. COUNCIL REGULATION No 440/2008 of 30 May 2008 laying down test methods pursuant
to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration,
Evaluation, Authorisation and Restriction of Chemicals (REACH). Official Journal of the European
Union No L 142 , p. 1 – 739.
EC 2008b. Regulation No 1272/2008 of 16 December 2008 of the European Parliament and of theCouncil on classification, labelling and packaging of substances and mixtures, amending andrepealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006.
Official Journal of the European Union L 353, p. 1-1355.
Galvao dos Santos G, Reinders J, Ouwehand K, Rustemeyer T, Schepper RJ & Gibbs S (2009) Progresson the development of human in vitro dendritic cell based assays for assessment of the sensitizing
potential of a compound. Toxicology and Applied Toxicology 236(3); 372-382.
Hartung T, Bremer S, Casati S, Coecke S, Corvi R, Fortaner S, Gribaldo L, Halder, M, Hoffmann S, Roi
AJ, Prieto P, Sabbioni E, Scott L, Worth A & Zuang V. (2004) A Modular Approach to the ECVAM
Principles on Test Validity. ATLA 32, 467-72.
Kimber I & Cumberbatch M (1992) Dendritic cells and cutaneous immune responses to chemicalallergens. Toxicology and Applied Pharmacology 117; 137-46.
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 (2010a) The Local Lymph Node
Assay. Guidelines for Testing of Chemicals No. 429, Paris.
OECD, Organisation for Economic Cooperation and Development (2010b) Skin Sensitization: Local
Lymph Node Assay: DA, Guidelines for Testing of Chemicals No. 442A, Paris.
OECD, Organisation for Economic Cooperation and Development (2010c) Skin Sensitization: Local
Lymph Node Assay: BrdU-ELISA, Guidelines for Testing of Chemicals No. 442B, Paris.
OECD, Organisation for Economic Cooperation and Development (2012a). The Adverse Outcome
Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins; Part 1: Scientific Evidence
Series on Testing and Assessment No.168 ENV/JM/MONO(2012)10/PART1. Paris.
OECD, Organisation for Economic Cooperation and Development (2012b). The Adverse Outcome
33

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ECVAM REQUEST FOR ESAC ADVICE

Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins; Part 2: Use of the AOP to
Develop Chemical Categories and Integrated Assessment and Testing Approaches Series on Testingand Assessment No.168 ENV/JM/MONO(2012)10/PART2. Paris.
UN, United Nations (2011). Globally Harmonised System of Classification and Labelling of Chemicals(GHS). Part 3: Health and Environmental Hazards. New York, NY, USA, and Geneva, Switzerland.

4.

OBJECTIVES, QUESTIONS, TIMELINES

4.1

OBJECTIVE

Objective
Why does ECVAM
require advice on
the current issue?

4.2

The opinion of ESAC should support EURL ECVAM with respect to thedevelopment of an EURL ECVAM recommendation on the h-CLAT assay
outlining (1) the scientific basis of the assay, (2) its overall performance asassessed during the study and based on other (e.g. published) information,(3) its applicability and limitations. Furthermore, the advice of ESAC should
support ECVAM with respect to the analysis of possible data gaps that need
to be addressed in view determining the test method's potential use andusefulness within integrated approaches for skin sensitisation hazard andrisk assessment.

QUESTION(S) TO BE ADDRESSEDQuestions

1) DESIGN & CONDUCT OF STUDY: The ESAC is requested to review

What are thequestions andissues that should
be addressed inview of achieving
the objective of
the advice?

whether the study was conducted appropriately in view of the objective of
the study. The study objective was to assess(1) the reproducibility of the h-CLAT method within one laboratory (WLR)(2) its transferability to other laboratories(3) its reproducibility between laboratories (BLR)(4) Furthermore, the study aimed at assessing, in a preliminary manner, thepredictive capacity of the test method for distinguishing between
sensitisers and non-sensitisers and, where possible, to appraise its potential
to contribute to a further sub-categorisation of sensitisers into two
subcategories (1A and 1B).
When reviewing the design and conduct of the study, the following issues
should be addressed in particular:(a) Clarity of the test definition (module 1)(b) Clarity of the definition of the study objective and study
management(c) Appropriateness of the study design & execution in view of thestudy objectives, inter alia:
o Is the number of tested chemicals (24) sufficient for the
purposes of the study?
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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICEo

Are the reference data used for assessing in particular thepredictive capacity appropriate and of good quality?
o Was the identification of chemicals conducted in an
appropriate manner (i.e. presence or absence of selection
criteria, justification etc.)?
o Is the adverse effect range of the selected chemicals
appropriate for the purpose of the studyo In case of gaps (chemical class etc.) – are these justified?
o Is the number of laboratories sufficient?
(d) Appropriateness of the study execution (e.g. were there predefined test acceptance criteria, were these respected? How were
exceptions / deviations handled? Were provisions specified forretesting? Was the number of repetitions sufficient? etc.)(e) Appropriateness of the statistical analysis used for analysing WLR,
transferability, BLR and (preliminary) predictive capacity.

2) CONCLUSIONS OF STUDY: The ESAC is requested to assess whether theconclusions, as presented in the Validation Study Report, are substantiatedby the information generated in the study and are plausible with respect toexisting information and current views (e.g. literature).
In particular:(a) Are the conclusions on reproducibility (WLR and BLR) as well astransferability justified and plausible?
(b) Are the conclusions on preliminary predictive capacity justified andplausible with respect to existing information(c) 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)?
(d) Do the data generated with this defined set of chemicals together
with available existing data provide sufficient information on theapplicability and possible limitations of the test method, inparticular in view of its potential use within an ITS for sensitisation?

3) SUGGESTED USE OF THE TEST METHOD: The ESAC is requested (a) toevaluate, on the basis of the data summarised in the validation study
report, the possible use of the test method (also within a strategy) toidentify skin sensitisers, (b) to make additional recommendations (asrequired) on the proper scientific use of the test method within such a
strategy taking specific aspects of this method into account (e.g.
applicability, limitations, technical limitations etc.) and (c) to identify
possible further information required (i.e. are there gaps) to be able todetermine the potential use and usefulness of the test method within
integrated approaches.

4.3

TIMELINES

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ANNEX 2

Timelines
concerning thisrequestWhen does
ECVAM require
the advice?

ECVAM REQUEST FOR ESAC ADVICE

Timeline

Indication

Finalised ESAC Opinion required by:

4Q 2013 (probably
written procedure)

Request to be presented to ESAC bywritten procedure (e.g. due tourgency) prior to the next ESACNO

Request to be presented to ESAC at
ESAC plenary meeting

YES Final request presented at
ESAC 38, 18/19 June 2013

through

5.

ECVAM PROPOSALS ON HOW TO ADDRESS THE REQUEST WITHIN ESAC

5.1

ECVAM PROPOSAL REGARDING REQUEST-RELATED STRUCTURES REQUIREDSpecific
structures
required within
ESAC to address
the request
Does the advice
require an ESAC
working group, an
ESAC rapporteur
etc.?

Structure(s) required

Required(YES/NO)accordingto

ECVAM?

S1 ESAC RapporteurNO

S2 ESAC Working Group

YES.
However, no WG needs to be established,
as EURL ECVAM has taken the decision toemploytheexisting
ESAC
WG"Sensitisation" (set up in 2011) also for theh-CLAT review. The WG has alreadyprepared detailed reviews/draft opinions
on the DPRA and the Keratinosens test
methods. This will add consistency to thereview of these three sensitisation test
methods and expedite progress as, at thetime of issuing this request (June 2013),
the VSR is already available and the WG
can therefore commence with the review
work.
Present ESAC WG:
x Dr. Erwin ROGGEN (ESAC member,
Chair of ESAC WG and rapporteur; 3Rs
Management
andConsultancy,
Denmark)x Prof. A. Wallace HAYES (external
expert; Harvard University, USA)x Dr. Maja ALECSIC (external expert,
Unilever, UK)
x Dr. Emanuela CORSINI (external expert;
36

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ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE
Dipartimento di scienze farmacologichee biomoleculari, Università Degli Studi
di Milano, Italy)
x Dr. David LOVELL (external expert;
University of Surrey, UK)
x Dr. Michael WOOLHISER (external
expert; Dow Chemical Company, USA)x Prof. Yong HEO (external expert,
ICATM nomination (KoCVAM); Collegeof Natural Sciences, Catholic Universityof Deagu, South Korea)

S3 Invited Experts
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):

5.2

DELIVERABLES AS PROPOSED BY ECVAM

Deliverables
What deliverables(other than the
ESAC opinion) are
required for
addressing therequest?

Title of deliverable otherthan ESAC opinion

Required? (YES/NO)

D1 ESAC Rapporteur Reportand draft opinion

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

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ECVAM REQUEST FOR ESAC ADVICE

6.

LIST OF DOCUMENTS TO BE MADE AVAILABLE TO THE ESACCount

Description of document

Already available?
(YES/NO)

File name

0

EURL ECVAM Validation Study Report

YES

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Appendices 1-15 to EURL ECVAM
Validation Study Report
EURL ECVAM Strategy for Replacementof Animal Testing for Skin Sensitisation
Hazard Identification and Classification
OECD Report:
The Adverse Outcome Pathway for Skin
Sensitisation Initiated by Covalent Binding
to Proteins. Part 1
Publication:
Progress on the development of human invitro dendritic cell based assays forassessment of the sensitizing potential of acompoundPublication:
A Comparative Evaluation of In Vitro Skin
Sensitisation Tests: The Human Cell-line
Activation Test (h-CLAT) versus
the Local Lymph Node Assay (LLNA)
Publication:
Predicting skin sensitization potential andinter-laboratory reproducibilityof a human Cell Line Activation Test (hCLAT) in the European Cosmetics
Association (COLIPA) ring trials
Publication
Predictive performance for human skinsensitizing potentialof the human cell line activation test (hCLAT)
Publication:
Prediction of skin sensitization potency ofchemicals by human Cell Line
Activation Test (h-CLAT) and an attempt atclassifying skin sensitization potency
Publication:
Data integration of non-animal tests for thedevelopment of a test battery
to predict the skin sensitizing potential andpotency of chemicals
Publication:
Predictive performance of the human Cell
Line Activation Test (h-CLAT) for lipophilic
chemicals with high octanol-water partition
coefficients

YES
YES

h-CLAT Validation Study
Report.pdf
h-CLAT appendices to
VSR.pdf
EURL ECVAM strategy .pdf

YES

OECD AOP-part1.pdf

YES

dos Santos 2009.pdf

YES

Ashikaga 2010.pdf

YES

Sakaguchi 2010.pdf

YES

Nukada 2011

YES

Nukada 2012a.pdf

YES

Nukada 2012b.pdf

NOWill be made available
as soon as possible

Takenouchi et al.Submitted for publication

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92

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE7.

TERMS OF REFERENCE OF THE ESAC WORKING GROUP

7.1

ESTABLISHMENT OF THE ESAC WORKING GROUP

During its 38th meeting on 18/19 June 2013 the ESAC plenary decided to employ the ESAC Working
Group "Sensitisation" for preparing a detailed scientific review of the study on the h-CLAT test
method for skin sensitisation testing.

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 WG is requested to conduct a scientific review of the ECVAM-coordinated validation study
focusing on an assessment of reliability of the h-CLAT test method. 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) 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 ESAC Chair and the ESAC Coordinator 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
July 2013
1.
Discussion of the mandate
and first appraisal of the
VSR.
2.
Agreement on further
timelines and possible
work distribution
2
Friday 20. September 2013
Forwarding of initial
39

93

ANNEX 2

ECVAM REQUEST FOR ESAC ADVICE3

1 & 2 October 20134

End of November/mid
December 2013

7.6

observations (within ESAC WG
template) to ECVAM
ESAC WG meeting at JRC
campus in Ispra, Italy
Forwarding final report to
ESAC Chair and ESAC
Coordinator

Draft ESACWG report
Finalreport,adopted by
WG

QUESTIONS WHICH SHOULD BE ADDRESSED BY THE ESAC WG

The ESAC WG is requested to address the questions posed to the ESAC which have been broken
down further in more specific questions (see section 4.2).
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. The template allows moreover for addressing the issues specific studies
outlined in section 4.2. The Coordinator will provide guidance if necessary.

40

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European Commission
EUR 27022 EN – Joint Research Centre – Institute for Health and Consumer Protection
Title: EURL ECVAM Recommendation on the human Cell Line Activation Test (h-CLAT) for skin sensitisation testing
Author(s): European Union Reference Laboratory for Alternatives to Animal Testing
Luxembourg: Publications Office of the European Union
2015 – 40 pp. – 21.0 x 29.7 cm
EUR – Scientific and Technical Research series – ISSN 1831-9424 (online)
ISBN 978-92-79-44706-8 (PDF)
doi:10.2788/29986

95

LB-NA-27022-EN-N

JRC Mission
As the Commission’s
in-house science service,
the Joint Research Centre’s
mission is to provide EU
policies with independent,
evidence-based scientificand technical support
throughout the wholepolicy cycle.
Working in close
cooperation with policy
Directorates-General,
the JRC addresses key
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Stimulating innovation
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doi:10.2788/29986
ISBN 978-92-79-44706-8

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