ᣂⷙ⹜㛎ᴺឭ᩺ᦠ             ᐔᚑ 26 ᐕ 1 ᦬ 20 ᣣ
                                  No. 2013-03
In vitro ⊹⤏ㅘㆊ⹜㛎ߦ㑐ߔࠆឭ᩺ ᐔᚑ 25 ᐕ 10 ᦬ 21 ᣣߦ᧲੩‫ޔ‬࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲߦߡ㐿௅ߐࠇߚᣂⷙ⹜㛎ᴺ⹏ଔળ⼏㧔ㅢ
⒓㧦JaCVAM ⹏ଔળ⼏㧕ߦ߅޿ߡએਅߩឭ᩺߇ߥߐࠇߚ‫ޕ‬   ឭ᩺ౝኈ㧦In vitro ⊹⤏ㅘㆊ⹜㛎ߪ‫ޔ‬ൻቇ‛⾰ߩ⊹⤏߆ࠄߩㅘㆊᕈࠍ᷹ቯߔࠆ߽ߩߢ޽ࠅ‫ޔ‬Ქᕈࠍ⹏ଔ޽ࠆ޿ߪ੍᷹ߔࠆ߽ߩߢߪߥ޿߇‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩోりᦑ㔺㊂ࠍផቯߔࠆઍᦧ⹜㛎ᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪ߔࠆߎߣߪน⢻ߢ޽ࠆ‫ޕ‬ ߎߩឭ᩺ᦠߪ‫ޔ‬OECD (Organisation for Economic Co-operation and Development) Test
Guideline 428߅ࠃ߮EUߩSCCP (Scientific Committee on Consumer Products) opinion on basic criteria
for the in vitro assessment of dermal absorption of cosmetic ingredients╬ࠍ߽ߣߦ‫⤏⊹ޔ‬ㅘㆊᕈ⹜㛎⹏ଔᆔຬળߦࠃࠅ߹ߣ߼ࠄࠇߚᢥᦠࠍ↪޿ߡJaCVAM⹏ଔળ⼏߇⹏ଔ߅ࠃ߮ᬌ⸛ߒߚ⚿ᨐ‫↪᦭ߩߘޔ‬ᕈ߇⏕⹺ߐࠇߚߎߣ߆ࠄ૞ᚑߐࠇߚ‫ޕ‬ એ਄ߩℂ↱ߦࠃࠅ‫ⴕޔ‬᡽ᒰዪߩ቟ోᕈ⹏ଔᣇᴺߣߒߡ‫ޟ‬In vitro⊹⤏ㅘㆊ⹜㛎‫ࠍ↪૶ߩޠ‬ឭ᩺ߔࠆ߽ߩߢ޽ࠆ‫ޕ‬ ศ↰ᱞ⟤                        ⷏Ꮉ⑺૫ศ↰ᱞ⟤JaCVAM ⹏ଔળ⼏ ⼏㐳               JaCVAM ㆇ༡ᆔຬળ ᆔຬ㐳

i

,C%8#/ ⹏ଔળ⼏ศ↰ᱞ⟤㧔ᣣᧄᲥᕈቇળ㧕㧦ᐳ㐳

ᵻ㊁ື⑲

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

⷏Ꮉ⑺૫

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

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

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

,C%8#/ ㆇ༡ᆔຬળ
⷏Ꮉ⑺૫

㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ቟ోᕈ↢‛⹜㛎⎇ⓥ࠮ࡦ࠲࡯㧕㧦ᆔຬ㐳Ꮉ⷏ ᔀ

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

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

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

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

iii

iv

JaCVAM statementon skin absorption in vitro method
At the meeting concerning the above method, held on 21 October 2013 at the National
Institute of Health Sciences (NIHS), Tokyo, Japan, the members of the Japanese Center for
the Validation of Alternative Methods (JaCVAM) Regulatory Acceptance Board unanimously
endorsed the following statement:
The skin absorption in vitro method is considered to be useful to guess the exposed doseof substances or products to whole body for regulatory use.
Following the review of the results of OECD (Organisation for Economic Co-operation andDevelopment) Test Guideline (TG䯵 428 and EU ߩ SCCP (Scientific Committee on Consumer
Products) opinion on basic criteria for the in vitro assessment of dermal absorption of cosmetic
ingredients, etc. it is concluded that skin absorption in vitro method are clearly beneficial.
The JaCVAM Regulatory Acceptance Board has been regularly kept informed of the
progress of the study, and this endorsement is based on an assessment of various documents,
including, in particular, the evaluation report prepared by the JaCVAM ad hoc peer review
panel for skin absorption testing.

Takemi Yoshida
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

20 January, 2014v

The JaCVAM Regulatory Acceptance Board was established by the JaCVAM Steering
Committee, and is composed of nominees from the industry and academia.
This statement was endorsed by the following members of the JaCVAM Regulatory
Acceptance Board:
Mr. Takemi Yoshida (Japanese Society of Toxicology): Chairperson
Mr. Norihide Asano (Japanese Environmental Mutagen Society)
Mr. Tsutomu Ichiki (Japan Chemical Industry Association)*
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Tsutomu Miki Kurosawa (Japanese Society for Animal Experimentation)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Mitsuteru Masuda (nominee by Chairperson)
Mr. Akiyoshi Nishikawa (NIHS)
Mr. Yasuo Ohno (nominee by Chairperson)*
Mr. Hiroshi Onodera (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Tomoko Tanita (Pharmaceuticals and Medical Devices Agency)*
Mr. Takashi Yamada (National Institute of Technology and Evaluation)*
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact
Dermatitis)
Ms. Midori Yoshida (NIHS)
Mr. Isao Yoshimura (nominee by Chairperson)
Mr. Kazuto Watanabe (Japan Pharmaceutical Manufacturers Association)
Term: From 1st April 2012 to 31st March 2014*: From 1st April 2013 to 31st March 2014vi

This statement was endorsed by the following members of the JaCVAM steering Committee
after receiving the report from JaCVAM Regulatory Acceptance Board:
Mr. Akiyoshi Nishikawa (BSRC, NIHS): Chairperson
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Jun Kanno (Division of Cellular and Molecular Toxicology, BSRC, NIHS)
Mr. Toru Kawanishi (NIHS)
Mr. Kenji Kuramochi (Ministry of Health, Labour and Welfare)*
Mr. Toshinari Mitsuoka (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Kazuyuki Saito (Pharmaceutical & Medical Devices Agency)
Mr. Masahiro Sasaki (Ministry of Health, Labour and Welfare)
Ms. Yuko Sekino (Division of Pharmacology, BSRC, NIHS)
Mr. Atsuya Takagi (Animal Management Section of the Division of Cellular andMolecular Toxicology, BSRC, NIHS)
Mr. Junji Yamamoto (Ministry of Health, Labour and Welfare)*Mr. Hajime Kojima (Section for the Evaluation of Novel Methods, Division of
Pharmacology, BSRC, NIHS): Secretary* Arrival at post day: 1st August 2013

vii

viii

In vitro ⊹⤏ㅘㆊ⹜㛎⋡ ᰴ⹏ଔળ⼏ႎ๔ᦠ -----------------------------------------------------------------------------------1
⹏ଔႎ๔ᦠ -----------------------------------------------------------------------------------------7
OECD TG428 --------------------------------------------------------------------------------19
SCIENTIFIC COMMITTEE ON CONSUMER PRODUCTS (SCCP) Opinion-------------------------------------------------------------------------------------------------------- 27
Guidelines for Percutaneous Absorption/Penetration㩷 ------------------------------39ix

x

In vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ߩ⹏ଔળ⼏ႎ๔ᦠJaCVAM ⹏ଔળ⼏ᐔᚑ 25 ᐕ 10 ᦬ 21 ᣣ





1

1


JaCVAM ⹏ଔળ⼏ศ↰ᱞ⟤㧔ᣣᧄᲥᕈቇળ㧕㧦ᐳ㐳

ᵻ㊁ື⑲

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

⷏Ꮉ⑺૫

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

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

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

એ਄

2

2

ᒰ⹥⹜㛎ᴺߪ‫ޔ‬એਅߩㅢ⍮߿ࠟࠗ࠼࡜ࠗࡦߦⷙቯߐࠇࠆࡅ࠻߹ߚߪታ㛎േ‛߆ࠄ៰಴ߒߚ⊹⤏ࠍ↪޿ߚൻቇ‛⾰ߩ⊹⤏ㅘㆊᕈࠍ᷹ቯߔࠆ⹜㛎ᴺߢ޽ࠆ‫ޕ‬ኻᔕߔࠆᓥ᧪⹜㛎ߪ‫ߪߚ߹࠻ࡅޔ‬ታ㛎േ‛ߩ⊹⤏ߦൻቇ‛⾰ࠍ⋥ធႣᏓ෶ߪ⾍ઃߒߡߘߩⴊਛỚᐲࠍ᷹ቯߔࠆ⹜㛎ߢ޽ࠆ߇‫․ޔ‬ቯߩࡊࡠ࠻ࠦ࡞ࠍⷙቯߒߚ
⹜㛎ᴺߢߪߥ޿‫ޕ‬৻ᣇ‫ߦ⤏⊹ߩ࠻࠶࡜ޔ‬᡼኿ᕈ࡜ࡌ࡞ߒߚൻቇ‛⾰ࠍ⋥ធႣᏓ෶ߪ⾍ઃߒߡⴊਛ‫ޔ❱⚵ޔ‬ዩਛߩ᡼኿ᵴ
ᕈࠍ᷹ቯߔࠆ⹜㛎ᴺߪ OECD TG 427 (Skin Absorption: In Vivo Method)ߣߒߡⷙቯߐࠇߡ޿ࠆ߇‫ޔ‬ൻቇ‛⾰ߩઍ⻢‛߽฽ࠎߛ᷹ቯߢ޽ࠆߚ߼‫ޔ‬ᒰ⹥⹜㛎ᴺߦኻᔕߔࠆᓥ᧪⹜㛎ᴺߦߪ⹥ᒰߒߥ޿‫ޕ‬
Ԙ Opinion on Basic Criteria for the in vitro assessment of dermal absorption of cosmetic
ingredients - updated March 2006 (Adopted by the SCCP during the 7th plenary of 28 March
2006)
ԙ Test guideline 428: OECD Guideline for the Testing of chemicals: Skin Absorption: in vitro
Method (Adopted 13 April, 2004)
Ԛ COLIPA GUIDELINES: Guidelines for percutaneous Absorption㧛Penetration (Edition of1997) ੹࿁‫ࠆߥ⇣ޔ‬ᯏ㑐߆ࠄ␜ߐࠇߡ޿ࠆ਄⸥ߩ⊹⤏ㅘㆊᕈ⹜㛎ᴺߦߟ޿ߡ‫⤏⊹ޟ‬ㅘㆊ⹜㛎⹏ଔᆔຬળ‫ޠ‬߇ߣࠅ߹ߣ߼ߚႎ๔㧝㧕‫ޔ‬㧞㧕ࠍฃߌ‫ޔ‬એਅߩ 10 㗄⋡ߦߟ޿ߡ⹏ଔߒߚߩߢႎ๔ߔࠆ‫ޕ‬㧨ክ⼏ౝኈ㧪
1㧚ᒰ⹥⹜㛎ᴺߪ‫ߥ߁ࠃߩߤޔ‬ᓥ᧪⹜㛎ᴺࠍઍᦧߔࠆ߽ߩ߆‫ߥ߁ࠃߩߤޔߪߚ߹ޕ‬Ქᕈࠍ⹏ଔ޽ࠆ޿ߪ
੍᷹ߔࠆ߽ߩ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ࡅ࠻߹ߚߪታ㛎േ‛ߩ⊹⤏ߦൻቇ‛⾰ࠍ⋥ធႣᏓ෶ߪ⾍ઃߒߡోりᦑ㔺㊂ࠍᛠីߔࠆᓥ᧪ߩ in vivo ߦࠃࠆ㧔⊹⤏߆ࠄߩๆ෼㊂ࠍ᷹ቯߔࠆ㧕ᣇᴺࠍઍᦧߔࠆ߽ߩߢ޽ࠆ‫ޕ‬
ᒰ⹥⹜㛎ᴺߪൻቇ‛⾰ߩ⊹⤏ࠍ੺ߔࠆㅘㆊᕈࠍ᷹ቯߔࠆ߽ߩߢ޽ࠅ‫ޔ‬Ქᕈࠍ⹏ଔ޽ࠆ޿ߪ੍᷹ߔࠆ
߽ߩߢߪߥ޿‫ޕ‬
2. ᒰ⹥⹜㛎ᴺߣᓥ᧪⹜㛎ᴺߩ㑆ߦߤߩࠃ߁ߥ⑼ቇ⊛ߥߟߥ߇ࠅ߇޽ࠆ߆㧫
ᒰ⹥⹜㛎ᴺߪࡅ࠻߹ߚߪታ㛎േ‛߆ࠄ៰಴ߒߚ⊹⤏ࠍ↪޿ߡൻቇ‛⾰ߩ⊹⤏ㅘㆊ㊂ࠍ⹏ଔߔࠆ invitro ߩࡕ࠺࡞♽ߢ޽ࠅ‫⤏⊹ޔ‬ㅘㆊ㊂߆ࠄోりᦑ㔺㊂ߩផቯ߇น⢻ߢ޽ࠆ‫ޕ‬ᓥ᧪ߩ⹜㛎ߪࡅ࠻߹ߚߪታ㛎േ‛ߩ⊹⤏ߦൻቇ‛⾰ࠍႣᏓ߹ߚߪ⾍ઃߒ‫ޔ‬ๆ෼ߐࠇߚൻቇ‛⾰ߩ㊂ ⴊਛỚᐲ ࠍ᷹ቯߔࠆ KPXKXQ ⹜㛎ߢ޽ࠆ‫ޕ‬ਔ⹜㛎ߣ߽ߦ‫ߩࠄ߆⤏⊹ޔ‬ൻቇ‛⾰ߩๆ෼ߦኻߔࠆਥⷐߥࡃ࡝ࠕߣߥࠆⷺ⾰ጀࠍ᦭ߔࠆὐߢ⑼ቇ
⊛ߟߥ߇ࠅ߇޽ࠆ‫ޕ‬33

3㧚ᒰ⹥⹜㛎ᴺߣߘߩ࠺࡯࠲ߪ‫ޔ‬ㅘ᣿ߢ⁛┙ߥ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔࠇߙࠇߘޔ‬EU‫ޔ‬OECD ߅ࠃ᰷߮Ꮊൻ♆ຠදળ߆ࠄ⊒ⴕߐࠇߚ߽ߩߢ‫ߪ࠲࡯࠺ޔ‬ㅘ᣿ᕈ߇޽ࠅ⁛┙ߒߚ⹏ଔ߇ⴕࠊࠇߡ޿ࠆ‫ޕ‬ᬌ⸛ኻ⽎ߩ 3 ⹜㛎ᴺߦ㑐ߔࠆ౏ᑼߩᬌ⸽ߪߥߐࠇߡ޿ߥ޿߇‫ޔ‬OECD ߩኾ㐷ኅߪ OECD TG 428
ࠍᡰᜬߔࠆߦචಽߥ࠺࡯࠲ߩ޽ࠆߎߣߦวᗧߒߡ޿ࠆ㧟㧕‫ޕ‬
4. ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߩઍᦧᴺߣߒߡ‫⾰‛ߥ߁ࠃߩߤޔ‬෶ߪ⵾ຠࠍ⹏ଔߔࠆߎߣࠍ⋡⊛ߣߒߡ޿ࠆ߆‫ޕ‬ᓥ᧪⹜㛎ᴺߪ⊹⤏ߦㆡ↪෶ߪᦑ㔺ߒߚൻቇ‛⾰ߩⴊਛỚᐲ̅ᤨ㑆ᦛ✢ਅ㕙Ⓧ㧔AUC㧕ࠍ᳞߼ࠆᣇᴺ
ߢ޽ࠆߩߦኻߒ‫ޔ‬ᒰ⹥⹜㛎ᴺߪ⊹⤏ㆡ↪෶ߪᦑ㔺ߒߚൻቇ‛⾰ߩ⊹⤏ࠍ੺ߔࠆๆ෼㊂ࠍ᳞߼ࠆ⹜㛎ߢ޽ࠆ‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫⥸৻ޔ‬ൻቇ‛⾰ߩ⊹⤏ㅘㆊᕈࠍᛠីߔࠆ߶߆‫ޔ‬ൻ♆ຠ‫⤏⊹ޔ‬ㆡ↪ߩᄖ↪ක⮎ຠ㧔⾍ઃ೷㧕߅ࠃ߮ක⮎ㇱᄖຠ╬‫ޔ‬෶ߪߘࠇࠄ⵾ຠߦ฽߹ࠇࠆᣂⷙේᢱ‛⾰ߩߚ߼ߦ‫ో⊛⊹⚻ޔ‬りᦑ㔺ࠍ⹏ଔߔࠆᓥ᧪⹜㛎ߩઍᦧᴺߣߒߡ೑↪߇น⢻ߢ޽ࠆ‫ޕ‬
5㧚ᒰ⹥⹜㛎ᴺߪ‫⹏࠼࡯ࠩࡂޔ‬ଔ޽ࠆ޿ߪ࡝ࠬࠢ⹏ଔߩߤߜࠄߦ᦭↪ߢ޽ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫⹜ᧄޔ‬㛎⚿ᨐ߆ࠄᓧࠄࠇࠆൻቇ‛⾰ߩ⊹⤏ㅘㆊ㊂ߦၮߠ߈‫ޔ‬in vivo ߢߩ⚻⊹ᛩਈߦࠃࠆోりᦑ㔺㊂ࠍផቯߔࠆߎߣ߇น⢻ߢ޽ࠆߩߢ‫ޔ‬ᒰ⹥ൻቇ‛⾰ߩ࡝ࠬࠢ⹏ଔߦ᦭↪ߢ޽ࠆ‫ޕ‬
6㧚ᒰ⹥⹜㛎ᴺߪ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏ଔߢ߈ࠆ߆‫ߩߘޕ‬႐ว‫ޔ‬ᒰ⹥⹜㛎ᴺߩㆡ↪᧦ઙ߇᣿⏕ߦߥߞߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ኻ⽎ߣߔࠆൻቇ‛⾰෶ߪ⵾ຠߩోりᦑ㔺ߦࠃࠆๆ෼㊂ࠍផቯ⹏ଔߔࠆ߽ߩߢ‫ޔ‬Ქᕈ⹏ଔࠍ⋥ធ⋡⊛ߣߔࠆ߽ߩߢߪߥ޿‫ޕ‬ߥ߅‫ޔ‬ኻ⽎ߣߔࠆൻቇ‛⾰ߩ‛ᕈߦᔕߓߚᒰ⹥⹜㛎ᴺߩㆡ↪᧦ઙ╬ߪ᣿⏕ߦߥߞߡ޿ࠆ‫ޕ‬㧣㧚ᒰ⹥⹜㛎ᴺߪࡊࡠ࠻ࠦ࡞ߩᓸ⚦ߥᄌᦝߦኻߒߡචಽ㗎ஜߢ޽ࠆ߆‫ޕ‬ࡊࡠ࠻ࠦ࡞ߩᓸ⚦ߥᄌᦝߦߪ㗎ஜߢ޽ࠆ߇‫⹜ޔ‬㛎ߦ↪޿ࠆ៰಴⊹⤏ߩㇱ૏߿ᕈ⁁ߦᔕߓߡ‫⚿ޔ‬ᨐߩ⸃㉼ࠍᘕ㊀ߦⴕ߁ᔅⷐ߇޽ࠆ㧔ᣂ㞲⊹⤏߆ಓ⚿⊹⤏߆‫ޔ‬Ძⓣߩ᦭ήઁ㧕‫ޕ‬8㧚ᒰ⹥⹜㛎ᴺߩᛛⴚ⠌ᓧߪ‫ޔ‬ㆡಾߥ⸠✵ߣ⚻㛎ࠍ⚻ߡ޿ࠆᜂᒰ⠪ߦߣߞߡኈᤃߥ߽ߩߢ޽ࠆ߆‫⹜ޕ‬㛎
ᴺߩታᣉߦ․ᱶߥ⸳஻߇ᔅⷐ߆‫ޕ‬ᒰ⹥⹜㛎ߦ↪޿ࠆ៰಴⊹⤏ߪ‫ޔ‬㉂⚛߿ᾲ߅ࠃ߮ൻቇಣℂ╬ߦࠃࠅ೸㔌ߒߚ⴫⊹‫ߪ޿ࠆ޽ޔ‬ෘߐ 200
㨪400 Ǎm ߦ⭯ಾߒߚ⊹⤏⚵❱ߢ޽ࠆ‫ޕ‬Ꮢ⽼ߩᮡຠࠍ↪޿ࠆ㒢ࠅ‫ߩߘޔ‬ᓟߩၭ㙃߿⹜㛎ᚻᛛ╬ߩᛛⴚ⠌ᓧߪኈᤃߥ߽ߩߢ޽ࠆ߇‫⭯ࠍ⤏⊹ߢࡓ࡯࠻ࡑ࡯࠳ޔ‬ಾߒߡ⹜㛎ᮡຠࠍ⥄ࠄ૞⵾ߔࠆᛛⴚߪᾫ✵ࠍⷐߔࠆ‫ޕ‬
߹ߚ‫⤏⊹ޔ‬ᮡຠࠍ⥄ࠄ૞⵾ߔࠆ႐วߦߪ࠳࡯ࡑ࠻࡯ࡓ߇ᔅⷐߣߥࠆ‫ࡓ࠙࠴࡝࠻ޕ‬᳓ࠍ↪޿ߚ⊹⤏ࡃ࡝ࠕߩஜోᕈߩ⏕⹺㧔Integrity ᷹ቯ㧕߅ࠃ߮ᮡ⼂ߐࠇߚⵍ㛎‛⾰ࠍ↪޿ࠆ႐วߦߪ RI ⸳஻߇ᔅⷐߣߥࠆ‫ޕ‬44

9㧚ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߣᲧߴߡᤨ㑆⊛⚻⾌⊛ߦఝࠇߡ޿ࠆ߆‫ޕ‬
ᒰ⹥⹜㛎ᴺߢߪ‫ޔ‬േ‛ታ㛎ߦ઻߁⚻⾌߇೥ᷫߢ߈‫ߦ⊛⾌⚻ޔ‬ఝࠇߡ޿ࠆ‫ޔߒߛߚޕ‬RI ࠍ૶↪ߔࠆ႐วߪ⚻⾌⊛ߦഠࠆ‫ޕ‬ ৻ᣇ‫ᤨޔ‬㑆⊛ߥఝ૏ᕈߦ㑐ߒߡߩᬌ⸛ߪߥߐࠇߡ޿ߥ޿‫ޕ‬10. ᒰ⹥⹜㛎ᴺߪ‫ޔ‬േ‛⑔␩ߩⷰὐ෸߮⑼ቇ⊛⷗࿾߆ࠄ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏ଔߔࠆઍ
ᦧᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪ߔࠆߎߣߪน⢻߆‫ޕ‬ᒰ⹥⹜㛎ᴺߪേ‛⑔␩ߩ 3R ߩⷰὐ߆ࠄផᅑߐࠇ‫ޔ‬᰷☨ߢߪ৻⥸⊛ߦ૶ࠊࠇ‫ޔ‬ᚒ߇࿖ߢ߽⚻⊹ๆ෼⹜㛎ߦᐢߊ↪޿ࠄࠇߡ޿ࠆ‫ޕ‬ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ൻቇ‛⾰ߩ⊹⤏߆ࠄߩㅘㆊᕈࠍ᷹ቯߔࠆ߽ߩߢ޽ࠅ‫ޔ‬Ქᕈࠍ⹏ଔ޽ࠆ޿ߪ੍᷹ߔࠆ߽ߩߢߪߥ޿߇‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩోりᦑ㔺㊂ࠍផቯߔࠆઍ
ᦧ⹜㛎ᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪ߔࠆߎߣߪน⢻ߢ޽ࠆ‫ޕ‬ෳ⠨ᢥ₂㧝㧕‫ޟ‬In vitro⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕‫⤏⊹ ޠ‬ㅘㆊ⹜㛎⹏ଔᆔຬળ✬㧞㧕‫ޟ‬In vitro ⊹⤏ㅘㆊᕈ⹜㛎ᴺߩ⏕┙ߩ⚻✲‫⤏⊹ ޠ‬ㅘㆊ⹜㛎⹏ଔᆔຬળ✬㧟㧕 OECD (2000). Report of the Meeting of the OECD Extended Steering Committee for

Percutaneous Absorption Testing, Annex 1 to ENV/JM/TG(2000)5. OECD, Paris.

5

5

6

In vitro ⚻⊹ๆ෼⹜㛎㧔In vitro ⊹⤏ㅘㆊ⹜㛎㧕⹏ଔႎ๔ᦠ

ᐔᚑ 22 ᐕ 9 ᦬ 11 ᣣ

7⊹⤏ㅘㆊ⹜㛎⹏ଔᆔຬળ
ᆔຬฬ᧖ᨋၷᰴ㧔ၔ⷏ᄢቇ㧕⮮੗߹߈ሶ㧔ᤘ๺⮎⑼ᄢቇ㧕⮮ၴᶈ᣿㧔ၔ⷏ᄢቇ㧕ዊፉ⡸㧔࿖┙ⴡ⎇㧕㧔ࠕ࠼ࡃࠗࠩ࡯㧕

8

㧝㧚⋡⊛
ൻ♆ຠ߿ක⮎ㇱᄖຠߩ቟ోᕈ⹏ଔߩߚ߼ߦ↪޿ࠄࠇߡ޿ࠆ in vitro ⚻⊹ๆ෼⹜㛎㧔invitro ⊹⤏ㅘㆊ⹜㛎㧕ߦ㑐ߔࠆฦ⒳ࠟࠗ࠼࡜ࠗࡦࠍᲧセߒ‫ޔ‬ା㗬ᕈ߿㐳⍴ᚲ╬ߦߟ޿ߡ⼏⺰ߒ‫ޔ‬ណุߩนุࠍᬌ⸛ߔࠆ‫ޕ‬㧞㧚ᬌ⸛ߒߚ⾗ᢱߩฬ⒓ߣߘߩℂ↱࡮SCCP opinion on basic criteria for the in vitro assessment of dermal absorption of
cosmetic ingredients (2006) 1).
࡮OECD guideline for the testing of chemicals, TG428, skin absorption: In vitro
method (2004) 2).
࡮Colipa regulatory, Guidelines for percutaneous absorption㧛penetration (1997) 3).
ዘWHO, Environmental Health Criteria 235,, Dermal Absorption (2006) 4).
޿ߕࠇߩ⹏ଔᴺ߽ࠟࠗ࠼࡜ࠗࡦ‫౏ߡߒߣࠬࡦ࠳ࠗࠟޔ‬⍮ߩ߽ߩߢ޽ࠅ‫ޔ‬᳢↪ᕈ߽㜞޿ߚ߼ߦᬌ⸛ߒߚ‫ޕ‬㧟㧚⹜㛎ᴺߩ᭎ⷐ㧔ᚻ㗅ߥߤ㧕೎⚕ 1 ෳᾖ
㧠㧚⹜㛎ᴺߣߒߡណ↪ߩนุ޿ߕࠇߩ⹜㛎߽⑼ቇ⊛‫୶ޔ‬ℂ⊛ߦ߽⹏ଔߔࠆߦߪචಽߢ޽ࠆ‫ޕ‬ૉߒ‫ޔ‬ฦ⹜㛎ߩㆡ↪
㒢⇇ߣታᣉߔࠆ਄ߢߩ໧㗴ὐࠍ⠨ᘦߔࠆᔅⷐ߇޽ࠆ‫ޕ‬4-1

In vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ߩㆡ↪㒢⇇ߣࠟࠗ࠼࡜ࠗࡦߦߘߞߡ

ታᣉߔࠆ਄ߢߩ໧㗴ὐ
4-1-1 ⚻⊹ๆ෼⹜㛎⚿ᨐߩౕ૕⊛ߥ೑↪ᴺߩ⸥タൻ♆ຠේᢱߩ⚻⊹ๆ෼⹜㛎ߩ⋡⊛ߪ‫ޔ‬ታ૶↪᧦ઙߦ߅޿ߡࡅ࠻ోりᓴⅣ♽ߦ౉ࠆน⢻ᕈ߇޽ࠆⵍ㛎‛⾰ߩ⾰⊛߅ࠃ߮㧔޽ࠆ޿ߪ㧕㊂⊛ߥᖱႎࠍᓧࠆߎߣߢ޽ࠆ‫ߦ․ޕ‬ᓟ⠪ߪ‫ޔ‬୘‫ߩߡ޿ߟߦ⾰‛ߩޘ‬෻ᓳᛩਈᲥᕈ⹜㛎ߩήᲥᕈ㊂㧔NOAEL㧕ߣߣ߽ߦ‫ޔ‬቟ోଥᢙ㧔MOS‫ޔ‬margin of safety㧕ࠍ▚಴ߔࠆ਄ߢᔅⷐߢ޽ࠆ㧔SCCP/0970/06㧕1)‫ޕ‬OECD TG 4282)ߪ⸥タߥߒ‫ޕ‬ൻ♆ຠᚑಽߩ⹜㛎߅ࠃ߮቟ోᕈ⹏ଔߦ㑐ߔࠆ SCCP ࠟࠗ࠳ࡦࠬ╙ 65)ߦ⸥タߐࠇ

ߡ޿ࠆ቟ోଥᢙߩ᳞߼ᣇߣᵈᗧὐࠍ⴫㧝ߦ␜ߔ‫ޕ‬9⴫㧝 ቟ోଥᢙ㧔Margin of Safety: MOS㧕5)ൻ♆♆ຠᚑಽߩ቟ోᕈ⹏ଔߩᦨ⚳Ბ㓏ߢ޽ࠆ࡝ࠬࠢߩ⹏ଔߢߪ‫ޔ‬ਇ⏕ታᕈߩⷐ࿃߇
ㆡ↪ߐࠇࠆ‫ޕ‬ൻ♆ຠߩ႐ว‫ⷐߩߎޔ‬࿃ߪ቟ోଥᢙ㧔MOS㧕ߣ๭߫ࠇࠆ‫ޔߦ⥸৻ޕ‬MOS
ߪᦨዊήᓇ㗀㊂㧔ήᲥᕈ㊂㧕㧔NO(A)EL㧕ࠍ‫ޔ‬ᗐቯߐࠇࠆൻ♆ຠᚑಽߩోり᥸㔺㊂㧔Systemic Exposure Dosage: SED㧕ߢ㒰ߔߎߣߦࠃߞߡMOS

NO(A)EL
SEDߣߒߡ⸘▚ߢ߈ࠆ‫ޕ‬MOS ୯ߪ⹜㛎േ‛⟲߆ࠄᐔဋ⊛ߥࡅ࠻߳‫ޔ‬ᰴߦᐔဋ⊛ߥࡅ࠻߆ࠄᗵฃᕈߩ㜞޿ࠣ࡞࡯ࡊ߳ߣᄖᝌߔࠆߩߦ↪޿ࠄࠇࠆ‫߇⾰‛ࠆ޽ޔߦ⥸৻ޕ‬቟ోߦ૶↪ߢ߈ࠆߣት⸒ߔࠆߚ߼ߦߪ MOS ߪ 100 એ਄ߢߥߌࠇ߫ߥࠄߥ޿ߣߐࠇߡ޿ࠆ‫ޕ‬
SED ߩ⸘▚ߪ‫੍ޔ‬ᗐߐࠇࠆᦨ㜞Ớᐲߦၮߠߊ৻ቯᤨ㑆ߩ↢‛ቇ⊛೑↪⢻ߩ⛘ኻ㊂㧔Ǎg/cm2㧕ߦၮߠߊߎߣ߇ᦸ߹ߒ޿߇‫⊹⚻ޔ‬ๆ෼₸ߦၮߠ޿ߡⴕ߁ߎߣ߽ߢ߈ࠆ‫ޕ‬ᓟ⠪ߩ႐ว‫ޔ‬ᓧࠄࠇࠆᢙ୯ߪ⊹⤏ߦㆡ↪ߐࠇࠆ↪㊂ߦଐሽߔࠆ‫⹏ޔ߈ߣߩߎޕ‬ଔߔࠆỚᐲ߇ૐ޿߶ߤ⚻⊹ๆ෼₸ߪ㜞ߊߥࠆน⢻ᕈ߇޽ࠆߎߣ߆ࠄ㧘੍ᗐߐࠇࠆᦨૐỚᐲࠍ฽߹ߥߌࠇ߫ߥࠄߥ޿‫ޕ‬
 OECD ࠟࠗ࠼࡜ࠗࡦ 428 7)㧔⚻⊹ๆ෼㧦in vitro ᴺ㧕ߦᓥ޿‫ޔ‬in vitro ⹜㛎ߢߪ‫ޔ‬࿕૕ߢㅢᏱ 1㨪5 mg/cm2‫ޔ‬ᶧ૕ߢᦨ㜞 10 ǍL/cm2 ߣ޿߁ࡅ࠻߳ߩ᥸㔺ࠍࠪࡒࡘ࡟࡯࠻ߒ
ߚㆡ↪㊂ࠍ૶↪ߒߥߌࠇ߫ߥࠄߥ޿‫ޕ‬ ⹜㛎‛⾰ߩㆡ↪㊂߇ 2 mg/cm2 ᧂḩߩ in vitro ⹜㛎ࠍታᣉߔࠆߎߣߪᛛⴚ⊛ߦਇน⢻ߢ޽ࠆ߇‫ޔ‬ㅢᏱ⊹⤏ߦㆡ↪ߐࠇࠆൻ♆ຠ㊂ߪታ૶↪᧦ઙਅߢ 1 mg/cm2 ߦḩߚߥ޿ߎߣ߇⚻㛎߆ࠄ␜ߐࠇߡ޿ࠆ‫ޔߡߞ߇ߚߒޕ‬in vitro ⹜㛎ߢߪታ૶↪᧦ઙࠍ਄࿁ࠆ㊂߇ㆡ↪ߐࠇ‫⹜ޔ‬㛎↪㊂ߩ⚻⊹ๆ෼₸%ࠍ SED ߩ⸘▚ߦ૶↪ߔࠆߣ‫ోޔ‬り᥸㔺㊂߇ㆊዊ⹏ଔߐࠇࠆߎߣߦߥࠆ‫⊹⚻ޔߡߞ߇ߚߒޕ‬ๆ෼ࠍࡄ࡯࠮ࡦ࠹࡯ࠫߢ⴫ߔ႐วߦߪ‫ޔ‬in vitro ⹜㛎߆ࠄᓧࠄࠇߚๆ෼㊂߽ታ૶↪᧦ઙਅߢㆡ↪ߒߚ↪㊂ߩࡄ࡯࠮ࡦ࠹࡯ࠫߢ
⴫ߔᔅⷐ߇޽ࠆ‫ߪࠇߎޕ‬ታ૶↪᧦ઙਅߢㆡ↪ߔࠆ⵾೷ߩᣢቯ㊂ߣ‫⵾ߔ␜ߦ⴫ޔ‬ຠ⒳㘃
೎ߩ⊹⤏⴫㕙Ⓧ㧔SSA㧕ߩᣢቯ୯㧔6-2 㗄ෳᾖ㧕ߩᲧߦࠃߞߡផቯߔࠆߎߣ߇ߢ߈ࠆ‫ޕ‬એ਄ߩࠃ߁ߦ‫ޔ‬ൻቇ‛⾰ߩ⚻⊹ๆ෼߇ߤߩࠃ߁ߦ⸥タႎ๔ߐࠇߡ޿ࠆ߆ߦࠃߞߡ‫ޔ‬
SED ߩ⸘▚ᣇᴺߦߪ 2 ⒳㘃޽ࠆߣ⚿⺰ߠߌࠄࠇࠆ‫ޕ‬10

4-1-2 In vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ߩㆡ↪㒢⇇

In vivo ᧦ઙਅߢߪ‫ޔ‬ᓸዋᓴⅣ♽㧔ⴊ▤߅ࠃ߮࡝ࡦࡄ▤㧕߇ൻว‛ࠍ⊹⤏⚵❱߆ࠄ૕
ᓴⅣࠦࡦࡄ࡯࠻ࡔࡦ࠻߳ㆇࠎߢ޿ࠆ㧔ๆ෼‫ޔ‬resorption㧕‫ޕ‬In vitro ߩ᧦ઙਅߢߪ‫ߩߘޔ‬᭽ߥๆ෼ㆊ⒟ߪ⹏ଔߢ߈ߥ޿㧔SCCP/0970/06㧕1)‫ޕ‬⴫⊹ߪ⛘߃ߕჇᱺ‫ޔ‬ಽൻ‫⪭ޔ‬ድࠍ➅ࠅ㄰ߒߡ߅ࠅ‫৻ޔ‬ᣣᒰߚࠅ⚂৻ጀಽߩⷺ⾰⚦⢩ጀ߇ขࠅ㒰߆ࠇߡ޿ࠆ‫ޕ‬ዪᚲㆡ↪ߒߚ႐ว‫ޔ‬in vitro ⹜㛎ߦ߅ߌࠆ⊹⤏਄‫ⷺߦ․ޔ‬ጀ߿Ძྙ⢽⣼ߢᬌ಴ߐࠇߚ⇣‛ߪ‫ޔ‬in vivo ߦ߅޿ߡߪ‫⪭ࠇߙࠇߘޔ‬ድ޽ࠆ޿ߪ⊹⢽ಽᴲߦࠃࠅขࠅ㒰߆ࠇࠆ‫ߩࠄࠇߎޕ‬ㆊ⒟߽ in vitro ߢߪౣ⃻ߢ߈ߥ޿ߩߢ‫ޔ‬in vitro ♽ߢߩᦨ⚳⊛ߥ⴫⊹㧔ⷺጀ㧕ਛỚᐲߪ‫ޔ‬in vivo ࡟ࡌ࡞ߣᲧセߒߡ㜞ߊߥࠆ㧔SCCP/0970/06㧕1)‫ޕ‬⹜㛎ൻว‛߇⴫⊹ߩ⚵❱ߦਇนㅒ⊛ߦ⚿วߔࠆߎߣ߇޽ࠆ߇‫ ߪࠇߎޔ‬in vivo ߦ߅޿ߡߪ⊹⤏⴫㕙ߩ⪭ድߦࠃߞߡ㒰෰ߐࠇࠆ‫␜߇⽎⃻ߩߎޕ‬ໂߐࠇߚ႐วߦߪ‫ޔ‬೎ߩ⹜㛎ߦࠃࠅታ⸽ߒߥߌࠇ߫ߥࠄߥ޿㧔SCCP/0970/06㧕1)‫ޕ‬⹜㛎ൻว‛ߪ‫ޔ‬᜛ᢔ࠮࡞ߦⵝ⌕ߐࠇߚ⊹⤏ࠨࡦࡊ࡞ߩ਄ߦ‫ޔ‬ㆡಾߥ⵾೷ࠍ↪޿ߡㆡ↪ߐࠇߥߌࠇ߫ߥࠄߥ޿㧔SCCP/0970/06㧕1)‫ޕ‬න⚐ṁᇦ♽ߢߥߊ⵾೷ߢߩታᣉ߇ਇน
ᰳߢ޽ࠆ‫ޕ‬4-1-3 In vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ߩ໧㗴ὐ⹜㛎ߩේℂ
៰಴ߒߚ⊹⤏ࠍ↪޿ߚ in vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ߩᱜᒰᕈߪ‫ޔ‬⴫⊹‫ⷺߦ․ޔ‬ጀ߇↢૕߳ߩ⇣‛ߩๆ෼߿ขࠅㄟߺߦኻߔࠆਥⷐߥࡃ࡝ࠕ࡯ߣߥߞߡ޿
ࠆߣ޿߁੐ታߦၮߠ޿ߡ޿ࠆ㧔SCCP/0970/06㧕1)‫ޕ‬໧㗴ὐ㧦᳓ṁᕈൻว‛ߩ⊹⤏ㅘㆊᕈߦߟ޿ߡߪⷺጀ߇ࡃ࡝ࠕ࡯ᯏ⢻ߣߥࠆߎߣ߇⍮ࠄࠇߡ޿ࠆ߇‫ޔ‬ൻ♆ຠ⚛᧚ߦ߅޿ߡߪᴤ╬ߩ⢽ṁᕈൻว‛߇߆ߥࠅߩᢙሽ࿷ߒ‫ࠄࠇߘޔ‬ߩ⊹⤏ㅘㆊᕈߦߪⷺጀߛߌߢߥߊߘࠇએਅߩ⴫⊹߿⌀⊹߽߹ߚࡃ࡝ࠕ࡯ߣߥࠆߣ⠨߃ࠄࠇࠆ‫ⷺޔߡߞ߇ߚߒޕ‬ጀએਅߩጀߩࡃ࡝ࠕ࡯⢻ߩነਈ߇㜞޿‛⾰ߩ in vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ߦߪ․ߦᵈᗧ߇ᔅⷐߢ޽ࠆ‫ޕ‬૶↪ߔࠆ⊹⤏WHO ߪᦨㆡᮡḰ㧔gold standard㧕ߣߒߡࡅ࠻⊹⤏ߩ૶↪ࠍ൘߼ߡ޿ࠆ 4)‫ࠈߜ߽ޕ‬ࠎ‫⊹⚻߇⤏⊹࠻ࡅޔ‬ๆ෼⹜㛎ߦᦨ߽ㆡߒߚ⹜ᢱߢ޽ࠆ߇‫߽ߟ޿ߪࠄࠇߘޔ‬ኈᤃߦ౉ᚻ

11ߢ߈ࠆߣߪ㒢ࠄߥ޿‫ޔߢߎߘޕ‬ઍࠊࠅߦࡅ࠻⊹⤏ߣห᭽ߩㅘㆊᕈࠍ␜ߔࡉ࠲⊹⤏߇૶↪ߐࠇࠆߎߣ߽޽ࠆ㧔SCCP/0970/06㧕1)‫ޕ‬ࡅ࠻㧘ࡉ࠲߹ߚߪ࡜࠶࠻⊹⤏ࠍ↪޿ࠆߎߣ߇ߢ߈ࠆ㧔COLIPA㧕 3)ࡅ࠻޽ࠆ޿ߪേ‛ߩ⊹⤏ࠍ↪޿ࠆߎߣ߇ߢ߈ࠆ㧔OECD TG 428㧕2)‫ޕ‬ⵍ㛎‛⾰߇‫ޔ‬in vivo ⹜㛎ߦ߅޿ߡ߆ߥࠅ⊹⤏ઍ⻢ࠍฃߌࠆ႐วߦߪ‫ߦࠄߐޔ‬ᬌ⸛߇ᔅⷐߢ޽ࠆ‫ޕ‬ಓ⚿⊹⤏ࠍ↪޿ࠆ႐วߦߪ‫ޔ‬ઍ⻢⢻߇ᰳ៊ߒߡ޿ࠆน⢻ᕈ߇޽ࠆߚ߼‫ޔ‬ⵍ㛎‛⾰ߩઍ⻢߇⿠ߎࠄߥ޿ߎߣ߿‫ߪ޿ࠆ޽ޔ‬ઍ⻢‛ߩ᭴ㅧ߅ࠃ߮ߘߩ⚻⊹ๆ෼ᕈ߇ᱜ⏕ߦߪࠊ߆ࠄߥ޿ߎߣߦ߽ᵈᗧߒߥߌࠇ߫ߥࠄߥ޿‫ޔߡߞ߇ߚߒޕ‬ಓ⚿⊹⤏ࠍ↪޿ߚ in vitro ታ㛎ߢߪ‫⤏⊹ޔ‬ਛߢઍ⻢ࠍฃߌࠆൻว‛ߩ⚻⊹ๆ෼߿‫ߩߘޔ‬ઍ⻢‛ߦߟ޿ߡ߽ᱜ⏕ߥᖱႎࠍਈ߃ߥ޿ߣ⠨߃ࠄࠇࠆ㧔SCCP/0970/06㧕1)‫ޕ‬⹜㛎ൻว‛ߩ⊹⤏ઍ⻢߇㊀ⷐߢ޽ࠆ႐วߦߪ‫ޔ‬ᣂ㞲ߥ⊹⤏ࠍ૶↪ߒߥߌࠇ߫ߥࠄߥ
޿㧔SCCP/0970/06㧕1)‫ޕ‬໧㗴ὐ㧦ᣣᧄߢ߽ᦨㄭ‫ࠍ⤏⊹࠻ࡅޔ‬቟ቯ⊛ߦଏ⛎ߢ߈ࠆ૕೙߇ᢛ޿ߟߟ޽ࠅ‫⹜ޔ‬㛎⸘↹ߣ⹜㛎ᣉ⸳ߩ୶ℂ㕙߇᣿⏕ߢ޽ࠇ߫ଏ⛎น⢻ߣߥߞߚ‫ߩߘޔࠄ߇ߥߒ߆ߒޕ‬႐ว߽ⓨャߩ㑐ଥߢ‫ޔ‬ಓ⚿⊹⤏ࠍ↪޿ࠆ႐ว߇߶ߣࠎߤߢ޽ࠆ‫ޔߡߞ߇ߚߒޕ‬ᣣᧄ࿖ౝߦ߅
޿ߡ‫ޔ‬ᣂ㞲ߥࡅ࠻⊹⤏ࠍ↪޿ߡ in vitro ⚻⊹ๆ෼⹜㛎㧔in vitro ⊹⤏ㅘㆊ⹜㛎㧕ࠍታᣉߒ‫⤏⊹ޔ‬ઍ⻢߹ߢ᣿ࠄ߆ߦߔࠆߎߣߪ㕖Ᏹߦ㔍ߒ޿ߣ⠨߃ࠆ‫ޕ‬૶↪น⢻ߥ⊹⤏ߪ‫ޔ‬split-thickness (200㨪500 μm) ߹ߚߪ full-thickness㧔500㨪
1000 μm㧕ߢ޽ࠆ [Sanco/222/2000]6㧕‫⭯ߢࡓ࡯࠻ࡑ࡯࠳ޕ‬ಾߐࠇߚ⊹⤏߽૶↪ߔࠆߎߣ߇ߢ߈ࠆ‫⤏⊹ޕ‬ෘߪᢥ₂ߦ⸥タߐࠇߡ޿ࠆㆡಾߥᣇᴺߢ᷹ቯߔࠆ‫ߪ⤏⊹ޕ‬ታ㛎↪࠮࡞ߦ޽߁ࠃ߁ߦ↪ᗧߒߥߌࠇ߫ߥࠄߥ޿㧔SCCP/0970/06㧕1)‫ޕ‬⹜㛎⊹⤏ߦߪ‫ޔ‬㉂⚛‫ޔ‬ᾲ‫ޔ‬ൻቇಣℂߦࠃࠅ೸㔌ߒߚ⴫⊹‫╬ࡓ࡯࠻ࡑ࡯࠳ߪ޿ࠆ޽ޔ‬ߢෘߐ 200㨪400 Ǎm ߦ⭯ಾߒߚ⊹⤏ࠍ↪޿ࠆ㧔OECD TG 428㧕2)‫ޕ‬໧㗴ὐ㧦࠳࡯ࡑ࠻࡯ࡓߢ⊹⤏ࠍෘߐ 200㨪400 Ǎm ߦ⭯ಾߔࠆᛛⴚߪ㕖Ᏹߦ㔍ߒߊ‫߆ޔ‬ߥࠅߩᾫ✵ࠍⷐߔࠆ‫ޕ‬⴫⊹ࠪ࡯࠻ࠍ૶↪ߔࠆ႐วߦߪ‫ߩߘޔ‬ℂ↱߇ᔅⷐߢ޽ࠆ‫޽߇ߣߎ޿ࠈ߽ߪ⤑⊹⴫ޕ‬ࠅ‫ࠬࡦ࡜ࡃࠬࡑߪߢ࡞࠺ࡕߩߎޔ‬ᚻᴺ㧔଀㧦࠹࡯ࡊࠬ࠻࡝࠶ࡇࡦࠣ㧕ࠍㆡ↪ߔࠆߎߣ߇ߢ߈ߥ޿‫ ߩ࠻ࡅޔߪ↪૶ߩ⤑⊹⴫ޕ‬in vivo ⚻⊹ๆ෼ࠍㆊᄢ⹏ଔߔࠆน⢻ᕈ߇޽ࠆߎ
ߣ߽ᜰ៰ߐࠇߡ޿ࠆ㧔SCCP/0970/06㧕1)‫ޕ‬໧㗴ὐ㧦૕Ძߪ⌀⊹஥ߦሽ࿷ߔࠆᲫᩮ߆ࠄ⊹⤏⴫㕙ߦિ߮ߡ޿ࠆߚ߼‫ޔ‬೸㔌ߒߚ⴫⊹ߦ߅޿ߡߪᲫⓣ߇⽾ㅢߒߡ޿ࠆ‫ޕ‬Ძⓣࠍㅢߞߡൻቇ‛⾰߇ㅢㆊߔࠆน⢻ᕈ߽޽ࠆߚ߼‫ޔ‬12⚻⊹ๆ෼㊂ࠍㆊᄢߦ⹏ଔߔࠆෂ㒾ᕈ߇޽ࠆ‫ޕ‬ಽᨆ⊹⤏߅ࠃ߮/޽ࠆ޿ߪ࡟ࠪ࡯ࡃ࡯ṁᶧߩࠨࡦࡊ࡞ߪ‫ޔ‬ᶧ૕ࠪࡦ࠴࡟࡯࡚ࠪࡦࠞ࠙ࡦ࠲࡯‫ޔ‬
HPLC‫ޔ‬GC‫ߩઁߪ޿ࠆ޽ޔ‬ㆡᒰߥᣇᴺߢ‫ޔ‬ㆡಾߦߘߒߡࡃ࡝࠺࡯࠻ߐࠇߚᣇᴺࠍ↪޿ߡ‫ޔ‬ಽᨆߐࠇߥߌࠇ߫ߥࠄߥ޿‫ޕ‬ಽᨆᣇᴺߩᗵᐲ‫ޔ‬ౣ⃻ᕈ‫♖ޔ‬ᐲ߇⸽᣿ߐࠇߥߌࠇ߫
ߥࠄߥ޿㧔SCCP/0970/06㧕1)‫ޕ‬໧㗴ὐ㧦Cold ಽᨆࠍታᣉߔࠆ႐วߦߪ‫ޔ‬ಽᨆᣇᴺߩࡃ࡝࠺࡯࡚ࠪࡦ߇ⷐ᳞ߐࠇࠆ‫ޕ‬OECD ࠟࠗ࠼࡜ࠗࡦߦߪ‫ߦࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬㑐ߔࠆ⸥タߪሽ࿷ߒߥ޿‫࡚ࠪ࡯࠺࡝ࡃޕ‬ࡦߦ㑐ߒߡߪ‫ޔ‬FDA ߩࠟࠗ࠼࡜ࠗࡦ߇ሽ࿷ߒ 7)‫ޔ‬ㅢᏱ‫ߦࠇߎޔ‬Ḱ᜚ߒߡታᣉߐࠇࠆ‫ޕ‬
FDA ߩࠟࠗ࠼࡜ࠗࡦߩ㗄⋡ߪએਅߩㅢࠅߢ޽ࠆ‫ޕ‬
FDA ߩࠟࠗ࠼࡜ࠗࡦ ࡃ࡝࠺࡯࡚ࠪࡦߩ໧㗴ὐߣߒߡ‫ޔ‬OECD ޽ࠆ޿ߪ SCCP ߦ㗄⋡ߩ⸥タ߇ߥ޿ߎߣ߆ࠄ‫ޔ‬FDA ߩࠟࠗ࠼࡜ࠗࡦߦ

․⇣ᕈ(ࡉ࡜ࡦࠢ⹜ᢱ‫ޔ‬n=6)⌀ᐲ(3 Ớᐲ‫ޔ‬n=5)♖ᐲ(3 Ớᐲ‫ޔ‬n=5)Ḱ᜚ߔࠆߎߣߦߥࠆ߇‫ߩߘޔ‬႐ว‫ޔ‬਄⸥ߩో㗄⋡ࠍታᣉߔࠆ੐ߦߥࠅ‫ޔ‬㊄㗵㕙ߢ‫ޔ‬ታᣉ࿎㔍ߦߥࠆ႐ว߇޽ࠆ‫ޕ‬࿁෼₸(3 Ớᐲ)
ᬌ㊂✢㧔หߓࡑ࠻࡝ࠢࠬ㧕ቯ㊂ਅ㒢Ớᐲ࡟ࠬࡐࡦࠬ቟ቯᕈ ಓ⚿Ⲣ⸃቟ቯᕈ㧔3 ࿁ߩಓ⚿Ⲣ⸃‫ޔ‬ૐỚᐲߣ㜞Ớᐲߢฦ n=3㧕 ⍴ᦼቶ᷷቟ቯᕈ㧔ૐỚᐲߣ㜞Ớᐲߢฦ n=3㧕 㐳ᦼ଻ሽ቟ቯᕈ㧔ૐỚᐲߣ㜞Ớᐲߢฦ n=3㧕 ଻ሽṁᶧ቟ቯᕈ ᷹ቯታ᷹⹜ᢱਛ቟ቯᕈ













࡟࠮ࡊ࠲࡯ṁᶧ࡟࠮ࡊ࠲࡯ᶧߩ⚵ᚑߪ‫ⵍޔ‬㛎‛⾰ߩ᜛ᢔࠍᅹߍߥ޿ࠃ߁ㆬᛯߔࠆ‫⹜ޔ߫߃଀ޕ‬㛎᧦ઙਅߩⵍ㛎‛⾰ߩ࡟࠮ࡊ࠲࡯ᶧਛߢߩṁ⸃ᕈ߅ࠃ߮቟ቯᕈߪ଻⸽ߐࠇߥߌࠇ߫ߥࠄߥ
޿‫↢ޕ‬ℂ㘩Ⴎᶧ߿╬ᒛ✭ⴣᶧ߇৻⥸⊛ߦⷫ᳓ᕈൻว‛ߦ↪޿ࠄࠇࠆ‫ⷫޕ‬ᴤᕈ‛⾰ߦኻߒߡߪ‫ⴊޔ‬ᷡࠕ࡞ࡉࡒࡦ߹ߚߪㆡᒰߥนṁൻ೷࡮੃ൻ೷ࠍᷝടߔࠆߎߣ߇ߢ߈ࠆ߇‫ޔ‬ߘߩ㓙⊹⤏⁁ᘒ㧔membrane integrity㧕ࠍᖡൻߐߖߡߪߥࠄߥ޿‫࡯࠲ࡊ࠮࡟ޕ‬ᶧߪಽ

13ᨆᠲ૞ߦ߽ᅹኂߒߥ޿ࠃ߁ߦߔࠆߴ߈ߢ޽ࠆ㧔SCCP/0970/06㧕1)‫ޕ‬໧㗴ὐ㧦ൻ♆ຠ⚛᧚ߦ߅޿ߡߪ‫ޔ‬ᴤ╬ߩ⢽ṁᕈൻว‛߇߆ߥࠅߩᢙሽ࿷ߒ‫ߩߘޔ‬႐ว‫ޔ‬↢ℂ㘩Ⴎᶧߢߪṁ⸃ߒߥ޿‫ޔߡߞ߇ߚߒޕ‬ฎߊ߆ࠄ‫ޔ‬in vitro ⚻⊹ๆ෼⹜㛎㧔in vitro⊹⤏ㅘㆊ⹜㛎㧕ߩႎ๔ߪ‫ޔ‬ᄙߊ߇᳓ṁᕈൻว‛ߦ㑐ߔࠆ߽ߩߢ޽ࠆ‫৻ޕ‬ᣇ‫ޔ‬⢽ṁᕈൻว‛ߩႎ๔߽ሽ࿷ߔࠆ߇‫ߩߘޔ‬႐ว‫࡯࠲ࡊ࠮࡟ޔ‬ṁᶧߦ‫ޔ‬นṁൻ೷/੃ൻ೷ߣߒߡ‫ࡐޔ‬࡝ࠝࠠࠪࠛ࠴࡟ࡦ(20)ࠝ࡟ࠗ࡞ࠛ࡯࠹࡞߇↪޿ࠄࠇࠆ႐ว߇ᄙ޿㧠㧕‫ߎޔࠄ߇ߥߒ߆ߒޕ‬ߩ႐วߦ߽ൻว‛ߩ⢽ṁᕈߦࠃߞߡߪṁ⸃ᕈߦ㒢⇇߇޽ࠆ‫⹜ޕ‬㛎᧦ઙਅߩⵍ㛎‛⾰ߩ࡟࠮ࡊ࠲࡯ᶧਛߢߩṁ⸃ᕈ߇ⵍ㛎‛⾰ߩ᜛ᢔࠍᅹߍߥ޿ࠃ߁ㆬᛯߔࠆߣㅀߴࠄࠇߡ޿
ࠆ߇‫᥸ߪࠇߎޔ‬㔺ᤨ㑆⚳ੌᤨߩⵍ㛎‛⾰ߩㅘㆊ㊂߇‫ޔ‬චಽṁ⸃น⢻ߥṁᇦࠍㆬᛯߔࠆ
ߎߣߢ޽ࠆ‫᥸ޔࠄ߇ߥߒ߆ߒޕ‬㔺ᤨ㑆⚳ੌᤨߩⵍ㛎‛⾰ߩㅘㆊ㊂ߪ⹜㛎ࠍታᣉߒߥ޿
ߣࠊ߆ࠄߥ޿ߩߢ‫ޔ‬ታ㓙ߦ⹜㛎ࠍታᣉߒߡ‫ⵍޔ‬㛎‛⾰ߩ᜛ᢔࠍᅹߍߥ޿ṁᇦߢ޽ࠆ߆
ุ߆ࠍ್ᢿߔࠆߎߣߪ㔍ߒ޿‫ޔߡߞ߇ߚߒޕ‬⢽ṁᕈൻว‛ߩ in vitro ⚻⊹ๆ෼⹜㛎㧔invitro ⊹⤏ㅘㆊ⹜㛎㧕ߦ↪޿ࠆߎߣ߇ߢ߈ࠆ࡟࠮ࡊ࠲࡯ṁᶧߩ޿ߊߟ߆ߩ⸥タ߇ᔅⷐߣ⠨߃ࠆ‫ޕ‬
ේೣߣߒߡ࡟࠮ࡊ࠲࡯ᶧߪ‫↢ޔ‬ℂ⊛ pH ୯ߦߔߴ߈ߢ޽ࠆ‫ߦࠇߎޕ‬ㅺ⣕ߔࠆ႐วߦ
ߪᱜᒰൻߔࠆᔅⷐ߇޽ࠆ‫ޔ߫߃଀ޕ‬WHO 2354)ߢឭ᩺ߐࠇߡ޿ࠆ 50/50 ࠛ࠲ࡁ࡯࡞/᳓ ࠍ↪޿ࠆ႐วߦߪ‫ ⤏⊹߇ࠇߘޔ‬integrity ߦߪᓇ㗀ࠍ෸߷ߐߥ޿ߎߣࠍ␜ߒ‫ޔ‬Ქᕈቇ⊛㑐ଥᦠ㘃ߦ⸥タߒߥߌࠇ߫ߥࠄߥ޿‫ޕ‬ᣧᦼߩណขᤨ㑆ߢㅪ⛯⊛ߥ ND ୯ߣߥࠄߥ
޿ࠃ߁ߦߔࠆߚ߼‫࡯࠲ࡊ࠮࡟ޔ‬ᶧ㊂ߪᦨዊ㒢ߦߒߥߌࠇ߫ߥࠄߥ޿‫ޕ‬໧㗴ὐ㧦ࠛ࠲ࡁ࡯࡞߇ 5%એ਄฽߹ࠇࠆߣ‫⊹⚻ޔ‬ๆ෼ᕈߦᓇ㗀ࠍ෸߷ߔߎߣ߇⍮ࠄࠇߡ޿ࠆ‫ޕ‬WHO 2354)ߢឭ᩺ߐࠇߡ޿ࠆ 50/50 ࠛ࠲ࡁ࡯࡞/᳓ߪ૶↪ߢ߈ߥ޿ߣ⠨߃ࠆ‫ޕ‬࡟࠮ࡊ࠲࡯ᶧߦߪ‫ޔ‬ታ㛎ਛߩ᳇ᵃߩ⊒↢ࠍᛥ߃ࠆߚ߼⣕᳇ߒߚṁᶧࠍ↪޿ࠆ‫ޔߚ߹ޕ‬⹜㛎ᦼ㑆ਛ‫ޔ‬㕖ᵹ಴ဳ᜛ᢔ࠮࡞ߢߪචಽߦᠣᜈߒ‫ޔ‬ᵹ಴ဳ᜛ᢔ࠮࡞ߢߪ⛘߃ߕᶧࠍᵹ
ߒߡ߅߆ߥߊߡߪߥࠄߥ޿‫ޕ‬໧㗴ὐ㧦࡟࠮ࡊ࠲࡯ṁᶧߦ‫↢ޔ‬ℂ㘩Ⴎᶧࠍ↪޿ࠆ႐วߪ‫ޔ‬⣕᳇ߔࠆߎߣߪน⢻ߢ޽ࠆ߇‫ޔ‬⢽ṁᕈൻว‛ߢ‫࡯࠲ࡊ࠮࡟ޔ‬ṁᶧߦนṁൻ೷/੃ൻ೷ࠍᷝടߔࠆ႐วߪ‫ޔ‬⣕᳇ߔࠆߣᵃ┙ߟߚ߼‫ޔ‬⣕᳇ߔࠆߎߣߪ㕖Ᏹߦ㔍ߒ޿‫ޕ‬࡟࠮ࡊ࠲࡯ṁᶧߪ‫ޔ‬ಽᨆᚻᴺࠍᅹߍߡߪ޿ߌߥ޿‫⹜ޕ‬㛎ࠪࠬ࠹ࡓߩㆬᛯߪ⎇ⓥႎ๔
ᦠߢᱜᒰൻߐࠇࠆߴ߈ߢ޽ࠆ‫࡯࠲ࡊ࠮࡟ޔߚ߹ޕ‬ṁᶧߦㅘㆊߒߚൻว‛ߩ㊂ߪ‫ޔ‬ๆ෼ߩㆊዊ⹏ଔࠍᒁ߈⿠ߎߐߥ޿ߚ߼ߦ‫ޔ‬㘻๺࡟ࡌ࡞ߩ 10%ࠍ⿥߃ࠆߴ߈ߢߪߥ޿‫ޕ‬14໧㗴ὐ㧦ⵍ㛎‛⾰߇ᴤಽߩ᭽ߥ႐วߪ‫ޔ‬นṁൻ೷/੃ൻ೷ࠍ↪޿ߡ߽‫ߩߘޔ‬ṁ⸃ᕈߦߪ㒢⇇߇޽ࠆ‫࡯࠲ࡊ࠮࡟ޕ‬ṁᶧߦㅘㆊߒߚൻว‛ߩ㊂߇㘻๺࡟ࡌ࡞ߩ 10%એਅߣߥࠆߣ‫ޔ‬ᴤಽߩ⚻⊹ๆ෼⹜㛎ࠍታᣉߔࠆߎߣߪ㕖Ᏹߦ࿎㔍ߢ޽ࠆ‫ޕ‬⊹⤏ߩintegrity ⊹⤏ࡃ࡝ࠕ࡯ߩ࠴ࠚ࠶ࠢ㧔=integrity㧕ߪ⹜㛎ߦᔅ㗇ߢ޽ࠆ‫ޔߪࠇߎޕ‬ᜰᮡߣߥࠆൻ
ว‛㧔଀㧦࠻࡝࠴࠙ࡓ᳓‫ࠬ࡯ࡠࠢࠬޔࡦࠗࠚࡈࠞޔ‬㧕ߩ⊹⤏ㅘㆊᕈࠍ᷹ቯߔࠆ߆‫߽ޔ‬ߒߊߪ‛ℂ⊛ᣇᴺ㧔଀㧦TEWL(Transepidermal Water Loss)‫ޔ‬TER(Transcutaneous
Electrical Resistance)㧕ߦࠃߞߡ⏕⹺ߔࠆ‫ޔߚ߹ޕ‬ᓧࠄࠇߚ࠺࡯࠲ߪ⹜㛎ႎ๔ᦠߦ⸥タߒߥߌࠇ߫ߥࠄߥ޿‫ޕ‬໧㗴ὐ㧦⊹⤏ߩ integrity ࠍ࠴ࠚ࠶ࠢߔࠆᣇᴺߣߒߡ࠻࡝࠴࠙ࡓ᳓ࠍ↪޿ࠆ႐ว߇ᄙ޿‫ޕ‬ᣣᧄߩ᡼኿✢㓚ኂ੍㒐ⷙቯߦ߅޿ߡߪ‫⃻ޔ‬࿷‫ޔ‬࿖㓙ၮḰߦวࠊߖࠆᗧ๧ߢ㧘࠻࡝࠴࠙ࡓߪỚᐲߢ 1MBq/g‫ޔ‬ᢙ㊂ߢ 1GBq ࠍਅ㒢୯ߣߒߡ‫ޔ‬Radio Isotope㧔RI㧕ߣߒߡߩข
ࠅᛒ޿ࠍ఺㒰ߔࠆߎߣߦߥߞߡ޿ࠆ‫ޔࠄ߇ߥߒ߆ߒޕ‬ᱧผ⊛ߥ⢛᥊߆ࠄ‫ޔ‬ᣣᧄߩ᡼኿
⢻ߦኻߔࠆ⹺⼂ߪ᰷☨⻉࿖ߣߪ⇣ߥࠆ‫ⷙᧄޔߡߞ߇ߚߒޕ‬ቯ߇ㆇ↪ߐࠇࠆߎߣߪ߶ߣ
ࠎߤߥߊ‫ࠆ޿↪ࠍࡓ࠙࠴࡝࠻ޔ‬႐วߪ‫ޔ‬ᣉ⸳ߩⷙቯߢ‫ޔࠅߪ߿ޔ‬RI ታ㛎ߣߥࠅ‫ޔ‬RI ᣉ
⸳ߢⴕ߁ߎߣ߇⟵ോߠߌࠄࠇࠆ‫ࡓ࠙࠴࡝࠻ޔߚ߹ޕ‬᳓ߢ⊹⤏ߩ integrity ࠍ࠴ࠚ࠶ࠢߒ
ߚ⊹⤏ߪ‫ޔ‬RI ᣉ⸳߆ࠄᜬߜ಴ߔߎߣߪߢ߈ߥ޿ߎߣ߆ࠄ‫⹜ޔ‬㛎‛⾰ߩㅘㆊᕈ߽ RI ᣉ⸳ౝߢታᣉߔࠆߎߣߦߥࠆ‫⹜ޕ‬㛎‛⾰ߦ᡼኿ᕈᮡ⼂૕ࠍ↪޿ࠆ႐วߪ໧㗴ߥ޿߇‫ޔ‬Coldߩᯏེಽᨆࠍታᣉߔࠆ႐วߪ‫ޔ‬RI ቶߦಽᨆᯏེࠍᏱ⸳ߔࠆᔅⷐ߇޽ࠆ‫ޕ‬ಽᨆᯏེߦࠃ
ߞߡߪ‫ޔ‬LC/MS/MS‫ޔ‬ICP/MS ╬‫ࠅߥ߆ޔ‬㜞ଔߥ߽ߩ߇޽ࠆߚ߼‫ޔ‬ᔅߕߒ߽ RI ᣉ⸳ౝߦ⸳⟎ߔࠆߎߣ߇㔍ߒ޿⃻⁁ߢ޽ࠆ‫ࠍࠇߎޕ‬ㆱߌࠆᣇᴺߣߒߡ‫ ߩ⤏⊹ޔ‬integrity ߪ࠻࡝࠴࠙ࡓ᳓ߢ࠴ࠚ࠶ࠢߒ‫ߪߣ⤏⊹ߩߘޔ‬೎ߦ‫ߩߘޔ‬ㄭறߩ⊹⤏ࠍ↪޿ߡ‫ޔ‬RI ᣉ⸳ᄖߢ⹜㛎ൻว‛ߩ⹜㛎ࠍⴕ޿‫ޔ‬Cold ߢᯏེಽᨆࠍታᣉߔࠆᣇᴺ߇޽ࠆ‫ޕ‬ߒ߆ߒߥ߇ࠄ‫ߩߎޔ‬᭽ߥᣇᴺߪ‫⸥ߦࡦࠗ࡜࠼ࠗࠟޔ‬タߐࠇߡ߅ࠄߕ‫ߩߘޔ‬ᱜᒰᕈߦ㑐ߒߡߪਇ᣿ߢ޽ࠆ‫ޕ‬OECD TG428 2)ߢߪ࠻࡝࠴࠙ࡓ᳓ߩ૶↪ࠍផᅑߒߡ޿ࠆ‫ޕ‬㧡㧚✚᜝ േ‛ታ㛎ߩ㧟Rs㧔Replacement‫ޔ‬Refinement‫ޔ‬Replacemnet㧕ߩ᥉෸ࠍฃߌ 8,9)‫ޔ‬᰷☨ߢߪൻ♆ຠⷙ೙߿ REACH ߦኻᔕߔࠆߚ߼‫ޔ‬േ‛ታ㛎ઍᦧᴺߩ೑↪߇ផᅑߐࠇߡ
޿ࠆ‫ޔߡߞࠃޕ‬᰷☨ߢߪ቟ోᕈ⹏ଔߦ߅ߌࠆ⚻⊹ๆ෼⹜㛎߿⊹⤏ㅘㆊᕈߪ in vitro ⹜㛎߇ਛᔃߢ޽ࠆ 10)‫ޕ‬ ᧄᆔຬળߦ߅޿ߡߪฦ⒳‫ޔ‬in vitro ⹜㛎ᴺߦߟ޿ߡߩ㐳⍴ᚲࠍ⼏⺰ߒߡ߈ߚ߇‫ߕ޿ޔ‬15ࠇߩ⹜㛎߽ㆡ↪㒢⇇ࠍℂ⸃ߒ‫ޔ‬ታᣉߔࠆ਄ߢߩ໧㗴ὐࠍ⠨ᘦߒߥ߇ࠄታᣉߔࠆᔅⷐ߇
޽ࠆ‫ޕ‬ૉߒ‫ޔ‬ㆡ↪㒢⇇ࠍℂ⸃ߒߚ਄ߢ‫ߦࠬࡦ࠳ࠗࠟ߿ࡦࠗ࡜࠼ࠗࠟޔ‬Ḱ᜚ߒߚᱜߒ޿
⹜㛎ߢⴕߞߚ in vitro ⹜㛎߆ࠄ in vivo ߩ⚿ᨐࠍផቯߔࠆߎߣ߇น⢻ߢ޽ࠆߣᕁࠊࠇࠆ‫ޕ‬
ᱜߒ޿ᣇᴺߣ޿߁ߩߪ⊹⤏ߩㆬቯ‫ߩ⋧࡯࠲ࡊ࠮࡟ޔ‬ㆬቯ‫⤏⊹ޔ‬ઍ⻢ߩ᦭ή‫⹜ޔ‬㛎‛⾰ߩ‛ᕈ╬ࠍචಽߦ⠨ᘦߔࠆߎߣߢ޽ࠆ‫ޕ‬ ᧄᬌ⸛ળߢ⹏ଔߒߚ 4 ⾗ᢱߪ޿ߕࠇ߽౏⍮ߢ‫ޔ‬᰷☨ߢߪ৻⥸⊛ߦ↪޿ࠄࠇߡ޿ࠆ⹜
㛎ᴺߢ޽ࠆߎߣࠍ㐓ߺࠆߣ‫ޔ‬ㆡ↪㒢⇇ࠍචಽߦℂ⸃ߒߚ਄ߢ⹏ଔߔࠇ߫‫ޔ‬ൻቇ‛⾰ߩ࡝ࠬࠢ⹏ଔ߅ࠃ߮ല⢻⹏ଔߩ৻ߟߩ⹏ଔ♽ߣߒߡචಽߦᵴ↪ߢ߈ࠆ‫ޕ‬ෳ⠨ᢥ₂
1)

SCCP/0970/06 Opinion on basic criteria for the in vitro assessment of dermal

absorption of cosmetic ingredients (2006)
2) OECD428 Guideline for the testing of chemicals, skin absorption: In vitro method(2004)
3) Colipa regulatory, Guidelines for i percutaneous absorption/penetration (1997)
4)

WHO, Environmental Health Criteria 235,, Dermal Absorption (2006)

5) SCCP The sccp's notes of guidance for the testing of cosmetic ingredients and theirsafety evaluation 6th revision
6) Sanco/222/2000 rev. 7, Guidance document on dermal absorption (2004)
7)

Food and Drug Administration, Guidance for industry: Bioanalytical method

validation (2001).
8)േ‛ߩᗲ⼔෸߮▤ℂߦ㑐ߔࠆᴺᓞ
http://www.env.go.jp/nature/dobutsu/aigo/amend_law2/law.pdf9)ታ㛎േ‛ߩ㘺㙃෸߮଻▤ਗ߮ߦ⧰∩シᷫߦ㑐ߔࠆၮḰ
http://www.env.go.jp/nature/dobutsu/aigo/law_series/nt_h180428_88.html
10)Commission Staff Working Documents(2004)Time Tables for the phasing-out of animal
testing in the framework of the 7th Amendment to the Cosmetics Directive (Council
Directive 76/768/EEC); EN, SEC82004,1210

16

ᐔᚑ 25 ᐕ 6 ᦬ 12 ᣣ
JaCVAM ⹏ଔળ⼏߆ࠄߩ⾰໧ߦኻߔࠆ࿁╵᧖ᨋၷᰴ‫⮮ޔ‬ၴᶈ᣿㧔ၔ⷏ᄢ㧕‫⮮ޔ‬੗߹߈ߎ㧔ᤘ๺⮎⑼ᄢ㧕㩷㧝㧕‫⤏⊹ޟ‬ㅘㆊᕈ‫⊹⚻ޔ‬ๆ෼‫⤏⊹ޔ‬ๆ෼ߥߤߩ⸒⪲ߩቯ⟵‫ޠ‬ ৻⥸ߦ‫ޔ‬‫⤏⊹ޟ‬ㅘㆊᕈ‫ߪޠ‬නߦ⊹⤏ࠍㅘㆊߔࠆ㊂‫ޔ‬ㅦᐲ‫ߩߘ߮ࠃ߅ޔ‬ᕈ⾰ࠍ޿޿߹ߔ‫ޕ‬‫ޟ‬In vitro ⊹⤏ㅘㆊᕈ‫ޕߔ߹޿૶ߣߤߥޠ‬ ৻ᣇ‫ޟޔ‬ๆ෼‫ోߪߣޠ‬りᓴⅣ♽㧔߅߽ߦⴊ▤♽㧕ߦൻቇ‛⾰߇౉ࠆߎߣࠍᗧ๧ߒ߹ߔ
ߩߢ‫⊹⚻ޟޔ‬ๆ෼ᕈ‫ోߡ⚻ࠍ⤏⊹ߪޠ‬りᓴⅣ♽ߦ౉ࠆ㊂‫ޔ‬ㅦᐲ‫ߩߘ߮ࠃ߅ޔ‬ᕈ⾰ࠍ޿޿߹ߔ‫ ߪࠇߎޕ‬in vivo ߦኻᔕߔࠆ⸒⪲ߣℂ⸃ߔࠆߩ߇ㆡᒰߢߔ‫ޔߛߚޕ‬਎ߩਛߦߪㆡᒰ
ߦ૶ߞߡ޿ࠆ⎇ⓥ⠪ߩᣇ߇ᄙ޿ߣᕁ޿߹ߔ‫⤏⊹ߪ⑳ޕ‬ๆ෼ߣ޿߁↪⺆ߪ૶޿߹ߖࠎ‫ߤޕ‬ߞߜ߆ߣ޿߃߫‫⊹⚻ޔ‬ๆ෼ߣหᗧ⺆߆ߣᕁ޿߹ߔ‫ޕ‬᧖ᨋవ↢߇ࠦࠬࡔ࠹࠶ࠢࠫࡖࡄࡦߦㅪタߒߡ޿ࠆ‫⊹⚻ ࠭࡯࡝ࠪޟ‬ๆ෼ߦߟ޿ߡ⠨߃ࠆ‫ߩߘޠ‬㧤ߢ‫⊹⚻ޟ‬ๆ෼‫⤏⊹ޟޔޠ‬ㅘㆊ‫⤏⊹ޟޔޠ‬ᶐㅘ‫ߩޠ‬ቯ⟵ߦߟ޿ߡ‫ޔ‬ᢥ₂ࠍᒁ↪
ߒ␜ߒߡ޿߹ߔ㧔ᷝઃߒ߹ߔ㧕‫ޕ‬㧞㧕‫ޟ‬in vitro ⊹⤏ㅘㆊᕈ⹜㛎ߪߤߩ⹜㛎ᴺߩઍᦧᴺߢߒࠂ߁߆‫ޠޕ‬in vivo ⚻⊹ๆ෼⹜㛎㧔TG427㧕ߩઍᦧᴺߣ⠨߃ߡ޿߹ߔ‫ޕ‬㧟㧕‫ޟ‬AUC ߩ▚ቯᑼߢ‫ోޟޔ‬りࠢ࡝ࠕ࡜ࡦࠬ߇ᣢ⍮ߥࠄ‫ޔ߇ߔ߹ࠅ޽ߣޠ‬ᣢ⍮ߢ޽ࠆ႐วߪߤࠇߊࠄ޿޽ࠆߩߢߒࠂ߁߆‫ޠޕ‬ ‛⾰ߩ㕒⣂ౝᛩਈᓟߩⴊਛỚᐲ㧙ᤨ㑆ᦛ✢ߩ࠺࡯࠲߇޽ࠇ߫‫ޔ‬ᣢ⍮ߢߔ‫ޕ‬ක⮎ຠ߿ㇱᄖຠߢ㕒ᵈ⵾೷߇޽ࠇ߫ᣢ⍮ߛߣᕁ޿߹ߔ‫ޕ‬ല⢻ࠍ⊒ើߔࠆᯏ⢻ᕈൻ♆ຠߩ᦭ല‛⾰߽ࠊ߆ߞߡ޿ࠆࠤ࡯ࠬߩᣇ߇ᄙ޿ࠃ߁ߦᕁ޿߹ߔ‫ోޕ‬りࠢ࡝ࠕ࡜ࡦࠬߦߟ޿ߡߢߔ‫ޕ‬ㇱᄖຠ╬ߢ૶ࠊࠇߡ޿ࠆ᦭ലᚑಽߪ↳⺧ᤨߦ᣿ࠄ߆ߦߐࠇߡ޿ࠆߣᕁ޿߹ߔ߇㧘ߘߩ‛⾰ߩࠢ࡝ࠕ࡜ࡦࠬ߇᷹ቯߐࠇߡ޿ࠆ߆ߣ޿߁ߣ㧘ߐࠇߡ޿ߥ޿߶߁߇ᄙ޿ߩߢߪߥ޿߆ߣ
ᕁ޿߹ߔ‫ߒ߆ߒޕ‬㧘‛⾰߇หߓߢ޽ࠇ߫㧘AUC ߣๆ෼㊂ߪᲧ଀ߔࠆߩߢ㧘㑆ធ⊛ߦๆ෼㊂ࠍ㘃ផߒߡ޿ߚ AUC ᴺߦઍࠊࠅ㧘ๆ෼㊂ࠍᛠីߔࠆᣇᴺࠍណ↪ߔࠆߎߣߦ໧㗴ߪߥ޿ߣ⠨߃߹ߔ‫ޕ‬㧠㧕‫⤏⊹ޟ‬ㅘㆊᕈߩ⚿ᨐ߆ࠄ‫ో⊹⚻ޔ‬りᦑ㔺߇ផቯߢ߈ࠆߪߕ߇ߥ޿‫ޕ‬17⊹⤏ㅘㆊᕈ⹜㛎ߪ‫ޔ‬ൻ♆ຠ࡮ක⮎ㇱᄖຠ╬ߦ߅ߌࠆ⊹⤏ㅘㆊ㊂ࠍᛠីߒ‫ޔ‬቟ోଥᢙࠍ᳞
߼ࠆߚ߼ߩᣇᴺߢ޽ࠆ‫⤏⊹ޔߪ޿ࠆ޽ޕ‬ㅘㆊᕈ߇߶ߣࠎߤߥ޿ߎߣࠍ⸽᣿ߔࠆ⹜㛎ᴺߦ
ㆊ߉ߥ޿ߣߩᗧ⷗ߦߟ޿ߡ‫ޠ‬ ⊹⤏ㅘㆊߒߚൻቇ‛⾰ߩ߶ߣࠎߤ㧔ㅢᏱ 95%એ਄㧕ߪోりᓴⅣ♽ߦⴕ߈߹ߔ‫⤏⊹ޕ‬ㅘㆊ⹜㛎߆ࠄ⚻⊹ోり᥸㔺߇ផቯߢ߈ࠆߪߕ߇ߥ޿ߣ޿޿߈ࠆ⑼ቇ⠪߇਎ߩਛߦ޿ࠆ
ߎߣ⥄૕㛳߈ߢߔ‫᧲ޕ‬ᄢ↢↥⎇ߩ㈬੗వ↢ߥߤߪ‫ߩ⑳ޔ‬ᗧ⷗ࠍࠃߊℂ⸃ߒߡߊࠇࠆߣᕁ
޿߹ߔ‫⮎ޔߦ⥸৻ޕ‬ቇߩ‫⮎ޟ‬೷ቇ‫⵾ޟޠ‬೷ቇ‫ߩޠ‬ಽ㊁ߩవ↢‫ޔ‬ൻቇᎿቇߩవ↢ߥߤߪࠃ
ߊℂ⸃ߒߡ޿߹ߔ‫⮎ޕ‬೷Ꮷ࿖ኅ⹜㛎ߦ߽಴ࠆౝኈߢ޽ࠆ߆ߣᕁߞߡ޿߹ߔ‫ޔ߅ߥޕ‬invitro ⊹⤏ㅘㆊᕈ߆ࠄⴊਛỚᐲࠍ੍᷹ߔࠆ⸘▚࠰ࡈ࠻߽Ꮢ⽼ߐࠇߡ޿߹ߔ㧔਻ᎺᎿᬺᄢቇ ᧲᧦వ↢ߩ SkinCad ߇᦭ฬߢߔ‫ߦߺߥߜޕ‬ᓐ߽ൻቇᎿቇ಴りߢߔ㧕‫⮎⵾ޕ‬ડᬺߩ⎇ⓥ⠪ߩᄙߊ߽ℂ⸃ߔࠆߣᕁ޿߹ߔ‫৻ޕ‬ᣇ‫ޔ‬ൻ♆ຠ⎇ⓥ⠪ߪ߹ߛᖱႎ⑼ቇ㧔ࠗࡦࡈࠜࡑ
࠹ࠖࠢࠬ㧕߿ᢙℂ⸘▚ࠍ೑↪ߒߡ޿ࠆ⎇ⓥ⠪߇ዋߥ޿ߩ߇⃻⁁ߢߔ‫ޕ‬ઍᦧᴺ⎇ⓥߢ߽‫ޔ‬in silico ߿೨ㅀߒߚ㈬੗వ↢ߩᣇᴺ⺰ߪࠃߊℂ⸃ߐࠇߡ޿߹ߖࠎ‫ޕ‬ ࡜࠶࠻ߩ in vitro ⊹⤏ㅘㆊㅦᐲߣ in vivo ⚻⊹ๆ෼ㅦᐲߪ‫ޔ‬න⚐ߥၮ೷♽ߢᲧセߒߚߎߣ߇޽ࠅ߹ߔ߇‫ޕߔߢ✜৻߷߶ޔ‬㆑߁႐วߩℂ↱ࠍតߔᣇ߇㔍ߒ޿ߊࠄ޿ߢߔ‫ޕ‬ ߽ߜࠈࠎ‫⤏⊹ޔ‬ਛߩઍ⻢߿࠻࡜ࡦࠬࡐ࡯࠲੺࿷ߩ⊹⤏ㅘㆊ߽⷗ࠄࠇࠆน⢻ᕈ߇޽ࠆߩߢ‫ޔ‬ᱜߒߊ in vitro ⊹⤏ㅘㆊታ㛎ࠍߔࠆߎߣ߇㊀ⷐߢ޽ࠆߎߣߪ⸒߁߹ߢ߽޽ࠅ߹ߖࠎ‫ޕ‬ ‛⾰෼ᡰߪ in vitro ߽ in vivo ߽߶ߣࠎߤหߓߢߔ‫ࠍ⤏⊹ޔߪߢߎߎޕ‬ㅢࠆㅦᐲߒ߆Ყセߒߡ޿ߥ޿ߩߢߔ߆ࠄ‫᧲ޕ‬ᄢ߆ࠄℂ⎇ߦ⒖ߞߚ᧖ጊవ↢߇᧲ᄢᢎ᝼ߦߥࠄࠇߚߎࠈ
ߪ PK ⎇ⓥࠍߐࠇߡ޿߹ߒߚ‫⾰‛ߢ࡞ࡌ࡟ེ⤳ޕ‬෼ᡰࠍ⎇ⓥߒߚ⚿ᨐ‫߿ེ⤳ޔ‬૕ߩ↢ℂቇ⊛⮎‛ㅦᐲ⺰ࡕ࠺࡞ࠍ⚵ߺ┙ߡࠄࠇ߹ߒߚ‫ߩ⑳ޕ‬ᣇᴺ⺰߽ߘࠇࠄߣ‫ߤࠎߣ߶ޔ‬ᄌࠊࠅ߹ߖࠎ‫ޕ‬
એ਄

18

別紙 1

3.試験法の概要（手順など）

表1-1．ガイドラインの比較（in vitro 経皮吸収試験）

SCCP(2006.3)

Guideline

OECD(TG428, 2004.4)

Colipa(1999)

Flow through/Static cell
（選択は化合物次第）

Flow through/Static cell
（有限系：開放，無限系：閉塞）

チャンバー(ドナー，レセプター)で皮膚を挟んだもの．
セルの選択は薬剤による．

レセプター液親水性： 生食，緩衝生食液
脂溶性： アルブミン，可溶化剤/乳化剤の添加は可・対象物質が溶解すること
・皮膚に影響を与えないこと
・代謝試験では皮膚能を維持す
ること

親水性： 生食液，緩衝生食液
脂溶性： アルブミン，可溶化
剤を含む液

種

○ ヒト/ブタ，× げっ歯類（△ 培養皮膚/再構成皮膚）ヒト/動物（ヒトの場合は倫理下で）記載なし（Std protocol では
ヒト，ブタ，ラット）部位

ヒト：腹，脚，胸囲
ブタ：腹，胸，背，側，耳

記載なし

記載なし

皮膚厚

ヒト：○ 200-500 µm△ 500-1,000 µm（表皮膜は過大評価の恐れ）
ブタ：500-1,000 µm でもOK

○ 表皮膜（酵素/熱処理）/剥離
皮膚（200-400 µm）△ full-thickness skin× >1mm○ whole，split-thickness（< 1 mm とすること）

必須： 方法については記載なし

必須：

拡散セル

皮
膚
膜

3H O，カフェイン，
2Integrity test

必須：

被験物質

RI 体は大規模ロット（Cold体）とは若干異なる特徴を示す

RI ラベル化が理想的標準処方で1 濃度以上-

適用量

固形/半固形： 2-5 mg/cm2
液体：～10 µL/cm2

固形： 1-5 mg/cm2
液体： ～10 µL/cm2

実使用で（consumer use）

>6
（3ドナーx 2 以上）

最低4-

n 数(/1ｻﾝﾌﾟﾙ)

ショ糖，TER，TEWL

3H O，TEWL など
2

表1-2．ガイドラインの比較（in vitro 経皮吸収試験）
Guideline

SCCP(2006.3)

OECD(TG428, 2004.4)

Colipa(1999)

皮膚表面温度

32±1 ℃

32±1 ℃（湿度は 30-70 %）30-32 ℃

・一般的には 24 時間（対象物
質の透過性による）・適用途中でｻﾝﾌﾟﾘﾝｸﾞを実施（透過ﾌﾟﾛﾌｧｲﾙを図示する）

24 時間

適用時間

24 時間
（越える場合は皮膚状態に注
意）

測定部位

・皮膚表面
・角層
・表皮（角層を除く）
・真皮・レセプター液・ドナーチャンバー
・皮膚表面・皮膚・レセプター液場合によっては，塗布部位，塗布外部（セル密着部位），角層，表皮，真皮に分ける．

・皮膚表面
・角層
・表皮（角層を除く）
・真皮・レセプター液

回収率

100±15 %（逸脱なら調査/説明）

RI 使用時の目標は，100±10 %（逸脱なら理由を記載）

100±15 %絶対量（µg/cm2）
吸収率（% of dose）

有限系の場合：皮膚表面量，皮膚内量，レセ
プター液の速度および量ある
いはパーセンテージ．
無限系の場合：透過係数を算出．パーセン
テー ジは必要ない．

絶対量（µg/cm2）
吸収率（% of dose）

算出値

OECD/OCDE

428
Adopted :13 April 2004

OECD GUIDELINE FOR THE TESTING OF CHEMICALS

1.
This test guideline has been designed to provide information on absorption of a test substanceapplied to excised skin. It can either be combined with the OECD Test Guideline for Skin Absorption:
Method (1), or be conducted separately. It is recommended that the OECD Guidance Document for
the Conduct of Skin Absorption Studies (2) be consulted to assist in the design of studies based on this Test
Guideline. The OECD Guidance Document has been prepared to facilitate the selection of appropriate
procedures for use in specific circumstances, to ensure the reliability of results obtained by thismethod.

2.
The methods for measuring skin absorption and dermal delivery can be divided into two
and.
methods on skin absorption are well established and provide
categories:
pharmacokinetic information in a range of animal species. An
method is separately described inanother OECD guideline (1).
methods have also been used for many years to measure skinmethods covered by this Test Guideline haveabsorption. Although formal validation studies of the
not been performed, OECD experts agreed in 1999 that there was sufficient data evaluated to support the
Test Guideline (3). Further details that substantiate this support, including a significant number of
direct comparisons ofandmethods, are provided with the Guidance Document (2). There
are a number of monographs that review this topic and provide detailed background on the use of an
method (4)(5)(6)(7)(8)(9)(10)(11)(12).
methods measure the diffusion of chemicals into andacross skin to a fluid reservoir and can utilise non-viable skin to measure diffusion only, or fresh,
metabolically active skin to simultaneously measure diffusion and skin metabolism. Such methods havefound particular use as a screen for comparing delivery of chemicals into and through skin from different
formulations and can also provide useful models for the assessment of percutaneous absorption in humans.
The in vitro method may not be applicable for all situations and classes of chemicals. It may
be possible to use the in vitro test method for an initial qualitative evaluation of skin penetration. In
certain cases, it may be necessary to follow this up with in vivo data. The Guidance Document (2) should
be consulted for further elaboration of situations where the in vitro method would be suitable. Additional
detailed information to support the decision is provided in an OECD Expert Meeting report (3).
3.

4.
This guideline presents general principles for measuring dermal absorption and delivery of a test
substance using excised skin. Skin from many mammalian species, including humans, can be used. The
permeability properties of skin are maintained after excision from the body because the principal diffusion; active transport of chemicals through the skin has not beenbarrier is the non-viable
identified. The skin has been shown to have the capability to metabolise some chemicals duringpercutaneous absorption (6), but this process is not rate limiting in terms of actual absorbed dose, although
it may affect the nature of the material entering the bloodstream.

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19

428

OECD/OCDE5.
The test substance, which may be radiolabelled, is applied to the surface of a skin sample
separating the two chambers of a diffusion cell. The chemical remains on the skin for a specified time
under specified conditions, before removal by an appropriate cleansing procedure. The receptor fluid issampled at time points throughout the experiment and analysed for the test chemical and/or metabolites.
6.
When metabolically active systems are used, metabolites of the test chemical may be analysed byappropriate methods. At the end of the experiment the distribution of the test chemical and its metabolites
are quantified, when appropriate.
7.
Using appropriate conditions, which are described in this guideline and accompanying guidance
document (2), absorption of a test substance during a given time period is measured by analysis of thereceptor fluid and the treated skin. The test substance remaining in the skin should be considered asabsorbed unless it can be demonstrated that absorption can be determined from receptor fluid values alone.
Analysis of the other components (material washed off the skin and remaining within the skin layers)
allows for further data evaluation, including total test substance disposition and percentage recovery.
8.
To demonstrate the performance and reliability of the test system in the performing laboratory,
the results for relevant reference chemicals should be available and in agreement with published literature
for the method used. This requirement could be met by testing an appropriate reference substance(preferably of a lipophilicity close to the test substance) concurrently with the test substance or byproviding adequate historical data for a number of reference substances of different lipophilicity (e.g.
caffeine, benzoic acid, and testosterone).

Diffusion cell9.
A diffusion cell consists of a donor chamber and a receptor chamber between which the skin ispositioned (an example of a typical design is provided in Figure 1). The cell should provide a good sealaround the skin, enable easy sampling and good mixing of the receptor solution in contact with theunderside of the skin, and good temperature control of the cell and its contents. Static and flow-throughdiffusion cells are both acceptable. Normally, donor chambers are left unoccluded during exposure to a
finite dose of a test preparation. However, for infinite applications and certain scenarios for finite doses,
the donor chambers may be occluded.
Receptor fluid10.
The use of a physiologically conducive receptor fluid is preferred although others may also be
used provided that they are justified. The precise composition of the receptor fluid should be provided.
Adequate solubility of the test chemical in the receptor fluid should be demonstrated so that it does not act
as a barrier to absorption. In addition, the receptor fluid should not affect skin preparation integrity. In aflow-through system, the rate of flow must not hinder diffusion of a test substance into the receptor fluid.
In a static cell system, the fluid should be continuously stirred and sampled regularly. If metabolism isbeing studied, the receptor fluid must support skin viability throughout the experiment.

2/8

20

OECD/OCDE

428

11.
Skin from human or animal sources can be used. It is recognised that the use of human skin issubject to national and international ethical considerations and conditions. Although viable skin ispreferred, non-viable skin can also be used provided that the integrity of the skin can be demonstrated.
Either epidermal membranes (enzymically, heat or chemically separated) or split thickness skin (typically
200-400 μm thick) prepared with a dermatome, are acceptable. Full thickness skin may be used but
excessive thickness ( . > 1 mm) should be avoided unless specifically required for determination of thetest chemical in layers of the skin. The selection of species, anatomical site and preparative technique mustbe justified. Acceptable data from a minimum of four replicates per test preparation are required.
Skin preparation integrity
12.
It is essential that the skin is properly prepared. Inappropriate handling may result in damage to, hence the integrity of the prepared skin must be checked. When skin metabolism isthebeing investigated, freshly excised skin should be used as soon as possible, and under conditions known tosupport metabolic activity. As a general guidance, freshly excised skin should be used within 24 hrs, but
the acceptable storage period may vary depending on the enzyme system involved in metabolisation andstorage temperatures (13). When skin preparations have been stored prior to use, evidence should bepresented to show that barrier function is maintained.
Test substance13.
The test substance is the entity whose penetration characteristics are to be studied. Ideally, thetest substance should be radiolabelled.
Test preparation
14.
The test substance preparation (e.g., neat, diluted or formulated material containing the test
substance which is applied to the skin) should be the same (or a realistic surrogate) as that to whichhumans or other potential target species may be exposed. Any variation from the ‘in-use’ preparation mustbe justified.

15.
Normally more than one concentration of the test substance is used in typical formulations,spanning the realistic range of potential human exposures. Likewise, testing a range of typicalformulations should be considered.
Application to the skin
16.
Under normal conditions of human exposure to chemicals, finite doses are usually encountered.
Therefore, an application that mimics human exposure, normally 1-5 mg/cm2 of skin for a solid and up to
10 Pl/cm2 for liquids, should be used. The quantity should be justified by the expected use conditions, thestudy objectives or physical characteristics of the test preparation. For example, applications to the skinsurface may be infinite, where large volumes per unit area are applied.
Temperature
17.
The passive diffusion of chemicals (and therefore their skin absorption) is affected bytemperature. The diffusion chamber and skin should be maintained at a constant temperature close to
3/8

21

428

OECD/OCDE

normal skin temperature of 32 r 1°C. Different cell designs will require different water bath or heatedblock temperatures to ensure that the receptor/skin is at its physiological norm. Humidity should preferably
be between 30 and 70%.
Duration of exposure and sampling
18.
Skin exposure to the test preparation may be for the entire duration of the experiment or for
shorter times (i.e., to mimic a specific type of human exposure). The skin should be washed of excess test
preparation with a relevant cleansing agent, and the rinses collected for analysis. The removal procedureof the test preparation will depend on the expected use condition, and should be justified. A period ofsampling of 24 hours is normally required to allow for adequate characterisation of the absorption profile.
Since skin integrity may start to deteriorate beyond 24 hours, sampling times should not normally exceed
24 hours. For test substances that penetrate the skin rapidly this may not be necessary but, for test
substances that penetrate slowly, longer times may be required. Sampling frequency of the receptor fluid
should allow the absorption profile of the test substance to be presented graphically.
Terminal procedures
19.
All components of the test system should be analysed and recovery is to be determined. This
includes the donor chamber, the skin surface rinsing, the skin preparation and the receptor fluid/chamber.
In some cases, the skin may be fractionated into the exposed area of skin and area of skin under the cell
, epidermis and dermis fractions, for separate analysis.
flange, and into
Analysis20.
In all studies adequate recovery should be achieved (the aim should be a mean of 100 r10% of
the radioactivity and any deviation should be justified). The amount of test substance in the receptor fluid,skin preparation, skin surface washings and apparatus rinse should be analysed, using a suitable technique.

Data
21.
The analysis of receptor fluid, the distribution of the test substance chemical in the test systemand the absorption profile with time, should be presented. When finite dose conditions of exposure areused, the quantity washed from the skin, the quantity associated with the skin (and in the different skinlayers if analysed) and the amount present in the receptor fluid (rate, and amount or percentage of applieddose) should be calculated. Skin absorption may sometimes be expressed using receptor fluid data alone.
However, when the test substance remains in the skin at the end of the study, it may need to be included in
the total amount absorbed (see Guidance Document, paragraph 66). When infinite dose conditions of
exposure are used the data may permit the calculation of a permeability constant (Kp). Under the latterconditions, the percentage absorbed is not relevant.

4/8

22

OECD/OCDE

428

22.
The test report must include the requirements stipulated in the protocol, including a justification
for the test system used and should, comprise the following:
Test substance:

physical nature, physicochemical properties (at least molecular weight and log Pow), purity(radiochemical purity);
identification information (e.g. batch number);solubility in receptor fluid.

Test preparation:


formulation and justification of use;homogeneity.

Test conditions:









sources and site of skin, method of preparation, storage conditions prior to use, any
pre-treatment (cleaning, antibiotic treatments, etc.), skin integrity measurements, metabolicstatus, justification of use;cell design, receptor fluid composition, receptor fluid flow rate or sampling times andprocedures;details of application of test preparation and quantification of dose applied;
duration of exposure;details of removal of test preparation from the skin, for example, skin rinsing;details of analysis of skin and any fractionation techniques employed to demonstrate skin
distribution;
cell and equipment washing procedures;
assay methods, extraction techniques, limits of detection and analytical method validation.

Results:
 overall recoveries of the experiment (Applied dose { Skin washings + Skin + Receptor fluid +Cell washings);
 tabulation of individual cell recoveries in each compartment;
 absorption profile; tabulated absorption data (expressed as rate, amount or percentage).
Discussion of results.
Conclusions.

5/8

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428

OECD/OCDE

Method. OECD, Paris.

(1)

OECD (2004). Test Guideline 427: Skin absorption:(2)

OECD (2004). Guidance Document for the Conduct of Skin Absorption Studies. OECD, Paris.

(3)

OECD (2000). Report of the Meeting of the OECD Extended Steering Committee for
Percutaneous Absorption Testing, Annex 1 to ENV/JM/TG(2000)5. OECD, Paris.

(4)

Kemppainen BW and Reifenrath WG. (1990). Methods for skin absorption. CRC Press, Boca
Raton.

(5)

Bronaugh RL and Collier, SW. (1991). Protocol for

Percutaneous Absorption Studies, in, RL Bronaugh

and HI Maibach, Eds., CRC Press, Boca Raton, pp. 237-241.
(6)

Bronaugh RL and Maibach HI. (1991).
. CRC Press, Boca Raton.

(7)

European Centre for Ecotoxicology and Toxicology of Chemicals (1993). Monograph No. 20,
Percutaneous Absorption, ECETOC, Brussels.

(8)

Diembeck W, Beck H, Benech-Kieffer F, Courtellemont P, Dupuis J, Lovell W, Paye
Assessment of Dermal Absorption
M,Spengler J, Steiling W (1999). Test Guidelines for
and Percutaneous Penetration of Cosmetic Ingredients, Fd Chem Tox, 37, 191-205.

(9)

Recommended Protocol for
Register, Vol. 61, No. 65.

(10). (1996). Methods for assessing percutaneous
Howes D, Guy R, Hadgraft J, Heylings JR
absorption. ECVAM Workshop Report ATLA 24, 81 R10.

(11)

Schaefer H and Redelmeier TE. (1996). Skin barrier: principles of percutaneous absorption.
Karger, Basel.

(12)

Roberts MS and Walters KA. (1998). Dermal absorption and toxicity assessment. Marcel
Dekker, New York.

(13)

Jewell, C., Heylings, JR., Clowes, HM. And Williams, FM. (2000). Percutaneous absorption andmetabolism of dinitrochlorobenzene in vitro. Arch Toxicol 74: 356-365.

Percutaneous Absorption Rate Studies (1996). US Federal

6/8

24

25

Magnetic stirrer bar

Glass diffusion cell

Support grid

Skin membrane (2.54cm2)

Cell donor chamber(for volatile test substances)

Activated charcoal filterof fresh receptor fluid

Volume maintained by replacement

scintillation or HPLC vials(Auto)pipette/syringe sampling into

specific time-course

Programmed to sample(AUTO)SAMPLER

7/8

Receptor chamber/ fluid maintained at 32°C±1°C in atemperature-controlled water bathOECD/OCDE

428

428

OECD/OCDE

Unabsorbed dose: represents that washed from the skin surface after exposure and any present on the nonocclusive cover, including any dose shown to volatilise from the skin during exposure.
mass of test substance reaching the receptor fluid or systemic circulation within
Absorbed dose:a specified period of time.
The absorbable dose:

represents that present on or in the skin following washing.

8/8

26

SCCP/0970/06

EUROPEAN COMMISSIONHEALTH & CONSUMER PROTECTION DIRECTORATE-GENERAL
Directorate C - Public Health and Risk Assessment
C7 - Risk assessment

SCIENTIFIC COMMITTEE ON CONSUMER PRODUCTS(SCCP)

Opinion on
BASIC CRITERIA FOR THE IN VITRO ASSESSMENT OF
DERMAL ABSORPTION OF COSMETIC INGREDIENTS -updated March 2006

Adopted by the SCCP
during the 7th plenary of 28 March 200627

SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________TABLE OF CONTENTS

28

1.

BACKGROUND…………………………………………………

3

2.

GENERAL PRINCIPLES…………………………………………………

4

3.

PRINCIPLE OF THE TEST…………………………………………………

5

4.

FACTORS AFFECTING DERMAL ABSORPTION AND METHODOLOGY

5.

RESULTS…………………………………………………

10

6.

REFERENCES…………………………………………………

11

7.

ACKNOWLEDGEMENTS…………………………………………………

12

2

….

6

SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

1.

BACKGROUND

As early as June 1999, before any OECD guideline on percutaneous absorption was available,
important scientific articles on the methodology were published [Beck et al. 1995, Diembeck et
al. 1999]. The SCCNFP discussed the scientific progress with external experts in the field andsubsequently adopted its first "Basic criteria for the in vitro assessment of percutaneousabsorption of cosmetic ingredients" [SCCNFP/0167/99]. The publication of such a set of criteria
was seen as a pro-active action of the SCCNFP in order to support and speed up the introductionof in vitro dermal absorption studies of cosmetic ingredients in the evaluation of dossiers of
cosmetic ingredients belonging to Annexes III, IV, VI or VII to Dir. 76/768/EEC. Moreover, theopinion did not only provide basic criteria for the in vitro assessment of dermal absorption of
cosmetic ingredients, but also discussed some general principles and practical points of themethodology.
In December 2000, two draft OECD Guidelines on dermal absorption became available, namely
1) OECD Draft Guideline 427 on skin absorption: in vivo method [OECD 427], and 2) OECD
Draft Guideline 428 on skin absorption: in vitro method [OECD 428]. The combination of theOECD-publication with gained practical experience, has led to the incorporation of the in vitro
dermal absorption study in toxicological dossiers of cosmetic ingredients, plant protection
products, biocides, etc [98/8/EC, 2000/6/EC, Sanco/222/2000].
Subsequently, in order to provide an up to date guidance for the application of the in vitro
methodology for cosmetic ingredients, document SCCNFP/0167/99 was revised and updated in2003 [SCCNFP/0750/03]. Existing documents such as the "Proposed rule for in vitro dermal
absorption rate testing …" [US EPA 1999], the "Guidance document on dermal absorption"[Sanco/222/2000], the "Draft Guidance Document for the conduct of skin absorption studies"[OECD 2000], and the "Technical Guidance Document on Risk Assessment part 1" [ECB 2003]
were taken into consideration.
The SCCP has noticed that several dossiers have failed to fulfil the requirements as described in
document SCCNFP/0750/03. In particular, a recent revision of all hair dye dossiers revealed anumber of problems [Pauwels and Rogiers 2004].
The following actions were undertaken:-

The SCCP established a working group, consisting of SCCP members and external experts
in the field of dermal absorption. The present opinion results from the work of the expert
group, whose task was to reach a consensus concerning a revision of the "SCCP basic
criteria for the in vitro assessment of dermal absorption of cosmetic ingredients".

-

In order to make the SCCP criteria more widely known, it is intended to expand thechapter on dermal absorption in the SCCP Notes of Guidance, 6th revision.

329

SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

2.

GENERAL PRINCIPLESDefinitions
The definitions with respect to dermal absorption slightly diverge in different official documents.
Hereunder, the definitions as proposed by the WHO [WHO 2005] are given here:
The percutaneous/dermal absorption process is a global term which describes the passage ofcompounds across the skin. This process can be divided into three steps:-

penetration, which is the entry of a substance into a particular layer or structure such as
the entrance of a compound into the stratum corneum;-

permeation, which is the penetration through one layer into another, which is both
functionally and structurally different from the first layer;-

resorption which is the uptake of a substance into the vascular system (lymph and/or
blood vessel), which acts as the central compartment.

The purpose of dermal absorption studies of cosmetic ingredients is to obtain qualitative and/or
quantitative information on the amounts that may enter, under in-use conditions, into thesystemic compartment of the human body. These quantities can then be taken into consideration
to calculate the margin of safety using the NOAEL of an appropriate repeated dose toxicity study
with the respective substance.
Justification for the use of in vitro dermal absorption studies on isolated skin is based on the factthat the epidermis, in particular the stratum corneum, forms the principal in vivo barrier of theskin against penetration and uptake of xenobiotics in the body.
Under in vivo conditions, the microcirculatory system (blood and lymph vessels) may carrycompounds from the dermis into the central compartment (resorption). In vitro, themicrocirculation is compromised, thus the potential resorption of a compound cannot beadequately studied in such a setting.
The dermal tissue may retain penetrating compounds that, in vivo, would have been removed
into the systemic compartment. Thus, either the dermis must be removed prior to in vitro
investigations (so-called split-thickness skin) or possible in vitro retention in the dermis andliving epidermis (without stratum corneum) must be taken into account when interpreting the invitro results.
The epidermis renews by continuous outward proliferation, differentiation and desquamation.
About one layer of corneocytes is shed off per day. After topical application, xenobiotics
detected in vitro in the skin, particularly in the stratum corneum and the pilosebaceous units,
might in vivo have been lost from the skin via desquamation or sebum secretion, respectively.
Because these processes are not present in vitro, the final epidermal (stratum corneum) levels in
vitro could be elevated compared with the corresponding in vivo levels.

30

4SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

According to these principles, the following should be included in the protocol for in vitro
dermal absorption studies:i.

Studies should be performed on appropriate standardised skin preparations. The respective
choice should be justified in the protocol. The WHO recommends human skin as the gold
standard.

ii.

At the end of the experiment, a full mass balance should be performed.

iii.

When considerable cutaneous metabolism of the test compound occurs in vivo, further
studies may be necessary. It should be noticed that frozen skin preparations may lack the
enzyme systems for biotransformation of the test compound and may not provide an
accurate picture of the formation of metabolites and their dermal absorption.
Therefore in vitro studies using frozen skin may not provide complete information on thedermal absorption of compounds that undergo biotransformation in the skin nor on theirpotential metabolite(s) formed. The role of cutaneous biotransformation in the absorption
process is still a matter of scientific debate.

iv.

Sometimes an irreversible binding of an ingredient to the epidermis may occur, followedby elimination through in vivo desquamation of the skin surface. When this mechanism isassumed, it must be documented by separate experiments.

3.

PRINCIPLE OF THE TESTAt present only skin preparations of natural origin may be used. Although the quality of cultured
or reconstituted skin has improved importantly during the last years [Coquette et al. 2000], thesecultures do not possess a complete barrier function comparable to that of living skin.
OECD Guideline 428 should be followed as close as possible, taking into account the guidance
given here. Any deviation from the OECD or SCCP guidelines should be documented andjustified by appropriate scientific argumentation.
The test substance should be applied in an appropriate formulation on the skin sample which isplaced in a diffusion cell (cf. 4.1). The skin is then positioned between the upper and lower
chambers of the cell. Diffusion cells may be of static or flow-through design. The integrity of thebarrier should be checked by an appropriate method. The test sample should remain in contact
with the skin on the donor side for a defined period of time, corresponding to the typical use of
the cosmetic end product, such as leave-on or rinse-off conditions. The receptor fluid should besampled at an early time point (e.g. after 30 minutes), at the end of the experiment and at
appropriate time points in between in order to obtain an absorption-time profile. A justificationof the procedure used (static or flow-through conditions) should be provided. The skin and/or
fluid samples should be analysed by appropriate and validated analytical methods, such as liquid
scintillation counting, HPLC, GC or other suitable methods. Information on the sensitivity andrepeatability / time-different intermediate precision of the analytical method(s) should be
provided.

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

4.

FACTORS AFFECTING DERMAL ABSORPTION AND METHODOLOGY

Dermal absorption can be affected by several factors : e.g. physical and chemical properties ofthe substance, type and composition of the formulation, occlusion, concentration of thesubstance in the formulation, exposure pattern, skin site of the body and technical aspects of therespective in vitro test [Howes et al. 1996, Schaefer and Redelmeier 1996, ECETOC 1993].
In the following section, an overview is given of factors that may affect dermal absorption in in
vitro dermal absorption studies.
4.1. Diffusion cell design
The diffusion cell consists of an upper donor and a lower receptor chamber, separated by theskin preparation under investigation. The stratum corneum faces the donor chamber. Diffusion
cells should consist of inert non-adsorbing material. Temperature control of the receptor fluid is
crucial throughout the experiment. The skin surface temperature in the diffusion cell should be
kept at the in vivo skin temperature of 32 r 1°C. Additional dermal absorption studies may berequired in some specific cases, e.g. when substance exposure at a higher skin temperature may
be expected. The receptor fluid in static cells should be well-stirred throughout the study. The
advantage of using a flow-through system is continuous sampling; the advantage of using a static
system is the increase in sensitivity for test substances only poorly penetrating the skin.
4.2. Receptor fluid
The composition of the receptor fluid is chosen so that it does not limit the extent of diffusion of
the test substance, i.e. the solubility and the stability in the receptor fluid of the chemical underinvestigation have to be guaranteed. Saline or buffered saline solutions are commonly used forhydrophilic compounds. For lipophilic molecules, serum albumin or appropriate
solubilisers / emulsifiers are added in amounts which must not interfere with membrane
integrity. The fluid should not interfere with the analytical procedure.
As a general rule, the receptor fluid should have a physiological pH. Any deviation from thisprinciple should be justified; e.g. in the case of 50/50 ethanol/water (as proposed in the OECD
Guideline), evidence should be included in the dossier, showing that this does not significantly
affect the integrity of the skin.
In order to avoid serial non-detects at early sample points, the receptor fluid volume should be
kept to a minimum.
The receptor fluid, preferably degassed in order to avoid formation of air bubbles during theexperiment, should be thoroughly stirred (static cells) or continuously replaced (flow-throughcells) during the entire experiment. The choice of static or flow-through conditions in thereceptor cell should be made on a compound-by-compound basis, depending on its theoreticalabsorption properties and the objective of the study. The choice of the test system should bejustified in the study report. The amount of penetrated substance in the receptor fluid should notexceed 10% of its saturation level at any time, in order to minimise interference with the freediffusion process that could produce an underestimation of dermal absorption. The substanceshould be stable in the receptor fluid for the duration of the in vitro test and the subsequent
analysis.

32

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SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

4.3. Skin preparations
Human skin is obviously the best choice but is not always readily available. Alternatively, pig
skin may be used because it shares essential permeation characteristics with human skin. Rat
skin is 2 to 10 times more permeable than human skin [Ross et al. 2000] which may result in an
overestimation of dermal absorption. Mouse, guinea pig or rabbit skins have no adequate barrier
function comparable to that of human skin. The use of cultured or reconstructed skin models isunder development and those systems are not yet advised for in vitro testing on the basis of theirinsufficient barrier function [Coquette et al. 2000].
The origin of skin samples should be specified in the respective report. The following
information is required:--

Species: by preference human or pig skin should be used. Although rodent skin is not
representative for human skin [ECETOC 1993], the results of such studies will not bedisregarded, since these models produce an overestimation of the dermal absorption (worstcase).
Skin location: abdomen, leg or breast (human skin); abdomen, breast, back, flanks or ears
(pig skin).
Gender and age: although these factors are not believed to be important variables, theyshould be stated.
Fresh/frozen skin: when significant biotransformation of the test compound in the skin is
expected, freshly excised, viable skin should be used [Diembeck et al. 1999].
Details on preservation and storage conditions of the skin should be specified (e.g. skin canbe stored in aluminium foil at -20°C or lower) [Howes et al. 1996, Bronaugh et al. 1986].
During transport skin samples should be kept at or below 4°C.

Skin samples that may be used are split-thickness (200-500 μm) or full-thickness (500-1000 μm)
skin preparations [Sanco/222/2000]. Dermatomed skin is often used. Skin thickness should bemeasured by an appropriate method, which should be described in the report. The skin samples
should be prepared to fit the experimental cell.
-

For human skin: split-thickness skin should be the general rule. If for a specific reason,full-thickness is required, this should be justified.
For pig skin:since it is technically more difficult to obtain intact split-thickness skin,this could justify the use of full-thickness skin.

When epidermal membranes are used for the in vitro dermal absorption study, the reason for thisshould be justified. Epidermal membranes are sometimes quite fragile and some mass balancetechniques (e.g. tape stripping) cannot be applied to this model. It must also be mentioned thatepidermal membranes may overestimate human in vivo skin absorption [Van de Sandt et al.
2000].
The minimum skin area to be covered is considered to be 0.64 cm².

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

4.4. Skin integrity
Barrier integrity is crucial for the experiment, and must therefore be measured. This is achieved
by either measuring the penetration of a marker molecule, e.g. tritiated water, caffeine or
sucrose, or by physical methods, such as determination of TEWL (Transepidermal Water Loss)or TER (Transcutaneous Electrical Resistance). Data obtained should be reported.
4.5. Skin temperature
Because the rate and extent of skin absorption is temperature-dependent, the skin disctemperature should be maintained constant (32 ± 1°C, corresponding to the normal human skinsurface temperature). The method of temperature maintenance should be described in the report.
4.6. Test substance
The relevant physical and chemical data (e.g. MW, log Pow, solubility, stability, and pKa of thetest substance) should be given.
The purity of the test substance should be described and should be comparable to that of thesubstance in marketed products (see 4.10). It is recognised that radio-synthesis of [14C]- or [3H]labelled substances may result in a somewhat different purity and/or impurity profile than that ofsubstances produced by large-scale chemical production.
As mentioned under 4.2, the solubility and stability of the test substance in the receptor fluid forthe entire test duration should be documented.
A separate set of criteria dealing with dermal absorption issues related
nanoparticles/nanotubes will, if necessary, become available as an annex to this Opinion.

to

4.7. Preparation of the dose and vehicle / formulation
The dose and vehicle / formulation should be representative for the in use condition(s) of thefinished cosmetic product. The quantitative composition of every formulation used during theexperiment, should be given.
More than one concentration of the test substance, including the highest requested one, is used intypical formulations spanning the range of human exposure. These concentrations should beselected in such a way that the range of the linear curve of concentration versus dermal
absorption is demonstrated.
The stability of the test substance under the foreseeable conditions of application and usage must
be ascertained.

34

8

SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

4.8. Dose and volume of test substanceThe dose of the test formulation as well as its contact time (exposure) with the skin should
resemble use conditions. The amount of the formulation to be applied is adapted to the consumer
use values described in the Notes of Guidance [SCCNFP/0690/03], and usually is between
2-5 mg/cm2 for solids and semi-solid preparations, and up to 10 μl/cm2 for liquids. For oxidative
hair dye formulations, 20 mg/cm2 is applied. Deviations should be explained. The volume offormulation used should be appropriate to spread the sample homogeneously over the skinsurface. This depends on the viscosity and lipophilicity of the formulation. Both mass andvolume applied should be stated in the test report.
4.9. Study period and sampling
The exposure time and sampling period(s) should be defined in the protocol. The normal
exposure time is 24 hours with regular sampling intervals. Longer duration of the study may
result in membrane deterioration and requires careful control of membrane integrity. The
exposure time should be consistent with the intended use of the cosmetic formulation. E.g. foroxidative hair dye formulations, the time of contact could vary from 15 to 45 minutes or evenmore according to the in-market use. The skin surface will also be rinsed using a procedure
mimicking the consumer situation. Sampling of the receptor fluid is continued until e.g.
24 hours.
The frequency of sampling should be chosen adequately to allow the determination of theextent/rate of absorption and the absorption profile. Kinetic measurements have to be obtained
for at least 6 post-application time points, including one early time point (30 minutes), in order
to be able to estimate the absorption kinetics. For rinse-off products, measurements after rinsing
have to be taken. The full sampling procedure must be described in the report.
4.10. Analytical methods
Appropriate analytical techniques, e.g. scintillation counting, HPLC or GC, should be used.
Their validity, sensitivity and detection limits should be documented in the report. When an
increase of sensitivity is needed, the test substance should, whenever possible, be radio-labelled.
Qualitative or semi-quantitative methods, such as micro-autoradiography, may be useful tools
for skin distribution assessments.
4.11. Data collection
The test compound must be determined in the following compartments:-

Product excess on the skin (dislodgeable dose)
Stratum corneum (e.g. adhesive tape strips)
Living epidermis (without stratum corneum)
DermisReceptor fluid

For the reason of an appropriate mass balance, it is necessary to check for substance adsorbed tothe equipment (included in rinsing solutions and/or compartments).

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Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

4.12. Mass balance analysis / recovery
The mass balance of the applied dose must be determined.
The overall recovery of test substance (including metabolites) should be within the range of
85-115%. Lower or higher recovery rates should be investigated and/or explained.
4.13. Variability / validity / reproducibility
-

-

-

-

5.

The variability of dermal absorption studies depends on the penetration rate of a particularingredient; the lower the penetration rate, the higher the variability. This high variability is
due to known intra-individual and inter-individual characteristics of the stratum corneum
barrier. The relative variability of the method should be documented.
Experience has shown that the high variation in dermal absorption may partly be explainedby differences in skin samples. Therefore skin samples should be taken from a suitable
anatomical site and the study should use a sufficient number of samples and replicates.
The technical ability of the performing laboratory and the validity of the method usedshould be assessed at regular intervals, at least twice per year, by using referencecompounds like caffeine or benzoic acid. These data should be included in the study report[OECD 2004, Van de Sandt et al. 2004].
The results of the dermal absorption studies should be reproducible. A minimum of six
evaluable samples (human or pig skin), from each of at least three donors, should be used
per dose tested. The coefficient of variation should be less than 30 %. If statisticalevaluation is not possible, the highest observed penetration value will be used in thesystemic exposure dosage (SED) calculation.
For oxidative hair dyes, the relevant combination(s) of hair dye, coupler(s) anddeveloper(s) should be tested.

RESULTS

Dermal absorption should be expressed as an absolute amount [μg/cm2 of skin surface] and as apercentage of the amount of test substance contained in the intended dose applied per squarecentimetre of skin surface.
The amounts of penetrated substance(s) found in the receptor fluid are considered to besystemically available. The epidermis (except for the stratum corneum) and dermis are
considered as a sink, therefore the amounts found in these tissues are considered as absorbed andare added to those found in the receptor fluid. The amounts that are retained by the stratum
corneum at the time of sampling are not considered to be dermally absorbed, and thus they are
not expected to contribute to the systemic dose.
The absorption rate and mass balance should be calculated separately for each diffusion cell.
Only then, the mean r S.D. and median with 10% and 90% percentiles should be calculated.
All measurements, statistical processing and obtained kinetic curves have to be provided.
In case the results are derived from an inadequate in vitro study, the default value of 100%
absorption is applied.
36

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SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

6.

REFERENCES

2000/6/EC - Twenty-fourth Commission Directive 2000/6/EC of 29 February 2000 adapting totechnical progress Annexes II, III, VI and VII to Council Directive 76/768/EEC on theapproximation of the laws of the Member States relating to cosmetic products. Official Journal L
56, 01/03/2000 p.42.
Beck H., Benech-Kieffer F., Bracher M., Diembeck W., Dupuis J., Lovell W., Steiling W.
Cosmetic ingredients : Guidelines for percutaneous absorption / penetration.COLIPA
Guidelines, 1-36 (1995)
Bronaugh R.L., Stewart R., Simon M. Methods for in vitro percutaneous absorption studies
VII : use of excised human skin. Journal of Pharmaceutical Sciences 75, 1094-1097 (1986).
Coquette A., Berna N., Poumay Y., Pittelkow M.R. The keratinocyte in cutaneous irritation andsensitisation. In : Biomedical Modulations of Skin Reactions in Dermal and Transdermal Drug
Delivery, Chapter 8. CRC Press, pp.125-143 (2000)
Diembeck W., Beck H., Benech-Kieffer F., Courtellemont P., Dupuis J., Lovell W., Paye M.,
Spengler J. and Steiling W. Test guidelines for in vitro assessment of dermal absorption andpercutaneous penetration of cosmetic ingredients. Food & Chemical Tox., 37, 191-205 (1999).
Directive 98/8/EC of the European Parliament and of the Council of 16 February 1998
concerning the placing of biocidal products on the market Official Journal L 123, 24/4/98 p. 1.
ECB (European Chemicals Bureau) Technical Guidance Document on Risk Assessment insupport of Commission Directive 93/67/EEC on Risk Assessment for new notified substances,
Commission Regulation (EC) No 1488/94 on Risk Assessment for existing substances andDirective 98/8/EC of the European Parliament and of the Council concerning the placing of
biocidal products on the market. Doc. EUR 20418 EN/1, European Communities (2003).
ECETOC Percutaneous absorption. European Centre for Ecotoxicology and Toxicology ofChemicals (ECETOC), Monograph No 20, Brussels (1993).
Howes D., Guy R., Hadgraft J., Heylings J., Hoeck U., Kemper F., Maibach H., Marty J-P.,
Merk H., Parra J., Rekkas D., Rondelli I., Schaefer H., Täuber U. and Verbiese N. Methods forassessing percutaneous absorption, ECVAM Workshop Report n. 13. Alternatives To Laboratory
Animals 24, 81-106 (1996).
OECD 427 - OECD Guideline for testing of chemicals - Guideline 427 : Skin absorption: In vivo
method Organization for Economic Cooperation and Development, Paris, adopted
13 April 2004.
OECD 428 - OECD Guideline for testing of chemicals - Guideline 428 : Skin absorption: In
vitro method Organization for Economic Cooperation and Development, Paris, adopted
13 April 2004.
OECD Guidance Document for the Conduct of Skin Absorption Studies. Organization for
Economic Cooperation and Development (OECD), Environment Directorate, OECD
Environmental Health and Safety Publications, Series on Testing and Assessment No. 28, Paris(2004).

1137

SCCP/0970/06
Basic criteria for the in vitro assessment of dermal absorption of cosmetic ingredients
updated March 2006____________________________________________________________________________________________

Pauwels M. and Rogiers V. How to improve the quality of a European cosmetic ingredients'
dossier? Oral presentation given during the Scientific Forum of the COLIPA (The European
Cosmetic Toiletry and Perfume Association) General Assembly 2004, Amsterdam, 24/06/2004.
Ross J.H., Dong M.H. and Krieger M.I. Conservatism in pesticide exposure assessment.
Regulatory Toxicology and Pharmacology 31, 53-58 (2000).
Sanco/222/2000 – Guidance Document on Dermal Absorption European Commission, Healthand Consumer Protection Directorate-General Doc. Sanco/222/2000 rev 6, of 27 November
2002.
SCCNFP/0167/99, Final : Basic Criteria for the in vitro assessment of percutaneous absorptionof cosmetic ingredients, adopted by the SCCNFP during the 8th plenary meeting of
23 June 1999.
SCCNFP/0690/03, Final : Notes of Guidance for Testing of Cosmetic Ingredients for Their
Safety Evaluation, adopted by the SCCNFP during the 25th plenary meeting of 20 October 2003.
SCCNFP/0750/03, Final : Basic Criteria for the in vitro assessment of dermal absorption of
cosmetic ingredients, updated October 2003, adopted by the SCCNFP during the 25th plenary
meeting of 20 October 2003.
Schaefer H. and Redelmeier T.E. Skin Barrier, Principles of Percutaneous Absorption, Karger,
Basel (1996).
US EPA (United States Environmental Protection Agency) Proposed rule for in vitro dermal
absorption rate testing of certain chemicals of interest to occupational safety and healthadministration. Federal Register, Volume 64, Number 110, June 9 (1999).
Van de Sandt J.J.M., Meuling W.J.A., Elliott G.R., Cnubben N.H.P. and Hakkert B.C.
Comparative in vitro – in vivo percutaneous absorption of the pesticide propoxur. Toxicological
Sciences 58, 15-22 (2000).
Van de Sandt J.J.M., vanBurgsteden J.A., Carmichael P.L., Dick I., Kenyon S., Korinth G.,
Larese F., Limasset J.C., Maas W.J.M., Montomoli L., Nielsen J.B., Payan J.-P., Robinson E.,
Sartorelli P., Schaller K.H., Wilkinson S.C., Williams F.M. In vitro predictions of skinabsorption of caffeine, testosterone, and benzoic acid: a multi-centre comparison study. Regul
Toxicol Pharmacol 39, 271-281 (2004).
WHO (World Health Organisation) Kielhorn J., Melching-KollmuE S., Mangelsdorf I. Dermal
Absorption. WHO / IPCS Environmental Health Criteria, Draft February 2005, accessible
through http://www.who.int/ipcs/methods/dermal_absorption/en/ (consulted Dec 2005).

7.

ACKNOWLEDGEMENTS

Members of the working group are acknowledged for their valuable contribution to this opinion.
The members of the working group are:Prof. G. Degen
Dr. B. Jazwiec-Kanyion
Prof. V. Kapoulas
Prof. J.-P. Marty
Prof. T. Platzek
Dr. S.C. Rastogi
38

Prof. J. Revuz
Prof. V. Rogiers
Prof. T. SannerProf. G. Speit
Dr. J. van Engelen
Dr. I.R. White
12(chairman and rapporteur)Guidelines for Percutaneous
Absorption/Penetration

Edition of 1997

39

CONTENTS
1. BackgroundPage
1

2. Introduction

1

3. Initial considerations

2

4. Principle of the test2

5. Description of the method
5.1 Penetration cell design
5.2 Receptor fluid
5.3 Skin disks
5.4 Membrane integrity
5.5 Test substance
5.6 Preparation of the dose2

6. Procedure
6.1 Application of test substance
6.2 Fluid dynamics
6.3 Temperature
6.4 Study time
6.5 Sampling
6.6 Analysis

4

7. Data and Reporting
7.1 Data
7.2 Test report
7.3 Implications for safety evaluation

5

8. References

6

9. Standard Protocols
1. Excised human skin
2. Pig skin
3. Excised pig skin
4. Rat skin

7
7
15
23
32

Although every effort is made to ensure that the information and concepts presented here are correct to the best knowledge of theauthors and editors, neither they nor Colipa accept responsiblity for any actions taken on the basis of this information, nor accept
any liability for any omissions or errors this publication may contain.
All rights reserved. No part of this publication may be reproduced or translated in any form, mechanical, electronic or otherwise,
without prior permission of Colipa.

40

2nd edition - August 1997

1 . B AC KG RO U N D
The 6th Amendment to the Cosmetics Directive (93/35/EEC) includes a potential ban on the use ofanimals in the testing of cosmetic products and their ingredients, from January 1, 1998.
With regard to the assessment of cutaneous absorption/penetration properties of substances, in vitro
methodologies are recommended for ethical reasons and feasibility. Excised and reasonably
standardised skin is easily obtainable from various species whereas human skin from individuals isavailable only under favourable circumstances and in limited quantities from surgical or postmortem sources.
The value of organotypic skin models for penetration studies has to be demonstrated by further
research. The viability of the skin is not a pre-requisite for penetration testing, since the processdepends on passive diffusion and not on active transport. Furthermore, biotransformation is of minor
interest and importance in the majority of cases. In the exceptional case of relevance,
biotransformation can be monitored.

2 . I N T RO D U C T I O N
Percutaneous penetration is the passage of material from the stratum corneum surface of the skin to
the systemic circulation. Percutaneous absorption is the passage of topically applied materials into
the skin.
In vitro methods used now in many laboratories, utilise transport across full- or split-thickness
animal or human skin to a receptor fluid reservoir. It is possible to estimate in vivo absorption by
extrapolating from suitable in vitro data.
This guideline for the testing of percutaneous penetration in vitro describes a general procedure formeasuring the penetration of test substances through excised mammalian skin. The barrier propertiesof skin are usually maintained after excision from the body and appropriate storage in a freezer for
up to 3 months, since penetration is driven by passive diffusion and there is no evidence for active
transport (ref. 5). Therefore, skin viability is not a prerequisite for these studies. The principal
diffusion barrier has been identified as the stratum corneum, the integrity of which has to becontrolled.
Details of the procedures are presented in the form of examples, in the appendix (section 9). The
appendix contains examples of the following standard protocols, according to a ranking in terms of
relevance to mimic human conditions (ref. 6):- human skin
- pig skin
- rat skin141

3. INITIAL CONSIDERATIONS
The in vitro methods described in these guidelines provide information in all cases where the stratum
corneum is the rate limiting barrier to percutaneous absorption and are, therefore, applicable to thosecompounds which are sufficiently soluble in the intended receptor fluid. There is considerable
experience to allow these in vitro methods to be used for the assessment of percutaneousabsorption/penetration and for the prediction of the in vivo situation (ref. 1). The use of appropriately
prepared skin membranes (full thickness, split-thickness and isolated epidermis) may allow themeasurement of the amount of substance delivered to the systemic circulation and the amountdelivered to the epidermis or dermis (ref. 6).
The advantages of the in vitro method are that it can be used with skin from human and other species,
it does not need live animals and it is suitable for compounds extensively metabolised in the body.
The limited supply of human skin means that the routine use of this material is unlikely and hence
animal skin will normally be used. There are now many comparisons in the literature for predictionsof in vivo absorption/penetration from in vitro data to be made with some confidence for the majority
of compounds (ref. 1 to 4).

4. PRINCIPLE OF THE TESTThe test substance, either as such or dissolved in an appropriate solvent or galenic formulation,
thereby yielding the test sample, is applied to the intact surface of the skin disk separating the upperand lower chambers of a diffusion cell, which can be run in static or flow-through mode.The test
sample remains in contact with the skin on the donor side for a defined period of time (leave-on orrinse-off respectively, depending on the intended use conditions). The receptor fluid is sampled onceat the end of the experiment or at various time points in between. The skin and/or fluid samples areanalysed by an appropriate method (e.g. scintillation counting, HPLC, GC). The integrity of thebarrier should be checked by an appropriate method (see paragraph 5.4).

5. DESCRIPTION OF THE
METHOD
5.1 Penetration cell design
The penetration cell consists of the upper donor and the lower receptor chamber, separated by a skinmembrane. The epidermis faces the donor chamber and the lower surface the receptor chamber. The
cells must be made from an inert and non-absorbing material (e.g. glass or PTFE). Temperature
control is crucial throughout the experiment and it should be maintained at in vivo skin conditions(see paragraph 6.3). The receptor fluid must be well-mixed throughout the experiment. Samplingshould be feasible without interrupting the experiment, by appropriate cell design.

422

5.2 Receptor fluid
The composition of the receptor fluid should not limit the extent of penetration of the test substance,
i.e. the theoretical total solubility should be guaranteed. The receptor fluid must not affect the
integrity or alter the permeability properties of the skin. A saline or buffered saline solution isrecommended for hydrophilic compounds. For lipophilic molecules, addition of serum albumin orother appropriate solutors, such as non-ionic surfactants, is recommended. The appropriate receptor
fluid volume is determined by the solubility and analytical detectability of the test substance. It canbe adapted by choosing a receptor chamber of adequate volume (static) or by varying the setting of
the pump (flow-through).

5.3 Skin disks
The test should be carried out with an appropriate number (i.e. minimum six) of skin disks of similar
integrity. Split-thickness skin (epidermis and upper dermis obtained by dermatomisation or
epidermis only obtained by pre-treatment with heat, enzymes or chemicals) may be used. Fullthickness skin may be used provided that the skin compartments are analysed in detail. Details on
preparation are found in the standard protocols. In case of lipophilic compounds split-thickness skin(≤ 1mm thick layer) or epidermal preparations are to be preferred to limit the dermal retention invitro. The skin should be clipped before preparing membranes. Skin thickness should be measured
by an appropriate method.

5.4 Membrane integrity
Since barrier integrity is crucial for the experiment, it should be checked by measuring the skinpenetration of a marker molecule, e.g. tritiated water, for which suitable historical control data are
available. Alternatively, the integrity can also be tested with a non-radioactive probe, e.g. caffeine,or with physical methods like TEWL or TER (Transepidermal Water Loss or Transdermal Electrical
Resistance respectively). Details of such procedures are given in the appendix.

5.5 Test substance
Toxicological and physico-chemical data, purity, solubility, stability, octanol-water partition
coefficient and analytical methods and their detection limits should be known for the test substance.
Relevant information on the formulation in which the test substance is used should be available.

5.6 Preparation of the dose
Depending upon the purpose of the experiment, the test substance may be used as such, in solution
or in formulation, where appropriate, to allow good contact with the skin or to simulate the intended
use conditions. The stability of the test sample and substance under the proposed conditions ofadministration and usage should be known.

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6 . P RO C E D U R E
6.1 Application of test substanceThe dose as well as the contact time with the skin should mimic the intended use conditions as
closely as possible or be higher to obtain relevant safety data under exaggerated use conditions (forassessment of foreseeable misuse). The amount of the formulation to be applied should be adapted
whenever possible to the consumer use values published by Colipa (doc. 93/067). If permeability
constants are being determined then infinite dose conditions are required.

6.2 Fluid dynamics
The receptor fluid, preferably degassed (e.g. by sonication), must be thoroughly stirred at all times
or be continuously replaced in flow through chambers. The choice of static or flow-throughconditions in the receptor cell should be made on a compound-by-compound basis, depending on its
absorption properties and the goal of the study. It must be ensured that the amount of penetrant in
the receptor fluid is less than 10% of its saturation level, in order to prevent significant backpenetration and hence underestimation of absorption.

6.3 Temperature
Because the rate and extent of skin absorption is temperature dependent, the skin temperature should
be maintained constant (30-32°C ± 1°C) either by use of penetration chambers with water jackets or
temperature-controlled incubators.

6.4 Study time
The study time is determined by the subsequent reasonable characterisation of the absorption
profiles and the intended use conditions. The exposure time and sampling period should be definedin the protocol. A normal study time of 24 hours is recommended.

6.5 Sampling
The frequency of sampling will depend on the rate and extent of dermal penetration, but should be
sufficient to allow the extent or rate of penetration and/or the profile to be determined.
Where appropriate, the surplus of non-penetrated test substance should be determined in skinsamples, rinsings and cell washings. The overall recovery of test substance should be at least 85%.
If low recoveries of the test substance are obtained with all cells, a search should be conducted todetermine the cause(s) which may include binding to proteins, to penetration cell surface andtubings, as well as possible evaporation or loss by chemical reaction.

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6.6 Analysis
The receptor fluid must be analysed and, if possible, an analysis should be made of the amounts
found in individual skin layers and on the skin surface. Suitable analytical procedures must be used,
e.g. scintillation counting, HPLC or GC.

7 . D ATA R E P O R T I N G
7.1 Data
The absorption profile normally is determined up to 24 hours post application with usually six cellsof similar barrier integrity. When adequate data are available, the lag time and the absorption rates
may be calculated. Permeability constants can only be calculated from infinite dose experiments.

7.2 Test report
The test report should include the following information:• Test substance:- chemical structure, physico-chemical properties and purity;- identification data.
• Vehicle (if appropriate):- type of formulation;- justification for choice of formulation.
• Test conditions:- source and site of skin sample, method of preparation, integrity;- details of test sample preparation, final concentration, stability and homogeneity of the
preparation, especially when blended with radiolabelled test substance;- details of the administration of the test substance (exposure time and conditions, e.g. occlusion
or not, rinse-off, leave-on);- justification for choice of skin source and skin preparation;- analytical method, sample clean-up, recovery and detection;- overall recovery of material, if possible.
• Reporting of results:- tabulation of individual results at each time point;- lag times, absorption rates, absorption profiles, if appropriate.

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Data can be presented in various ways. Depending on the experimental set-up, percent dose,
micrograms, micromoles or a penetration2 constant can be displayed. The presentation of
cumulated percent dose or micrograms/cm in tables and graphs is recommended. Graphs areprepared with appropriate computer programs which may be equipped with functions forstatistical treatment of data.
• Statistical methods• Discussion of results and conclusions:
It should be remembered that percentage absorbed values are dependent on specific exposureand formulation conditions. In some cases, a permeability constant may provide usefulinterpretation of the results.

7.3 Implications for safety evaluation
Percutaneous absorption/penetration is a key parameter for the safety evaluation of cosmetic
ingredients, as well as of the finished products containing them. Determination ofabsorption/penetration allows the interpretation of systemic toxicity data in terms of exposure via
topical application. Furthermore, the utilisation of the procedures described in this guideline will
provide a suitable substitute for in vivo experiments to measure percutaneous absorption/penetration.
Depending on the outcome of such studies, with the context of the overall safety evaluation, further
animal toxicity studies would be minimised. The results of such studies may also provide guidanceon the optimisation of the design of further required tests.

8. REFERENCES
1. Scott R.C. & Ramsey J.D., J. Investig. Dermatol., 89, pp. 142-146, 1987.
2. Scott R.C., in Biological Monitoring for Pesticide Exposure: Measurement, Estimation, 1989.
3. Grissom R.E., in Prediction of Percutaneous Penetration - Methods, Measurements, Modelling,(Eds. Scott R.C., Guy R.H. and Hadgraft J.), IBC Technical Services, London, pp. 73-83, 1990.
4. Bronaugh R. L., Stewart R. & Simon M., Methods for in vitro percutaneous absorption studies
VII: use of excised human skin, J. Pharm. Sci., 75, 11, 1986.
5. Bartnik F.G., Reddy A.K., Klecak G., Zimmermann V., Hostynek J.J., Künstler K., Percutaneousabsorption, metabolism and hemolytic activity of n-Butoxyethanol, in Fundam. and Applied Tox.,
8, 59-70, 1987.
6. ECETOC Monograph N° 20.

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S TA N D A R D P R O T O C O L
P E R C U TA N E O U S A B S O R P T I O N /P E N E T R AT I O N I N V I T R O
EXCISED HUMAN SKIN
Author:Florence BENECH-KIEFFER - L'OREAL,
Aulnay sous Bois, FRANCE
April 1995

CONTENTS

Pages

I. Introduction

8

II. Materials

8

III. Methods
III.1 Preparation of the formulation

88

III.1.1 Unlabelled test compounds
III.1.2 Radiolabelled test compounds

III.2 Preparation of the samples of human skin9

III.2.1 Origin and storage of biological samples
III.2.2 Preparation of skin discs
III.2.3 Control of skin disc thickness
III.2.4 Specific recommendations for the use of human skinIII.3. Preparation of the diffusion cells

11

III.3.1 Static mode or dynamic mode
III.3.2 Receptor fluid

III.4. Test of skin integrity
III.5. Application of formulations
III.6. Start of the experimental setup and collection of fractions
III.7. Study time
III.8. Analysis

11
12
12
12
12

III.8.1 Unlabelled test compounds
III.8.2 Radiolabelled test compounds

III.9. Presentation of the results
III.10. Interpretation of the results for safety evaluations
III.11. Study report
III.12. Archives

IV. Literature references

13
13
13
13
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I . I N T RO D U C T I O NThis standard protocol describes in vitro percutaneous absorption/penetration studies of cosmetic
ingredients through human skin. Such studies are performed to measure the amount of test
compound penetrating through the skin barrier for safety evaluation. Recommendations published by
an AAPS-FDA panel on percutaneous absorption studies of drugs have been taken into account forthis protocol (ref. 1).
The in vitro percutaneous absorption/penetration of the test compound is assessed using samples ofisolated human skin placed in static or dynamic diffusion cells (ref. 2,3,4).
All the mentioned SOP (Standard Operating Procedure) have been specifically written up for therespective laboratory.

I I . M AT E R I A L S• Dynamic mode: flow-through diffusion cell (ref. 3)• Static mode: static Franz diffusion cell (ref. 2)
Usual laboratory equipment as indicated in a specific SOP.

III. METHODS
III.1. Preparation of the formulation
III.1.1. UNLABELLED TEST COMPOUNDSThe specifications of each test compound must be defined prior to the formulation: a recent analysisof the respective batch by the analytical department must be included in the final study report.
The qualitative controls of the formulation are performed by the formulator.
The quantitative controls are performed according to the SOP “Control of an unlabelledformulation”. This control is based on HPLC or another suitable method in order to ascertain theconcentration of the test compound in the formulation.
III.1.2. RADIOLABELLED TEST COMPOUNDSThe degree of radiolabelling is specific for each formulation: the radiolabelled test compound isdiluted according to its initial specific activity.

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The supplier of the radiolabelled compound must provide a complete analysis bulletin including theradiochemical purity and date of the analysis; radiochemical purity must be reassessed before
beginning the formulation and in the final formulation by the technical investigator; analyticalbulletins should be included in the final study report.
Since only small quantities of radiolabelled formulations can be prepared each time, unlabelled pilot
formulation assays on the respective scale should be performed and analytical results should be
provided by the study promotor/department requesting the study. The latter ascertains the feasabilityof the formulation and its stability. The formulation should remain stable for the duration of thestudy.
The qualitative controls of the formulation are performed by the formulator.
The quantitative controls are performed according to a specific SOP. This control is based on HPLCor another method suited to ascertaining the concentration of the test compound (radiolabelled andunlabelled) in the formulation.
Specific radioactivity of the formulation is determined by scintillation counting using an external
standard; the quench curves are established according to a specific SOP.
III.2. Preparation of the samples of human skinIII.2.1. ORIGIN AND STORAGE OF BIOLOGICAL SAMPLES
Human skin used for these experiments is obtained from surgery. The skin samples can be obtained
from a variety of anatomical sites including the breast (mastectomy and reduction mammoplasty)
and abdomen (cosmetic reductive surgery). Skin must be collected as soon as possible after surgery.
All skin samples are checked visually to ensure they are healthy and unaltered by clinical removal
conditions. For transportation from the hospital/clinic to the laboratory, the skin should be kept in an
isotherm container at 4°C. In the laboratory, each skin sample should be identified (identification
number, type, age, gender, date of operation) and have the subcutaneous fat removed. The wholeprocess of transporting and preparing the skin samples must be achieved as quickly as possible.
The skin samples are put into plastic bags, sealed and stored at - 20°C until use. Skin samples canbe kept in a freezer for up to 3 months. For in vitro percutaneous absorption/penetration experiments,
it has been shown that human skin permeability is unaffected by freezing (ref. 5, 6). These different
stages should be performed according to a specific SOP “including security measures for handlingof biological materials”.
III.2.2. PREPARATION OF SKIN DISCS
An in vitro study using isolated skin should simulate as closely as possible the in vivo conditions.
In vivo, a compound must diffuse up to the upper papillary dermis before being taken up by bloodvessels and then entering the systemic circulation. Thus, depending on the solubility of the test

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compound, to limit the effect of the dermal retention in vitro, especially with hydrophobic
compounds, split-thickness skin (dermatomed skin or isolated epidermis) should be used (ref. 7).
Each skin sample is thawed at room temperature and prepared the day of the experiment according
to a specific SOP:
• Dermatomed skin:An electric dermatome (Davies simplex, THACKRAY SURGERY) is used to cut horizontal slices
of skin. The thickness of the cut is controlled by a lever on the side of the dermatome head with theindicated calibrations. Full-thickness skin is fixed on a dissection board, epidermal side up, andsections are cut at 300 to 500 μm. The thickness of the membrane obtained is the result of thepressure applied and the angle of the dermatome as it is pushed across the skin. During thisprocedure, a constant angle of inclination of the oscillating blade relative to the skin must be
maintained. The dermatomed layer thus obtained includes the epidermis and some dermal tissue.
• Isolated epidermis:
Epidermal membranes are prepared from full-thickness skin by a heat separation method (ref. 8).
Water is heated in a beaker to 58-60°C. Full-thickness skin is suspended in the water with forceps
for 60 seconds. Then the epidermis is gently peeled off from the dermis using forceps. The remaining
epidermal membrane is cut to fit the diffusion cell area. Each epidermal membrane should bechecked for integrity with the aid of a stereomicroscope.
III.2.3. CONTROL OF SKIN DISC THICKNESSSkin thickness should be measured with a Digitaler Meßtaster (MT12, HEIDENHAIN) and abidirectional counter (VRZ 401, HEIDENHAIN) or with equivalent equipment. The measurements
should be performed according to the directions for use. Ten measurements per skin disc should be
made. This process should be performed according to a specific SOP.
III.2.4. SPECIFIC RECOMMENDATIONS FOR THE USE OF HUMAN SKIN
As there is wide variability in human skin permeability (ref. 9), not less than 3 skin donors should
be used to take account of interindividual variability besides the intraindividual variability; a proper
statistical design should be used.
Age and anatomic site influence percutaneous absorption (ref. 9,10). Thus, skin samples from thesame anatomical region and in the same age range (20-60 years) should be used to limit thesevariations.
To preserve the integrity of human skin membranes mounted in diffusion cells, the study time should
never exceed 24 hours.

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10III.3. Preparation of the diffusion cells
The number of diffusion cells per experiment and the number of runs per study, must be specified in
the protocol. The study should involve a minimum of six samples of similar integrity. This process
should be performed according to a specific SOP.
III.3.1.STATIC MODE OR DYNAMIC MODE
The choice of static mode or dynamic mode depends on the absorption/penetration properties of thetest compound and depends on the aim of the study.
The skin samples are placed as a barrier between the two halves of the diffusion cell, the stratum
corneum facing the donor chamber; the donor side of the cell is open to the air (non occlusive
conditions). The skin sample is kept at (32 +/- 1)°C by circulation of temperature controlled receptor
fluid through the cell (dynamic diffusion cell) or by emersing the cell (static diffusion cell) in atemperature-controlled water bath.
The receptor chamber is filled with receptor fluid, capped and allowed to attain the correcttemperature 1 hour before the beginning of the experiment. In dynamic mode, care is taken to ensurethat no air bubbles form on the underside of the skin throughout the experiment.
III.3.2. RECEPTOR FLUID
To maintain the integrity of the skin samples and to collect the test compound, the receptor fluid issaline or buffered saline solution; a non-ionic surfactant can be added to solubilise the test compound
especially for lipophilic compounds (ref. 11).
Before the experiments, it should be checked that the test compound is stable in the receptor fluid(in experimental conditions) for a period corresponding to the duration of the experiment.
III.4. Test of skin integrity
Each sample of human skin must be checked for integrity before the application of the test
compound. This test must not affect the quality of the skin samples and must not influence thepenetration of the test compound.
The integrity of the skin samples is checked with the the aid of a stereomicroscope and/or bymeasuring TEWL (Trans Epidermal Water Loss) (ref. 12). The procedure is performed according toa specific SOP.

11

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III.5. Application of formulations
The quantities to be applied are expressed as follows:• amount of formulation applied (mg/cm2);• amount of test compound applied (μg/cm2);• in case of radiolabelled compounds: amount of radioactivity applied (Bq/cm2).
A finite dose of the test compound is applied to the skin surface (ref. 13) i.e. about 2 mg/cm2 of asemisolid formulation (cream, ointment, gel, etc.) and about 5 μl/cm2 of liquid preparations(solutions, emulsions, etc.). If semisolid formulations are compared to liquid formulations, identicalamounts of 5 mg/cm2 .. When investigating rinse-off preparations (e.g. hair dyes), an infinite dose(> 10 mg/cm2) of the test compound is applied to the skin surface to mimic the use conditions. The
rinsing procedure must be specified in the protocol.
The application is performed according to a specific SOP.
III.6. Start of the experimental setup and collection of fractions
The experiments are started immediately after the application of the formulation, according to a
specific SOP.
In the static mode, aliquots of the receptor fluid are taken either throughout the experiment or at theend of the experiment. In the dynamic mode, the receptor fluid is pumped from a reservoir into andthrough the cell by a peristaltic pump. After exiting the cell, the receptor fluid is collected in an
automatic fraction collector.
III.7. Study time
The study time is chosen according to the nature and intended use of the test compound. Rinse-off
preparations are left on the skin for a restricted time (e.g. 30 minutes in the case of hair dyes). Thenthe skin and the upper part of the diffusion cell are rinsed and the experiment is continued for a
maximum of 24 hours. Leave-on preparations are left in place for the entire study time (maximum
24 hours).
The diffusion cells are then taken apart according to a specific SOP.

III.8. Analysis
In all cases, the receptor fluid samples are analysed. In case of dermatomed skin, the epidermis isseparated from the dermis and the remaining dermis sample is analysed.

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III.8.1. UNLABELLED TEST COMPOUNDS
If relevant and if a suitable analytical method exists (in terms of detection limit, sensitivity, etc) ananalysis will be made of the amounts of the test compound on the skin surface, in rinsings, cellwashings and skin layers. The preparation of samples, extraction steps and analysis by HPLC or
another suitable method depend on the test compound and should be described in the appendix ofthe study report.
This procedure should be performed according to a specific SOP.
III.8.2.RADIOLABELLED TEST COMPOUNDS
An analysis of the test compound on the skin surface, in rinsings, cell washings and skin layers
should be made. This procedure should be performed according to a specific SOP. The analyses arecarried out by means of scintillation counting according to a specific SOP. Scintillation counting is
performed using an external standard; the quench curves are established according to a specific SOP.
III.9. Presentation of the results
The results are expressed in μg/cm2 and % of the applied dose in the form of tables and graphs.
III.10. Interpretation of the results for safety evaluations
Classical in vitro percutaneous absorption studies only consider as penetrated the amount ofcompound entering into the receptor fluid. This interpretation is only correct when the skin samples
are completely devoid of dermal tissue, i.e. when the epidermis has been removed from the dermis
by heat (in vivo the dermis is vascularised, i.e. is part of the central compartment).
The dermatome-based separation technique leaves some upper dermal tissue underneath theepidermis. Hence, the epidermal and the dermal compartments have to be considered separately. The
amount of test compound found after the study time in the receptor fluid, plus that found in thedermis, must be considered to have crossed the skin barrier i.e. as penetrated.
III.11. Study report
The study report has to be finalized according to a specific SOP.
III.12. Archives
The protocol, raw data and study report are kept on file by the study director's office for at least 5
years.

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L I T E R AT U R E R E F E R E N C E S :1. Skelly J.P., Shah V.P., Maibach H.I., Guy R.H., Wester R.C., Flynn G. & Yacobi A., FDA and
AAPS report of the workshop on principles and practices of in vitro percutaneous penetration
studies: Relevance to bioavailability and bioequivalence, Pharm. Res., 4, p. 3, 1987.
2. Franz T.J., Percutaneous absorption: On the relevance of in vitro data, J. Invest. Dermatol., 64,
pp. 190-195, 1975.
3. Gummer C.L., Hinz R.S. & Maibach H.I., The skin penetration cell: a design update, Int. J.
Pharm., 40, pp. 101-104, 1987.
4. Addicks W.J., Flynn G.L. & Weiner N., Validation of a flow-through diffusion cell for use intransdermal research, Pharm. Res., 4, 1987.
5. Harrison S.M., Barry B.W. & Dugard P.H., Effects of freezing on human skin permeability,
J. Pharm. Pharmacol., 36, pp. 261-262, 1984.
6. Bronaugh R.L., Stewart R.F. & Simon M., Methods for in vitro percutaneous absorption studies
VII: Use of excised human skin, J. Pharm. Sci., 75, p. 11, 1986.
7. Collier S.W. & Bronaugh R.L., Preparation of human and animal skin, In vitro percutaneousabsorption: Principles, Fundamentals and applications, (Bronaugh R.L. & Maibach H.I., Eds.),
CRC Press, Boca Raton, 1991.
8. Kligman A.M. & Christophers E., Preparation of isolated sheets of human stratum corneum,Arch. Dermatol., 88, pp. 702-705, 1963.
9. Wester R.C. & Maibach H.I., Individual and regional variation with in vitro percutaneousabsorption, In vitro percutaneous absorption: Principles, Fundamentals and Applications,(Bronaugh R.L. and Maibach H.I., Eds.), CRC Press, Boca Raton, 1991.
10. Marzulli F.N.& Maibach H.I., Permeability and reactivity of skin related to age, J. Soc. Cosmet.
Chem., 35, pp. 95-102, 1984.
11. Collier S.W & Bronaugh R.L., Receptor fluids, In vitro percutaneous absorption: Principles,
Fundamentals and Applications, (Bronaugh R.L and Maibach H.I, Eds.), CRC Press, Boca Raton,
1991.
12. Lotte C., Rougier A., Wilson D.R. & Maibach H.I., In vivo relationship between transepidermal
water loss and percutaneous penetration of some organic compounds in man: effect of anatomic site,Arch. Dermatol. Res., 279, 1987.
13. Franz T.J., The finite dose technique as a valid in vitro model for the study of percutaneousabsorption in man, Currents problems in Dermatology, 7, (Simon G.A., Paster, Klingberg M.A. &Kaye M., Eds.), Karger, Basel, 1978.

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