ᣂⷙ⹜㛎ᴺឭ᩺ᦠ             ᐔᚑ 26 ᐕ 1 ᦬ 20 ᣣ                                  No. 2013-04
ᡷ⸓ OECD TG No.405㧦࠙ࠨࠡࠍ↪޿ࠆ⌒ೝỗᕈ⹜㛎ᴺ
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 ࠙ࠨࠡࠍ↪޿ࠆ⌒ೝỗᕈ⹜㛎ᴺߪ‫ޔ‬㤗㈮⮎ߩ૶↪߅ࠃ߮ੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߩ
⸳ቯ╬ࠍⴕߞߡ߽‫⹏ޔ‬ଔ⚿ᨐ߇ၮᧄ⊛ߦߪᄌࠊࠄߥ޿ߎߣ߆ࠄ‫ⴕޔ‬᡽਄ߢߩ೑↪
ߩน⢻ᕈߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߩ႐วߣห᭽ߢ޽ࠆ‫ޕ‬ ߎߩឭ᩺ᦠߪ‫ޔ‬OECD (Organisation for Economic Co-operation and Development) Revised Test
Guideline No. 405 ߅ࠃ߮ ICCVAM (Interagency Coordinating Committee on the Validation of
Alternative Methods )Test Method Evaluation Report: Recommendations for Routine Use of Topical
Anesthetics, Systemic Analgesics, and Humane Endpoints to Avoid or Minimize Pain and Distress in
Ocular Safety Testingࠍ߽ߣߦ‫⌒ޔ‬ೝỗᕈ⹜㛎⹏ଔᆔຬળߦࠃࠅ߹ߣ߼ࠄࠇߚᢥᦠࠍ↪޿ߡJaCVAM
⹏ଔળ⼏߇⹏ଔ߅ࠃ߮ᬌ⸛ߒߚ⚿ᨐ‫↪᦭ߩߘޔ‬ᕈ߇⏕⹺ߐࠇߚߎߣ߆ࠄ૞ᚑߐࠇߚ‫ޕ‬ એ਄ߩℂ↱ߦࠃࠅ‫ⴕޔ‬᡽ᒰዪߩ቟ోᕈ⹏ଔᣇᴺߣߒߡ‫ޟ‬ᡷ⸓ OECD TG No.405㧦࠙ࠨࠡࠍ↪޿ࠆ⌒ೝỗᕈ⹜㛎ᴺ‫ࠍ↪૶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 statement
on revised Test Guideline No. 405: Acute Eye Irritation/Corrosion
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 Acute Eye Irritation/Corrosion revised by OECD (Organisation for Economic
Co-operation and Development) in 2012 is considered to be able to predict eye irritating
substances as it always has been in the past by the use of anesthetics.
Following the review of the results of OECD Revised Test Guideline No. 405 and ICCVAM(Interagency Coordinating Committee on the Validation of Alternative Methods )Test Method
Evaluation Report: Recommendations for Routine Use of Topical Anesthetics, Systemic
Analgesics, and Humane Endpoints to Avoid or Minimize Pain and Distress in Ocular Safety
Testing, it is concluded that revised Acute Eye Irritation/Corrosion such as rritation testing areclearly 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 eye irritation 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

ᡷ⸓ OECD TG No.405㧦࠙ࠨࠡࠍ↪޿ࠆ⌒ೝỗᕈ⹜㛎ᴺ⋡ ᰴ⹏ଔળ⼏ႎ๔ᦠ ------------------------------------------------------------------------------- 1⹏ଔႎ๔ᦠ ------------------------------------------------------------------------------------- 5
OECD TG405㧔2012㧕 --------------------------------------------------------------------- 15
ICCVAM Test Method Evaluation Report: Recommendations for Routine
Use of Topical Anesthetics, Systemic Analgesics, and Humane Endpoints to
Avoid or Minimize Pain and Distress in Ocular Safety Testing -------------- 35

ix

x

 



⚻ᷣදജ㐿⊒ᯏ᭴㧔OECD㧕ߩ࠙ࠨࠡࠍ↪޿ߚ⌒ೝỗᕈ⹜㛎ᡷ⸓ࠟࠗ࠼࡜ࠗࡦ㧔TG405㧕
ߩ⹏ଔળ⼏ႎ๔ᦠ















JaCVAM ⹏ଔળ⼏















ᐔᚑ 25 ᐕ 10 ᦬ 21 ᣣ

11


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*: ᐔᚑ 25 ᐕ 4 ᦬ 1 ᣣ㨪ᐔᚑ 26 ᐕ 3 ᦬ 31 ᣣ
એ਄












2
2

⚻ᷣදജ㐿⊒ᯏ᭴㧔OECD㧕ߩ࠙ࠨࠡࠍ↪޿ߚ⌒ೝỗᕈ⹜㛎ᡷ⸓ࠟࠗ࠼࡜ࠗࡦ㧔TG405, 2012
ᐕ 10 ᦬ 2 ᣣᡷ⸓㧕ߦߟ޿ߡߪ⌒ೝỗᕈ⹏ଔᆔຬળ߆ࠄߩႎ๔ࠍฃߌ‫ޔ‬એਅߩ 10 㗄⋡ߦߟ޿ߡ⹏ଔߒߚߩߢႎ๔ߔࠆ‫ޕ‬ᧄࠟࠗ࠼࡜ࠗࡦߪㆊ෰ߦ߽േ‛⑔␩ߩⷰὐ߆ࠄᡷ⸓߇ⴕࠊࠇߡ޿ࠆ߇‫੹ޔ‬࿁ߩᡷ⸓ߢേ‛⑔␩ߩᦝߥࠆలታ߇࿑ࠄࠇߚ‫⌒ߚ޿↪ࠍࠡࠨ࠙ޕ‬ೝỗᕈ⹜㛎ߦ߅ߌࠆേ‛ߩ∩ߺߣ⧰∩ࠍシᷫߔࠆߚ߼ߦ‫ޔ‬ዪᚲ㤗㈮⮎෸߮ోり⊛㎾∩⮎ߦࠃࠆቯᏱ⊛ಣ⟎‫⁁∝∩⧰ߣߺ∩ޔ‬෸߮ߔߴߡߩ⌒்ኂߩⷰኤߦߟ޿ߡߩౕ૕⊛ᣇᴺ‫ޔ‬ਗ߮ߦ‫ੱޔ‬㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߩ⸳ቯߣߘߩ್ᢿߩ㓙ߩታ㛎േ‛₞කᏧߥߤߩ㑐ਈ߇ⷙቯߐࠇߚ‫ޕ‬㧨ክ⼏ౝኈ㧪
1.

ᒰ⹥⹜㛎ᴺߪ‫ߥ߁ࠃߩߤޔ‬ᓥ᧪⹜㛎ᴺࠍઍᦧߔࠆ߽ߩ߆‫ߥ߁ࠃߩߤޔߪߚ߹ޕ‬Ქᕈࠍ⹏ଔ
޽ࠆ޿ߪ੍᷹ߔࠆ߽ߩ߆‫ޕ‬ߎࠇ߹ߢ OECD ߇⌒ೝỗ⹜㛎㧔ᕆᕈೝỗᕈ࡮⣣㘩ᕈ⹜㛎㧕ࠟࠗ࠼࡜ࠗࡦߣߒߡ౏⴫ߒߡ޿ߚ

࠼࡟ࠗ࠭⹜㛎ࠍઍᦧߔࠆ߽ߩߢ޽ࠆ‫⹏ޕ‬ଔ޽ࠆ޿ߪ੍᷹ߔࠆᲥᕈߪ‫ޔ‬ᓥ᧪ߩ OECDTG405 ߣห᭽ߢ޽ࠆ‫ޕ‬2.

ᒰ⹥⹜㛎ᴺߣᓥ᧪⹜㛎ᴺߩ㑆ߦߤߩࠃ߁ߥ⑼ቇ⊛ߥߟߥ߇ࠅ߇޽ࠆ߆‫ޕ‬
ᓥ᧪⹜㛎ᴺߦ߅ߌࠆേ‛⑔␩߳ߩኻಣߣߒߡ‫ޔ‬േ‛ߩ∩ߺߣ⧰∩ࠍࠃࠅシᷫߔࠆ⹜㛎ᣇᴺ߇ⷙ

ቯߐࠇߡ޿ࠆ‫ⵍޔ߫߃଀ޕ‬㛎‛⾰ᛩਈ೨ߩዪᚲ㤗㈮⮎‫ోޔ‬りᕈ㎾∩⮎ߩᛩਈ‫ޔ‬ᛩਈᓟߩోりᕈ㎾∩⮎ߩᛩਈ‫ޔ‬േ‛ߩ∩ߺߣ⧰∩ߩ∝⁁ⷰኤߣ⹜㛎߆ࠄߩ㒰ᄖ‫ੱߦ߮ࠄߥޔ‬㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߩ
⸳ቯߢ޽ࠆ‫ޕ‬3.

ᒰ⹥⹜㛎ᴺߣߘߩ࠺࡯࠲ߪ‫ޔ‬ㅘ᣿ߢ⁛┙ߥ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆ߆‫ޕ‬ ☨࿖ ICCVAM ߩ࿖㓙⊛ߥ⑼ቇ⊛ࡇࡘࠕ࡟ࡆࡘ࡯ᆔຬળ㧔International Scientific Peer
Review Panel㧕ߦ߅޿ߡ⑼ቇ⊛⹏ଔࠍฃߌߡ޿ࠆ‫ޕ‬
4.

ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߩઍᦧᴺߣߒߡ‫⾰‛ߥ߁ࠃߩߤޔ‬෶ߪ⵾ຠࠍ⹏ଔߔࠆߎߣࠍ⋡⊛ߣߒߡ޿ࠆ߆‫ޕ‬
ᓥ᧪⹜㛎ᴺߢᗐቯߐࠇߡ޿ߚ‛⾰෶ߪ⵾ຠࠍ⹏ଔߔࠆߎߣࠍ⋡⊛ߣߒߡ޿ࠆ‫⹏ޕ‬ଔኻ⽎ߩ‛⾰෶ߪ⵾ຠߪ‫ޔ‬ᓥ᧪ߩ OECDTG405 ߣห᭽ߢ޽ࠆ‫ޕ‬
5.

ᒰ⹥⹜㛎ᴺߪ‫⹏࠼࡯ࠩࡂޔ‬ଔ޽ࠆ޿ߪ࡝ࠬࠢ⹏ଔߩߤߜࠄߦ᦭↪ߢ޽ࠆ߆‫ޕ‬
ᓥ᧪⹜㛎ᴺߣห᭽‫⹏࠼࡯ࠩࡂޔ‬ଔߦ᦭↪ߢ޽ࠅ‫ⵍޔ‬㛎‛⾰ߩỚᐲࠍ⺞ᢛߔࠆߎߣߦࠃࠅ࡝ࠬࠢ

⹏ଔߦ߽૶↪น⢻ߢ޽ࠆ‫ޕ‬
6.

ᒰ⹥⹜㛎ᴺߪ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏ଔߢ߈ࠆ߆‫ߩߘޕ‬႐ว‫ޔ‬ᒰ⹥⹜㛎ᴺߩ
ㆡ↪᧦ઙ߇᣿⏕ߦߥߞߡ޿ࠆ߆‫ޕ‬
ᓥ᧪⹜㛎ᴺߣห᭽‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈ㧔⌒ೝỗᕈ㧕ࠍ⹏ଔน⢻ߢ޽ࠅ‫ޔ‬ㆡ↪᧦ઙ߽᣿⏕ߢ޽ࠆ‫ޕ‬33

7.

ᒰ⹥⹜㛎ᴺߪࡊࡠ࠻ࠦ࡞ߩᓸ⚦ߥᄌᦝߦኻߒߡ㗎ஜߢ޽ࠆ߆‫ޕ‬ ࡊࡠ࠻ࠦ࡞ߩᓸ⚦ߥᄌᦝߦኻߒߡߩ㗎ஜᕈߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߣห᭽ߢ޽ࠆ‫ޕ‬8.

ᒰ⹥⹜㛎ᴺߩᛛⴚ⠌ᓧߪ‫ޔ‬ㆡಾߥ⸠✵ߣ⚻㛎ࠍ⚻ߡ޿ࠆᜂᒰ⠪ߦߣߞߡኈᤃߥ߽ߩߢ޽ࠆ
߆‫⹜ޕ‬㛎ᴺߩታᣉߦ․ᱶߥ⸳஻߇ᔅⷐ߆‫ޕ‬ᛛⴚ⠌ᓧߪᓥ᧪⹜㛎ᴺࠍ⚻㛎ߒߡ޿ࠆᜂᒰ⠪ߦߣߞߡኈᤃߢ޽ࠆ‫⹜ޕ‬㛎ᴺߩታᣉߦዪᚲ㤗㈮⮎‫ޔ‬ోりᕈ㎾∩⮎ߩᛩਈ߇ᔅⷐߢ޽ࠅ‫⮎ࠆ޿ߡࠇߐ␜଀ޔ‬೷ߪ޿ߕࠇ߽ක⮎ຠߣߒߡߩ૶↪߇ᗐቯߐࠇߡ߅ࠅ‫ޔ‬ታ㛎േ‛₞කᏧߩ㑐ਈ߇ᔅⷐߣߥࠆ‫ࠇߘޕ‬એᄖߩ․ᱶߥ⸳஻ߪਇⷐߢ޽ࠆ‫ޕ‬߹ߚ‫⹜ޔ‬㛎ਛߦേ‛ߦ⧰∩߇↢ߓߚ႐วߦߪㆬછ₞කᏧ‫ޔ‬ታ㛎േ‛කቇኾ㐷ක‫ߩઁߩߘޔ‬േ‛ߩ∔∛ࠍᾫ⍮ߒߡ޿ࠆ⠪߳ߩ⋧⺣߇⸥タߐࠇߡ޿ࠆߎߣ߆ࠄ‫ߦ․ߢ߹ࠇߎޔ‬ᔅⷐߣߪߐࠇߡ޿ߥ
߆ߞߚ‫ޔ‬ኾ㐷ኅߩ㑐ਈ߇ᔅⷐߣߥࠆ‫ޕ‬
9.

ᒰ⹥⹜㛎ᴺߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߣᲧߴߡᤨ㑆⊛⚻⾌⊛ߦఝࠇߡ޿ࠆ߆‫ޕ‬ 㤗㈮⮎‫ޔ‬㎾∩⮎ߩᛩਈ߇ᔅⷐߥ੐߆ࠄ‫ޔ‬ᓥ᧪⹜㛎ᴺࠃࠅ߽⧯ᐓߩᤨ㑆⊛߅ࠃ߮⚻⾌Ⴧߣߥࠆ‫ޕ‬߹ߚߎࠇࠄක⮎ຠߩᛒ޿‫್ߩ∩⧰ߦ߮ࠄߥޔ‬ቯߣ⹜㛎߆ࠄߩ㒰ᄖߦኾ㐷ኅߩ㑐ਈ߇⸥タߐࠇߡ߅ࠅ‫ߪ⾌⚻ޔ‬Ⴧߒߡ޿ࠆ‫ޕ‬
10. ᒰ⹥⹜㛎ᴺߪ‫ޔ‬േ‛⑔␩ߩⷰὐ෸߮⑼ቇ⊛⷗࿾߆ࠄ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏
ଔߔࠆઍᦧᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪ߔࠆߎߣߪน⢻߆‫ޕ‬㤗㈮⮎ߩ૶↪߅ࠃ߮ੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߩ⸳ቯ╬ࠍⴕߞߡ߽‫⹏ޔ‬ଔ⚿ᨐ߇ၮᧄ⊛ߦߪᄌࠊࠄ
ߥ޿ߎߣ߆ࠄ‫ⴕޔ‬᡽਄ߢߩ೑↪ߩน⢻ᕈߪ‫ޔ‬ᓥ᧪⹜㛎ᴺߩ႐วߣห᭽ߦน⢻ߢ޽ࠆ‫ޕ‬ෳ⠨ᢥ₂
1)

JaCVAM⌒ೝỗᕈ⹏ଔᆔຬળ㧦⚻ᷣදജ㐿⊒ᯏ᭴㧔OECD㧕ᡷ⸓ࠟࠗ࠼࡜ࠗࡦ㧔Test
Guideline No.405㧕 ᡷ⸓࠙ࠨࠡࠍ↪޿ߚ⌒ೝỗᕈ⹜㛎ߩ⹏ଔႎ๔ᦠ

2)

OECD Test Guideline, Revised TG405㧔2012㧕

44

⚻ᷣදജ㐿⊒ᯏ᭴㧔OECD㧕ᡷ⸓ࠟࠗ࠼࡜ࠗࡦ㧔Test Guideline No.405㧕
࠙ࠨࠡࠍ↪޿ߚ⌒ೝỗᕈ⹜㛎ߩ⹏ଔႎ๔ᦠ

2013ᐕ10᦬15⌒ೝỗᕈ⹜㛎ઍᦧᴺ⹏ଔᆔຬળᆔຬฬ㧦ዊဈ ᔘม㧔ᱷ⇐ㄘ⮎⎇ⓥᚲ㧕ศ᧛ ഞ 㧔᧲੩ℂ⑼ᄢቇ㧕ጊᧄ ⋥᮸㧔⮮↰଻ஜⴡ↢ᄢቇ㧕┻ౝ ዊ⧣㧔P&G ࠗࡁࡌ࡯࡚ࠪࡦวหળ␠㧕⚦੗ ৻ᒄ㧔ෳᄤ⵾⮎ᩣᑼળ␠㧕ട⮮ 㓷৻㧔ᩣᑼળ␠ J-TEC㧕☄ౝ ᩶ሶ㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ㧕Ⴧ↰ శノ26㧝㧚ᐨ ⺰OECD⹜㛎ࠟࠗ࠼࡜ࠗࡦߪ㧘ᦨㆡߩ⑼ቇᛛⴚ߇෻ᤋߐࠇࠆࠃ߁ߦቯᦼ⊛ߦ⷗⋥ߐࠇߡ߈ߚ‫ߩߘޕ‬ਛߢ㧘ߎࠇ߹ߢߩᡷ⸓ߢߪਇᔅⷐߥേ‛ታ㛎ࠍ࿁ㆱߔࠆߚ
߼ߩᡷༀ߇ⴕࠊࠇߡ߈ߚ‫੹ޕ‬࿁ߪ࠙ࠨࠡࠍ↪޿ߚᕆᕈೝỗᕈ࡮⣣㘩ᕈ⹜㛎ࠟࠗ࠼࡜ࠗࡦ1) ߦߟ޿ߡ㧘േ‛⑔␩߳ߩ㈩ᘦࠍ⋡⊛ߣߒߚᡷ⸓߇ⴕࠊࠇߚ‫ޕ‬ᧄᡷ⸓ߦవ┙ߜ㧘☨࿖ICCVAMߩ࿖㓙⊛ߥ⑼ቇ⊛ࡇࠕ࡟ࡆࡘ࡯ᆔຬળ
㧔International Scientific Peer Review Panel㧕2) ߦ߅޿ߡ㧘ዪᚲ㤗㈮⮎ߣోりᕈ㎾∩⮎ߩ૶↪ߦࠃࠅ㧘⹜㛎ᚑ❣ߦᓇ㗀ࠍਈ߃ࠆߎߣߥߊേ‛ߩ∩ߺߣ⧰∩ࠍ࿁ㆱߢ߈ࠆ߆㧘ߘߒߡߘߩ㓙ߩዪᚲ㤗㈮⮎ߣోりᕈ㎾∩⮎ߩಣᣇ෸߮ੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߦߟ޿ߡ⼏⺰ߐࠇߚ‫ᧄޕ‬ᡷ⸓ߦࠃࠅ‫⌒ߚ޿↪ࠍࠡࠨ࠙ޔ‬ೝỗᕈ⹜㛎ߦ߅޿ߡ‫⹜ޔ‬㛎ᚑ❣ߦᓇ㗀ࠍਈ߃ࠆߎߣߥߊേ‛ߩ∩ߺߣ⧰∩߇シᷫߐࠇࠆߣ⠨߃ࠄࠇߚ‫ޕ‬੹࿁ߩᡷ⸓ߩⷐὐࠍᰴߦ␜ߔ‫ޕ‬1)ⵍ㛎‛⾰ᛩਈ೨ߢߪ㧘ዪᚲ㤗㈮⮎㧔଀㧦ࡊࡠࡄ࡜ࠞࠗࡦ㧘࠹࠻࡜ࠞࠗࡦ㧕ߣోりᕈ㎾∩⮎㧔଀㧦ࡉࡊ࡟ࡁ࡞ࡈࠖࡦ㧕ߦࠃࠆቯᏱ⊛೨ಣ⟎‫ޕ‬2)ⵍ㛎‛⾰ᛩਈᓟߢߪ㧘ోりᕈ㎾∩⮎㧔଀㧦ࡉࡊ࡟ࡁ࡞ࡈࠖࡦ෸߮ࡔࡠࠠࠪࠞ
ࡓ㧕ߦࠃࠆቯᏱ⊛ಣ⟎‫ޕ‬3)േ‛ߩ∩ߺߣ⧰∩ߩ∝⁁ߩ⸘↹ⷰኤ㧘ࡕ࠾࠲࡝ࡦࠣ߅ࠃ߮⸥㍳‫ޕ‬4)ోߡߩ⌒்ኂߩ㧔ᕈ⾰㧘⒟ᐲ߅ࠃ߮ㅴⴕ⁁ᴫ㧕⸘↹ⷰኤ㧘ࡕ࠾࠲࡝ࡦࠣ㧘⸥
㍳߅ࠃ߮ੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻⸳ቯ‫ޕ‬
2㧚࠙ࠨࠡ⌒ೝỗᕈ⹜㛎ᚻ㗅
2.1. ଏ⹜േ‛ஜᐽߥ⧯㦂ᚑᾫߩ⊕⦡࠙ࠨࠡࠍଏ⹜േ‛ߣߒߡ૶↪ߔࠆ‫⹜ޕ‬㛎㐿ᆎ೨24ᤨ㑆એౝߦଏ⹜േ‛ߩਔ⌒ࠍᬌᩏߔࠆ‫ߩ⌒ޕ‬ೝỗ㧘⌒்ኂ㧘߹ߚߪⷺ⤑៊்߇⹺߼
ࠄࠇߚേ‛ߪ૶↪ߒߥ޿‫ޕ‬േ‛ߪ୘೎㘺⢒ߔࠆ‫ޕ‬േ‛㘺⢒ቶߩ᷷ᐲߪ20±3°C㧘⋧ኻḨᐲߪ50%㨪60%ߣ
ߒ㧘ᾖ᣿ߪ12ᤨ㑆᣿ᦼ㧘12ᤨ㑆ᥧᦼߣߔࠆ‫ޕ‬
2.2. ዪᚲ㤗㈮⮎෸߮ోりᕈ㎾∩⮎ߩㆡ↪
⌒ೝỗᕈ⹜㛎ߩታᣉߦ޽ߚࠅ㧘േ‛ߩ∩ߺ෸߮⧰∩ࠍ࿁ㆱߥ޿ߒᦨዊ㒢ߦߔࠆߎߣࠍ⋡⊛ߣߔࠆ‫ޕ‬ⵍ㛎‛⾰ᛩਈߩ60ಽ೨ߦ㧘ࡉࡊ࡟ࡁ࡞ࡈࠖࡦ0.01 mg/kg㧔㎾∩⮎ߣߒߡᴦ≮ߦ↪޿ࠄࠇࠆ↪㊂㧕ࠍ⊹ਅᛩਈߔࠆ‫ޕ‬ⵍ㛎‛⾰ᛩਈ5ಽ೨ߦ㧘ਔ⌒ߦዪᚲ㤗㈮⮎㧔଀߃߫㧘0.5㧑Ⴎ㉄ࡊࡠࡄ࡜ࠞࠗ
ࡦ޽ࠆ޿ߪ0.5% Ⴎ㉄࠹࠻࡜ࠞࠗࡦ㧕ࠍ1㧘2Ṣὐ⌒ߔࠆ‫ߚ߹ޕ‬㧘Ꮢ⽼ዪᚲ㤗㈮ὐ
3
7

⌒⮎ਛߩ㒐⣣೷߇⹜㛎⚿ᨐߦᓇ㗀ߔࠆน⢻ᕈࠍ࿁ㆱߔࠆߚ߼㧘㒐⣣೷߇ᷝടߐ
ࠇߡ޿ߥ޿ዪᚲ㤗㈮ὐ⌒⮎ߩ૶↪ࠍផᅑߔࠆ‫ޕ‬ኻᾖ⌒ߦߪⵍ㛎‛⾰ߪᛩਈߖߕ㧘ዪᚲ㤗㈮⮎ߩߺࠍᛩਈߔࠆ‫ⵍޕ‬㛎‛⾰ᛩਈ߇㗼⪺ߥ⧰∩߿∩ߺࠍ⺃⿠ߔࠆߣ੍
ᦼߐࠇࠆ႐วߦߪ㧘ㅢᏱin vivo⹜㛎ࠍታᣉߔߴ߈ߢߪߥ޿‫ࠄ߇ߥߒ߆ߒޕ‬㧘ೝỗᕈ߇ਇ᣿ߥ႐ว߿⹜㛎ታᣉ߇ᔅⷐߥ႐วߦߪ㧘ⵍ㛎‛⾰ᛩਈ೨ߦ5ಽ㑆㓒ߢዪᚲ㤗㈮⮎ߩㅊടὐ⌒ߩታᣉࠍ⠨ᘦߔߴ߈ߢ޽ࠆ‫ߩߘޕ‬㓙㧘ዪᚲ㤗㈮⮎ߩⶄᢙ࿁૶↪ࠃߞߡ⌒ೝỗߩ⒟ᐲ߇ࠊߕ߆ߦ㜞ߊߥࠅ‫ޔ‬࿁ᓳ߇ㆃߊߥࠆน⢻ᕈߦ⇐ᗧߒߥߌࠇ߫ߥࠄߥ޿‫ޕ‬ⵍ㛎‛⾰ᛩਈ8ᤨ㑆ᓟߦࡉࡊ࡟ࡁ࡞ࡈࠖࡦ0.01 mg/kg෸߮ࡔࡠࠠࠪࠞࡓ0.5
mg/kg ࠍ⊹ਅᛩਈߔࠆ‫߅ߥޕ‬㧘ࡔࡠࠠࠪࠞࡓ߇1ᣣ1࿁⊹ਅᛩਈߢ⌒ߦኻߔࠆ᛫
Ἳ∝ᕈ૞↪ࠍ␜ߔߣ޿߁࠺࡯࠲ߪߥ޿߇㧘ⵍ㛎‛⾰ᛩਈߩ8ᤨ㑆ᓟ߹ߢߪࡔࡠࠠ
ࠪࠞࡓࠍᛩਈߔߴ߈ߢߪߥ޿‫ޕ‬ⵍ㛎‛⾰ᛩਈ8ᤨ㑆એ㒠ߪ㧘⌒ᚲ⷗߿∩ߺ෸߮⧰∩߇ᶖᄬߔࠆ߹ߢ㧘ࡉࡊ࡟ࡁ࡞ࡈࠖࡦ0.01 mg/kgࠍ12ᤨ㑆㑆㓒ߢ⊹ਅᛩਈߒ㧘ࡔࡠࠠࠪࠞࡓࠍ0.5 mg/kg 24ᤨ㑆㑆㓒ߢ⊹ਅᛩਈߔࠆ‫ޕ‬వⴕᛩਈߩ㎾∩⮎෸߮ዪᚲ㤗㈮⮎ߩലᨐ߇ਇචಽߥ႐วߦߪ㧘ⵍ㛎‛⾰ᛩਈ⋥ᓟߦ㎾∩⮎ߩᛩਈࠍታᣉߔࠆ‫⹜ޕ‬㛎ᦼ㑆ਛߦേ‛߇∩ߺ߿⧰∩ࠍ␜ߒߚ㓙ߦߪ⋥ߜߦㅊടߩ㎾∩⮎㧔✕ᕆ↪㊂ߣߒߡ0.03 mg/kg ࡉࡊ࡟ࡁ࡞ࡈࠖࡦ, ⊹ਅᛩਈ㧕ࠍᛩਈߒ㧘ᔅⷐߦᔕߓߡ8ᤨ㑆Ფߦ✕ᕆ↪㊂ࠍᛩਈߔࠆ‫ޕ‬0.5 mg/kg ߩࡔࡠࠠࠪࠞࡓߪ✕ᕆ↪㊂ߩࡉࡊ࡟ࡁ࡞ࡈࠖࡦߣ૬↪ߒ㧘24ᤨ㑆㑆㓒ߢ⊹ਅᛩਈߔࠆ‫ޕ‬
2.3. ⵍ㛎‛⾰ߩㆡ↪
ਅ⌒⍘ࠍⓏ߿߆ߦᒁ߈㧘ฦേ‛ߩ ⌒ߩ⚿⤑ྙౝߦⵍ㛎‛⾰ࠍᛩਈߔࠆ‫ⵍޕ‬㛎‛⾰ߩᵹᄬࠍ㒐ߋߚ߼㧘⚂1 ⑽㑆਄ਅߩ⌒⍘ࠍ㕒߆ߦวࠊߖ㧘㐽ߓߚ߹߹ߦ
ߒߡ߅ߊ‫ޕ‬2.3.1. ᵞᵺⵍ㛎‛⾰߇࿕૕ߩ႐ว෸߮ᛩਈ⋥ᓟߦೝỗᕈ࡮⣣㘩ᕈ૞↪ࠍ␜ߔ႐วࠍ㒰޿ߡߪ㧘ⵍ㛎‛⾰ᛩਈᓟዋߥߊߣ߽24ᤨ㑆ߪⵍ㛎േ‛ߩ⌒ࠍᵞᵺߒߥ޿‫ޕ‬24ᤨ㑆ᓟߦㆡಾߣ⠨߃ࠄࠇࠆ႐วߪ㧘ᵞᵺߒߡ߽ࠃ޿‫ޕ‬⑼ቇ⊛ߦᅷᒰᕈ߇ߥ޿㒢ࠅ㧘ᵞᵺലᨐࠍ⺞ᩏߔࠆߚ߼ߩⴡᤊ⟲ߩ૶↪ߪផᅑߢ߈ߥ޿‫ߒ߽ޕ‬㧘ⴡᤊ⟲߇ᔅⷐߥ႐วߪ2଀ߩ࠙ࠨࠡࠍ૶↪ߔࠆ‫ޕ‬ᵞᵺߩ᧦ઙ㧘
଀߃߫ᵞᵺᤨ㑆㧘ᵞᵺᶧߩ⚵ᚑ㧘᷷ᐲ㧘૶↪ᤨ㑆㧘㊂෸߮ㅦᐲߥߤࠍ⸥㍳ߔࠆ‫ޕ‬

2.3.2. ↪㊂(1) ᶧ૕ߩ⹜㛎
48ᶧ૕ߩ႐วߪ㧘ᛩਈ㊂ߪ0.1 mL ߣߔࠆ‫ⵍޕ‬㛎‛⾰ࠍ⌒߳ᛩਈߔࠆߚ߼ߦ㧘ࡐࡦࡊࠬࡊ࡟࡯ߪ૶↪ߒߥ޿‫ޕ‬ᶧ૕ߩࠬࡊ࡟࡯↪ᬌ૕ߢߪ੍߼ኈེߦ⹜ᢱࠍណข
ߒ㧘ߘߎ߆ࠄ0.1 mLࠍណขߒ㧘⌒ߦṢਅߔࠆ‫ޕ‬(2) ࿕૕ߩ⹜㛎࿕૕㧘ࡍ࡯ࠬ࠻෸߮☸ሶ⁁‛⾰ߩ႐วߪ㧘ᛩਈ㊂ߪኈ㊂ߣߒߡ0.1 mL㧘޽ࠆ޿ߪ㊀㊂ߢ100 mgએਅߩ㊂ߣߔࠆ‫ⵍޕ‬㛎‛⾰ߪ៺⎈ߒߡᓸ☳ᧃൻߔࠆ‫ޕ‬࿕૕‛⾰ߩኈ㊂ߪኈེࠍシߊߚߚ޿ߡ⹣߼ߚᓟߦ᷹ቯߔࠆ‫ޕ‬࿕૕‛⾰߇ᛩਈᓟ㧝ᤨ㑆ߩⷰኤᤨὐߢ↢ℂ⊛ᯏ᭴ߦࠃߞߡ⌒߆ࠄ㒰෰ߐࠇߡ޿ߥ޿႐วߦߪ㧘⌒ࠍ↢ℂ㘩Ⴎᶧ߹ߚߪ⫳⇐᳓ߢᵞᵺߒߡ߽ࠃ޿‫ޕ‬(3) ࠛࠕ࠱࡯࡞ߩ⹜㛎ࡐࡦࡊࠬࡊ࡟࡯෸߮ࠛࠕ࠱࡯࡞⵾ຠߩ႐วߪ㧘੍߼ౝኈ‛ࠍណขߒ⌒ߦㆡ↪
ߔࠆߎߣ߇ផᅑߐࠇࠆ‫ߒߛߚޕ‬㧘ౝኈ‛߇᳇ൻߔࠆߚ߼ߦട࿶ࠛࠕ࠱࡯࡞ኈེ
ߦ౉ࠇࠄࠇߡ޿ࠆⵍ㛎‛⾰ߩ႐วߪ଀ᄖߢ޽ࠅ㧘ߘߩ႐วߪ⌒ࠍ㐿߈㧘⌒ߩ⋥
೨10 cm ߩ〒㔌߆ࠄ⚂1⑽㑆න࿁ྃ኿ߒߡⵍ㛎‛⾰ࠍ⌒ߦㆡ↪ߔࠆ‫〒ߩߎޕ‬㔌ߪ㧘ࠬࡊ࡟࡯ߩ኿಴࿶ജ෸߮ߘߩ฽㊂ߦᔕߓߡᄌൻߒߡ߽ࠃ޿‫ߩ࡯࡟ࡊࠬޕ‬኿಴࿶ജߢ⌒ࠍ៊்ߒߥ޿ࠃ߁ߦᵈᗧߔࠆ‫ߩࠄ߆࡞࡯࠱ࠕࠛޕ‬ᛩਈ㊂ߪ㧘એਅߩࠪࡒ
ࡘ࡟࡯࡚ࠪࡦߦࠃߞߡ᭎▚ߩᛩਈ㊂ࠍផቯߔࠆ‫ⵍޕ‬㛎‛⾰ࠍ⒊㊂↪⚕ߩ⋥೨ߦ⟎޿ߚ࠙ࠨࠡߩ⌒ߩࠨࠗ࠭ߩⓣࠍㅢߒߡࠬࡊ࡟࡯ߒ㧘⒊㊂↪⚕ߩ㊀㊂Ⴧട߆ࠄ⌒ߦࠬࡊ࡟࡯ߐࠇࠆ㊂ࠍ᭎▚ߔࠆ‫ޕ‬ើ⊒ᕈ‛⾰ߩᛩਈ㊂ߦߟ޿ߡߪ㧘ⵍ㛎‛⾰ࠍࠬࡊ࡟࡯ߔࠆ೨ߣᓟߩኈེ㊀㊂ࠍ⒊㊂ߔࠆߎߣߦࠃࠅផቯߔࠆ‫ޕ‬
2.4. ೋ࿁⹜㛎 (േ‛1඘ࠍ↪޿ࠆin vivo ⌒ೝỗᕈ࡮⣣㘩ᕈ⹜㛎㧕Ბ㓏⊛⹜㛎ᚢ⇛ߣߒߡ㧘in vivo⹜㛎ߢߪ㧘߹ߕ1඘ߩേ‛ࠍ↪޿ߡታᣉߔࠆ‫ޕ‬⏕⹺⹜㛎ߦ⒖ࠆ೨ߦ㧘ೝỗᕈߩ㊀ᐲߣนㅒᕈߩ᦭ήࠍⷰኤߔࠆ‫ⷰߩߎޕ‬ኤ⚿ᨐߦࠃࠅ㧘ⵍ㛎‛⾰߇⌒ߦኻߒߡ⣣㘩ᕈ߹ߚߪᒝ޿ೝỗᕈࠍ᦭ߔࠆߣ್ᢿߐࠇࠆ
႐วߦߪ㧘⏕⹺⹜㛎ߪታᣉߒߥ޿‫ޕ‬
2.5. ⏕⹺⹜㛎㧔ㅊടേ‛ࠍ↪޿ࠆin vivo ⌒ೝỗᕈ⹜㛎㧕ೋ࿁⹜㛎ߦ߅޿ߡ⣣㘩ᕈߥ޿ߒᒝ޿ೝỗᕈ૞↪߇ⷰኤߐࠇߥ޿႐วߪ㧘1඘ߥ
޿ߒ2඘ߩㅊടേ‛ߢೝỗ෻ᔕߩ᦭ήࠍ⏕⹺ߔࠆ‫ޕ‬వߩ⹜㛎ߢਛ╬ᐲߩೝỗᕈ߇⹺߼ࠄࠇߚ႐วߪ㧘2඘ߩㅊടേ‛ࠍหᤨߦᛩਈߔࠆࠃࠅ߽㧘1඘ߕߟߢ⏕⹺⹜㛎ࠍᲑ㓏⊛ߦታᣉߔࠆߎߣ߇ផᅑߐࠇࠆ‫ߡߒߘޕ‬2඘⋡ߩേ‛ߦ߅޿ߡ㧘⣣㘩ᕈ߹ߚߪᒝ޿ೝỗᕈ૞↪߇⹺߼ࠄࠇߚ႐วߪ⹜㛎ࠍ⛮⛯ߒߥ޿‫ߚ߹ޕ‬2඘⋡ߩേ‛ߩ⚿ᨐߢࡂࠩ࡯࠼ಽ㘃ߦචಽߣ್ᢿߐࠇࠆ႐ว㧘ߘߩᓟߩ⹜㛎ࠍ⛮⛯ߒߥ޿‫ޕ‬59

2.6. ⷰኤᦼ㑆ⷰኤᦼ㑆ߣߒߡߪ㧘ⷰኤߐࠇߚ૞↪ߩ⒟ᐲ෸߮นㅒᕈࠍචಽߦ⹏ଔߢ߈ࠆᦼ
㑆ߣߔࠆ‫ߒ߆ߒޕ‬㧘േ‛߇㊀ᐲߩ⧰∩߹ߚߪ㓚ኂࠍ␜ߒߚ႐วߪ㧘ߘߩᤨὐߢ
ታ㛎ࠍ⚳ੌߔࠆ‫ޕ‬૞↪ߩนㅒᕈࠍ᳿ቯߔࠆߦߪ㧘ⵍ㛎‛⾰ᛩਈᓟ㧘ㅢᏱ21ᣣ㑆ⷰኤߔࠆ‫ޕ‬นㅒᕈߢ޽ࠆߎߣ߇21ᣣએ೨ߦ⏕⹺ߢ߈ߚ႐วߪ㧘ታ㛎ࠍߘߩᤨὐ
ߢ⚳ੌߔࠆ‫ޕ‬
2.7. ⥃ᐥⷰኤ෸߮⌒ೝỗᕈࠬࠦࠕ㧔⹏ὐ㧕ᛩਈᓟ1ᤨ㑆෸߮ߘߩᓟᢙᣣ㑆Ფᣣⷰኤߒ㧘⌒ߩ៊்ߩ᦭ήࠍ൮᜝⊛ߦ⹏ଔߔࠆ‫ޕ‬ᛩਈᓟ3ᣣ㑆ߪ㧝ᣣߦᢙ࿁ⷰኤߒ㧘⹜㛎ਛᱛᤨᦼࠍㆡቱ᳿ቯߔࠆ‫⹜ޕ‬㛎േ‛ߪ⹜㛎ᦼ㑆ਛቯᦼ⊛ߦ⧰∩෸߮∩ߺ㧔଀㧧⸅ࠇࠆ߆ᡂࠆ㧘ㆊᐲߩ⍍߈ࠍߔࠆ㧘ㆊᐲߩᵹᶡߥߤ㧕ࠍዋߥߊߣ߽1ᣣ2࿁㧘6ᤨ㑆એ਄ߩ㑆㓒ߢⷰኤߒ㧘߹ߚᔅⷐߦ
ᔕߓߡߘࠇએ਄ߩ㗫ᐲߢⷰኤߔࠆ‫ࡦࠗ࠮࡟ࠝ࡞ࡈޕ‬ᨴ⦡ߪቯᦼ⊛ߦⴕ߁ߴ߈ߢ
޽ࠅ㧘ᔅⷐߣ⠨߃ࠄࠇߚ႐วߪ⚦㓗Ἦ↢૕㗼ᓸ㏜ࠍ૶↪ߒ㧘⌒ߩ៊்ၞߩᬌ಴
෸߮⸘᷹ࠍㆡಾߦ್ᢿߔࠆ㧔଀㧧ⷺ⤑ẩ≌߇⹺߼ࠄࠇࠆᤨߩ៊்ߩᷓߐߩᬌ಴㧕‫ޕ‬߹ߚߎࠇࠄᣇᴺߪੱ㆏⊛቟ᭉᲕಣಽߩߚ߼ߩ೙ቯߐࠇߚࠛࡦ࠼ࡐࠗࡦ࠻ⷐઙࠍ
ḩߚߔ߆ߩ⹏ଔߦ߽૶↪ߐࠇࠆ‫▸ߩࠫ࡯ࡔ࠳ߩ⌒ޕ‬࿐ࠍᕡਭ⊛ߦ⸥㍳ߔࠆ⋡⊛ߢ㧘ෳ⠨⾗ᢱߣߒߡ்ኂㇱ૏ࠍ࠺ࠫ࠲࡞ࠞࡔ࡜߽↪޿ߡ᠟ᓇߔࠆ‫⏕ޕ‬ታߥ࠺࡯࠲߇ᓧࠄࠇߚߩߜߪ㧘ਇᔅⷐߦ㐳ߊേ‛ࠍ⛽ᜬߒߡߪߥࠄߥ޿‫ޕ‬േ‛߇㊀◊ߥ⧰∩ࠍ␜ߒߚ႐วߪㆃṛߥߊੱ㆏⊛቟ᭉᲕಣಽߒߥߌࠇ߫ߥࠄߕ‫ⵍޔ‬㛎‛⾰ߩ⹏ଔߪએਅߩࠃ߁ߦⴕ߁‫ޕ‬ᛩਈᓟ㧘ᰴߩࠃ߁ߥ⌒்ኂࠍ⿠ߎߒߚേ‛ߪ቟ᭉᲕࠍታᣉߔࠆ‫⓫ߩ⤑ⷺޕ‬ሹ߿߱ߤ߁⣲㧔Staphyloma㧕ࠍ฽߻ⷺ⤑ߩ㊀ᐲߩẩ≌ᒻᚑ㧘೨⌒ᚱߩ಴ⴊ㧘⒟ᐲ
4ߩⷺ⤑ᷙỘ㧘72ᤨ㑆⛮⛯ߔࠆኻశ෻኿ߩᶖᄬ㧔Ⰲᓀ෻ᔕ㧧⒟ᐲ2㧕㧘⚿⤑ߩẩ≌㧘⚿⤑߹ߚߪ⍍⤑ߩუᱫ㧘⣕⪭߇᜼ߍࠄࠇࠆ‫∛ߥ߁ࠃߩߎޕ‬ᄌߪ৻⥸⊛ߦਇนㅒᕈߢ޽ࠆߎߣߦࠃࠆ‫ߦࠄߐޕ‬㧘21ᣣ㑆ߩⷰኤ⚳ੌ߹ߢߦᰴߩ⌒∛ᄌ߇⹺߼ࠄࠇߚ႐วߦߪੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߣߒߡ⹜㛎ਛᱛߣ್ᢿߔࠆߎߣࠍផᅑߔ
ࠆ‫∛⌒ߩࠄࠇߘޕ‬ᄌߪ㧘ᷓ޿៊்㧔଀㧦ⷺ⤑ታ⾰ጀߦ㆐ߔࠆⷺ⤑៊்㧕㧘ⷺ⤑ベㇱߩ៊்߇50㧑એ਄㧔⚿⤑⚵❱ߩ⫷⊕ൻߦࠃࠅ⹏ଔ㧕㧘㊀ᐲߩ⌒ᗵᨴ㧔ൻ⤥ᕈߩಽᴲ‛㧕ߢ޽ࠅ㧘ߎࠇࠄߩ⌒∛ᄌߪ21ᣣ㑆ߩⷰኤ⚳ੌᤨ߹ߢߦቢోߦ࿁ᓳ
ߔࠆߎߣ߇ᦸ߼ߥ޿㧘޽ࠆ޿ߪ⣣㘩ᕈ߹ߚߪᒝ޿ೝỗᕈࠍ␜ߔ៊்ࠍᒁ߈⿠ߎ
ߔߣ⠨߃ࠄࠇࠆ߆ࠄߢ޽ࠆ‫ߚ߹ޕ‬㧘ⷺ⤑⴫㕙߳ߩⴊ▤ᣂ↢㧔ࡄࡦ࠿ࠬ㧕㧘ࡈ࡞ࠝ࡟࠮ࠗࡦߦࠃࠆᨴ⦡ၞ㧔Ფᣣߩⷰኤߦࠃߞߡ❗ዊߒߥ޿㧕㧘޽ࠆ޿ߪⵍ㛎‛⾰ᛩਈᓟ5ᣣએ㒠ߦ⷗ࠄࠇࠆⷺ⤑਄⊹ౣ↢ߩᰳ⪭ߥߤߩⶄว⊛ᚲ⷗߽߹ߚᣧᦼ
⹜㛎⚳ੌߩⷰኤ㗄⋡ߣߒߡ⠨߃ࠄࠇࠆ‫ޕ‬ߒ߆ߒߥ߇ࠄ㧘਄⸥⌒ᚲ⷗ߩ1ߟ1ߟߢߪᣧᦼ⹜㛎⚳ੌߩ್ᢿ᧚ᢱߣߒߡචಽ
610ߢߪߥ޿‫ޕ‬ᒝᐲߩ⌒߳ߩᓇ㗀߇⹺߼ࠄࠇߚ႐ว㧘ᣧᦼ⹜㛎⚳ੌߔߴ߈߆ߤ߁߆
ߩ್ᢿߩߚ߼㧘ㆬછ₞කᏧ޽ࠆ޿ߪ⾗ᩰߩ޽ࠆታ㛎േ‛₞කᏧ‫⥃ߪ޿ࠆ޽ޔ‬ᐥళ୥ࠍ⏕⹺ߢ߈ࠆࠃ߁⸠✵ߐࠇߚ⠪ߦ₞කቇ⊛⹜㛎ߦ㑐ߒߡഥ⸒ࠍ᳞߼ߨ߫ߥ
ࠄߥ޿‫ߩ⌒ޕ‬ೝỗᕈ෻ᔕ㧔⚿⤑㧘ⷺ⤑෸߮Ⰲᓀ㧕ߪⵍ㛎‛⾰ᛩਈᓟ1㧘24㧘48
෸߮72ᤨ㑆ߦណὐߔࠆ‫்ߩ⌒ޕ‬ኂ߇⹺߼ࠄࠇߥ޿ᤨߪ㧘ᛩਈᓟ3ᣣએౝߦ⹜㛎ࠍ⚳ੌߐߖߡߪߥࠄߥ޿‫ޕ‬シᐲ߆ࠄਛ╬ᐲߩೝỗᕈ߇᣿⍎ߦ⹺߼ࠄࠇࠆ႐วߪᛩ
ਈᓟ21ᣣ߹ߢⷰኤߒ㧘ߘߩᤨὐߢ⹜㛎ࠍ⚳ੌߔࠆ‫ⷰޕ‬ኤߩታᣉ෸߮⸥㍳ߪዋߥߊߣ߽ᛩਈᓟ1㧘24㧘48㧘72ᤨ㑆㧘7㧘14෸߮21ᣣߦ៊்ߩ⁁ᘒࠍ⹏ଔߒߡ㧘นㅒᕈ߆㕖นㅒᕈ߆ࠍ್ᢿߔࠆ‫⹜ޕ‬㛎േ‛߇ੱ㆏⊛ߥ㈩ᘦ߆ࠄ቟ᭉᲕಣಽߦߐࠇ
ࠆߴ߈߆㧘ุቯ⊛ߥ⚿ᨐߦࠃࠅ⹜㛎߆ࠄ㒰ᄖߔࠆߴ߈߆ߦߟ޿ߡߪ㧘ࠃࠅ㗫❥ߥⷰኤߦࠃߞߡ್ᢿߔߴ߈ߢ޽ࠆ‫ޕ‬⌒ೝỗᕈߩࠬࠦࠕ㧔⹏ὐ㧕ࠍฦ⹜㛎ߢ⸥㍳ߔࠆ‫ߩ⌒ߩઁߩߘޕ‬ᚲ⷗㧔଀߃߫ࡄࡦ࠿ࠬ㧘ᨴ⦡㧘೨⌒ᚱߩᄌൻ㧕߿ోりᕈߩ᦭ኂ૞↪߽߹ߚ⸥㍳ߔࠆ‫ޕ‬෻ᔕߩᬌᩏߪ㧘෺⌒࡞࡯ࡍ㧘ᚻᜬߜᑼ⚦㓗Ἦ↢૕㗼ᓸ㏜߹ߚߪઁߩㆡಾߥౕེ
ߩ૶↪ߦࠃߞߡ౞Ṗߦታᣉߢ߈ࠆ‫ޕ‬24ᤨ㑆⋡ߩⷰኤࠍ⸥㍳ᓟ㧘ࡈ࡞ࠝ࡟࠮ࠗࡦ
ࠍ↪޿ߡ⌒ࠍߐࠄߦᬌᩏߒߡ߽ࠃ޿‫⌒ޕ‬ೝỗᕈߩࠬࠦࠕ㧔⹏ὐ㧕ߪਥⷰ⊛ߦߥࠅ߿ߔߊ⌒ೝỗᕈ෻ᔕ⹏ଔߩ৻⥌ࠍଦㅴߐߖࠆߚ߼㧘߹ߚ⹜㛎ᣉ⸳෸߮ታ㛎⠪߇ⷰኤߩታᣉ෸߮ℂ⸃ࠍኈᤃߣߔࠆߚ߼㧘ណὐᣇᴺߦߟ޿ߡⷰኤ⠪ߩㆡಾߥ⸠
✵߇ᔅⷐߢ޽ࠆ‫ޕ‬
3. ⚿ᨐߩ⹏ଔ
⌒ೝỗᕈࠬࠦࠕࠍណὐߒ㧘ߘߩᕈ⾰෸߮㊀∝ᐲ㧘ߥࠄ߮ߦߘߩนㅒᕈߩ᦭ή
ߦߟ޿ߡGHSߩၮḰߦᓥ޿⹏ଔߔࠆ‫ߚ߹ޕ‬㧘୘೎ߩࠬࠦࠕߪೝỗᕈ⹏ଔߩ⛘ኻ⊛ߥᮡḰߣߪߺߥߐߥ޿‫ޕ‬
4. ⌒ೝỗᕈ෸߮⣣㘩ᕈ⹏ଔߩߚ߼ߩᲑ㓏⊛⹜㛎ᚢ⇛㧔⹜㛎ࠟࠗ࠼࡜ࠗࡦ405ߩઃዻᢥᦠ㧕⑼ቇ⊛ᅷᒰᕈ෸߮േ‛⑔␩ߩታᣉࠍ⋡⊛ߣߒߡ㧘േ‛ߩ⌒ߦ㊀ᐲߩೝỗᕈࠍ⺃⿠ߔࠆน⢻ᕈ߇㜞޿ൻቇ‛⾰ߩ⌒ೝỗᕈ⹜㛎ࠍ࿁ㆱ޽ࠆ޿ߪᦨዊ㒢ߦߒߡ㧘ਇᔅⷐߥേ‛ߩ૶↪ࠍㆱߌࠆߎߣ߇㊀ⷐߥ໧㗴ߢ޽ࠆ‫ߚ޿↪ࠍࠡࠨ࠙ޕ‬in vivo⹜㛎ߩታᣉ೨ߦ㧘Ბ㓏⊛⹜㛎ᚢ⇛ߩ৻ㇱߣߒߡൻቇ‛⾰ߩẜ࿷⊛⌒ೝỗᕈ࡮⣣㘩ᕈߦ㑐ߔࠆߔߴߡߩᖱႎࠍ෼㓸ߒ㧘⹏ଔߔࠆᔅⷐ߇޽ࠆ‫ޕ‬એਅߩᲑ㓏⊛⹜㛎ᚢ⇛ߦࠃࠆᖱႎࠍ⹏ଔߒߡ㧘ᒝᐲߩೝỗᕈ߿⣣㘩ᕈ߇⹺߼ࠄࠇࠆ႐วߦߪin vivo⹜㛎ࠍታᣉߔߴ߈ߢߪߥ޿‫ޕ‬(1) ࡅ࠻߹ߚߪേ‛ߩᣢሽ࠺࡯࠲෸߮࿖㓙⊛ߦᛚ⹺ߐࠇߚᣇᴺߦࠃࠆin vitro
711⹜㛎࠺࡯࠲ߩ⹏ଔ
(2) ᭴ㅧᵴᕈ⋧㑐㧔SAR㧕ߩಽᨆ(3) ‛ℂൻቇ⊛․ᕈ෸߮ൻቇ෻ᔕᕈ㧔଀߃߫㧘2.0એਅ߹ߚߪ11.5એ਄ߩᭂ┵ߥ
pHࠍ␜ߔ‛⾰㧘ᒝ޿ዪᚲ૞↪ߩน⢻ᕈ㧕(4) ਄⸥એᄖߩᣢሽᖱႎ㧔⊹⤏⚻〝߆ࠄߩోりᲥᕈߦߟ޿ߡߩ೑↪น⢻ߥߔߴߡߩᖱႎ㧕(5) ⊹⤏⣣㘩ᕈߩ⹏ଔ㧔⊹⤏⣣㘩ᕈ෸߮ᒝ޿ೝỗᕈᓇ㗀㧧OECD⹜㛎ࠟࠗ࠼࡜
ࠗࡦ404޽ࠆ޿ߪ࿖㓙⊛ߦᛚ⹺ߐࠇߚin vitro ⊹⤏⣣㘩ᕈ⹜㛎ᴺߢߩ⹏ଔ㧕(6) in vitro߹ߚߪex vivo⹜㛎߆ࠄߩ⚿ᨐ㧔࿖㓙⊛ߦᛚ⹺ߐࠇߚin vitro߹ߚߪ
ex vivo⹜㛎ߢߩ⣣㘩ᕈߥ޿ߒᒝ޿ೝỗᕈ㧕
5. ⌒ೝỗᕈ⹏ଔᆔຬળߩ⷗⸃ ᧄᆔຬળߪࠊ߇࿖߽ᧄᡷ⸓⹜㛎ࠟࠗ࠼࡜ࠗࡦࠍฃߌ౉ࠇࠆߴ߈ߢ޽ࠆߣ⠨߃ߚ‫ࠄ߇ߥߒ߆ߒޕ‬㧘ࠟࠗ࠼࡜ࠗࡦߩㆡ↪ߦ㓙ߒߡߪᰴߩὐࠍ⠨ᘦߔߴ߈ߢ޽
ࠆ‫ޕ‬㧔㧝㧕ᧄ⹜㛎ࠟࠗ࠼࡜ࠗࡦߢ଀␜ߐࠇߡ޿ࠆዪᚲ㤗㈮⮎ߪࠊ߇࿖ߢߪක⮎ຠߣߒߡᵹㅢߒߡ޿ߥ޿‫߃ࠁࠇߘޕ‬㧘ࠊ߇࿖ߢ౉ᚻน⢻ߥዪᚲ㤗㈮⮎ࠍ↪޿ࠆߴ߈ߢ޽ࠆ‫ޕ‬㒐⣣೷ࠍᷝടߒߡ޿ߥ޿ዪᚲ㤗㈮ὐ⌒⮎ߣߒߡ㧘‫ࠗࠞࡠࡊࡉࠪࠠࠝޟ‬ࡦႮ㉄Ⴎࡒ࠾ࡓࠬὐ⌒ᶧ0.4%‫ޠࡘࠫࡦ࠮ޟ‬㧔ජኼ⵾⮎ᩣᑼળ␠㧕‫߹ޕࠆ޽߇ޠ‬ߚ㧘ዪᚲ㤗㈮⮎ේ૕ࠍ↪޿ߡ㧘0.5㧑Ⴎ㉄ࡊࡠࡄ࡜ࠞࠗࡦ޽ࠆ޿ߪ0.5% Ⴎ㉄࠹࠻࡜ࠞࠗࡦࠍ⺞⵾ߔࠆ႐วߦߪ㧘㤗㈮⮎ߦ㑐ߔࠆᖱႎࠍᾫ⍮ߒߚ₞කᏧ޽ࠆ޿
ߪ⮎೷Ꮷߩᜰዉߩਅߢή⩶⊛ߦታᣉߔࠆᔅⷐ߇޽ࠆ‫ޕ‬㧔㧞㧕ᧄࠟࠗ࠼࡜ࠗࡦߦ⸥タߐࠇߡ޿ࠆታ㛎േ‛₞කᏧߪ㧘᰷☨ߢߪታ㛎േ‛⑔␩ࠍᜂ଻ߔࠆߚ߼ߦቯ⟵ߐࠇߡ޿ࠆ߇㧘ᚒ߇࿖ߩ㑐ㅪᴺ઎ᜰ㊎ߦߪߘߩቯ⟵߇ߥ޿‫߃ࠁࠇߘޕ‬㧘ታ㛎േ‛₞කᏧߣห╬ߥ₞කᏧ޽ࠆ޿ߪߘࠇߦḰߕࠆታ㛎േ‛ߩ↢ℂ㧘∔ᖚ㧘∝⁁╬ߦᾫ⍮ߒߚ⠪߇₞කቇ⊛⹜㛎ࠍⴕ޿ੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻ߩ್ቯࠍⴕ߁߽ߩߣߔߴ߈ߢ޽ࠆ‫ޕ‬㧔㧟㧕ᧄࠟࠗ࠼࡜ࠗࡦߦ⸥タߐࠇߡ޿ࠆᲑ㓏⊛⹜㛎ᚢ⇛㧔਄⸥㧠㗄㧕ߢߪ㧘ൻቇ‛⾰ߩẜ࿷⊛⌒ೝỗᕈ࡮⣣㘩ᕈߦ㑐ߔࠆߔߴߡߩᖱႎ㧘⊹⤏ೝỗᕈ⹜㛎ᖱႎ෸߮⌒ೝỗᕈin vitro߹ߚߪex vivo⹜㛎ᖱႎࠍ෼㓸࡮⹏ଔߒ㧘ߎࠇࠄߩᖱႎ෸߮
⹜㛎ᖱႎߦࠃࠅᒝᐲߩೝỗᕈ߿⣣㘩ᕈ߇⹺߼ࠄࠇࠆߣ್ᢿߐࠇࠆ႐วߦߪ㧘࠙ࠨࠡࠍ↪޿ࠆin vivo⹜㛎㧔OECDࠟࠗ࠼࡜ࠗࡦTG405㧕ࠍታᣉߔߴ߈ߢߪߥ޿
ߣផᅑߒߡ޿ࠆ‫⌒ᧄޕ‬ೝỗᕈ⹏ଔᆔຬળߪ㧘࠙ࠨࠡࠍ↪޿ߚ⌒ೝỗᕈ⹜㛎ߩታᣉߦ޽ߚࠅ਄⸥Ბ㓏⊛⹜㛎ᚢ⇛ࠍචಽ⠨ᘦߒߚ਄ߢᒰ⹥⌒ೝỗᕈ⹜㛎ࠍታᣉߔߴ߈ߢ޽ࠆߣ್ᢿߒߚ‫ޕ‬812

6㧚 ᢥ₂
1) OECD (2012), Test No. 405: Acute Eye Irritation/Corrosion. OECD
Guidelines for the Testing of Chemicals, Section 4, OECD Publishing.
2) ICCVAM (2010), ICCVAM Test Method Evaluation Report:
Recommendations for Routine Use of Topical Anesthetics, Systemic
Analgesics, and Humane Endpoints to Avoid or Minimize Pain and Distressin Ocular Safety Testing, NIH Publication No. 10-7514, Research Triangle
Park, NC, USA.

9
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ෳ⠨⾗ᢱ
↪⺆⸃⺑
ㆬછ₞කᏧ㧦⹜㛎⎇ⓥᯏ㑐ߦ߅޿ߡ૶↪ߐࠇࠆߔߴߡߩേ‛ߩஜᐽߣ⑔␩ߦ
ኻߒߡ⽿છࠍ᦭ߔࠆ‫⽿ߩߎޕ‬છࠍㆀⴕߔࠆߚ߼⎇ⓥᯏ㑐߆ࠄචಽߥᮭ
㒢ߣ₞කቇ⊛ࠤࠕ⸘↹ࠍㆇ↪ߔࠆߚ߼ߩ⾗Ḯ㧔ੱ⊛‫⾗ޔ⊛‛ޔ‬㊄⊛㧕ࠍਈ߃ࠄࠇߡ޿ࠆ‫ޕ‬ታ㛎േ‛₞කᏧ㧔Laboratory Animal Veterinarian㧕㧦ᚒ߇࿖ߢߪ㧘࿖㓙ታ㛎േ‛කቇኾ㐷කදળߦዻߔࠆᣣᧄታ㛎േ‛කቇኾ㐷කදળ߇‫ޟ‬ታ㛎േ‛කቇኾ㐷ක‫⾗߁޿ߣޠ‬ᩰࠍ⹺ቯߒߡ޿ࠆ‫ߪࠇߎޕ‬ታ㛎േ‛ߩ∛᳇㧘ᴦ≮㧘ಣ⟎ߦᾫ⍮ߒߚ₞කᏧߢ޽ࠆ‫ޕ‬⥃ᐥళ୥ࠍ⏕⹺ߢ߈ࠆࠃ߁⸠✵ߐࠇߚ⠪㧦ᚒ߇࿖ߢߪ㧘₞කᏧએᄖߦ⹥ᒰߔ
ࠆ⹺ቯ⾗ᩰߪߥ޿‫⃻ޕ‬Ბ㓏ߢߪ㧘േ‛⋴⼔Ꮷ⛔৻⹺ቯᯏ᭴߇⹺ቯߒߚ
‫⹺ޟ‬ቯേ‛⋴⼔Ꮷ‫߿ޠ‬ᣣᧄታ㛎േ‛ᛛⴚ⠪දળߢᚲቯߩᢎ⢒⸠✵ࠍฃߌߚ⠪ߥߤ߇㧘ߎࠇߦ⹥ᒰߔࠆ଀ߣߒߡ᜼ߍࠄࠇࠆ‫ޕ‬ੱ㆏⊛ࠛࡦ࠼ࡐࠗࡦ࠻㧔Humane Endpoint㧕㧦 േ‛ታ㛎ߦ߅ߌࠆᒝ޿∩ߺ㧘⧰∩㧘ᗵᨴ∝⟕ᖚ㧘߹ߚߪἄᱫᦼࠍ್ᢿߔࠆߚ߼ߩ⹏ଔᜰᮡ‫ޕ‬

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OECD/OCDE

405
Adopted:
2 October 2012

OECD GUIDELINE FOR THE TESTING OF CHEMICALSAcute Eye Irritation/Corrosion

INTRODUCTION
1. OECD Guidelines for Testing of Chemicals are periodically reviewed to ensure that they reflect thebest available science. In previous reviews of this Test Guideline, special attention was given to possible
improvements through the evaluation of all existing information on the test substance in order to avoid
unnecessary testing in laboratory animals and thereby address animal welfare concerns. This Test
Guideline (adopted in 1981 and updated in 1987, 2002, and 2012) includes the recommendation that prior
to undertaking the described in vivo test for acute eye irritation/corrosion, a weight-of-the-evidence
analysis be performed (1) on the existing relevant data. Where insufficient data are available, it isrecommended that they be developed through application of sequential testing (2) (3). The testing strategy
includes the performance of validated and accepted in vitro tests and is provided as a Supplement to the
Guideline. Testing in animals should only be conducted if determined to be necessary after considerationof available alternative methods, and use of those determined to be appropriate. At the time of drafting of
this updated TG 405, there are instances where using this Test Guideline is still necessary or required bysome regulatory authorities.
2. The latest update mainly focused on the use of analgesics and anesthetics without impacting the basic
concept and structure of the Test Guideline. ICCVAM and an independent international scientific peer
review panel reviewed the usefulness and limitations of routinely using topical anesthetics, systemic
analgesics, and humane endpoints during in vivo ocular irritation safety testing (12). The review concluded
that the use of topical anesthetics and systemic analgesics could avoid most or all pain and distress without
affecting the outcome of the test, and recommended that these substances should always be used. This Test
Guideline takes this review into account. Topical anesthetics, systemic analgesics, and humane endpoints
should be routinely used during acute eye irritation and corrosion in vivo testing. Exceptions to their use
should be justified. The refinements described in this proposal will substantially reduce or avoid animal
pain and distress in most testing situations where in vivo ocular safety testing is still necessary.
3. Balanced preemptive pain management should include (i) routine pretreatment with a topical anesthetic(e.g., proparacaine or tetracaine) and a systemic analgesic (e.g. buprenorphine), (ii) routine post-treatment
schedule of systemic analgesia (e.g., buprenorphine and meloxicam), (iii) scheduled observation,
monitoring, and recording of animals for clinical signs of pain and/or distress, and (iv) scheduled
observation, monitoring, and recording of the nature, severity, and progression of all eye injuries. Further
detail is provided in the updated procedures described below. Following test substance administration, no
additional topical anesthetics or analgesics should be applied in order to avoid interference with the study.

© OECD, (2012)
You are free to use this material for personal, non-commercial purposes without seeking prior consent fromthe OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written
permission from the OECD.

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Analgesics with anti-inflammatory activity (e.g., meloxicam) should not be applied topically, and doses
used systemically should not interfere with ocular effects.
4. Definitions are set out in the Annex to the Guideline.
INITIAL CONSIDERATIONS
5. In the interest of both sound science and animal welfare, in vivo testing should not be considered until
all available data relevant to the potential eye corrosivity/irritation of the substance have been evaluated ina weight-of-the-evidence analysis. Such data include evidence from existing studies in humans and/or
laboratory animals, evidence of eye corrosivity/irritation of one or more structurally related substances or
mixtures of such substances, data demonstrating high acidity or alkalinity of the substance (4) (5), andresults from validated and accepted in vitro or ex vivo tests for skin corrosion and eye corrosion/irritation(6) (13) (14) (15) (16) (17). The studies may have been conducted prior to, or as a result of, a weight-ofthe-evidence analysis.
6. For certain substances, such an analysis may indicate the need for in vivo studies of the ocularcorrosion/irritation potential of the substance. In all such cases, before considering the use of the in vivo
eye test, preferably a study of the in vitro and/or in vivo skin corrosion effects of the substance should be
conducted first and evaluated in accordance with the sequential testing strategy in Test Guideline 404 (7).
7. A preferred sequential testing strategy, which includes the performance of validated in vitro or ex vivoeye corrosion/irritation tests, is included as a Supplement to this Guideline. It is recommended that thistesting strategy be followed prior to undertaking in vivo testing. For new substances, it is the recommended
stepwise testing approach for developing scientifically sound data on the corrosivity/irritation of thesubstance. For existing substances with insufficient data on skin and eye corrosion/irritation, the strategy
can be used to fill missing data gaps. The use of a different testing strategy or procedure, or the decision
not to use a stepwise testing approach, should be justified.
PRINCIPLE OF THE IN VIVO TEST8. Following pretreatment with a systemic analgesic and induction of appropriate topical anesthesia, thesubstance to be tested is applied in a single dose to one of the eyes of the experimental animal; theuntreated eye serves as the control. The degree of eye irritation/corrosion is evaluated by scoring lesions ofconjunctiva, cornea, and iris, at specific intervals. Other effects in the eye and adverse systemic effects arealso described to provide a complete evaluation of the effects. The duration of the study should be
sufficient to evaluate the reversibility or irreversibility of the effects.
9. Animals showing signs of severe distress and/or pain at any stage of the test or lesions consistent withthe humane endpoints described in this Test Guideline (see Paragraph 26) should be humanely killed, andthe substance assessed accordingly. Criteria for making the decision to humanely kill moribund andseverely suffering animals are the subject of a separate Guidance Document (8).
PREPARATIONS FOR THE IN VIVO TEST
Selection of species
10. The albino rabbit is the preferable laboratory animal and healthy young adult animals are used. A
rationale for using other strains or species should be provided.

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Preparation of animals
11. Both eyes of each experimental animal provisionally selected for testing should be examined within 24
hours before testing starts. Animals showing eye irritation, ocular defects, or pre-existing corneal injury
should not be used.
Housing and feeding conditions
12. Animals should be individually housed. The temperature of the experimental animal room should be
20°C (± 3°C) for rabbits. Although the relative humidity should be at least 30% and preferably not exceed
70%, other than during room cleaning, the aim should be 50-60%. Lighting should be artificial, thesequence being 12 hours light, 12 hours dark. Excessive light intensity should be avoided. For feeding,conventional laboratory diets may be used with an unrestricted supply of drinking water.
TEST PROCEDURE
Use of topical anesthetics and systemic analgesics
13. The following procedures are recommended to avoid or minimize pain and distress in ocular safety
testing procedures. Alternate procedures that have been determined to provide as good or better avoidanceor relief of pain and distress may be substituted.
x

Sixty minutes prior to test substance application (TSA), buprenorphine 0.01 mg/kg is administered by
subcutaneous injection (SC) to provide a therapeutic level of systemic analgesia. Buprenorphine andother similar opiod analgesics administered systemically are not known or expected to alter ocularresponses (12).

xFive minutes prior to TSA, one or two drops of a topical ocular anesthetic (e.g. 0.5% proparacainehydrochloride or 0.5% tetracaine hydrochloride) are applied to each eye. In order to avoid possible
interference with the study, a topical anesthetic that does not contain preservatives is recommended.
The eye of each animal that is not treated with a test article, but which is treated with topicalanesthetics, serves as a control. If the test substance is anticipated to cause significant pain and distress,
it should not normally be tested in vivo. However, in case of doubt or where testing is necessary,consideration should be given to additional applications of the topical anesthetic at 5-minute intervals
prior to TSA. Users should be aware that multiple applications of topical anesthetics could potentiallycause a slight increase in the severity and/or time required for chemically-induced lesions to clear.

x

Eight hours after TSA, buprenorphine 0.01 mg/kg SC and meloxicam 0.5 mg/kg SC are administeredto provide a continued therapeutic level of systemic analgesia. While there are no data to suggest that
meloxicam has anti-inflammatory effects on the eye when administered SC once daily, meloxicam
should not be administered until at least 8 hours after TSA in order to avoid any possible interferencewith the study (12).

xAfter the initial 8-hour post-TSA treatment, buprenorphine 0.01 mg/kg SC should be administered
every 12 hours, in conjunction with meloxicam 0.5 mg/kg SC every 24 hours, until the ocular lesions
resolve and no clinical signs of pain and distress are present. Sustained-release preparations ofanalgesics are available that could be considered to decrease the frequency of analgesic dosing.

x“Rescue” analgesia should be given immediately after TSA if pre-emptive analgesia and topicalanesthesia are inadequate. If an animal shows signs of pain and distress during the study, a “rescue”
dose of buprenorphine 0.03 mg/kg SC would be given immediately and repeated as often as every 8hours, if necessary, instead of 0.01 mg/kg SC every 12 hours. Meloxicam 0.5 mg/kg SC would be
administered every 24 hours in conjunction with the “rescue” dose of buprenorphine, but not until atleast 8 hours post-TSA.
3© OECD, (2012)

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Application of the test substance
14. The test substance should be placed in the conjunctival sac of one eye of each animal after gently
pulling the lower lid away from the eyeball. The lids are then gently held together for about one second inorder to prevent loss of the material. The other eye, which remains untreated, serves as a control.
Irrigation
15. The eyes of the test animals should not be washed for at least 24 hours following instillation of the test
substance, except for solids (see paragraph 18), and in case of immediate corrosive or irritating effects. At
24 hours a washout may be used if considered appropriate.
16. Use of a satellite group of animals to investigate the influence of washing is not recommended unless it
is scientifically justified. If a satellite group is needed, two rabbits should be used. Conditions of washingshould be carefully documented, e.g., time of washing; composition and temperature of wash solution;
duration, volume, and velocity of application.
Dose level(1) Testing of liquids
17. For testing liquids, a dose of 0.1 mL is used. Pump sprays should not be used for instilling thesubstance directly into the eye. The liquid spray should be expelled and collected in a container prior to
instilling 0.1 mL into the eye.
(2) Testing of solids
18. When testing solids, pastes, and particulate substances, the amount used should have a volume of 0.1
mL or a weight of not more than 100 mg. The test material should be ground to a fine dust. The volume ofsolid material should be measured after gently compacting it, e.g. by tapping the measuring container. If
the solid test substance has not been removed from the eye of the test animal by physiological mechanisms
at the first observation time point of 1 hour after treatment, the eye may be rinsed with saline or distilledwater.
(3) Testing of aerosols
19. It is recommended that all pump sprays and aerosols be collected prior to instillation into the eye. The
one exception is for substances in pressurised aerosol containers, which cannot be collected due tovaporisation. In such cases, the eye should be held open, and the test substance administered to the eye in a
simple burst of about one second, from a distance of 10 cm directly in front of the eye. This distance mayvary depending on the pressure of the spray and its contents. Care should be taken not to damage the eyefrom the pressure of the spray. In appropriate cases, there may be a need to evaluate the potential for“mechanical” damage to the eye from the force of the spray.
20. An estimate of the dose from an aerosol can be made by simulating the test as follows: the substance issprayed on to weighing paper through an opening the size of a rabbit eye placed directly before the paper.
The weight increase of the paper is used to approximate the amount sprayed into the eye. For volatile
substances, the dose may be estimated by weighing a receiving container before and after removal of thetest material.
Initial test (in vivo eye irritation/corrosion test using one animal)
21. It is strongly recommended that the in vivo test be performed initially using one animal (seeSupplement to Guideline 405: A Sequential Testing Strategy for Eye Irritation and Corrosion).
Observations should allow for determination of severity and reversibility before proceeding to aconfirmatory test in a second animal.
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22. If the results of this test indicate the substance to be corrosive or a severe irritant to the eye using theprocedure described, further testing for ocular irritancy should not be performed.
Confirmatory test (in vivo eye irritation test with additional animals)
23. If a corrosive or severe irritant effect is not observed in the initial test, the irritant or negative response
should be confirmed using up to two additional animals. If an irritant effect is observed in the initial test, it
is recommended that the confirmatory test be conducted in a sequential manner in one animal at a time,rather than exposing the two additional animals simultaneously. If the second animal reveals corrosive or
severe irritant effects, the test is not continued. If results from the second animal are sufficient to allow fora hazard classification determination, then no further testing should be conducted.
Observation period
24. The duration of the observation period should be sufficient to evaluate fully the magnitude andreversibility of the effects observed. However, the experiment should be terminated at any time that the
animal shows signs of severe pain or distress (8). To determine reversibility of effects, the animals should
be observed normally for 21 days post administration of the test substance. If reversibility is seen before 21
days, the experiment should be terminated at that time.
Clinical observations and grading of eye reactions
25. The eyes should be comprehensively evaluated for the presence or absence of ocular lesions one hourpost-TSA, followed by at least daily evaluations. Animals should be evaluated several times daily for the
first 3 days to ensure that termination decisions are made in a timely manner. Test animals should beroutinely evaluated for the entire duration of the study for clinical signs of pain and/or distress (e.g.
repeated pawing or rubbing of the eye, excessive blinking, excessive tearing) (9) (10) (11) at least twice
daily, with a minimum of 6 hours between observations, or more often if necessary. This is necessary to (i)adequately assess animals for evidence of pain and distress in order to make informed decisions on theneed to increase the dosage of analgesics and (ii) assess animals for evidence of established humane
endpoints in order to make informed decisions on whether it is appropriate to humanely euthanize animals,
and to ensure that such decisions are made in a timely manner. Fluorescein staining should be routinely
used and a slit lamp biomicroscope used when considered appropriate (e.g., assessing depth of injury when
corneal ulceration is present) as an aid in the detection and measurement of ocular damage, and to evaluate
if established endpoint criteria for humane euthanasia have been met. Digital photographs of observed
lesions may be collected for reference and to provide a permanent record of the extent of ocular damage.
Animals should be kept on test no longer than necessary once definitive information has been obtained.
Animals showing severe pain or distress should be humanely killed without delay, and the substanceassessed accordingly.
26. Animals with the following eye lesions post-instillation should be humanely killed (refer to Table 1 fora description of lesion grades): corneal perforation or significant corneal ulceration including staphyloma;
blood in the anterior chamber of the eye; grade 4 corneal opacity; absence of a light reflex (iridial response
grade 2) which persists for 72 hours; ulceration of the conjunctival membrane; necrosis of the conjunctivae
or nictitating membrane; or sloughing. This is because such lesions generally are not reversible.
Furthermore, it is recommended that the following ocular lesions be used as humane endpoints to terminate
studies before the end of the scheduled 21-day observation period. These lesions are considered predictiveof severe irritant or corrosive injuries and injuries that are not expected to fully reverse by the end of the
21-day observation period: severe depth of injury (e.g., corneal ulceration extending beyond the superficial
layers of the stroma), limbus destruction >50% (as evidenced by blanching of the conjunctival tissue), andsevere eye infection (purulent discharge). A combination of: vascularization of the cornea surface (i.e.,pannus); area of fluorescein staining not diminishing over time based on daily assessment; and/or lack ofre-epithelialization 5 days after test substance application could also be considered as potentially useful5© OECD, (2012)

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criteria to influence the clinical decision on early study termination. However, these findings individually
are insufficient to justify early study termination. Once severe ocular effects have been identified, an
attending or qualified laboratory animal veterinarian or personnel trained to identify the clinical lesions
should be consulted for a clinical examination to determine if the combination of these effects warrants
early study termination. The grades of ocular reaction (conjunctivae, cornea and iris) should be obtained
and recorded at 1, 24, 48, and 72 hours following test substance application (Table 1). Animals that do notdevelop ocular lesions may be terminated not earlier than 3 days post instillation. Animals with ocularlesions that are not severe should be observed until the lesions clear, or for 21 days, at which time the study
is terminated. Observations should be performed and recorded at a minimum of 1 hour, 24 hours, 48 hours,
72 hours, 7 days, 14 days, and 21 days in order to determine the status of the lesions, and their reversibility
or irreversibility. More frequent observations should be performed if necessary in order to determine
whether the test animal should be euthanized out of humane considerations or removed from the study due
to negative results
27. The grades of ocular lesions (Table 1) should be recorded at each examination. Any other lesions in theeye (e.g. pannus, staining, anterior chamber changes) or adverse systemic effects should also be reported.
28. Examination of reactions can be facilitated by use of a binocular loupe, hand slit-lamp, biomicroscope,or other suitable device. After recording the observations at 24 hours, the eyes may be further examinedwith the aid of fluorescein.
29. The grading of ocular responses is necessarily subjective. To promote harmonisation of grading ofocular response and to assist testing laboratories and those involved in making and interpreting theobservations, the personnel performing the observations need to be adequately trained in the scoring
system used.
DATA AND REPORTING
Evaluation of results
30. The ocular irritation scores should be evaluated in conjunction with the nature and severity of lesions,and their reversibility or lack of reversibility. The individual scores do not represent an absolute standard
for the irritant properties of a material, as other effects of the test material are also evaluated. Instead,individual scores should be viewed as reference values and are only meaningful when supported by a fulldescription and evaluation of all observations.
Test report
31. The test report should include the following information:
Rationale for in vivo testing: weight-of-the-evidence analysis of pre-existing test data, including
results from sequential testing strategy:–

description of relevant data available from prior testing;–data derived in each step of testing strategy;–

description of in vitro tests performed, including details of procedures, results obtained withtest/reference substances;–

description of in vivo dermal irritation / corrosion study performed, including results obtained;–

weight-of-the-evidence analysis for performing in vivo study
Test substance:
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identification data (e.g. chemical name and if available CAS number, purity, known impurities, source,
lot number);–physical nature and physicochemical properties (e.g. pH, volatility, solubility, stability, reactivity withwater);–

in case of a mixture, components should be identified including identification data of the constituent
substances (e.g. chemical names and if available CAS numbers) and their concentrations;–dose applied;
Vehicle:–identification, concentration (where appropriate), volume used;–justification for choice of vehicle.
Test animals:–

species/strain used, rationale for using animals other than albino rabbit;–

age of each animal at start of study;–

number of animals of each sex in test and control groups (if required);–

individual animal weights at start and conclusion of test;–

source, housing conditions, diet, etc.
Anaesthetics and analgesics–

doses and times when topical anaesthetics and systemic analgesics were administered;–

if local anaesthetic is used, identification, purity, type, and potential interaction with test substance.
Results:–

description of method used to score irritation at each observation time (e.g., hand slitlamp,
biomicroscope, fluorescein);–tabulation of irritant/corrosive response data for each animal at each observation time up to removal ofeach animal from the test;–

narrative description of the degree and nature of irritation or corrosion observed;–

description of any other lesions observed in the eye (e.g., vascularization, pannus formation, adhesions,staining);–

description of non-ocular local and systemic adverse effects, record of clinical signs of pain anddistress, digital photographs, and histopathological findings, if any.
Discussion of results.

Interpretation of the results
32. Extrapolation of the results of eye irritation studies in laboratory animals to humans is valid only to alimited degree. In many cases the albino rabbit is more sensitive than humans to ocular irritants or
corrosives.
33. Care should be taken in the interpretation of data to exclude irritation resulting from secondary
infection.

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OECD/OCDELITERATURE(1)Barratt, M.D., et al. (1995), The Integrated Use of Alternative Approaches for Predicting Toxic
Hazard, ECVAM Workshop Report 8, ATLA 23, 410 - 429.

(2)

de Silva, O., et al. (1997), Evaluation of Eye Irritation Potential: Statistical Analysis and Tier
Testing Strategies, Food Chem. Toxicol 35, 159 - 164.

(3)

Worth A.P. and Fentem J.H. (1999), A general approach for evaluating stepwise testing strategies
ATLA 27, 161-177.

(4)Young, J.R., et al. (1988), Classification as Corrosive or Irritant to Skin of Preparations Containing
Acidic or Alkaline Substance Without Testing on Animals, Toxicol. In Vitro, 2, 19 - 26.

(5)

Neun, D.J. (1993), Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at aSingle pH, J. Toxicol. Cut. Ocular Toxicol. 12, 227 - 231.

(6)

Fentem, J.H., et al. (1998), The ECVAM international validation study on in vitro tests for skincorrosivity. 2. Results and evaluation by the Management Team, Toxicology in Vitro 12, pp.483 –
524.

(7)

OECD (2002), Test No. 404: Acute Dermal Irritation/Corrosion, OECD Guidelines for the Testingof Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264070622-en(8)OECD (2000), Guidance Document on the Recognition, Assessment and Use of Clinical Signs as
Humane Endpoints for Experimental Animals Used in Safety Evaluation. OECD Environmental
Health and Safety Publications, Series on Testing and Assessment No. 19,(http://www.oecd.org/ehs/test/monos.htm ).

(9)Wright EM, Marcella KL, Woodson JF. (1985), Animal pain: evaluation and control, Lab Animal,
May/June:20-36.

(10)National Research Council (NRC) (2008), Recognition and Alleviation of Distress in Laboratory
Animals, Washington, DC: The National Academies Press.

(11)

National Research Council (NRC) (2009), Recognition and Alleviation of Pain in Laboratory
Animals, Washington, DC: The National Academies Press.

(12)

ICCVAM (2010), ICCVAM Test Method Evaluation Report: Recommendations for Routine Useof Topical Anesthetics, Systemic Analgesics, and Humane Endpoints to Avoid or Minimize Pain
and Distress in Ocular Safety Testing, NIH Publication No. 10-7514,
Research Triangle Park,
NC, USA: National Institute of Environmental Health Sciences.
Available: http://iccvam.niehs.nih.gov/methods/ocutox/OcuAnest-TMER.htm(13)

OECD (2004), Test No. 430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test
(TER), OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264071124-en(14)OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines
for the Testing of Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264071148-en(15)

OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines
for the Testing of Chemicals, Section 4, OECD Publishing.
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405

doi: 10.1787/9789264071148-en
OECD (2009),Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying
Ocular Corrosives and Severe Irritants, OECD Guidelines for the Testing of Chemicals, Section 4,
OECD Publishing.
doi: 10.1787/9789264076303-en(17)

OECD (2009b), Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe
Irritants, Test Guideline No. 438, OECD Guidelines for the Testing of Chemicals, OECD, Paris(18)

U.S. EPA (2003), Label Review Manual: 3rd Edition, EPA737-B-96-001, Washington, DC: U.S.,
Environmental Protection Agency.

(19)

UN (2011), Globally Harmonized System of Classification and Labelling of Chemicals (GHS),
Fourth revised edition, New York & Geneva: United Nations Publications.

(20)

EC (2008), Regulation (EC) No. 1272/2008 of the European Parliament and of the Council of 16
December 2008 on Classification, Labelling and Packaging of Substances and Mixtures, amending
and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No.
1907/2006. Official Journal of the European Union L353, 1-1355.

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OECD/OCDETABLE 1: GRADING OF OCULAR LESIONS
CorneaGrade

Opacity: degree of density (readings should be taken from most dense area)*
No ulceration or opacity .............................................................................................................. 0
Scattered or diffuse areas of opacity (other than slight dulling of normal lustre); detailsof iris clearly visible ................................................................................................................... 1
Easily discernible translucent area; details of iris slightly obscured ........................................... 2
Nacrous area; no details of iris visible; size of pupil barely discernible ..................................... 3
Opaque cornea; iris not discernible through the opacity ............................................................. 4
Maximum possible: 4* The area of corneal opacity should be noted
Iris
Normal......................................................................................................................................... 0Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia;
or injection; iris reactive to light (a sluggish reaction is considered to be an effect.................... 1
Hemorrhage, gross destruction, or no reaction to light ............................................................... 2
Maximum possible: 2
Conjunctivae
Redness (refers to palpebral and bulbar conjunctivae; excluding cornea and iris)
Normal......................................................................................................................................... 0Some blood vessels hyperaemic (injected) ................................................................................. 1
Diffuse,crimson colour; individual vessels not easily discernible............................................... 2
Diffuse beefy red ......................................................................................................................... 3
Maximum possible: 3
Chemosis
Swelling (refers to lids and/or nictating membranes)
Normal......................................................................................................................................... 0Some swelling above normal....................................................................................................... 1
Obvious swelling, with partial eversion of lids ........................................................................... 2
Swelling, with lids about half closed........................................................................................... 3
Swelling, with lids more than half closed.................................................................................... 4
Maximum possible: 4

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ANNEX
DEFINITIONS
1. Acid/alkali reserve: For acidic preparations, this is the amount (g) of sodium hydroxide/100 g ofpreparation required to produce a specified pH. For alkaline preparations, it is the amount (g) of sodiumhydroxide equivalent to the g sulphuric acid/100 g of preparation required to produce a specified pH(Young et al. 1988).
2. Non irritants: Substances that are not classified as EPA Category I, II, or III ocular irritants; or GHS
eye irritants Category 1, 2, 2A, or 2B; or EU Category 1 or 2 (18) (19) (20).
3. Ocular corrosive: (a) A substance that causes irreversible tissue damage to the eye; (b) Substances thatare classified as GHS eye irritants Category 1, or EPA Category I ocular irritants, or EU Category 1 (18)(19) (20).
4. Ocular irritant: (a) A substance that produces a reversible change in the eye; (b) Substances that areclassified as EPA Category II or III ocular irritants; or GHS eye irritants Category 2, 2A or 2B ; or EU
Category 2 (18) (19) (20).
5. Ocular severe irritant: (a) A substance that causes tissue damage in the eye that does not resolve within
21 days of application or causes serious physical decay of vision; (b) Substances that are classified as GHS
eye irritant Category 1, or EPA Category I ocular irritants, or EU Category 1 (18) (19) (20).
6. Tiered approach: A stepwise testing strategy where all existing information on a test substance isreviewed, in a specified order, using a weight-of-evidence process at each tier to determine if sufficient
information is available for a hazard classification decision, prior to progression to the next tier. If theirritancy potential of a test substance can be assigned based on the existing information, no additional
testing is required. If the irritancy potential of a test substance cannot be assigned based on the existing
information, a step-wise sequential animal testing procedure is performed until an unequivocal
classification can be made.
7. Weight-of-the-evidence (process): The strengths and weaknesses of a collection of information areused as the basis for a conclusion that may not be evident from the individual data.

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OECD/OCDE
SUPPLEMENT TO TEST GUIDELINE 405
A Sequential Testing Strategy for Eye Irritation and Corrosion

GENERAL CONSIDERATIONS
1.
In the interests of sound science and animal welfare, it is important to avoid the unnecessary useof animals, and to minimise testing that is likely to produce severe responses in animals. All information
on a substance relevant to its potential ocular irritation/corrosivity should be evaluated prior to consideringin vivo testing. Sufficient evidence may already exist to classify a test substance as to its eye irritation or
corrosion potential without the need to conduct testing in laboratory animals. Therefore, utilizing a weightof-the-evidence analysis and sequential testing strategy will minimise the need for in vivo testing,especially if the substance is likely to produce severe reactions.
2.
It is recommended that a weight-of-the-evidence analysis be used to evaluate existing
information pertaining to eye irritation and corrosion of substances and to determine whether additional
studies, other than in vivo eye studies, should be performed to help characterise such potential. Where
further studies are needed, it is recommended that the sequential testing strategy be utilised to develop therelevant experimental data. For substances which have no testing history, the sequential testing strategy
should be utilised to develop the data needed to evaluate its eye corrosion/irritation. The initial testing
strategy described in this Supplement was developed at an OECD workshop (1). It was subsequently
affirmed and expanded in the Harmonised Integrated Hazard Classification System for Human Health andEnvironmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the Chemicals
Committee and the Working Party on Chemicals, in November 1998 (2), and updated by an OECD expert
group in 2011.
3.
Although this testing strategy is not an integrated part of Test Guideline 405, it expresses therecommended approach for the determination of eye irritation/corrosion properties. This approach
represents both best practice and an ethical benchmark for in vivo testing for eye irritation/corrosion. The
Guideline provides guidance for the conduct of the in vivo test and summarises the factors that should be
addressed before considering such a test. The sequential testing strategy provides a weight-of-the-evidence
approach for the evaluation of existing data on the eye irritation/corrosion properties of substances and atiered approach for the generation of relevant data on substances for which additional studies are needed or
for which no studies have been performed. The strategy includes the performance first of validated andaccepted in vitro or ex vivo tests and then of Guideline 404 skin irritation/corrosion studies under specificcircumstances (3) (4).
DESCRIPTION OF THE STEPWISE TESTING STRATEGY
4.
Prior to undertaking tests as part of the sequential testing strategy (Figure), all available
information should be evaluated to determine the need for in vivo eye testing. Although significant
information might be gained from the evaluation of single parameters (e.g., extreme pH), the totality ofexisting information should be assessed. All relevant data on the effects of the substance in question, andits structural analogues, should be evaluated in making a weight-of-the-evidence decision, and a rationale
for the decision should be presented. Primary emphasis should be placed upon existing human and animal
data on the substance, followed by the outcome of in vitro or ex vivo testing. In vivo studies of corrosive
substances should be avoided whenever possible. The factors considered in the testing strategy include:
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5.
Evaluation of existing human and/or animal data and/or in vitro data from validated andinternationally accepted methods (Step 1). Existing human data, e.g. clinical and occupational studies, andcase reports, and/or animal test data from ocular studies and/or in vitro data from validated andinternationally accepted methods for eye irritation/corrosion should be considered first, because they
provide information directly related to effects on the eyes. Thereafter, available data from human and/or
animal studies investigating dermal corrosion/irritation, and/or in vitro studies from validated andinternationally accepted methods for skin corrosion should be evaluated. Substances with known
corrosivity or severe irritancy to the eye should not be instilled into the eyes of animals, nor should
substances showing corrosive or severe irritant effects to the skin; such substances should be considered tobe corrosive and/or irritating to the eyes as well. Substances with sufficient evidence of non-corrosivity
and non-irritancy from previously performed ocular studies should also not be tested in in vivo eye studies.
6.
Analysis of structure activity relationships (SAR) (Step 2). The results of testing of structurally
related chemicals should be considered, if available. When sufficient human and/or animal data are
available on structurally related substances or mixtures of such substances to indicate their eye
corrrosion/irritancy potential, it can be presumed that the test substance will produce the same responses.
In those cases, the substance may not need to be tested. Negative data from studies of structurally related
substances or mixtures of such substances do not constitute sufficient evidence of non-corrosivity/nonirritancy of a substance under the sequential testing strategy. Validated and accepted SAR approaches
should be used to identify the corrosion and irritation potential for both dermal and ocular effects.
7.
Physicochemical properties and chemical reactivity (Step 3). Substances exhibiting pH extremes
such as d2.0 or t11.5 may have strong local effects. If extreme pH is the basis for identifying a substanceas corrosive or irritant to the eye, then its acid/alkaline reserve (buffering capacity) may also be taken into
consideration (5)(6)(7). If the buffering capacity suggests that a substance may not be corrosive to the eye(i.e., substances with extreme pH and low acid/alkaline reserve), then further testing should be undertaken
to confirm this, preferably by the use of a validated and accepted in vitro or ex vivo test (see paragraph 10).
8.
Consideration of other existing information (Step 4). All available information on systemic
toxicity via the dermal route should be evaluated at this stage. The acute dermal toxicity of the test
substance should also be considered. If the test substance has been shown to be highly toxic by the dermalroute, it may not need to be tested in the eye. Although there is not necessarily a relationship between acutedermal toxicity and eye irritation/corrosion, it can be assumed that if an agent is highly toxic via the dermalroute, it will also exhibit high toxicity when instilled into the eye. Such data may also be consideredbetween Steps 2 and 3.
9.
Assessment of dermal corrosivity of the substance if also required for regulatory purposes (Step5). The skin corrosion and severe irritation potential should be evaluated first in accordance with Guideline
404 (4) and the accompanying Supplement (8), including the use of validated and internationally acceptedin vitro skin corrosion test methods (9) (10) (11). If the substance is shown to produce corrosion or severe
skin irritation, it may also be considered to be a corrosive or severely irritant to the eye. Thus, no further
testing would be required. If the substance is not corrosive or severely irritating to the skin, an in vitro or
ex vivo eye test should be performed.
10.
Results from in vitro or ex vivo tests (Step 6). Substances that have demonstrated corrosive or
severe irritant properties in an in vitro or ex vivo test (12) (13) that has been validated and internationallyaccepted for the assessment specifically of eye corrosivity/irritation, need not be tested in animals. It canbe presumed that such substances will produce similar severe effects in vivo. If validated and accepted invitro/ex vivo tests are not available, one should bypass Step 6 and proceed directly to Step 7.
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OECD/OCDE

11.
In vivo test in rabbits (Steps 7 and 8): In vivo ocular testing should begin with an initial test using
one animal. If the results of this test indicate the substance to be a severe irritant or corrosive to the eyes,
further testing should not be performed. If that test does not reveal any corrosive or severe irritant effects, aconfirmatory test is conducted with two additional animals. Depending upon the results of the confirmatory
test, further tests may be needed. [see TG 405]

14
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LITERATURE(1)OECD (1996) OECD Test Guidelines Programme: Final Report of the OECD Workshop on
Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Test Methods.
Held in Solna, Sweden, 22 - 24 January 1996 (http://www.oecd.org/ehs/test/background.htm).

(2)

OECD (1998) Harmonized Integrated Hazard Classification System for Human Health andEnvironmental Effects of Chemical Substances, as endorsed by the 28th Joint Meeting of the
Chemicals Committee and the Working Party on Chemicals, November 1998
(http://www.oecd.org/ehs/Class/HCL6.htm).

(3)Worth, A.P. and Fentem J.H. (1999). A General Approach for Evaluating Stepwise Testing
Strategies. ATLA 27, 161-177.

(4)

OECD (2002), Test No. 404: Acute Dermal Irritation/Corrosion, OECD Guidelines for the Testingof Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264070622-en(5)Young, J.R., How, M.J., Walker, A.P., Worth W.M.H. (1988) Classification as Corrosive or
Irritant to Skin of Preparations Containing Acidic or Alkaline Substance Without Testing on
Animals. Toxicol. In Vitro, 2, 19 - 26.

(6)

Fentem, J.H., Archer, G.E.B., Balls, M., Botham, P.A., Curren, R.D., Earl, L.K., Edsail, D.J.,
Holzhutter, H.G. and Liebsch, M. (1998) The ECVAM international validation study on in vitro
tests for skin corrosivity. 2. Results and evaluation by the Management Team. Toxicology in Vitro
12, pp.483 – 524.

(7)

Neun, D.J. (1993) Effects of Alkalinity on the Eye Irritation Potential of Solutions Prepared at aSingle pH. J. Toxicol. Cut. Ocular Toxicol. 12, 227 - 231.

(8)OECD (2001) Supplement to Test Guideline 404: A Sequential Testing Strategy for Skin Irritation
and Corrosion.

(9)OECD (2004), Test No. 430: In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test
(TER), OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264071124-en(10)OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines
for the Testing of Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264071148-en(11)

OECD (2004), Test No. 431: In Vitro Skin Corrosion: Human Skin Model Test, OECD Guidelines
for the Testing of Chemicals, Section 4, OECD Publishing.
doi: 10.1787/9789264071148-en
15© OECD, (2012)

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(12)

OECD/OCDEOECD (2009),Test No. 437: Bovine Corneal Opacity and Permeability Test Method for Identifying
Ocular Corrosives and Severe Irritants, OECD Guidelines for the Testing of Chemicals, Section 4,
OECD Publishing.
doi: 10.1787/9789264076303-en(13 ) OECD (2009b), Isolated Chicken Eye Test Method for Identifying Ocular Corrosives andSevere Irritants, Test Guideline No. 438, OECD Guidelines for the Testing of Chemicals, OECD, Paris

16© OECD, (2012)

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OECD/OCDEFIGURETESTING AND EVALUATION STRATEGY FOR EYE IRRITATION/CORROSION

1

2

Activity

Finding

Conclusion

Existing human and/or animal data,
and/or in vitro data from validated
and internationally accepted methods
showing effects on eyes

Severe damage to eyes

Apical endpoint; consider corrosive toeyes. No testing is needed.

Eye irritant

Apical endpoint; consider irritating toeyes. No testing is needed.

Not corrosive/not
irritating to eyes

Apical endpoint; considered noncorrosive and non-irritating to eyes.
No testing required.

Existing human and/or animal data
and/or in vitro data from validated
and internationally accepted methods
showing corrosive effects on skin

Skin corrosive

Assume corrosivity to eyes. No testingis needed.

Existing human and/or animal data
and/or in vitro data from validated
and internationally accepted methods
showing severe irritant effects on
skinp
no information available, oravailable information is notconclusivep
Perform SAR for eye
corrosion/irritation

Severe skin irritant

Assume irritating to eyes. No testingis needed

Predict severe damage toeyesAssume corrosivity to eyes. No testingis needed.

Predict irritation to eyes

Assume irritating to eyes. No testingis needed.

Predict skin corrosivity

Assume corrosivity to eyes. No testingis needed.

pH d 2 or t 11.5 (withhigh buffering capacity,
if relevant)Assume corrosivity to eyes. No testingis needed.

Consider SAR for skin corrosion3p
No predictions can be made, orpredictions are not conclusive or
negativepMeasure pH (buffering capacity, if
relevant)p2 pH < 11.5, or pHd2.0 or t11.5
with low/no buffering capacity, if

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OECD/OCDE
relevant
p

4

5

6

7

Consider existing systemic toxicity
data via the dermal routep
Such information is not available, or
substance is not highly toxic
p
Experimentally assess skin corrosion
potential according to the testing
strategy in OECD Guideline 404 if
also required for regulatory purposesp
Substance is not corrosive or
severely irritating to skinp
Perform validated and accepted invitro or ex vivo ocular test(s)pValidated and accepted in vitro or exvivo ocular test(s) cannot be used toreach a conclusionp
Perform initial in vivo rabbit eye test
using one animal

Highly toxic at
Substance would be too toxic forconcentrations that would testing. No testing is needed.
be tested in the eye.

Corrosive or severe
irritant response

Assume corrosive to eyes. No further
testing is needed.

Corrosive or severe
irritant response

Assume corrosive or severe irritant toeyes, provided the test performed canbe used to identify corrosives/severe
irritants and the substance is within
the applicability domain of the test.
No further testing is needed.

Irritant response

Assume irritant to eyes, provided thetest(s) performed can be used tocorrectly identify corrosive, severe
irritants, and irritants, and thesubstance is within the applicability
domain of the test(s). No further
testing is needed.

Non-irritant response

Assume non-irritant to eyes, provided
the test(s) performed can be used tocorrectly
identify
non-irritants,
correctly distinguish these fromsubstances that are irritants, severe
irritants, or ocular corrosives, and thesubstance is within the applicability
domain of the test. No further testingis needed.

Severe damage to eyes

Consider corrosive to eyes. No further
testing is needed.

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8p
No severe damage, or no responsepPerform confirmatory test using one
or two additional animals

405

Corrosive or irritating

Consider corrosive or irritating toeyes. No further testing is neededNot corrosive or
irritating

Consider non-irritating and noncorrosive to eyes. No further testing isneeded.

19© OECD, (2012)

33

34

ICCVAM Test Method Evaluation Report:
Recommendations for Routine Use of Topical Anesthetics,
Systemic Analgesics, and Humane Endpoints to Avoid or
Minimize Pain and Distress in Ocular Safety Testing

Interagency Coordinating Committee on the
Validation of Alternative Methods

National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods

National Institute of Environmental Health Sciences
National Institutes of Health
U.S. Public Health Service
Department of Health and Human Services

2010

NIH Publication No. 10-7514

National Toxicology Program
P.O. Box 12233
Research Triangle Park, NC 27709

35

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

This document is available electronically at

http://iccvam.niehs.nih.gov/methods/ocutox/AAHE-TMER.htm
When referencing this document, please cite as follows:
ICCVAM. 2010. ICCVAM Test Method Evaluation Report: Recommendations for Routine Use of
Topical Anesthetics, Systemic Analgesics, and Humane Endpoints to Avoid or Minimize Pain andDistress in Ocular Safety Testing. NIH Publication No. 10-7514. Research Triangle Park,
NC:National Institute of Environmental Health Sciences.

ii

36

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Table of Contents
List of Tables......................................................................................................................................... v
List of Abbreviations and Acronyms ................................................................................................. vi
Interagency Coordinating Committee on the Validation of Alternative Methods: Agency
Representatives ........................................................................................................................ vii
Acknowledgements ............................................................................................................................ viii
Preface ................................................................................................................................................ xiii
Executive Summary ........................................................................................................................... xv
1.0

Introduction................................................................................................................................. 1

2.0

ICCVAM Recommendations for the Routine Use of Topical Anesthetics andSystemic Analgesics to Avoid or Minimize Pain and Distress in Ocular Safety
Testing......................................................................................................................................... 3
2.1

ICCVAM Recommendations: Test Method Usefulness and Limitations ......................... 3

2.2

ICCVAM Recommendations: Test Method Protocol for the Routine Use of
Topical Anesthetics and Systemic Analgesics .................................................................. 4

2.3

ICCVAM Recommendations: Future Studies for the Routine Use of Topical
Anesthetics and Systemic Analgesics................................................................................ 4

3.0

Validation Status: Routine Use of Topical Anesthetics and Systemic Analgesics in
Ocular Safety Testing ................................................................................................................ 6

4.0

ICCVAM Recommendations for the Use of Humane Endpoints to Avoid or
Minimize Pain and Distress in Ocular Safety Testing ............................................................ 8
4.1

ICCVAM Recommendations: Test Method Usefulness and Limitations ......................... 8

4.2

ICCVAM Recommendations: Changes to the Ocular Safety Testing Protocol toInclude the Use of Humane Endpoints .............................................................................. 9

4.3

ICCVAM Recommendations: Future Studies for the Use of Humane Endpoints............. 9

5.0

Validation Status of the Use of Humane Endpoints to Avoid or Minimize Pain andDistress in Ocular Safety Testing ........................................................................................... 11

6.0

ICCVAM Consideration of Public and SACATM Comments ............................................ 13
6.1

Public Comments in Response to 70 FR 13512 (March 21, 2005): Request for
Data on Non-Animal Methods and Approaches for Determining Skin and Eye
Irritation Potential of Antimicrobial Cleaning Product Formulations; Request for
Nominations for an Independent Expert Panel................................................................ 14

6.2

Public Comments in Response to 72 FR 26396 (May 9, 2007): Request for Dataon the Use of Topical Anesthetics and Systemic Analgesics for In Vivo Eye
Irritation Testing.............................................................................................................. 14

6.3

Public Comments in Response to 72 FR 31582 (June 7, 2007): Request for
Ocular Irritancy Test Data From Human, Rabbit, and In Vitro Studies Using
Standardized Testing Methods ........................................................................................ 14

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

7.0

6.4

Public Comments in Response to 73 FR 18535 (April 4, 2008): Non-Animal
Methods and Approach for Evaluating Eye Irritation Potential for Antimicrobial
Cleaning Products (AMCPs): Request for Nominations for an Independent Expert
Panel and Submission of Relevant Data ..........................................................................15

6.5

Public Comments in Response to 74 FR 14556 (March 31, 2009): Announcementof an Independent Scientific Peer Review Panel on Alternative Ocular Safety
Testing Methods; Availability of Draft Background Review Documents (BRD);
Request for Comments.....................................................................................................15

6.6

Public Comments in Response to 74 FR 19562 (April 29, 2009): Meeting of the
Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) ......................................................................................................................16

6.7

Public Comments in Response to 74 FR 33444 (July 13, 2009): Independent
Scientific Peer Review Panel Report: Evaluation of the Validation Status of
Alternative Ocular Safety Testing Methods and Approaches; Notice of
Availability and Request for Public Comments...............................................................16

References...................................................................................................................................17

Appendix A

ICCVAM Evaluation Timeline............................................................................. A-1

Appendix B

ICCVAM-Recommended Test Protocol: Revised OECD Test Guideline
405 (Draize Test for Acute Eye Irritation/Corrosion) .........................................B-1

Appendix C

Background Review Document: Use of Topical Anesthetics, Systemic
Analgesics, and Earlier Humane Endpoints to Minimize Pain and Distressin Ocular Toxicity Testing..................................................................................... C-1

Appendix D

Independent Scientific Peer Review Panel Assessment ...................................... D-1

Appendix E

Appendix F

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D1

Summary Minutes from the Peer Review Panel Meeting on
May 19-21, 2009......................................................................................... D-3

D2

Independent Scientific Peer Review Panel Report: Evaluation of the
Validation Status of Alternative Ocular Safety Testing Methods andApproaches ............................................................................................... D-31

Federal Register Notices and Public Comments ...................................................E-1
E1

Federal Register Notices ............................................................................. E-3

E2

Public Comments Received in Response to Federal Register Notices...... E-19

E3

Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) Comments: SACATM Meeting on June 25-26, 2009 ......... E-109

Relevant U.S. Federal and International Ocular Toxicity Regulations,Labeling and Test Guidelines ................................................................................ F-1
F1

Table of Relevant U.S. Federal and International Ocular Testing
Regulations for Hazard Classification and Labeling ................................... F-3

F2

EPA OPPTS Guidance Document 870.2400 (August 1998)....................... F-9

F3

EPA Office of Pesticide Programs Label Review Manual(August 2003) ............................................................................................ F-19

F4

Organisation for Economic Co-operation and Development (OECD)
Test Guideline 405 (Adopted April 2002) ................................................. F-21

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

List of Tables
Table 6-1

Opportunities for Public Comment ............................................................................ 13v

39

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

List of Abbreviations and Acronyms
ºC
AHT
BRD
CPSC
CV
ECVAM
EPA
ESAC
EU
FDA
FR
g
GHS
GLP
ICCVAMILSIS
JaCVAM
kg
LVET
MAS
MeSH
mg
mL
NICEATM
NIEHS
NSAID
NTP
OECD
OTWG
SACATM
SC
SRD
TG
TSA
UN

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40

Degrees centigrade
Animal Health Technologist
Background review document
U.S. Consumer Product Safety Commission
Coefficient of variation
European Centre for the Validation of Alternative Methods
U.S. Environmental Protection Agency
European Centre for the Validation of Alternative Methods Scientific Advisory
Committee
European Union
U.S. Food and Drug Administration
Federal Register
Gram
United Nations Globally Harmonized System of Classification and Labelling ofChemicals
Good Laboratory PracticeInteragency Coordinating Committee on the Validation of Alternative Methods
Integrated Laboratory Systems, Inc.
Irritation score
Japanese Center for the Validation of Alternative Methods
KilogramLow volume eye test
Maximum average score
Medical Subject Headings
MilligramMilliliter
National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods
National Institute of Environmental Health Sciences
Nonsteroidal anti-inflammatory drug
U.S. National Toxicology Program
Organisation for Economic Co-operation and Development
ICCVAM Ocular Toxicity Working Group
Scientific Advisory Committee on Alternative Toxicological Methods
Subcutaneous
Summary review document
Test guideline
Test substance administration
United Nations

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Interagency Coordinating Committee on the Validation of
Alternative Methods: Agency Representatives
Agency for Toxic Substances and Disease
Registry* Moiz Mumtaz, Ph.D.
Bruce Fowler, Ph.D.
Edward Murray, Ph.D.
Eric Sampson, Ph.D.
Consumer Product Safety Commission* Marilyn L. Wind, Ph.D. (Chair)+Kristina Hatlelid, Ph.D.
Joanna Matheson, Ph.D.
Department of Agriculture* Jodie Kulpa-Eddy, D.V.M. (Vice-Chair)+Elizabeth Goldentyer, D.V.M.
Department of Defense* Robert E. Foster, Ph.D.
+Patty Decot
Harry Salem, Ph.D.
Peter J. Schultheiss, D.V.M., DACLAM
Department of Energy* Michael Kuperberg, Ph.D.
+Marvin Stodolsky, Ph.D.
Department of the Interior* Barnett A. Rattner, Ph.D.
+Sarah Gerould, Ph.D. (to Feb. 2009)
Department of Transportation* George Cushmac, Ph.D.
+Steve Hwang, Ph.D.
Environmental Protection Agency
Office of Pesticide Programs* John R. “Jack” Fowle III, Ph.D., DABT+Vicki Dellarco, Ph.D.
+Tina Levine, Ph.D.
Deborah McCall
Christine Augustyniak, Ph.D. (U.S. Coordinator,
OECD Test Guidelines Program)
Office of Pollution Prevention and Toxics
Jerry Smrchek, Ph.D. (U.S. Coordinator, OECD
Test Guidelines Program, to July 2009)
Office of Research and Development
Suzanne McMaster, Ph.D. (to Dec. 2008)
Julian Preston, Ph.D. (to July 2009)
Stephanie Padilla, Ph.D. (to July 2009)
Office of Science Coordination and Policy
Karen Hamernik, Ph.D. (to July 2009)

Food and Drug Administration
Office of the Commissioner* Suzanne Fitzpatrick, Ph.D., DABTCenter for Biologics Evaluation and Research
Richard McFarland, Ph.D., M.D.
Ying Huang, Ph.D.
Center for Devices and Radiological Health
Melvin E. Stratmeyer, Ph.D.
Vasant G. Malshet, Ph.D., DABTCenter for Drug Evaluation and Research+ Abigail C. Jacobs, Ph.D.
Paul C. Brown, Ph.D.
Center for Food Safety and Applied Nutrition
David G. Hattan, Ph.D.
Robert L. Bronaugh, Ph.D.
Center for Veterinary Medicine
Devaraya Jagannath, Ph.D.
M. Cecilia Aguila, D.V.M.
National Center for Toxicological ResearchPaul Howard, Ph.D.
Donna Mendrick, Ph.D.
William T. Allaben, Ph.D. (to Jan. 2009)
Office of Regulatory Affairs
Lawrence D'Hoostelaere, Ph.D.
National Cancer Institute* T. Kevin Howcroft, Ph.D.
Chand Khanna, D.V.M., Ph.D.
Alan Poland, M.D. (to Oct. 2008)
National Institute of Environmental Health
Sciences* William S. Stokes, D.V.M., DACLAM+ Raymond R. Tice, Ph.D.
Rajendra S. Chhabra, Ph.D., DABT
Jerrold J. Heindel, Ph.D.
National Institute for Occupational Safety and
Health* Paul Nicolaysen, V.M.D.
+ K. Murali Rao, M.D., Ph.D.
National Institutes of Health* Margaret D. Snyder, Ph.D.
National Library of Medicine* Pertti (Bert) Hakkinen, Ph.D.
+ Jeanne Goshorn, M.S.
Occupational Safety and Health Administration* Surender Ahir, Ph.D.

* Principal agency representative+ Alternate principal agency representative

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Acknowledgements
Interagency Coordinating Committee on the Validation of
Alternative Methods (ICCVAM) Ocular Toxicity Working Group (OTWG)
U.S. Consumer Product Safety Commission
Marilyn L. Wind, Ph.D.
Adrienne Layton, Ph.D.
U.S. Department of Defense
Harry Salem, Ph.D.
U.S. Department of Transportation
Steve Hwang, Ph.D.
U.S. Environmental Protection Agency
Office of Pesticide Programs
Meta Bonner, Ph.D.
Jonathan Chen, Ph.D.
John R. “Jack” Fowle III, Ph.D., DABT
Masih Hashim, D.V.M., Ph.D.
Karen Hicks
Marianne Lewis
Debbie McCall
Timothy McMahon, Ph.D.
Mark PerryJohn Redden
Jenny Tao, Ph.D.
Office of Research and Development
Andrew Geller, Ph.D.
Office of Science Coordination and Policy
Karen Hamernik, Ph.D.

U.S. Food and Drug Administration
Center for Drug Evaluation and ResearchPaul Brown, Ph.D.
Wiley Chambers, M.D.
Abigail (Abby) Jacobs, Ph.D.
Jill Merrill, Ph.D., DABT (OTWG Chair)Center for Food Safety and Applied Nutrition
Robert Bronaugh, Ph.D.
Donnie Lowther
Office of the Commissioner
Suzanne Fitzpatrick, Ph.D., DABTNational Institute Environmental Health
Sciences
Warren Casey, Ph.D., DABTMark F. Cesta, D.V.M, DACVP
Raymond (Buck) Grissom, Ph.D.
William Stokes, D.V.M., DACLAM
Occupational Safety and Health Administration
Surender Ahir, Ph.D.
European Centre for the Validation of
Alternative Methods – LiaisonJoão Barroso, Ph.D.
Thomas Cole, Ph.D.
Valerie Zuang, Ph.D.
Japanese Center for the Validation of
Alternative Methods – Liaison
Hajime Kojima, Ph.D.

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Alternative Ocular Safety Testing Methods and Approaches
Independent Scientific Peer Review Panel(May 19-21, 2009)
Hongshik Ahn, Ph.D.
Professor
Stony Brook University
Stony Brook, NYPaul T. Bailey, Ph.D.
Bailey & Associates Consulting
Neshanic Station, NJ
Richard Dubielzig, D.V.M.
Professor
School of Veterinary Medicine
University of Wisconsin–Madison
Madison, WIHenry Edelhauser, Ph.D.1
Professor of Ophthalmology and Director of
Ophthalmic Research
Emory University School of Medicine
Atlanta, GA
Mark Evans, D.V.M., Ph.D., DACVP
Pathology Lead for Ophthalmology Therapeutic
Area
Pfizer Global Research and Development
La Jolla Drug Safety Research and Development
San Diego, CA
A. Wallace Hayes, Ph.D., DABT, FATS, ERT
Visiting Scientist (Harvard)
Principal Advisor
Spherix Incorporated
Bethesda, MD
Harvard School of Public HealthAndover MA
James V. Jester, Ph.D.
Professor of Ophthalmology and Biomedical
Engineering
Endowed Chair
University of California–Irvine
Orange, CA

1

Tadashi Kosaka, D.V.M., Ph.D.
Associate Director
Chief, Laboratory of Immunotoxicology and
Acute Toxicology
Toxicology Division
The Institute of Environmental Toxicology
Ibaraki, Japan
Alison McLaughlin, MSc, DABT
Health Canada
Environmental Impact Initiative
Office of Science and Risk Management
Health Products and Food Branch
Ottawa, Ontario
CanadaJ. Lynn Palmer, Ph.D.
Associate Professor
Dept. of Palliative Care &Rehabilitation Medicine
University of Texas M.D. Anderson
Cancer Center
Houston, TX
Robert Peiffer, Jr., D.V.M., Ph.D., DACVO
Senior Investigator
Merck Research Laboratories
Safety Assessment Toxicology
Doylestown, PA
Denise Rodeheaver, Ph.D., DABTAssistant Director
Alcon Research Ltd.
Dept. of Toxicology
Fort Worth, TX
Donald Sawyer, D.V.M., Ph.D., DACVA
Professor Emeritus
Retired, Michigan State University(Summer Residence)
Okemos MI(Winter Residence)Tucson, AZ

Drs. Edelhauser, Thake, and Tseng were unable to attend the public meeting on May 19–21, 2009. However,they were involved in the peer review of the background review documents and concur with the conclusions
and recommendations included in the Independent Scientific Peer Review Panel Report – Evaluation of the
Validation Status of Alternative Ocular Safety Testing Methods and Strategies.

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Kirk Tarlo, Ph.D., DABTScientific Director
Comparative Biology and Safety Sciences
Amgen, Inc.
One Amgen Center Drive
Thousand Oaks, CA
Daryl C. Thake, D.V.M., DACVP
Midwest ToxPath Sciences Inc.
Chesterfield, MO

1

Scheffer Tseng, M.D., Ph.D.1
Director, Ocular Surface (OS) Center
Medical Director OS Research & Education
Foundation
Director R&D Department
Tissue Tech, Inc.
Ocular Surface Center, P.A.
Miami, FL
Jan van der Valk, Ph.D.
Senior Scientist
Departments of Animals, Science and Society
Faculty of Veterinary Medicine
Utrecht University
Netherlands Centre Alternatives to Animal Use(NCA)
Utrecht, Netherlands
Daniel Wilson, Ph.D., DABT
Mammalian Toxicology Consultant
Toxicology and Environmental ResearchConsulting
The Dow Chemical Co.
Midland, MIx

44

Philippe Vanparys, Ph.D., DABTManaging Director
Cardam, Centre for Advanced Research &Development
Mol, Belgium
Maria Pilar Vinardell, Ph.D.
Director, Department of Physiology
Professor of Physiology and Pathology
Department Fisologia
Facultat de Farmacia
Universitat de Barcelona
Barcelona, Spain
Fu-Shin Yu, Ph.D.
Director of Research
Department of Ophthalmology & Anatomy
School of Medicine
Wayne State University
Detroit, MI
Sherry Ward, Ph.D., MBA
In Vitro Toxicology Consultant
BioTred Solutions
Science Advisor
International Foundation for Ethical Research(IFER)New Market, MD

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)
National Institute of Environmental Health Sciences
William Stokes, D.V.M., DACLAM
Director; Project Officer
Warren Casey, Ph.D., DABT
Deputy Director
Deborah McCarley
Special Assistant; Assistant Project Officer
NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)David Allen, Ph.D.
Jonathan Hamm, Ph.D.
Nelson Johnson
Brett Jones, Ph.D.
Elizabeth Lipscomb, Ph.D.
Linda Litchfield
Steven Morefield, M.D.
Catherine Sprankle
James Truax, M.A.
Linda Wilson
Statistical Consultant for ILS, Inc.
Joseph Haseman, Ph.D.

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Other Acknowledgements
ICCVAM and NICEATM gratefully acknowledge the following individuals and institutions that
submitted data to NICEATM for the evaluation of alternative ocular safety testing methods andapproaches.
Access Business Group
Luann Potts and Tom Truszkowski
Washington, DC
Clorox Company
Pleasanton, CA
Colgate-Palmolive Company
Piscataway, NJ
Cosmetics, Toiletry, and Fragrance
Association
Carol Eisenmann, Ph.D.
Washington, DC
The Dial Corporation
Scottsdale, AZ
ECOLabs
St. Paul, MN
ECVAM
Chantra Eskes, Ph.D.
Ispra, Italy
ExxonMobil Biomedical Sciences, Inc.
James Freeman, Ph.D.
Annandale, NJ
Institute for In Vitro Sciences, Inc.
Rodger Curren, Ph.D., Jennifer Nash, Angela
Sizemore, and John Harbell, Ph.D. (to March
2006)
Gaithersburg, MD
Johnson Diversey, Inc.
John Hamilton, Ph.D. and Sarah Willems
Sturdivant, WI

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Johnson & Johnson Pharmaceutical R&D
Phillipe Vanparys, Ph.D., and Freddy van
Goethem, Ph.D.
Beerse, Belgium
L’OrealChristine Van den Berge, Ph.D.
Paris, France
MatTek Corporation
Patrick Hayden, Ph.D.
Ashland. MA
Merck
Joseph Sina, Ph.D.
West Point, PA
National Institute of Health Sciences
Yasuo Ohno, Ph.D.
S.C. Johnson & Son
Nicole Cuellar and Judith Swanson
Racine, WI
The Procter & Gamble Company
Len Sauers, Ph.D.
Dan Marsman, D.V.M., Ph.D., DABTCincinnati, OH
TNO Nutrition and Food Research Institute
Menk Prinsen, Ph.D.
Zeist, The Netherlands
U.S. Food and Drug Administration
Donnie Lowther
College Park, MD
ZEBET
Horst Spielmann, Dr. med., and Manfred
Liebsch, Ph.D.
Berlin, Germany

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

PrefaceEye injury is a leading cause of visual impairment in the United States with 40,000 to 50,000 new
cases of impaired vision reported each year. 1 Many eye injuries occur due to contact with workplaceor household products or chemicals. Accidents involving common household products (e.g., ovencleaner and bleach) cause about 125,000 eye injuries each year. 2 These products often result inchemical burns and emergency room visits. 3 Each day about 2,000 U.S. workers have a job-related
eye injury that requires medical treatment. Although the majority of these eye injuries result frommechanical sources, chemical burns from industrial chemicals or cleaning products are common. 4
To prevent eye injuries, regulatory agencies require testing to determine if chemicals and products
may cause eye damage. This testing information is used to classify the ocular hazard and determine
appropriate labeling to warn consumers and workers of the potential hazard. Appropriate labelingtells users how to avoid exposure that could damage the eye and what emergency procedures should
be followed if there is accidental exposure. Nearly all ocular safety testing has been conducted using
the Draize rabbit eye test, although in vitro methods can now be used to identify whether substances
cause severe irritation or permanent eye damage. The Draize rabbit eye test (Draize et al. 1944)
involves instillation of 0.1 mL of the test substance into the conjunctival sac of one eye. The other eye
serves as the untreated control. The eye is examined at least daily for up to 21 days. The presence andseverity of any injuries to the cornea, conjunctiva, and the iris (tissues inside the eye) are scored, andthe duration that the injuries persist is recorded.
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
recently evaluated the routine use of topical anesthetics, systemic analgesics, and earlier humane
endpoints to avoid or minimize pain and distress during ocular safety testing. As a part of thisevaluation, ICCVAM and the National Toxicology Program Interagency Center for the Evaluation of
Alternative Toxicological Methods (NICEATM) requested the submission of data and experience
with topical anesthetics and systemic analgesics to alleviate pain and distress in rabbits during eye
irritation testing (72 FR 26396). 5
ICCVAM carefully compiled and assessed all available data and arranged an independentinternational scientific peer review. ICCVAM and the Ocular Toxicity Working Group (OTWG)
solicited and considered public comments and stakeholder involvement throughout the evaluation
process. As part of their ongoing collaboration with ICCVAM, scientists from the European Centre
for the Validation of Alternative Methods (ECVAM) and the Japanese Center for the Validation of
Alternative Methods (JaCVAM) served as liaisons to the OTWG. ICCVAM, NICEATM, and the
OTWG prepared (1) a draft background review document (BRD) on the use of topical anesthetics,
systemic analgesics, and humane endpoints to avoid or minimize pain and distress during ocularsafety testing and (2) draft test method recommendations for their usefulness and limitations.
ICCVAM released this document to the public for comment on March 31, 2009. ICCVAM alsoannounced a meeting of the independent international scientific peer review panel (Panel)(74 FR 14556). 6
The Panel met in public session on May 19–21, 2009, to review the ICCVAM draft BRD forcompleteness and accuracy. The Panel then evaluated (1) the extent to which the draft BRD addressed
established validation and acceptance criteria and (2) the extent to which the draft BRD supported
1

Available at http://www.preventblindness.org/resources/factsheets/Eye_Injuries_FS93.pdf
Available at http://www.geteyesmart.org/eyesmart/injuries/home.cfm
3From the CPSC NEISS database, 2007
4
Available at http://www.cdc.gov/niosh/topics/eye/
5
Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_8898.pdf
6Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/E9-7220.pdf
2

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

ICCVAM’s draft test method recommendations. Before concluding their deliberations, the Panelconsidered written comments and comments made at the meeting by public stakeholders. The Panel
prepared a report summarizing their conclusions and recommendations. 7
ICCVAM provided the Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) with the Topical Anesthetics/Systemic Analgesics/Humane Endpoints draft BRD anddraft test method recommendations, the Panel report, and all public comments for discussion at their
meeting on June 25–26, 2009, where public stakeholders were given another opportunity to comment.
A detailed timeline of the evaluation is included with this report.
ICCVAM solicited and considered public comments and stakeholder involvement throughout the test
method evaluation process. ICCVAM considered the SACATM comments, the conclusions of the
Panel, and all public comments before finalizing the ICCVAM test method recommendations. The
recommendations and the BRD, which is provided as an appendix to this report, are incorporated inthis ICCVAM test method evaluation report. As required by the ICCVAM Authorization Act,ICCVAM will forward its recommendations to U.S. Federal regulatory agencies for consideration.
Federal agencies must respond to ICCVAM within 180 days after receiving the ICCVAM test method
recommendations. ICCVAM recommendations are available to the public on the NICEATM–ICCVAM website, and agency responses will also be made available on the website as they arereceived.
We gratefully acknowledge the many individuals who contributed to the preparation, review, andrevision of this report. We especially recognize the Panel members for their thoughtful evaluations
and generous contributions of time and effort. Special thanks are extended to Dr. A. Wallace Hayes
for serving as the Panel Chair and to Dr. Paul Bailey, Dr. Donald Sawyer, Dr. Kirk Tarlo, andDr. Daniel Wilson for their service as Evaluation Group Chairs. We thank the OTWG for assuring ameaningful and comprehensive review. We especially thank Dr. Jill Merrill (U.S. Food and Drug
Administration Center for Drug Evaluation and Research) and Dr. Karen Hamernik (U.S.
Environmental Protection Agency, until April 2009) for serving as Co-Chairs of the OTWG.
Integrated Laboratory Systems, Inc., the NICEATM support contractor, provided excellent scientificsupport, for which we thank Dr. David Allen, Dr. Jonathan Hamm, Nelson Johnson, Dr. Brett Jones,
Dr. Elizabeth Lipscomb, and James Truax. Finally, we thank European Centre for the Validation of
Alternative Methods liaisons Dr. João Barroso, Dr. Thomas Cole, and Dr. Valerie Zuang andJapanese Center for the Validation of Alternative Methods liaison Dr. Hajime Kojima for their
participation and contributions.
Marilyn Wind, Ph.D.
Deputy Associate Executive Director
Directorate for Health Sciences
U.S. Consumer Product Safety Commission
Chair, ICCVAM
William S. Stokes, D.V.M., DACLAM
Rear Admiral/Assistant Surgeon General, U.S. Public Health Service
Director, NICEATM
Executive Director, ICCVAM

7

Available at http://iccvam.niehs.nih.gov/docs/ocutox_docs/OcularPRPRept2009.pdf
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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

Executive Summary
The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
recently evaluated the routine use of topical anesthetics, systemic analgesics, and earlier humane
endpoints to avoid or minimize pain and distress during in vivo ocular safety testing. This test method
evaluation report provides ICCVAM’s recommendations. The report also includes (1) ICCVAM’s
recommended changes to the protocol for the Draize rabbit eye test and (2) a final background review
document (BRD) on the use of topical anesthetics, systemic analgesics, and earlier humane endpoints
in the Draize rabbit eye test.
The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological
Methods (NICEATM), ICCVAM, and ICCVAM’s Ocular Toxicity Working Group prepared a draft
BRD on the use of topical anesthetics, systemic analgesics, and earlier humane endpoints to minimize
pain and distress in ocular safety testing. The BRD is based upon published studies and forms thebasis for the draft ICCVAM test method recommendations. NICEATM provided the draft BRD and
ICCVAM recommendations to an independent international scientific peer review panel (Panel) andthe public for comment. A detailed timeline of the ICCVAM evaluation process is appended to thisreport.
The Panel met in public session on May 19–21, 2009, to discuss its review of the ICCVAM draft
BRD and to provide conclusions and recommendations on these proposed changes to the Draize
rabbit eye test protocol. The Panel also reviewed how well the information in the draft BRD
supported ICCVAM’s draft test method recommendations. In finalizing this test method evaluation
report and the BRD, which is included as an appendix, ICCVAM considered (1) the conclusions andrecommendations of the Panel, (2) comments from ICCVAM’s Scientific Advisory Committee on
Alternative Toxicological Methods, and (3) public comments.
Routine Use of Topical Anesthetics and Systemic Analgesics in the Draize Rabbit Eye Test
Specific ICCVAM Test Method Recommendations
Balanced preemptive pain management should be provided whenever the Draize rabbit eye test is
conducted for regulatory safety testing. Pain management should include (1) treating the animals witha topical anesthetic and a systemic analgesic before applying test substances; (2) following a routineschedule of systemic analgesia after applying test substances; (3) scheduled observation, monitoring,and recording of animals for clinical signs of pain and/or distress; and (4) scheduled observation,
monitoring, and recording of the nature, severity, and progression of all eye injuries. ICCVAM
further recommends that ocular safety testing protocols include a pain management procedure andschedule.
Changes to Ocular Safety Testing Protocol to Include the Routine Use of Topical Anesthetics andSystemic Analgesics
When required for regulatory safety assessment of potential ocular hazards (EPA 1998; OECD 2002),
the current Draize rabbit eye test should be conducted with the following changes unless pain
response monitoring is required (e.g., pharmaceutical tolerability testing). Alternative pain
management procedures may be considered if they provide analgesia and anesthesia as good or better
than the following pain management procedure:xx

Sixty minutes before test substance application (TSA), provide a therapeutic level ofsystemic analgesia by administering 0.01 mg/kg buprenorphine by subcutaneous
injection.
Five minutes before applying the test substance, apply one or two drops of a topicalocular anesthetic (e.g., 0.5% proparacaine hydrochloride or 0.5% tetracaine

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

x

x

x

hydrochloride) to each eye. For each animal, the eye that is treated with topicalanesthetics and no test substance will serve as a control. If the test substance isanticipated to cause significant pain and distress, consider applying more than one doseof topical anesthetic at 5-minute intervals before TSA. Be aware that multiple
applications of topical anesthetics could increase the severity of chemically induced
lesions and/or extend the time required for them to heal.
If a test subject shows signs of pain and distress during the test interval, immediately give
additional analgesia (i.e., a “rescue” dose of 0.03 mg/kg subcutaneous buprenorphine).
Repeat 0.03 mg/kg buprenorphine every 8 hours (+/- 30 minutes) instead of 0.01 mg/kg
subcutaneously every 12 hours. Continue meloxicam with the same dose and interval
described below. If preemptive analgesia is inadequate, give the “rescue” analgesia
immediately after TSA.
Eight hours (+/- 30 minutes) after TSA, administer 0.01 mg/kg buprenorphine and
0.5 mg/kg meloxicam subcutaneously to provide a continued therapeutic level ofsystemic analgesia.
If ocular lesions and/or clinical signs of pain and distress are present following thebuprenorphine and meloxicam treatment that was administered 8 hours after TSA,
continue to administer 0.01 mg/kg buprenorphine subcutaneously every 12 hours(+/- 30 minutes) in conjunction with 0.5 mg/kg meloxicam subcutaneously every
24 hours. If the “rescue dose” described above is needed, administer buprenorphine at
0.03 mg/kg every 8 hours instead of 0.01 mg/kg every 12 hours.

Future Studies on the Routine Use of Topical Anesthetics and Systemic Analgesics
ICCVAM recommends routinely observing and recording lesions and clinical signs during ocularsafety studies in order to evaluate the effectiveness of pain management and to determine if theenhanced “rescue” analgesia procedure should be implemented. These data should be reviewed todetermine whether adjustments are needed to (1) improve the effectiveness of analgesia before andafter treatment and (2) optimize dosages and treatment intervals. Data should be analyzed periodicallyto determine the effectiveness of the pain management procedures for specific types of lesions andclinical signs of pain and distress associated with ocular safety testing.
To support the development of improved pain management strategies, ICCVAM recommends
evaluating detailed animal injury and pain response data collected from animals used for regulatory
safety testing. This could help gauge the adequacy of the recommended pain management proceduresand help identify the need for modifications to dosages and dosing intervals for anesthetics and/or
analgesics. Additionally, where possible, ICCVAM recommends that the eyes of test animals becollected for histopathology to more thoroughly evaluate depth and area of ocular damage, as well as
to provide a reference against which to compare effects produced in vitro. ICCVAM emphasizes that
new animal studies should be considered only when absolutely necessary in developing new pain
management strategies for testing.
Use of Earlier Humane Endpoints — Test Method Usefulness and Limitations
ICCVAM recognizes that current ocular testing guidelines include criteria for study termination in thecase of certain types of severe ocular injuries or evidence of severe pain and distress (EPA 1998;
OECD 2002). There is also international guidance on general humane endpoints that can be used as
the basis for ending an experiment (OECD 2000). In addition to these currently accepted endpoints,
and consistent with the recommendations of the Panel, ICCVAM recommends that the following
ocular lesions be used as earlier humane endpoints to terminate studies before the end of thescheduled 21-day observation period. These lesions are considered predictive of severe irritant or
corrosive injuries and injuries that are not expected to fully reverse by the end of the 21-day
observation period after treatment:

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Reportxxx

Severe depth of injury (e.g., corneal ulceration extending beyond the superficial layers)
Destruction of more than 50% of the limbus, as evidenced by blanching of the
conjunctival tissue
Severe eye infection (purulent discharge)

A combination of the following endpoints may be useful in clinical decisions on study termination.
However, these endpoints cannot be used individually to justify early study termination:xx
x

Vascularization of the cornea surface (i.e., pannus)
Area of fluorescein staining not diminishing over time based on daily assessment
Lack of re-epithelialization 5 days after test substance application

ICCVAM emphasizes that, once severe ocular effects have been identified, a qualified laboratory
animal veterinarian should perform a clinical exam to determine if the combination of these effects
warrants early study termination.
Changes to the Ocular Safety Testing Protocol to Include the Use of Humane Endpoints
The current protocol for the Draize rabbit eye test, as used for regulatory safety testing (EPA 1998;
OECD 2002), should be updated to incorporate ICCVAM’s recommended use of humane endpoints.
ICCVAM recommends that test animals be comprehensively evaluated for the presence or absence ofocular lesions one hour after TSA, followed by at least daily evaluations. Animals should be
evaluated once daily for the first 3 days, or more often if necessary, to ensure that termination
decisions are made promptly. ICCVAM also recommends that test animals should be routinely
evaluated for clinical signs of pain and/or distress at least twice daily with at least 6 hours between
observations. Examples of relevant clinical signs include (Wright et al. 1985; NRC 2008, 2009)xx
x

repeated pawing or rubbing of the eye
excessive blinking
excessive tearing

Decisions to end a study based on humane endpoints should ensure that reversal of the clinical signs
is not expected or that no further useful information can be obtained from the study. A written recordof all observations should be kept, including evidence of an infection and/or pain and distress. Such
records can facilitate decisions on the progression or resolution of ocular lesions. ICCVAM
emphasizes that fluorescein staining should be used routinely. A slit-lamp biomicroscope should also
be used, when considered appropriate (e.g., assessing depth of injury when corneal ulceration ispresent), to help detect and measure ocular endpoints. Digital photographs should be taken to
document ocular lesions and to help assess their severity, progression, and resolution.
Future Studies on the Use of Humane Endpoints
ICCVAM recommends that additional data should be collected on the use of fluorescein staining tomonitor wound healing. These data should be evaluated to identify criteria that may be useful as
humane endpoints to terminate studies.
ICCVAM encourages users to provide NICEATM with detailed data and observations collected inocular safety studies that can be used to create a database to (1) further characterize the usefulness
and limitations of proposed humane endpoints and (2) identify potential new endpoints. Such datasubmissions will contribute to efforts to find ways to further prevent and minimize pain and distressin ocular safety assessments.

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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

1.0

Introduction

Current U.S. Environmental Protection Agency (EPA) and Organisation for Economic Co-operation
and Development (OECD) test guidelines for the Draize rabbit eye test provide for the use of topicalanesthetics only when the user demonstrates that such pretreatments do not interfere with the test
results (EPA 1998; OECD 2002). 8 Topical anesthetics are seldom used because a separate study
would likely be necessary to meet this requirement. EPA (1998), European Union (EU 2001), and the
Globally Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2007)
regulatory guidelines recognize and accept certain humane endpoints for ocular hazard assessment.
These include (1) severe and enduring signs of pain or distress and (2) eye lesions considered to beirreversible. However, current testing guidelines underemphasize the routine use of such endpoints.
Consequently, the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) recently evaluated the routine use of topical anesthetics, systemic analgesics, and earlier
humane endpoints to avoid or minimize pain and distress during in vivo ocular safety testing.
The ICCVAM Authorization Act of 2000 (Public Law 106-545, 42 United States Code 285l-3)
charged ICCVAM with coordinating the technical evaluations of new, revised, and alternative test
methods with regulatory applicability. The National Toxicology Program Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM) administers ICCVAM and provides
scientific support for ICCVAM activities. The ICCVAM Ocular Toxicity Working Group (OTWG)
worked with NICEATM in evaluating alternative methods and testing strategies. Drs. João Barroso,
Tom Cole, and Valerie Zuang were the European Centre for the Validation of Alternative Methods(ECVAM) liaisons, and Dr. Hajime Kojima was the Japanese Center for the Validation of Alternative
Methods (JaCVAM) liaison to the OTWG.
To facilitate peer review, the OTWG and NICEATM prepared a comprehensive draft backgroundreview document (BRD). The BRD provided information and data from published and unpublisheddata on the use of topical anesthetics, systemic analgesics, and humane endpoints in ocular safety
testing.
ICCVAM and NICEATM requested the submission of data and experience with topical anesthetics
and systemic analgesics for alleviating pain and distress in rabbits during ocular safety testing(72 FR 26396). 9 One individual provided comments supporting the use of anesthetics to minimize
pain and distress in rabbit eye irritation studies. No additional data were received.
On April 4, 2008, NICEATM published a Federal Register notice (73 FR 18535) 10 requesting
relevant data and nominations of individuals to serve on an independent international scientific peer
review panel (Panel). The request was also disseminated via the ICCVAM electronic mailing list andthrough direct requests to over 100 stakeholders. Twenty individuals were nominated as potential
panelists for consideration. No additional data were received (see Section 6.0).
The BRD forms the basis for these ICCVAM test method recommendations. The ECVAM and
JaCVAM liaisons to the OTWG provided input and contributed throughout the evaluation process. A
detailed timeline of the ICCVAM evaluation is provided in Appendix A. The ICCVAMrecommended test method protocol and final BRD are provided in Appendices B and C, respectively.

8OECD Test Guideline 405 states: “The type, concentration, and dose of a local anesthetic should be carefully
selected to ensure that differences in reaction to the test substance will not result from its use.” Similarly,
EPA (1998) states that “The type and concentration of the local anesthetic should be carefully selected toensure that no significant differences in reaction to the test substance will result from its use.”
9
Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR_E7_8898.pdf
10Available at http://iccvam.niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E8-6969.pdf
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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

On March 31, 2009, ICCVAM announced the availability of the ICCVAM draft BRD. ICCVAM alsoannounced a public Panel meeting to review the routine use of topical anesthetics, systemic
analgesics, and earlier humane endpoints in ocular safety testing (74 FR 14556). The ICCVAM draft
BRD and draft test method recommendations were posted on the NICEATM–ICCVAM website(http://iccvam.niehs.nih.gov/). All of the information provided to the Panel and all public comments
received before the Panel meeting were made available on the NICEATM–ICCVAM website.
The Panel met in public session from May 19–21, 2009, to review a proposal for the routine use of
topical anesthetics, systemic analgesics, and earlier humane endpoints in ocular safety testing. The
Panel also reviewed the completeness and accuracy of the ICCVAM draft BRD. They then evaluated(1) the extent to which the draft BRD addressed established validation and acceptance criteria and(2) the extent to which the BRD supported ICCVAM’s draft test method recommendations. Public
stakeholders were provided opportunities to comment at the Panel meeting. The Panel considered allcomments during their deliberations. On July 13, 2009, ICCVAM posted the final report of the
Panel’s recommendations (Appendix D) on the NICEATM–ICCVAM website for public review andcomment (announced in 74 FR 33444). 11
ICCVAM provided the Scientific Advisory Committee on Alternative Toxicological Methods(SACATM) with the draft BRD, draft test method recommendations, the Panel report, and all publiccomments. SACATM discussed this material at their meeting on June 25–26, 2009. Public
stakeholders were given another opportunity to comment.
After the SACATM meeting, ICCVAM and the OTWG considered the SACATM comments, thePanel report, and all public comments before finalizing the ICCVAM test method evaluation report
and the BRD, provided as an appendix to this report. As required by the ICCVAM Authorization Act,ICCVAM will make this test method evaluation report and the accompanying final BRD available tothe public and to U.S. Federal agencies for consideration. Federal agencies must respond to ICCVAM
within 180 days after receiving ICCVAM test method recommendations. Agency responses to the
ICCVAM test method recommendations will be made available to the public on the NICEATM–ICCVAM website at http:///www.iccvam.niehs.nih.gov as they are received.

11

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Announcement available at http://niehs.nih.gov/SuppDocs/FedDocs/FR/FR-E9-16388; report available at
http://iccvam.niehs.nih.gov/docs/ocutox_docs/OcularPRPRept2009.pdf

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

2.0

ICCVAM Recommendations for the Routine Use of Topical
Anesthetics and Systemic Analgesics to Avoid or Minimize Pain andDistress in Ocular Safety Testing

2.1

ICCVAM Recommendations: Test Method Usefulness and Limitations

ICCVAM recommends that balanced preemptive pain management should always be provided when
the Draize rabbit eye test is conducted for regulatory safety testing. Pain management should include(1) pretreatment with a topical anesthetic and systemic analgesic prior to test substanceadministration; (2) routine post-treatment with systemic analgesics, with additional treatments asnecessary; (3) scheduled observation, monitoring, and recording of animals for clinical signs of pain
and/or distress; and (4) scheduled observation, monitoring, and recording of the nature, severity, andprogression of all eye injuries. ICCVAM further recommends that ocular safety testing protocols
include a pain management plan and schedule consistent with that outlined below.
When required for ocular safety testing, the Draize rabbit eye test protocol currently used forregulatory safety assessments of potential ocular hazards (EPA 1998; OECD 2002) should be
conducted with the following modifications unless there is a requirement for monitoring the pain
response (e.g., pharmaceutical tolerability testing). Alternative pain management procedures may also
be considered that provide as good or better analgesia and anesthesia than the recommended pain
management procedure below:
x

x

x

x
x

Sixty minutes before test substance administration (TSA), buprenorphine 0.01 mg/kg isadministered by subcutaneous injection (SC) to provide a therapeutic level of systemic
analgesia.
Five minutes pre-TSA, one or two drops of a topical ocular anesthetic (e.g., 0.5%
proparacaine hydrochloride or 0.5% tetracaine hydrochloride) is applied to each eye. Theeye of each animal that is not treated with a test article, but which is treated with topicalanesthetics, serves as a control. If the test substance is anticipated to cause significant
pain and distress, consideration should be given to more than one application of topicalanesthetic at 5-minute intervals pre-TSA. Users should be aware that multiple
applications of topical anesthetics could increase the severity and/or extend the timerequired for chemically induced lesions to clear.
If a test subject shows signs of pain and distress during the test interval, additional
analgesia (i.e., a “rescue” dose of 0.03 mg/kg SC buprenorphine) is given immediately
and repeated every 8 hours, 12 instead of 0.01 mg/kg SC every 12 hours. Meloxicam
would continue with the same dose and interval described below. The “rescue” analgesia
should be given immediately after TSA if preemptive analgesia is inadequate.
Eight hours post-TSA, buprenorphine 0.01 mg/kg SC and meloxicam 0.5 mg/kg SC areadministered to provide a continued therapeutic level of systemic analgesia.
After the initial 8-hour post-TSA treatment, if ocular lesions and/or clinical signs of painand distress are present, buprenorphine 0.01 mg/kg SC should be administered every
12 hours (0.03 mg/kg every 8 hours if the “rescue” dose is needed), in conjunction withmeloxicam 0.5 mg/kg SC every 24 hours.

Independent Peer Review Panel Conclusions and Recommendations
Following the Panel’s review of the BRD and draft recommendations developed by ICCVAM, thePanel proposed an alternative preemptive pain management protocol for rabbits used for ocular safety
12Time intervals are +/- 30 minutes.
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ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Reporttesting. This protocol (hereafter, the alternative protocol or the Panel’s protocol) was proposed by the
Panel to be applied to all in vivo rabbit ocular safety tests intended for regulatory safety testing, unlessthere is a requirement for monitoring the pain response (e.g., pharmaceutical tolerability testing). The
only differences in the ICCVAM-recommended plan and the Panel’s protocol are that the ICCVAMrecommended plan (1) allows for either tetracaine or proparacaine as a topical anesthetic and (2)recommends only one dose of topical anesthetic unless there is reason to believe that this will beinsufficient to relieve pain and distress, at which time additional pre-TSA applications can beconsidered. The basis for these differences arise from previous studies showing that multiple doses of
proparacaine can result in significant differences in hazard classification due to the increased severity
and/or prolonged appearance of ocular lesions.

2.2

ICCVAM Recommendations: Test Method Protocol for the Routine Use of
Topical Anesthetics and Systemic Analgesics

When required for ocular safety testing, the Draize rabbit eye test protocol currently used forregulatory safety assessments of potential ocular hazards (EPA 1998; OECD 2002) should be
conducted with the modifications as outlined in Section 2.1 unless pain-response monitoring isrequired (e.g., pharmaceutical tolerability testing).
Independent Peer Review Panel Conclusions and Recommendations
The Panel considered its proposal (Section 2.1) more appropriate in terms of the type and frequencyof dosing for topical anesthetics and systemic analgesics.
The Panel noted that the available guidance on measuring fluorescein staining as presented in thedraft ICCVAM recommendations is not adequate for laboratories to obtain consistent results, and themethod of fluorescein staining will have to be standardized in order to be useful. In addition, theguidelines lack details about potential preservatives in the dye, anesthesia requirements, or physicalrestraint that may need to be considered.

2.3

ICCVAM Recommendations: Future Studies for the Routine Use of Topical
Anesthetics and Systemic Analgesics

The routine observation and recording of lesions and clinical signs is recommended during ocular
irritation safety studies to evaluate the effectiveness of pain management and to determine if theenhanced “rescue” analgesia procedure should be implemented. Furthermore, periodic retrospective
reviews of these data should be performed to determine if adjustments are needed to improve theeffectiveness of pretreatment and post-treatment analgesia and to optimize dosages and treatment
intervals. Ideally, data collected during routine safety testing should be analyzed periodically todetermine the effectiveness of the pain management plan for specific types of lesions and clinicalsigns of pain and distress associated with ocular irritation/corrosivity testing.
ICCVAM recommends the following studies and activities to support the development of improved
pain management strategies, recognizing that some involve research that would be conductedindependent of regulatory safety testing.
x
x

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New animal studies should be considered only when absolutely necessary in developing
new pain management strategies for testing.
Detailed ocular injury and pain response data should be collected from animals used forrequired regulatory testing and evaluated to assess the adequacy of the recommended
pain management procedures. This data will help identify the need for modifications to
dosages and dosing intervals for anesthetics and/or analgesics.

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

x

x
x

x
x
x

Where possible, eyes should be collected for histopathology to more thoroughly evaluate
depth and area of ocular damage, as well as to provide a reference against which tocompare effects produced in vitro.
Digital photographs of observed lesions should be collected for reference and to provide
a permanent record of the extent of ocular damage.
Studies should be conducted to determine whether the timing and dosing of systemic
analgesics together with topical anesthetics might alter the ocular defense sufficient tochange the classification of test substances.
Studies should be conducted to investigate other topical anesthetics that might provide
longer duration of action or other advantages.
Studies should be conducted to evaluate the impact of using other systemic analgesics
that might provide longer duration of action, improved analgesia, or other advantages.
ICCVAM encourages users to provide data generated using the recommended pain
management procedures to NICEATM to create a database that can be periodicallyevaluated to further characterize the usefulness and limitations of such procedures foravoiding or minimizing pain and distress in ocular safety assessments.

Independent Peer Review Panel Conclusions and Recommendations
The Panel agreed with the draft ICCVAM recommendations for future studies related to the routine
use of topical anesthetics and systemic analgesics. The Panel also recommended a number of
additional studies, which have been incorporated into the ICCVAM recommendations listed above.

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3.0

Validation Status: Routine Use of Topical Anesthetics and Systemic
Analgesics in Ocular Safety Testing

Since 1984, the U.S. Consumer Product Safety Commission has recommended preapplication oftetracaine ophthalmic anesthetic in all rabbit ocular safety studies. However, current EPA and OECD
test guidelines for the Draize rabbit eye test provide for the use of topical anesthetics only when theuser demonstrates that such pretreatments do not interfere with the test results (EPA 1998; OECD
2002). 13 Topical anesthetics are seldom used because a separate study would likely be necessary toprovide the necessary information.
In 2005, a symposium entitled “Minimizing Pain and Distress in Ocular Toxicity Testing” evaluatedthe use of topical ophthalmic anesthetics and/or systemic analgesics during the conduct of the Draize
rabbit eye test. ICCVAM, NICEATM, and the European Centre for the Validation of Alternative
Methods (ECVAM) organized the symposium. Experts acknowledged that a single treatment with a
topical anesthetic to anesthetize the surface of the cornea before application of the test article could
cause slight physiologic changes. However, the consensus was that such changes in the irritantresponse would be slight if any. Furthermore, the predominant view was that if there were any effecton the irritant response, it would tend to slightly increase the severity of the response.
Participants recommended routine use of topical anesthetics. The anesthetics at least prevent thediscomfort caused by installation of the test article on the eye. They also temporarily prevent or
minimize pain and distress that might result from immediate ocular damage.
NICEATM recently evaluated the effects of pretreatment with tetracaine hydrochloride (0.5% w/v) on
the ocular irritancy potential of 97 formulations. The results indicate that such pretreatments have nostatistically significant impact on the hazard classification severity category of observed ocular
irritation (Annex II of Appendix C). For most of the formulations tested, topical anesthetic
pretreatment had little or no impact on:xxx

The hazard classification severity category of observed ocular irritation
The variability in ocular irritation responses among animals treated with the same test
articleThe number of days required for an ocular lesion to clear

When a difference in ocular irritation response was observed, the more severe response was usuallyobserved in the animals pretreated with topical anesthesia. However, none of the observed differences
was statistically significant. Differences included both increases and decreases in the irritancy level,
which suggests that they are related to the inherent inter-individual biological variability of response
rather than topical anesthetic pretreatment.
Scientific experts at the 2005 workshop also recommended (Annex I of Appendix C) that animals be
routinely pretreated with topical anesthetics and systemic analgesics to prevent pain. Animals thatshow signs of pain or distress and those with ocular lesions associated with painful conditions shouldbe treated with systemic analgesics. Similarly, a recently convened independent international
scientific peer review panel recommended the routine use of topical anesthetics and systemic
analgesics to avoid or minimize pain and distress during in vivo ocular safety testing. The Panelrecommended a protocol that includes pretreatment with systemic analgesics in conjunction with

13

6

58

OECD Test Guideline 405 states: “The type, concentration, and dose of a local anesthetic should be carefully
selected to ensure that differences in reaction to the test substance will not result from its use.” Similarly,
EPA (1998) states that “the type and concentration of the local anesthetic should be carefully selected toensure that no significant differences in reaction to the test substance will result from its use.”ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Report

topical anesthetics prior to test substance administration. The protocol also includes treatment withsystemic analgesics after test substance administration.
A therapeutic analgesic protocol conducted before the onset of pain is referred to as preemptive pain
management (Polomano et al. 2008). The Panel recommended a balanced preemptive pain
management protocol for all animals used for ocular safety testing. For routine safety testing, thePanel considered proparacaine preferable to tetracaine because the initial application to the eye is lesspainful (Bartfield et al. 1994). The relative merits of proparacaine and tetracaine are detailed in
Annex III of Appendix C. Multiple applications of topical anesthetics before test substanceadministration maximize effective penetration of the epithelial layer (Sasaki et al. 1995). A 5-minuteinterval between the last topical anesthetic dose and test substance administration minimizes thepossibility of any volume dilution (Maurice 1995).
The Panel recommended buprenorphine as the systemic analgesic of choice. Buprenorphine is an
opioid agonist–antagonist analgesic that has been effective in managing pain in rabbits and othersmall animals (Roughan and Flecknell 2002; Sawyer 2008). It has a wide safety margin in rabbits,
causes minimal sedation, and provides a long duration of analgesia (6–12 hours) (Flecknell 1984;
Flecknell and Liles 1992; Roughan and Flecknell 2002). Increasing buprenorphine dose rates inrabbits has little effect on the maximum degree of analgesia produced (Flecknell and Liles 1990). For
this reason, the recommended dose range in rabbits is 0.01–0.05 mg/kg (Dobromylskyj et al. 2006;
Flecknell 1984, 1995; Flecknell and Liles 1990).
The Panel recommended treatment with systemic analgesics after test substance administration tomaintain the prior level of analgesia. A well-tested approach to balanced analgesia is to use an opioid(e.g., buprenorphine) in combination with a cyclooxygenase-sparing nonsteroidal anti-inflammatory
drug such as meloxicam (Cooper et al. 2009; Roughan and Flecknell 2002; Sawyer 2008). Meloxicam
has been used for postoperative or chronic pain in humans (Akarsu et al. 2004; Aoki et al. 2006) anddogs for over 10 years. Its effectiveness has been demonstrated in rabbits (Cooper et al. 2009; Sawyer
2008). The Panel recommended a low dose of meloxicam once daily in conjunction with thebuprenorphine.

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4.0

ICCVAM Recommendations for the Use of Humane Endpoints to
Avoid or Minimize Pain and Distress in Ocular Safety Testing

4.1

ICCVAM Recommendations: Test Method Usefulness and Limitations

ICCVAM recognizes that current ocular testing guidelines include criteria for study termination in thecase of certain types of severe ocular injuries or evidence of severe pain and distress (EPA 1998;
OECD 2002). These include:x

Draize corneal opacity score of 4 that persists for 48 hours
–

x
x
x
x
x
x

Corneal score of 4 is defined as: Opaque cornea, iris not discernable through theopacityCorneal perforation or significant corneal ulceration including staphyloma
Blood in the anterior chamber of the eye
Absence of a light reflex (iridial response grade 2) that persists for 72 hours
Ulceration of the conjunctival membrane
Necrosis of the conjunctiva or nictitating membrane
Sloughing (separation of necrotic tissue from the living structure)

There is also international guidance on general humane endpoints that can be used as the basis forending an experiment (OECD 2000). In addition to these currently accepted endpoints and consistent
with the recommendations of the Panel, ICCVAM recommends that the following ocular lesions also
be used as earlier humane endpoints to terminate studies before the end of the scheduled 21-day
observation period. These lesions are considered predictive of severe irritant or corrosive injuries andinjuries that are not expected to fully reverse by the end of the 21-day observation period after
treatment:xxx

Severe depth of injury (e.g., corneal ulceration extending beyond the superficial layers)
Destruction of more than 50% of the limbus, as evidenced by blanching of the
conjunctival tissue
Severe eye infection (purulent discharge)

The following endpoints, in combination, may be useful in clinical decisions on early study
termination:xx
x

Vascularization of the corneal surface (i.e., pannus)
Area of fluorescein staining not diminishing over time based on daily assessment
Lack of re-epithelialization 5 days after test substance application

However, these endpoints cannot be used individually to justify early study termination. ICCVAM
emphasizes that, once severe ocular effects have been identified, a qualified laboratory animal
veterinarian should perform a clinical exam to determine if the combination of these effects warrants
early study termination.
Conclusions and Recommendations of the Independent Peer Review Panel
The Panel concluded that the current and proposed humane endpoints should be used routinely as
humane endpoints. The Panel considered them predictive enough of irreversible or severe effects (i.e.,
EPA Category I, GHS Category 1, EU R41) that a study should be terminated as soon as they areobserved. To ensure that termination decisions are made promptly, the Panel recommended that test
animals be examined at least daily and the presence or absence of these lesions recorded. For the first
three days, test animals should be examined at least twice daily, or more often if necessary. The Panel

860

ICCVAM Anesthetics, Analgesics, & Humane Endpoints Evaluation Reportemphasized the need for a slit-lamp examination to ensure accurate measurement of most of theocular endpoints.
The Panel did not consider some of the endpoints adequate for early study termination when taken
individually (e.g., pannus, area of fluorescein staining, lack of re-epithelialization). They can,however, be considered together. With this in mind, the Panel emphasized that decisions to terminate
a study should be based on multiple endpoints when possible. Only very severe endpoints (e.g.,corneal perforation) would be adequate alone to terminate a study.

4.2

ICCVAM Recommendations: Changes to the Ocular Safety Testing Protocol toInclude the Use of Humane Endpoints

Ocular safety assessment studies should be conducted using the ICCVAM-recommended
modifications to the current Draize eye test protocol for regulatory safety assessments of potential
ocular hazards (EPA 1998; OECD 2002). ICCVAM recommends that test animals becomprehensively evaluated for the presence or absence of ocular lesions one hour after test substanceadministration, followed by at least daily evaluations. Animals should be evaluated once daily for the
first 3 days, or more often if necessary, to ensure that termination decisions are made in a timely
manner. ICCVAM also recommends that test animals be routinely evaluated for clinical signs of pain
and/or distress at least twice daily with a minimum of 6 hours between observations, or more often if
necessary. Examples of relevant clinical signs include (Wright et al. 1985; NRC 2008, 2009):
xxx

Repeated pawing or rubbing of the eye
Excessive blinking
Excessive tearing

Study termination based on humane endpoints should ensure that reversal is not expected and that nofurther useful information can be obtained from the study. A written record of all observations should
be kept for determinations on the progression or resolution of ocular lesions. ICCVAM emphasizes
that fluorescein staining should be used routinely to help detect and objectively measure ocular
endpoints. A slit-lamp biomicroscope should be used when considered appropriate (e.g., assessing
depth of injury when corneal ulceration is present). Digital photographs should be taken to document
ocular lesions and help assess their severity, progression, and resolution.

4.3

ICCVAM Recommendations: Future Studies for the Use of Humane Endpoints

ICCVAM recommends that additional data should be collected on the use of fluorescein staining tomonitor wound healing. These data should be evaluated to identify criteria that may be useful as
humane endpoints to terminate studies. ICCVAM recommends that guidelines should be developedfor (1) the frequency of fluorescein staining that can be conducted without significant impacts on
wound healing that would affect classification categories and (2) the usefulness of the area, intensity,and progression/regression of fluorescein staining for identifying specific hazard classification
categories.
ICCVAM also recommends the following:xxx

Studies should be conducted to identify earlier, more predictive endpoints such as thosequantifying area and intensity of fluorescein staining.
Data should be collected during current testing to support the identification of potential
earlier endpoints and to facilitate development of a database that can be used to identify
useful earlier endpoints.
Data should be collected to further evaluate pannus as a potential earlier humane
endpoint. (ICCVAM did not consider the BRD data sufficient to determine the adequacyof pannus as a recommended humane endpoint for terminating a test.)

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Improved guidance should be developed on clinical signs of pain and distress in rabbits.
Pain assessment training is also an important part of an effective pain management
program and should be routinely provided to relevant personnel.
Users should provide NICEATM with detailed data and observations collected fromocular safety studies that can be used to create a database to (1) further characterize theusefulness and limitations of proposed humane endpoints and (2) identify potential new
endpoints. Such data submissions will contribute to efforts to find ways to further avoid
or minimize pain and distress during ocular safety assessments.

Independent Peer Review Panel Conclusions and Recommendations
The Panel agreed with the draft ICCVAM recommendations for future studies related to the routine
use of humane endpoints to avoid or minimize pain and distress in ocular safety testing. The Panelalso recommended a number of additional studies, which have been incorporated into the ICCVAM
recommendations listed above. The Panel emphasized that Animal Health Technologist (AHT)training requirements are an important part of a successful humane endpoint program.

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5.0

Validation Status of the Use of Humane Endpoints to Avoid or
Minimize Pain and Distress in Ocular Safety Testing

Public Health Service policy and U.S. Department of Agriculture regulations on pain and distress inlaboratory animals state that more than momentary or light pain and distress (1) must be limited tothat which is unavoidable for the conduct of scientifically valuable research or testing, (2) must be
conducted with appropriate pain relief medication unless justified in writing by the principal
investigator, and (3) will continue for only a necessary amount of time. These regulations also statethat animals suffering severe or chronic pain or distress that cannot be relieved should be humanely
killed after or, if appropriate, during the procedure. Finally, Institutional Animal Care and Use
Committees must ensure that the principal investigator complies with the requirements. Of theanimals reported to the Department of Agriculture as experiencing unrelieved pain and distress, themajority are justified by regulatory testing requirements.
The OECD published a guidance document on the recognition, assessment, and use of clinical signs
as humane endpoints for experimental animals used in safety assessment tests (OECD 2000).
According to this document, guiding principles for humane endpoints include:
x
x
x
x
x

Designing studies to minimize any pain, distress, or suffering, consistent with thescientific objective of the study
Sacrificing animals at the earliest indication of severe pain, distress, or impending death,and avoiding severe pain, suffering, or death as endpoints
Terminating animal studies once study objectives are achieved or when it is realized thatthese objectives will not be achieved
Including knowledge about the test substance in the study design
Defining in the protocol or standard operating procedure the conditions under whichauthorized personnel should intervene to alleviate pain and distress by humane killing

Accordingly, humane endpoints recognized and accepted by current EPA (2003), Globally
Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2007), and EU (2001)
regulatory guidelines for ocular hazard assessment include severe and enduring signs of pain or
distress or eye lesions considered to be irreversible.
A recent report of the National Research Council Committee on Recognition and Alleviation of Painin Laboratory Animals emphasized the need for increased efforts to identify appropriate humane
endpoints (NRC 2009).
During the 2005 symposium “Minimizing Pain and Distress in Ocular Toxicity Testing,” panelists
recommended early adverse responses that could serve as early humane endpoints to terminate
animals on a study. Among the invited participants were human and veterinary ophthalmologists andanesthesiologists, scientific experts in ocular hazard testing, research scientists, and industrial
toxicologists. The following ocular lesions are predictive of maximal severity, that of a severe irritantor corrosive with irreversible effects, including EPA Category I (2003) GHS Category 1 (UN 2007),and EU Category R41 (2001). They could be used routinely as humane endpoints to terminate astudy.
x

Endpoints currently accepted for study termination (OECD 2002)– Draize corneal opacity score of 4 that persists for 48 hours– Corneal perforation or significant corneal ulceration including
staphyloma– Blood in the anterior chamber of the eye– Absence of light reflex that persists for 72 hours– Ulceration of the conjunctival membrane

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–
–
x
x
x
x
x

Necrosis of the conjunctiva or nictitating membrane
Sloughing

Vascularization of the corneal surface (i.e., pannus)
Destruction of more than 75% of the limbus
No diminishment in area of fluorescein staining and/or increase in depth of injury overtime
Lack of re-epithelialization 5 days after application of the test substance
Depth of injury to the cornea (routinely using slit-lamp and fluorescein staining) in which
corneal ulceration extends beyond superficial layers of the stroma

The Panel discussed other endpoints that might allow for early termination of a study. These included
destruction of the limbus and the relationship to re-epithelialization of the cornea, and positive resultsin Shirmer’s test. Shirmer’s test measures moisture content of the corneal tear film. A positive resultin Shirmer’s test suggests that conjunctival redness is likely to return to normal within 21 days. Afterthese discussions, the endpoints described above were recommended for routine use. As discussed in
Section 4.0, the Panel also recommended many of these endpoints (see the Panel’s full report at
http://iccvam.niehs.nih.gov/methods/ocutox/PeerPanel09.htm).

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6.0

ICCVAM Consideration of Public and SACATM Comments

The ICCVAM evaluation process provides numerous opportunities for public stakeholder
involvement, including submission of written comments and oral comments at ICCVAM independent
peer review panel and SACATM meetings. Table 6-1 lists the nine different opportunities for publiccomments that were provided during the ICCVAM evaluation of the validation status of alternative
ocular safety testing methods and approaches. The number of public comments received in responseto each of the opportunities is also indicated. Thirty-seven comments were received. Comments
received in response to or related to the Federal Register notices are accessible on the NICEATM–ICCVAM website. The following sections, delineated by Federal Register notice, briefly discuss the
public comments received.
Table 6-1

Opportunities for Public Comment
Opportunities for Public Comment

Date

Number of
PublicComments
Received

70 FR 13512: Request for Data on Non-Animal Methods andApproaches for Determining Skin and Eye Irritation Potential of
Antimicrobial Cleaning Product Formulations; Request for
Nominations for an Independent Expert Panel

March 21, 2005

072 FR 26396: Request for Data on the Use of Topical
Anesthetics and Systemic Analgesics for In Vivo Eye Irritation
Testing

May 9, 2007

1

72 FR 31582: Request for Ocular Irritancy Test Data From
Human, Rabbit, and In Vitro Studies Using Standardized
Testing Methods

June 7, 2007

073 FR 18535: Non-Animal Methods and Approach for
Evaluating Eye Irritation Potential for Antimicrobial Cleaning
Products (AMCPs): Request for Nominations for an
Independent Expert Panel and Submission of Relevant Data

April 4, 2008

12

74 FR 14556: Announcement of an Independent Scientific Peer
Review Panel on Alternative Ocular Safety Testing Methods;
Availability of Draft Background Review Documents (BRD);
Request for Comments

March 31, 20098

74 FR 19562: Meeting of the Scientific Advisory Committee on
Alternative Toxicological Methods (SACATM)

April 29, 20092

Independent Scientific Peer Review Panel Meeting: Alternative
Ocular Safety Testing Methods

May 19–21, 200912

SACATM Meeting, Arlington Hilton, Arlington, VA

June 25–26, 20092

July 13, 2009

074 FR 33444: Independent Scientific Peer Review Panel Report:
Evaluation of the Validation Status of Alternative Ocular Safety
Testing Methods and Approaches; Notice of Availability andRequest for Public Comments

13

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6.1

Public Comments in Response to 70 FR 13512 (March 21, 2005):
Request for Data on Non-Animal Methods and Approaches for Determining
Skin and Eye Irritation Potential of Antimicrobial Cleaning Product
Formulations; Request for Nominations for an Independent Expert Panel

NICEATM requested (1) submission of data that would assist in evaluating the validation status of
non-animal methods and approaches used for determining the skin and eye irritation potential of
AMCP formulations to meet regulatory hazard classification and labeling purposes and(2) nominations of expert scientists to serve as members of an independent peer review panel.
No data or nominations were received in response to this Federal Register notice.

6.2

Public Comments in Response to 72 FR 26396 (May 9, 2007):
Request for Data on the Use of Topical Anesthetics and Systemic Analgesics for
In Vivo Eye Irritation Testing

NICEATM requested submission of (1) data and information on the use of topical anesthetics andsystemic analgesics for alleviating pain and distress in rabbits during eye irritation testing and(2) information about other procedures and strategies that may reduce or eliminate pain and distressassociated with in vivo eye irritation methods.
Public Response
NICEATM received one comment in response to this Federal Register notice.
Comment:
The commenter supported the use of anesthetics to minimize pain and distress in rabbit eye irritation
studies and offered assistance in the evaluation. However, the commenter noted that data from theirstudies involving the use of local anesthetics could not be shared without permission of its sponsors.
ICCVAM Response:
ICCVAM encourages users to provide data that are generated from future studies, as they could be
used to further characterize the usefulness and limitations of topical anesthetics and systemic
analgesics for avoiding or minimizing pain and distress in ocular safety assessments.

6.3

Public Comments in Response to 72 FR 31582 (June 7, 2007):
Request for Ocular Irritancy Test Data From Human, Rabbit, and In Vitro
Studies Using Standardized Testing Methods

NICEATM requested data on substances tested for ocular irritancy in humans, rabbits, and/or in vitro
to be used to:xx

Review the state of the science in regard to the availability of accurate and reliable invitro test methods for assessing the range of potential ocular irritation activity, including
whether ocular damage is reversible or not
Expand NICEATM’s high-quality ocular toxicity database. In vitro test methods forwhich data are sought include but are not limited to (1) the bovine corneal opacity andpermeability test, (2) the isolated rabbit eye test, (3) the isolated chicken eye test, and (4)the hen’s egg test–chorioallantoic membrane.

No data or information was received in response to this Federal Register notice.

14

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6.4

Public Comments in Response to 73 FR 18535 (April 4, 2008):
Non-Animal Methods and Approach for Evaluating Eye Irritation Potential for
Antimicrobial Cleaning Products (AMCPs): Request for Nominations for an
Independent Expert Panel and Submission of Relevant Data

NICEATM requested the following:xxx

Nominations of expert scientists to serve as members of an independent peer review
panel
Submission of relevant data and information on AMCPs or related substances obtained
from (1) human testing or experience, including reports from accidental exposures, and(2) rabbit testing using the standard eye test or the LVET
In vitro ocular safety test methods such as the bovine corneal opacity and permeability
test method, the Cytosensor® Microphysiometer test method, and the EpiOcular test
method, including data supporting the accuracy and reproducibility of these methods

In response to this Federal Register notice, NICEATM received 12 comments, including nominationsof 20 potential panelists. The nominees were included in the database of experts from which the Panelwas selected. No additional data were received.

6.5

Public Comments in Response to 74 FR 14556 (March 31, 2009):
Announcement of an Independent Scientific Peer Review Panel on Alternative
Ocular Safety Testing Methods; Availability of Draft Background Review
Documents (BRD); Request for Comments

NICEATM requested public comments on the draft BRDs, SRDs, and draft ICCVAM test method
recommendations that were provided to an independent scientific peer review panel meeting(May 19–21, 2009). These documents summarized the current validation status of several test
methods and testing strategies for identifying potential ocular irritants. The test methods and testing
strategies included the following:
x
x

x
x

A testing strategy that proposes the use of three in vitro test methods to assess the eyeirritation potential of AMCPs
Four in vitro test methods for identifying moderate (EPA Category II, UN Globally
Harmonized System of Classification and Labelling of Chemicals [GHS] Category 2A)
and mild (EPA Category III, GHS Category 2B) ocular irritants and substances notclassified as ocular irritants (EPA Category IV, GHS Not Classified)
The in vivo LVET
A proposal for the routine use of topical anesthetics, systemic analgesics, and earlier
humane endpoints to avoid and minimize pain and distress during in vivo ocular irritation
testing

NICEATM received 20 comments in response to this Federal Register notice. Eight writtencomments were received before the Panel meeting, and 12 oral comments were provided at the Panelmeeting.
No written comments were relevant to the use of topical anesthetics, systemic analgesics, or earlier
humane endpoints to minimize pain and distress in ocular safety testing.
None of the 12 oral public comments provided at the Panel meeting was relevant to the use of topicalanesthetics, systemic analgesics, or earlier humane endpoints to avoid or minimize pain and distressin ocular safety testing.

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6.6

Public Comments in Response to 74 FR 19562 (April 29, 2009):
Meeting of the Scientific Advisory Committee on Alternative Toxicological
Methods (SACATM)NICEATM announced the SACATM meeting (June 25–26, 2009) and requested written and public
oral comments on the agenda topics.
Public Response:
NICEATM received four comments. Two written comments were received before the meeting, andtwo oral comments were provided at the SACATM meeting.
SACATM Response:
In general, SACATM was pleased with the Panel report. One SACATM member expressed the needfor harmonization in the assessment of performance standards. Another SACATM member said thefocus should be on the GHS system because it will ultimately be adopted. Another SACATM
member expressed concern regarding the availability of the Cytosensor® Microphysiometer.

6.7

Public Comments in Response to 74 FR 33444 (July 13, 2009):
Independent Scientific Peer Review Panel Report: Evaluation of the Validation
Status of Alternative Ocular Safety Testing Methods and Approaches; Notice of
Availability and Request for Public Comments

NICEATM requested submission of written public comments on the independent scientific peer
review panel report.
No public comments were received.

16

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7.0

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