ᣂⷙ⹜㛎ᴺឭ᩺ᦠ             ᐔᚑ 26 ᐕ 1 ᦬ 20 ᣣ                                  No. 2013-05
ᡷ⸓ OECD TG No.437 ‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ
㧔BCOP ᴺ㧦Bovine Corneal Opacity and Permeability Test㧕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BCOP ᴺߪ‫ޔ‬ൻቇ‛⾰ߦࠃࠆ⋥ធ⊛ߥ⌒ೝỗᕈࠍ⹏ଔߢ߈‫ޔ‬ߘߩ▸࿐ߪ‫ޔ‬
ԘUN GHS඙ಽ 1㧔㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔ㧕෸߮ ԙUN GHS ඙ಽᄖ⌒ߩ்ኂࠍᒁ߈⿠ߎ

ߐߥ޿㧕ߢ޽ࠆ‫ ࠄࠇߎޕ‬Ԙ෸߮ԙߩ▸࿐ߦ߅޿ߡ‫ⴕޔ‬᡽⊛ߥ೑↪ߪน⢻ߢ޽ࠆ‫ޕ‬ ߎߩឭ᩺ᦠߪ‫ޔ‬OECD (Organisation for Economic Co-operation and Development) Test Guideline 437
߅ࠃ߮Streamed Summary Document Supporting OECD Guideline 437 on the Bovine Corneal Opacityand Permeability for Eye Irritation/Corrosionࠍ߽ߣߦ‫⌒ޔ‬ೝỗᕈ⹜㛎⹏ଔᆔຬળߦࠃࠅ߹ߣ߼ࠄࠇߚ
ᢥᦠࠍ↪޿ߡJaCVAM⹏ଔળ⼏߇⹏ଔ߅ࠃ߮ᬌ⸛ߒߚ⚿ᨐ‫↪᦭ߩߘޔ‬ᕈ߇⏕⹺ߐࠇߚߎߣ߆ࠄ૞
ᚑߐࠇߚ‫ޕ‬ એ਄ߩℂ↱ߦࠃࠅ‫ⴕޔ‬᡽ᒰዪߩ቟ోᕈ⹏ଔᣇᴺߣߒߡ‫ޟ‬ᡷ⸓ OECD TG No.437 ‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜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

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JaCVAM statement
on Bovine Corneal Opacity and Permeability Test Method for Identifyingi) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage
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:
Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals
Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye
Irritation or Serious Eye Damage is considered to have sufficient accuracy andreliability for prediction of eye irritating test substances for regulatory use.
Following the review of the results of OECD (Organisation for Economic Co-operation andDevelopment) Test Guideline revised No. 437 and STREAMLINED SUMMARY
DOCUMENT SUPPORTING OECD GUIDELINE 437 ON THE BOVINE CORNEAL
OPACITY AND PERMEABILITY FOR EYE IRRITATION/CORROSION, it is concluded
that Bovine Corneal Opacity and Permeability Test Method 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

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ᡷ⸓ OECD TG No.437 ‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ(BCOP ᴺ㧦Bovine Corneal Opacity and Permeability Test)⋡ ᰴ⹏ଔળ⼏ႎ๔ᦠ --------------------------------------------------------------------------------1
⹏ଔႎ๔ᦠ ------------------------------------------------------------------------------------- 5
OECD TG437㧔2013㧕---------------------------------------------------------------------- 15
STREAMLINED SUMMARY DOCUMENT SUPPORTING OECDGUIDELINE 437 ON THE BOVINE CORNEAL OPACITY ANDPERMEABILITY FOR EYE IRRITATION/CORROSION --------------------- 43ix

xᡷ⸓ OECD TG No.437 ‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ
㧔BCOP ᴺ㧦Bovine Corneal Opacity and Permeability Test㧕ߩ⹏ଔળ⼏ႎ๔ᦠJaCVAM ⹏ଔળ⼏ᐔᚑ 25 ᐕ 10 ᦬ 21 ᣣ





1

1


JaCVAM ⹏ଔળ⼏ศ↰ᱞ⟤㧔ᣣᧄᲥᕈቇળ㧕㧦ᐳ㐳

ᵻ㊁ື⑲

㧔ᣣᧄⅣႺᄌ⇣ේቇળ㧕੖ච፲⦟᣿ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ ↢ᵴⴡ↢ൻቇㇱ㧕৻㝩 ീ㧔ᣣᧄൻቇᎿᬺදળ㧕*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 ᣣ
એ਄

2

2

‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ㧔BCOP ᴺ㧦Bovine Corneal Opacity and Permeability
Test㧕ߪ‫⌒࠭ࠗ࡟࠼ޔ‬ೝỗᕈ⹜㛎ߩઍᦧᴺߣߒߡ‫ࠆߌ߅ߦ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰㧔UN GHS ඙ಽ 1‫ޔ‬US EPA ඙ಽ 1‫ޔ‬EU DSD R43㧕ࠍหቯߔࠆߚ߼ߩ⹜㛎ᴺ
ߣߒߡ 2009 ᐕߦ OECD㧔Organization for Economic Co-operation and Development㧕ߦ࠹ࠬ࠻ࠟࠗ࠼࡜ࠗࡦ㧔TG㧕No.437 ߣߒߡណᛯߐࠇߚ‫ޔߦࠄߐޕ‬2013 ᐕߦߪ‫ޔ‬BCOP ᴺ߇ࡏ࠻ࡓࠕ࠶ࡊ
ࠕࡊࡠ࡯࠴ߢߩ⌒ߩ்ኂࠍᒁ߈⿠ߎߐߥ޿ൻቇ‛⾰㧔GHS ╬ߩ඙ಽᄖ‛⾰㧕ߩหቯᴺߣߒߡᱜᑼ
ߦណᛯߐࠇߚ‫ޕ‬JaCVAM ⌒ೝỗᕈ⹏ଔᆔຬળߪ‫੹ޔ‬࿁ߩᡷ⸓ߩᅷᒰᕈߦߟ޿ߡᬌ⸛ߒߚ‫ޕ‬㧝㧚⹜㛎ᴺߩቯ⟵ฬ⒓㧦 ᡷ⸓‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ㧔BCOP ᴺ㧦Bovine Corneal Opacity andPermeability Test㧕ઍᦧߔࠆኻ⽎Ქᕈ⹜㛎㧦࠼࡟ࠗ࠭⌒ೝỗᕈ⹜㛎⹜㛎ᴺߩ᭎⇛㧦BCOP ᴺߪ‫៰ࠄ߆⃿⌒‐ޔ‬಴ߒߚⷺ⤑ߦⵍ㛎‛⾰ࠍ৻ቯᤨ㑆ᦑ㔺ߐߖ‫ߩ⤑ⷺޔ‬ᄌᕈߩᜰᮡߣߥࠆⷺ⤑ᷙỘᐲߣⷺ⤑਄⊹ߩࡃ࡝ࠕᯏ⢻ߩᜰᮡߣߥࠆⷺ⤑ㅘㆊᐲࠍ᷹ቯߒ‫ࠄࠇߘޔ‬᷹ቯ୯߆ࠄ⸘▚ᑼߦࠃߞߡ᳞߼ࠄࠇࠆ IVIS㧔in vitro ೝỗᕈࠬࠦࠕ㧕ࠍၮߦ࠙ࠨࠡࠍ↪޿ߚ Draize ᴺߢߩ⌒ೝỗᕈࠍ੍᷹ߔࠆઍᦧᴺߢ޽ࠆ‫ޕ‬㧞㧚⹏ଔߦ↪޿ߚ⾗ᢱ߅ࠃ߮⹏ଔౝኈߩ⑼ቇ⊛ᅷᒰᕈNICEATM㧔NTP Interagency Center for the Evaluation of Alternative Toxicological
Methods 㧕/ICCVAM㧔Interagency Coordinating Committee on the Validation of Alternative
Methods㧕ߦࠃࠆ࿁㘈⊛ߥࡃ࡝࠺࡯࡚ࠪࡦࠍ⚻ߡ‫ޔ‬2009 ᐕߦ NICEATM/ICCVAM ߩ╙ਃ⠪⹏ଔᆔຬળߢ⹏ଔߐࠇߡ߅ࠅ‫⹏ߩߘޔ‬ଔౝኈߦ⑼ቇ⊛ᅷᒰᕈ߇޽ࠆ‫⹏ޔߪߢࡦ࡚ࠪ࡯࠺࡝ࡃޕ‬ଔ‛⾰ࠍ
213 ‛⾰㧔න৻‛⾰㧛ᷙว‛⾰㧦113 ‛⾰㧛100 ‛⾰㧕ߦჇ߿ߒߡ߅ࠅචಽߥᢙ߇⹏ଔߐࠇߡ޿ࠆ‫ޕ‬㧟㧚ᧄ⹜㛎ᴺߩ᦭↪ᕈߣㆡ↪㒢⇇
2009 ᐕߩ TG ߢߪ‫ޔ‬ᣉ⸳㑆࡮ᣉ⸳ౝߩౣ⃻ᕈߪ㜞ߊ‫்ߩ⌒ࠆߌ߅ߦ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬ኂࠍ⿠ߎߔൻቇ‛⾰ࠍหቯߢ߈ࠆ⹜㛎ᴺߢ޽ࠆߣߐࠇߚ‫ࡦ࠻ࠤ࡮࡞࡯ࠦ࡞ࠕޕ‬㘃߅ࠃ߮࿕૕‛⾰ߩㆡ↪ߦߟ޿ߡߪ೨࿁ߩ࠻࠶ࡊ࠳࠙ࡦࠕࡊࡠ࡯࠴ߩ⹏ଔߢߪᧄ⹜㛎ᴺߩ㒢⇇ߣߒߡ᜼ߍࠄࠇߡ޿ߚ
߇‫੹ޔ‬࿁ߩ TG ᡷ⸓ߢߪ‫ࡦ࠻ࠤ࡮࡞࡯ࠦ࡞ࠕޔ‬㘃ߢ㓁ᕈ⚿ᨐ߇ᓧࠄࠇߚ႐วߪ‫ޔ‬㜞޿ன㓁ᕈ₸ࠍ⠨ᘦߒ‫⚿ߩߘޔ‬ᨐߪᘕ㊀ߦᬌ⸛ߔࠆᔅⷐ߇޽ࠆߣߩᵈ㉼߇ઃߐࠇߚ਄ߢ‫ޔ‬ㆡ↪㒢⇇ߪߥߊߥߞߚ‫ޕ‬࿕
33

૕‛⾰ߩன㒶ᕈ߽ઁߩ⹜㛎ᴺߢ⵬ቢߢ߈ࠆߎߣ߆ࠄ‫ߪߢ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬⠨ᘦߔࠆᔅⷐ߇
ߥ޿ߣߐࠇߚ‫ޕ‬੹࿁ߩ TG ᡷ⸓ߢ‫்ߩ⌒ߩߢ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޔ‬ኂࠍᒁ߈⿠ߎߐߥ޿ൻቇ‛⾰ߩหቯࠍⴕ
߁ߚ߼ߩ⹜㛎ᴺߢ޽ࠆߣߐࠇߡ޿ࠆ‫⚿ߩߘޕ‬ᨐ߆ࠄ‫ޔ‬㜞޿ன㓁ᕈ₸ࠍ␜ߒߚ߇‫ޔ‬ㅊട⹜㛎ߦࠃࠆಽ㘃್ᢿ߇ᔅⷐߣߥࠆߎߣ߆ࠄ‫ޔ‬ᔅߕߒ߽໧㗴ߪߥࠄߥ޿ߣ್ᢿߐࠇߚ‫ߩ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޕ‬႐วߣ⇣ߥࠅ‫ޔ߽ߢ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޔ‬ㆡ↪㒢⇇ߪߥ޿ߣߐࠇߚ‫ޕ‬㧠㧚ᒰ⹥⹜㛎ᴺߪ‫⾰‛ࠆߔߣ⊛⋡ޔ‬෶ߪ⵾ຠߩᲥᕈࠍ⹏ଔߔࠆ⹜㛎ᴺߣߒߡ‫ⴕޔ‬᡽਄೑↪෸߮␠ળ⊛ฃߌ౉ࠇߩน⢻ᕈ ␠ળ⊛ฃߌ౉ࠇᕈ㧦ర᧪ BCOP ᴺߪ‫ޔ‬㘩↪ߥߤߩ↪ㅜߢዼᲕߐࠇߚ࠙ࠪߩⷺ⤑ࠍ↪޿ߚേ‛ታ㛎ઍᦧᴺߢ޽ࠆߚ߼‫ޔ‬࠼࡟ࠗ࠭⌒ೝỗᕈ⹜㛎ᴺࠃࠅ߽୶ℂ⊛ߦఝࠇߡ߅ࠅ␠ળ⊛ฃߌ౉ࠇᕈߪ㜞޿‫੹ޕ‬࿁ߩᡷ⸓ߦ߅޿ߡ‫ޔ‬േ‛⑔␩ߩⷰὐ߆ࠄߩᄌᦝߪߥ޿ߎߣ߆ࠄ‫ᧄޔ‬ᡷ⸓ᴺߩ␠ળ⊛ฃߌ౉ࠇᕈ߽‫ޔ‬ේᴺߣห᭽㜞޿߽ߩߣ⠨߃ࠄࠇࠆ‫ޔߒߛߚޕ‬ᣣᧄߢߪ‐ⷺ⤑ߩ౉ᚻ߇ኈᤃߢߪߥߊ‫ߩߎޔ‬ὐ߇᥉෸ߩᅹߍߦߥࠆน⢻ᕈ߇␜ໂߐࠇߚ‫ޕ‬ ⴕ᡽਄ߩ೑↪ᕈ㧦   BCOP ᴺߪ‫ޔ‬ൻቇ‛⾰ߦࠃࠆ⋥ធ⊛ߥ⌒ೝỗᕈࠍ⹏ଔߢ߈‫⹏ޔ‬ଔߢ߈ࠆ⌒ೝỗᕈߩ▸࿐ߪ‫ޔ‬
ԘUN GHS ඙ಽ 1㧔㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔ㧕෸߮ԙUN GHS ඙ಽᄖ (⌒ߩ்ኂࠍᒁ߈⿠ߎ
ߐߥ޿㧕ߢ޽ࠆ‫ࠄࠇߎޕ‬Ԙ෸߮ԙߩ▸࿐ߦ߅޿ߡ‫ⴕޔ‬᡽⊛ߥ೑↪ߪน⢻ߢ޽ࠆ‫ޕ‬ෳ⠨ᢥ₂1) JaCVAM ⌒ೝỗᕈ⹏ଔᆔຬળ㧦⌒ೝỗᕈઍᦧᴺߩ╙ਃ⠪⹏ଔႎ๔ᦠ‫ޔ‬ᡷ⸓ OECD TG No.437‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ㧔BCOP ᴺ㧦Bovine Corneal Opacity and Permeability
Test㧕ᐔᚑ 23 ᐕ 10 ᦬ 11 ᣣ
 ‐៰಴ⷺ⤑ࠍ↪޿ߚ⌒ೝỗᕈ⹜㛎ઍᦧᴺ BCOPᴺ㧦Bovine Corneal Opacity and Permeability
Test ߩ⹏ଔળ⼏ႎ๔ᦠ ᐔᚑ21ᐕ12᦬17ᣣ‫ޔ‬ᐔᚑ23ᐕ4᦬20ᣣᡷቯ 3) JaCVAM⌒ೝỗᕈ⹏ଔᆔຬળ㧦⌒ೝỗᕈઍᦧᴺߩ╙ਃ⠪⹏ଔႎ๔ᦠ‫៰‐ޔ‬಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ㧔BCOPᴺ㧦Bovine Corneal Opacity and Permeability Test㧕ᐔᚑ21ᐕ10᦬14ᣣ
4) OECD Test Guideline, Revised TG437㧔2013㧕

44⌒ೝỗᕈઍᦧᴺ ᡷ⸓ OECD TG No.437 ‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ
㧔BCOP ᴺ㧦Bovine Corneal Opacity and Permeability Test㧕ߩ⹏ଔႎ๔ᦠ

ᐔᚑ 25 ᐕ 7 ᦬ 19 ᣣ15⌒ೝỗᕈઍᦧᴺ⹏ଔᆔຬળᆔຬฬ㧦┻ౝ ዊ⧣ 㧔P&G ࠗࡁࡌ࡯࡚ࠪࡦวหળ␠㧕ዊဈ ᔘม 㧔ᱷ⇐ㄘ⮎⎇ⓥᚲ㧕ട⮮ 㓷৻ 㧔ᩣᑼળ␠ࠫࡖࡄࡦ࡮࠹ࠖ࠶ࠪࡘ࡮ࠛࡦࠫ࠾ࠕ࡝ࡦࠣ㧕☄ౝ ᩶ሶ 㧔࿖┙ක⮎ຠ㘩ຠⴡ↢⎇ⓥᚲ㧕⚦੗ ৻ᒄ 㧔ෳᄤ⵾⮎ᩣᑼળ␠㧕Ⴧ↰ శノጊᧄ ⋥᮸ 㧔⮮↰଻ஜⴡ↢ᄢቇ㧕ศ᧛ ഞ 㧔᧲੩ℂ⑼ᄢቇฬ⹷ᢎ᝼㧕

2

6

⇛⺆
BCOP:

Bovine Corneal Opacity and Permeability Test

BRD:

Background Review Document

DSD:

Dangerous Substances Directive

EPA:

Environmental Protection Agency

EU:

European Union

GHS:

Globally Harmonized Systems of Classification and Labeling

ICCVAM: Interagency Coordinating Committee on the Validation of Alternative
Methods
IVIS:

In Vitro Irritancy Score

NICEATM: NTP Interagency Center for the Evaluation of Alternative Toxicological
Methods
OECD:

Organization for Economic Co-operation and Development

UN:

United Nations

US:

United States

37㧝㧚ᡷ⸓ߩ⢛᥊‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺ㧔Bovine Corneal Opacity and Permeability Test;એਅ‫ޔ‬BCOP ᴺ㧕ߪ‫ ߚ޿↪ࠍࠡࠨ࠙ޔ‬Draize ⌒ೝỗᕈ⹜㛎ᴺ㧔એਅ‫ޔ‬Draize ᴺ㧕ߩઍᦧ⹜㛎ᴺߢ޽ࠆ‫ޕ‬2003 ᐕ߆ࠄ 2006 ᐕߩ NICEATM㧔NTP Interagency Center for the
Evaluation of Alternative Toxicological Methods㧕/ICCVAM 㧔Interagency Coordinating
Committee on the Validation of Alternative Methods㧕ߦࠃࠆ࿁㘈⊛ߥࡃ࡝࠺࡯࡚ࠪࡦࠍ
⚻ߡ‫ࠆߌ߅ߦ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰㨇UN㧔United
Nations㧕 GHS㧔Globally Harmonized Systems of Classification and Labeling㧕඙ಽ 1‫ޔ‬US㧔United States㧕 EPA㧔Environmental Protection Agency㧕඙ಽ 1‫ޔ‬EU㧔European
Union㧕 DSD㧔Dangerous Substances Directive㧕 R43㨉ࠍหቯߔࠆߚ߼ߩ⹜㛎ᴺߣߒߡ
2009 ᐕߦ OECD㧔Organization for Economic Co-operation and Development㧕࠹ࠬ࠻ࠟࠗ࠼࡜ࠗࡦ㧔TG㧕No.437 ߣߒߡណᛯߐࠇߚ‫ޕ‬ߐࠄߦ‫ޔ‬2006 ᐕ߆ࠄ 2009 ᐕߦ߆ߌ‫ࠆߌ߅ߦ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޔ‬㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔ‛⾰߅ࠃ߮⌒ೝỗᕈ‛⾰ߣߪಽ㘃ߐࠇߥ޿‛⾰㧔એਅ‫ޔ‬඙ಽᄖ‛⾰㧕ߩหቯᴺߣ
ߒߡߩㆡ↪ᕈߦߟ޿ߡ‫ޔ‬࿁㘈⊛ߥࡃ࡝࠺࡯࡚ࠪࡦ߇ⴕࠊࠇߚ‫⚿ߩߘޕ‬ᨐ‫ޔ‬2009 ᐕ‫ޔ‬NICEATM/ICCVAM ߩ╙ਃ⠪⹏ଔᆔຬળߢ‫ޔ‬UN GHS ߅ࠃ߮ EU DSD ߩಽ㘃ߦ߅ߌࠆ඙
ಽᄖ‛⾰ࠍหቯߔࠆᣇᴺߣߒߡ᦭↪ߢ޽ࠆߣ޿߁⚿⺰ࠍᓧߚ‫⚿ߩߎޕ‬ᨐࠍฃߌ‫ޔ‬OECD ߢߪ BCOP ᴺࠍ㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰ߛߌߢߥߊ‫ޔ‬඙ಽᄖ‛⾰ߩหቯᣇᴺߣߒߡ
↪޿ࠆߎߣࠍᬌ⸛ߔࠆߚ߼‫ޔ‬TG No.437 ߩᡷ⸓ᬌ⸛ࡊࡠࠫࠚࠢ࠻߇ߚߜ޽ߍࠄࠇߚ‫ߩߎޕ‬ࡊࡠࠫࠚࠢ࠻ߢ⚵❱ߐࠇߚ⌒ೝỗᕈኾ㐷ኅળ⼏ߦ߅޿ߡ‫߼ߚߩ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬ߩ BRD㧔Background Review Document㧕ߦᣂߚߥ࠺࡯࠲ࠍㅊടߒߚ Issue Paper ߦၮߠ޿ߡ‫ޔ‬BCOP ᴺߩࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴ߢߩᕈ⢻߇ᬌ⸛ߐࠇߚ‫⚿ߩߘޕ‬ᨐ‫ޔ‬2013 ᐕ‫ޔ‬BCOP
ᴺ߇ࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴ߢߩ඙ಽᄖ‛⾰ߩหቯᴺߣߒߡᱜᑼߦᡷቯ TG No.437 ߦណᛯߐࠇߚ‫ޔ߈ߣߩߎޕ‬ㆡ↪㒢⇇ߣ㓁ᕈኻᾖ‛⾰╬ߦߟ޿ߡߩᡷ⸓߽หᤨߦⴕࠊࠇߚ‫ߩߎޕ‬ࠃ߁ߦߒߡߢ߈ߚߩ߇ᡷቯ TG No.437 ߢ޽ࠆ‫⹏ᧄޕ‬ଔᆔຬળߪ‫ߩߎޔ‬ᡷቯ TG No.437
ߩᅷᒰᕈࠍᬌ⸛ߒߚ‫ޕ‬એਅߦߘߩᬌ⸛⚿ᨐࠍႎ๔ߔࠆ‫ޕ‬㧞㧚⹜㛎ߩ᭎ⷐBCOP ᴺߪ‫៰ࠄ߆⃿⌒‐ޔ‬಴ߒߚⷺ⤑ߦⵍ㛎‛⾰ࠍ৻ቯᤨ㑆ᦑ㔺ߐߖ‫ߩ⤑ⷺޔ‬ᄌᕈߩᜰᮡߣߥࠆⷺ⤑ᷙỘᐲߣⷺ⤑਄⊹ߩࡃ࡝ࠕᯏ⢻ߩᜰᮡߣߥࠆⷺ⤑ㅘㆊᐲࠍ᷹ቯߒ‫ࠄࠇߘޔ‬᷹ቯ୯߆ࠄ⸘▚ᑼߦࠃߞߡ᳞߼ࠄࠇࠆ IVIS㧔In Vitro Irritancy Score 㧦in vitro ೝỗᕈࠬࠦࠕ㧕ࠍၮߦ࠙ࠨࠡࠍ↪޿ߚ Draize ᴺߢߩ⌒ೝỗᕈࠍ੍᷹ߔࠆઍᦧ⌒ೝỗᕈ⹜㛎ᴺߢ޽ࠆ‫ޕ‬⹜㛎⟲޽ߚࠅ 3 ୘ߩⷺ⤑ࠍ૶↪ߒ‫ⵍޔ‬㛎‛⾰߇ᶧ૕ߩ႐วߪේᶧ㧔100%‫⇇ߒߛߚޔ‬㕙ᵴᕈ೷ߩ႐วߪ 10%Ꮧ㉼ᶧ㧕ࠍ‫ޔ‬࿕૕ߩ႐วߪ 20%ߩṁᶧ߹ߚߪ Ộᶧࠍ‫ ࠇߙࠇߘޔ‬10 ಽ㑆‫ ߪߚ߹ޔ‬4 ᤨ㑆ᦑ㔺ߐߖࠆ‫ߩߘޕ‬ᓟ‫ޔ‬ᷙỘᐲߣߒߡࠝࡄࠪ࠻ࡔ࡯࠲ߢⷺ⤑ߩశㅘㆊᐲࠍ‫ޔ‬߹ߚ‫ޔ‬ㅘㆊᐲߣߒߡࡈ࡞ࠝ࡟࠮ࠗࡦ࠽࠻࡝࠙ࡓߩㅘㆊᐲ㧔OD490㧕ࠍ᷹ቯߔࠆ‫ ࠄࠇߎޕ‬2 ߟ

48ߩ᷹ቯ୯ࠍએਅߩ⸘▚ᑼߦᒰߡߪ߼ IVIS ୯ࠍ▚಴ߔࠆ‫ޕ‬ 㧔⸘▚ᑼ㧕IVIS = mean opacity + (15 x mean OD490)
IVIS ߩ୯߇ 3 એਅߩ႐ว‫ⵍߩߘޔ‬㛎‛⾰ߪ GHS ඙ಽᄖ‛⾰ߣ್ᢿߐࠇ㧔ㅊടߐࠇߚၮ
Ḱ㧕‫ޔ‬IVIS ߩ୯߇ 55 ࠍ⿥߃ࠆ႐วߪ඙ಽ㧝ߣ್ᢿߐࠇࠆ‫ޕ‬IVIS ߩ୯߇ 3 < IVIS < 55 ߩ႐ว‫⹜ᧄޔ‬㛎ᴺߩ⚿ᨐߩߺߢߪ⌒ೝỗᕈߩ੍᷹ߪߢ߈ߥ޿ߚ߼‫ޔ‬ㅊടߩ⹜㛎ࠍᔅⷐߣߔࠆ‫ޕ‬㧟㧚ᡷ⸓ὐ㧟㧙㧝㧚ࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴ߢߩ GHS ඙ಽᄖ‛⾰ߩหቯ㧟㧙㧝㧙㧝㧚 ࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴ߢߩᱜ⏕ᕈ೨࿁⹏ଔࠍⴕߞߚ ICCVAM ߩࡃ࡝࠺࡯࡚ࠪࡦ࠺࡯࠲ࡌ࡯ࠬߦ 66 ߩ᛫⩶ᵞᵺ⵾ຠߩ⚿ᨐࠍᣂߚߦㅊടߒߚ࠺࡯࠲ࡌ࡯ࠬࠍ↪޿ߚᱜ⏕ᕈ⹏ଔߩ⚿ᨐࠍ⴫ 1 ߦ␜ߔ‫ޕ‬⴫ 1㧚BCOP ᴺߩᱜ⏕ᕈ㧙GHS ಽ㘃ᴺߢߩ඙ಽᄖ‛⾰ߩหቯ

59඙ಽᄖ‛⾰ߩหቯߦ߅޿ߡ‫ޔ‬Draize ᴺߩ⚿ᨐࠍᲧセኻᾖߦߔࠆߣ‫ޔ‬BCOP ᴺߩᱜ⏕ᐲߪ
69%㧔135/196㧕‫ޔ‬ன㓁ᕈ₸ 69%㧔61/89㧕‫ޔ‬ன㒶ᕈ₸ 0%㧔0/107㧕ߣߥߞߚ‫ޕ‬ன㓁ᕈ₸ߪ㜞޿߇‫ߪߢ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޔ‬㓁ᕈ⚿ᨐ߇ᓧࠄࠇߚ႐ว‫ߜࠊߥߔޔ‬඙ಽᄖ‛⾰ߣ್ቯߐࠇߥ߆ߞߚ႐วߪ‫ߩઁޔ‬ᣇᴺߦࠃࠆㅊട⹜㛎߇ᔅⷐߣߥࠆߎߣ߆ࠄ‫ޔ‬ன㓁ᕈ₸߇㜞޿ߎߣߪᔅߕߒ߽໧㗴ߦߪߥࠄߥ޿ߣ್ᢿߒߚ‫ޕ‬㧟㧙㧝㧙㧞㧚ࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴ߢߩᣉ⸳ౝ࡮ᣉ⸳㑆ౣ⃻ᕈ
BCOP ᴺߩ IVIS ୯ߩᣉ⸳ౝౣ⃻ᕈߪචಽߢ޽ࠆߣ޿߁್ᢿߪ೨࿁ߩ⹏ଔߢߐࠇߡ޿ࠆ‫ޕ‬ࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴ߦ߅ߌࠆ඙ಽᄖ‛⾰ࠍหቯߔࠆ႐วߩᣉ⸳㑆ౣ⃻ᕈߦߟ޿ߡ‫ޔ‬3ߟߩࡃ࡝࠺࡯࡚ࠪࡦ⹜㛎ߩ⚿ᨐࠍ⴫ 2 ߦ␜ߔ‫⚿ߩߘޕ‬ᨐ‫ޔ‬80%㧔103/128㧕ߩ‛⾰ߦ߅޿ߡ‫ޔ‬ߘࠇߙࠇߩ⹜㛎ෳടᣉ⸳ߔߴߡߩ್ቯ߇ 100%৻⥌ߒߚ‫ߩߢ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޕ‬ᣉ⸳㑆ౣ⃻ᕈߪචಽߢ޽ࠆߣ್ᢿߒߚ‫ޕ‬⴫ 2㧚BCOP ᴺߩᣉ⸳㑆ౣ⃻ᕈ㧙GHS ಽ㘃ᴺߢߩ඙ಽᄖ‛⾰ߩหቯ́ICCVAM(2010)ࠃࠅᛮ☴Data Source
Balls et al. (1995)
Gautheron et al. (1994)

No. ofTesting Labs

No. ofSubstances

Substances with 100%
Agreement among Labs

5

60

55 (92%)52

34 (65%)

11
12

Southee (1998)

3

16

14 (88%)

Total

-

128

103 (80%)㧟㧙㧞㧚࠻࠶ࡊ࠳࠙ࡦࠕࡊࡠ࡯࠴ߢߩ GHS ඙ಽ㧝㧔㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰㧕ߩหቯ࠻࠶ࡊ࠳࠙ࡦࠕࡊࡠ࡯࠴ߦ߅ߌࠆ㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰㧔඙ಽ 1㧕ࠍหቯߔࠆ
႐วߩᱜ⏕ᕈߦߟ޿ߡ߽‫ޔ‬ᣂߚߥࡃ࡝࠺࡯࡚ࠪࡦ࠺࡯࠲ࡌ࡯ࠬߢߩ⚿ᨐࠍ⴫ 3 ߦ␜ߔ‫ߎޕ‬ࠇߦࠃࠆߣ‫ޔ‬ᱜ⏕ᐲ‫ޔ‬ன㓁ᕈ₸‫ޔ‬ன㒶ᕈ₸ߣ߽‫ޔ‬೨࿁⹏ଔߩ⚿ᨐ߇ᄌࠊࠆ߶ߤߩᄢ߈ߥ㆑
޿ߪߥ޿‫ޕ‬ߥ߅‫ߩ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬ᣉ⸳ౝ࡮ᣉ⸳㑆ౣ⃻ᕈߪචಽߢ޽ࠆߣ೨ႎߦߡ್ᢿߒ
ߚ‫ޕ‬

6

10

⴫ 3㧚BCOP ᴺߩᱜ⏕ᕈ㧙GHS ಽ㘃ᴺߢߩ඙ಽ 1 ‛⾰ߩหቯ㧟㧙㧟㧚ㆡ↪㒢⇇ࠕ࡞ࠦ࡯࡞࡮ࠤ࠻ࡦ㘃߅ࠃ߮࿕૕‛⾰ߩㆡ↪ߦߟ޿ߡߪ೨࿁ߩ࠻࠶ࡊ࠳࠙ࡦࠕࡊࡠ࡯࠴
ߩ⹏ଔߢߪᧄ⹜㛎ᴺߩ㒢⇇ߣߒߡ᜼ߍࠄࠇߡ޿ߚ߇‫ޔ‬એਅߩℂ↱߆ࠄࠕ࡞ࠦ࡯࡞࡮ࠤ࠻ࡦ
㘃߅ࠃ߮࿕૕‛⾰߽ㆡ↪ߔࠆߎߣ߇ߢ߈ࠆߣ್ᢿߒߚ‫ޕ‬y㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰ࠍหቯߔࠆߩߦ↪޿ࠆ႐ว㧔࠻࠶ࡊ࠳࠙ࡦࠕࡊࡠ࡯
࠴㧕‫ߚࠇࠄ޿↪ߦࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬ൻቇ‛⾰ߩ࠺࡯࠲ࡌ࡯ࠬߩਛߢ‫ࠤ࡮࡞࡯ࠦ࡞ࠕޔ‬࠻ࡦ㘃ߦኻߒ㜞޿ன㓁ᕈ₸‫ޔ‬࿕૕‛⾰ߦኻߒ㜞޿ன㒶ᕈ₸ࠍ␜ߒߚ‫ࠄࠇߎޔߛߚޕ‬ൻቇ‛⾰㘃ߩਛߢ߽ᱜ⏕ߦ੍᷹ߐࠇߚ߽ߩߪ޽ࠆ‫ޔߚ߹ޕ‬ன㒶ᕈߩ⚿ᨐࠍ␜ߒߚ࿕૕‛⾰ߪߔߴߡ 3 < IVIS < 55㧔⌒ೝỗᕈ੍᷹ਇน㧕ߣߥࠅ‫⚿ޔ‬ᨐߣߒߡ೎ߩ⹜㛎ᴺߢߩ
ᬌ⸛߇ᔅⷐߣߐࠇࠆ‫ࡦ࠻ࠤ࡮࡞࡯ࠦ࡞ࠕޕ‬㘃ߢ㓁ᕈ⚿ᨐ߇ᓧࠄࠇߚ႐วߪ‫ޔ‬㜞޿ன㓁ᕈ₸ࠍ⠨ᘦߒ‫⚿ߩߘޔ‬ᨐߪᘕ㊀ߦᬌ⸛ߔࠆᔅⷐ߇޽ࠆ‫ࠄࠇߎޔߪߡߒߣ⺰⚿ޕ‬ൻቇ‛⾰㘃ࠍ‫ޔ‬ㆡ↪㗔ၞ߆ࠄ㒰ᄖߔࠆᔅⷐߪߥ޿ߣ್ᢿߐࠇߚ‫ޕ‬඙ಽᄖ‛⾰ࠍหቯߔࠆߩߦ
↪޿ࠆ႐ว㧔ࡏ࠻ࡓࠕ࠶ࡊࠕࡊࡠ࡯࠴㧕‫ޔ‬㜞޿ன㓁ᕈ₸ࠍ␜ߒߚ‫ޔࠄ߇ߥߒ߆ߒޕ‬3 <IVIS < 55㧔⌒ೝỗᕈ੍᷹ਇน㧕ߣߥߞߚ‛⾰ߪㅊട⹜㛎ߦࠃࠆಽ㘃್ᢿ߇ᔅⷐߣߥ
ࠆߎߣ߆ࠄ‫ޔ‬ᔅߕߒ߽໧㗴ߪߥࠄߥ޿ߣ್ᢿߒߚ‫ߩ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޕ‬႐วߣ⇣ߥࠅ‫ࡦ࠻ࠤ࡮࡞࡯ࠦ࡞ࠕޔߪߢ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޔ‬㘃‫ޔ‬࿕૕‛⾰ߩ⚿ᨐ߇ᱜ

7

11⏕ᕈߦᓇ㗀ࠍਈ߃ߡߪ޿ߥ޿‫ޕ‬߹ߚ‫⹜ࡦ࡚ࠪ࡯࠺࡝ࡃޔ‬㛎ߢߪන৻‛⾰࡮ᷙว‛⾰߇ߘࠇߙࠇ 113 ‛⾰‫ޔ‬100 ‛⾰ߣචಽߥᢙ߇⹏ଔߐࠇߡ߅ࠅ‫߽ࠄߜߤޔ‬ㆡ↪น⢻ߢ޽ࠆߣ್ᢿߐࠇߡ޿ࠆ‫ޕ‬㧟㧙㧠㧚 㓁ᕈኻᾖ2009 ᐕߦᧄ⹏ଔᆔຬળ߇⹏ଔߒߚ BCOP ᴺߪᶧ૕ⵍ㛎‛⾰ߩ㓁ᕈኻᾖߣߒߡ 100%ࠛ࠲
ࡁ࡯࡞ࠍ↪޿ߡ޿ߚ߇‫ޔ‬OECD ࠟࠗ࠼࡜ࠗࡦൻߩᤨὐߢ 100%ࠛ࠲ࡁ࡯࡞ߦᦧࠊࠅ 1%᳓㉄ൻ࠽࠻࡝࠙ࡓ߇ណ↪ߐࠇߡ޿ߚ‫ޕ‬੹࿁ߩᡷ⸓ TG No.437 ߢߪ㓁ᕈኻᾖߦߟ޿ߡએਅߩ᧦ઙ߇ᔅⷐߣߐࠇߚ‫ޔߜࠊߥߔޕ‬หߓ⹜㛎♽ߢ㓁ᕈ෻ᔕࠍ⺃⊒ߔࠆߎߣ߇ᣢ⍮ߢ޽ࠆߎߣߣ‫ޔ‬෻ᔕߩᄌേࠍ⹏ଔߔࠆߚ߼ߦ෻
ᔕ߇ㆊᐲߢߥ޿ߎߣߢ޽ࠆ‫ޔࠅࠃߦࠇߎޕ‬1%᳓㉄ൻ࠽࠻࡝࠙ࡓ߇㓁ᕈኻᾖ୥⵬‛⾰࡝ࠬ࠻߆ࠄ㒰ᄖߐࠇ‫ޔ‬100%ࠛ࠲ࡁ࡯࡞߇ߎߩ࡝ࠬ࠻ߦട߃ࠄࠇߚ‫ޕ‬100%ࠛ࠲ࡁ࡯࡞ߪ‫ޔ‬BCOP
ᴺߢߩ⌒ೝỗᕈಽ㘃߇ Draize ᴺߣ৻⥌ߒߡ޿ߥ޿߇‫ޔ‬ᡷ⸓ TG No.437 ߩ㓁ᕈኻᾖߩᔅⷐ
᧦ઙߦߪวߞߡ߅ࠅ‫ޔ‬BCOP ᴺߢߩ⢛᥊࠺࡯࠲߇ᄙߊ޽ࠆߎߣ߆ࠄ‫⹏ᧄޔ‬ଔᆔຬળߪ 100%ࠛ࠲ࡁ࡯࡞ࠍᶧ૕ⵍ㛎‛⾰ߩ㓁ᕈኻᾖ‛⾰ߣߔࠆߎߣߦ໧㗴ߪߥ޿ߣ್ᢿߒߚ‫ޕ‬㧟㧙㧡㧚ߘߩઁ ⹜㛎ណ↪ၮḰߦߟ޿ߡ߽ᡷ⸓߇ߥߐࠇߡ޿ࠆ߇‫ߥ⊛⾰ᧄޔ‬ㇱಽߢߪߥߊ‫ౕޔ‬૕⊛ߥ࠺࡯࠲߽ߥ޿ߎߣ߆ࠄ޽߃ߡ⹏ଔߪታᣉߒߥ߆ߞߚ‫ޕ‬㧠㧚⚿⺰ᧄ⹏ଔᆔຬળߪߘߩᱜ⏕ᕈ‫ޔ‬ౣ⃻ᕈߩ⚿ᨐࠃࠅ‫ޔ‬BCOP ᴺࠍ GHS ߩ⌒ೝỗᕈಽ㘃ߦ߅޿ߡ࠻࠶ࡊ࠳࠙ࡦࠕࡊࡠ࡯࠴ߢߩ㊀◊ߥ⌒ߩ்ኂࠍ⿠ߎߔൻቇ‛⾰ࠍหቯߔࠆߚ߼ߩᣇᴺߛ
ߌߢߥߊ‫⌒ߩߢ࠴࡯ࡠࡊࠕࡊ࠶ࠕࡓ࠻ࡏޔ‬ೝỗᕈ඙ಽᄖ‛⾰ࠍหቯߔࠆᣇᴺߣߒߡ߽↪޿ࠆߎߣ߇น⢻ߢ޽ࠆߣ್ᢿߒߚ‫⹜ᧄޔ߽ߡ޿߅ߦ࠴࡯ࡠࡊࠕߩࠄߜߤޔߚ߹ޕ‬㛎ᴺߩㆡ↪
‛⾰ߦ೙㒢ࠍ⸳ߌࠆᔅⷐߪߥߊ‫ޔ‬ᶧ૕ⵍ㛎‛⾰ߩ㓁ᕈኻᾖߣߒߡ 100%ࠛ࠲ࡁ࡯࡞ࠍ↪޿ࠆߎߣ߇ߢ߈ࠆߣ⠨߃ߚ‫ޕ‬ ⚿⺰ߣߒߡ‫ ࠆߌ߅ߦ࠴࡯ࡠࡊࠕࡦ࠙࠳ࡊ࠶࠻ޔ‬GHS ඙ಽ 1 ‛⾰‫ࠕࡊ࠶ࠕࡓ࠻ࡏ߮ࠃ߅ޔ‬ࡊࡠ࡯࠴ߦ߅ߌࠆ GHS ඙ಽᄖ‛⾰ߩหቯߦ‫ޔ‬Draize ᴺߩઍᦧᴺߣߒߡᡷ⸓ TG No.437 ࠍ↪޿ࠆߎߣߪน⢻ߢ޽ࠆߣ್ᢿߒߚ‫ޕ‬

8

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㧡㧚ᢥ₂
y

JaCVAM (2009), ⌒ೝỗᕈ⹜㛎ઍᦧᴺߩ╙ਃ⠪⹏ଔႎ๔ᦠ́⹏ଔኻ⽎⹜㛎㧦⌒ߦኻߔࠆ⣣㘩ᕈ߅ࠃ߮ᒝೝỗᕈ⹏ଔߩߚ߼ߩ‐៰಴ⷺ⤑ߩᷙỘ߅ࠃ߮ㅘㆊᕈ⹜㛎ᴺy

ICCVAM (2010), ICCVAM Test Method Evaluation Report: Current Validation
Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard
Potential of Chemicals and Products. NIH Publication No. 10-7553.

yOECD (2009), Bovine Corneal Opacity and Permeability Test Method for
Identifying Ocular Corrosives and Severe Irritants, TG437yOECD (2013), Updated TG 437 on the BCOP Test Method, ENV/JM/TG(2013)14yOECD (2013), Issue Paper on the Use of the BCOP Test Method to Identify
Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage,
EVN/JM/TG(2013)15

yOECD (2013), Streamlined Summary Document Supporting OECD Guideline 437
on the Bovine Corneal Opacity and Permeability for Eye Irritation/Corrosion,
ENV/JM/MONO(2013)13

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OECD/OCDE

437
Adopted:26 July 2013

OECD GUIDELINES FOR THE TESTING OF CHEMICALSBovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious
Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye
DamageINTRODUCTION
1.
The Bovine Corneal Opacity and Permeability (BCOP) test method was evaluated by theInteragency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), in
conjunction with the European Centre for the Validation of Alternative Methods (ECVAM) and theJapanese Center for the Validation of Alternative Methods (JaCVAM), in 2006 and 2010 (1)(2). In the firstevaluation, the BCOP test method was evaluated for its usefulness to identify chemicals (substances andmixtures) inducing serious eye damage (1). In the second evaluation, the BCOP test method wasevaluated for its usefulness to identify chemicals (substances and mixtures) not classified for eye irritationor serious eye damage (2). The BCOP validation database contained 113 substances and 100 mixtures intotal (2)(3). From these evaluations and their peer review it was concluded that the test method cancorrectly identify chemicals (both substances and mixtures) inducing serious eye damage as well as those
not requiring classification for eye irritation or serious eye damage, as defined by the United Nations(UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (4), and it wastherefore endorsed as scientifically valid for both purposes. Serious eye damage is the production of tissue
damage in the eye, or serious physical decay of vision, following application of a test chemical to theanterior surface of the eye, which is not fully reversible within 21 days of application. Test chemicals
inducing serious eye damage are classified as UN GHS Category 1. Chemicals not classified for eye
irritation or serious eye damage are defined as those that do not meet the requirements for classification as
UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category. This Test
Guideline (adopted in 2009 and updated in 2013) includes the recommended use and limitations of the
BCOP test method based on its evaluations. The main differences between the original 2009 version andthe 2013 updated version concern, but are not limited to: the use of the BCOP test method to identify
chemicals not requiring classification according to UN GHS (paragraphs 2 and 7); clarifications on theapplicability of the BCOP test method to the testing of alcohols, ketones and solids (paragraphs 6 and 7)
1© OECD, (2013)
You are free to use this material for personal, non-commercial purposes without seeking prior consent fromthe OECD, provided the source is duly mentioned. Any commercial use of this material is subject to written
permission from the OECD.

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and of substances and mixtures (paragraph 8); clarifications on how surfactant substances and surfactantcontaining mixtures should be tested (paragraph 28); updates and clarifications regarding the positive
controls (paragraphs 39 and 40); an update of the BCOP test method decision criteria (paragraph 47); an
update of the study acceptance criteria (paragraph 48); an update to the test report elements (paragraph49); an update of Annex 1 on definitions; the addition of Annex 2 for the predictive capacity of the BCOP
test method under various classification systems; an update of Annex 3 on the list of proficiency
chemicals; and an update of Annex 4 on the BCOP corneal holder (paragraph 1) and on the opacitometer(paragraphs 2 and 3).
2.
It is currently generally accepted that, in the foreseeable future, no single in vitro eye irritation
test will be able to replace the in vivo Draize eye test to predict across the full range of irritation fordifferent chemical classes. However, strategic combinations of several alternative test methods within a(tiered) testing strategy may be able to replace the Draize eye test (5). The Top-Down approach (5) is
designed to be used when, based on existing information, a chemical is expected to have high irritancy
potential, while the Bottom-Up approach (5) is designed to be used when, based on existing information, achemical is expected not to cause sufficient eye irritation to require a classification. The BCOP test method
is an in vitro test method that can be used under certain circumstances and with specific limitations for eye
hazard classification and labeling of chemicals. While it is not considered valid as a stand-alone replacement
for the in vivo rabbit eye test, the BCOP test method is recommended as an initial step within a testing strategy
such as the Top-Down approach suggested by Scott et al. (5) to identify chemicals inducing serious eye
damage, i.e. chemicals to be classified as UN GHS Category 1, without further testing (4). The BCOP test
method is also recommended to identify chemicals that do not require classification for eye irritation orserious eye damage, as defined by the UN GHS (UN GHS No Category) (4) within a testing strategy suchas the Bottom-up approach (5). However, a chemical that is not predicted as causing serious eye damage or
as not classified for eye irritation/serious eye damage with the BCOP test method would require additional
testing (in vitro and/or in vivo) to establish a definitive classification.
3.
The purpose of this Test Guideline is to describe the procedures used to evaluate the e ye
h a z a r d potential of a test chemical as measured by its ability to induce opacity and increased
permeability in an isolated bovine cornea. Toxic effects to the cornea are measured by: (i) decreased lighttransmission (opacity), and (ii) increased passage of sodium fluorescein dye (permeability). The opacityand permeability assessments of the cornea following exposure to a test chemical are combined to derive
an In Vitro Irritancy Score (IVIS), which is used to classify the irritancy level of the test chemical.
4.

Definitions are provided in Annex 1.

INITIAL CONSIDERATIONS AND LIMITATIONS
5.
This Test Guideline is based on the ICCVAM BCOP test method protocol (6)(7), which was
originally developed from information obtained from the Institute for In Vitro Sciences (IIVS) protocol and
INVITTOX Protocol 124 (8). The latter represents the protocol used for the European Communitysponsored prevalidation study conducted in 1997-1998. Both of these protocols were based on the BCOP
test method first reported by Gautheron et al. (9).
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6.
The BCOP test method can be used to identify chemicals inducing serious eye damage as definedby UN GHS, i.e. chemicals to be classified as UN GHS Category 1 (4). When used for this purpose, theBCOP test method has an overall accuracy of 79% (150/191), a false positive rate of 25% (32/126), and afalse negative rate of 14% (9/65), when compared to in vivo rabbit eye test method data classified
according to the UN GHS classification system (3) (see Annex 2, Table 1). When test chemicals withincertain chemical (i.e., alcohols, ketones) or physical (i.e., solids) classes are excluded from the database,the BCOP test method has an overall accuracy of 85% (111/131), a false positive rate of 20% (16/81), anda false negative rate of 8% (4/50) for the UN GHS classification system (3). The potential shortcomings ofthe BCOP test method when used to identify chemicals inducing serious eye damage (UN GHS
Category 1) are based on the high false positive rates for alcohols and ketones and the high false negative
rate for solids observed in the validation database (1)(2)(3). However, since not all alcohols and ketones areover-predicted by the BCOP test method and some are correctly predicted as UN GHS Category 1, these
two organic functional groups are not considered to be out of the applicability domain of the test method. It
is up to the user of this Test Guideline to decide if a possible over-prediction of an alcohol or ketone can be
accepted or if further testing should be performed in a weight-of-evidence approach. Regarding the false
negative rates for solids, it should be noted that solids may lead to variable and extreme exposure
conditions in the in vivo Draize eye irritation test, which may result in irrelevant predictions of their true
irritation potential (10). It should also be noted that none of the false negatives identified in the ICCVAM
validation database (2)(3), in the context of identifying chemicals inducing serious eye damage (UN GHS
Category 1), resulted in IVIS ≤ 3, which is the criterion used to identify a test chemical as a UN GHS No
Category. Moreover, BCOP false negatives in this context are not critical since all test chemicals that
produce an 3 < IVIS ≤ 55 would be subsequently tested with other adequately validated in vitro tests, or asa last option in rabbits, depending on regulatory requirements, using a sequential testing strategy in aweight-of-evidence approach. Given the fact that some solid chemicals are correctly predicted by the
BCOP test method as UN GHS Category 1, this physical state is also not considered to be out of theapplicability domain of the test method. Investigators could consider using this test method for all types ofchemicals, whereby an IVIS > 55 should be accepted as indicative of a response inducing serious eye
damage that should be classified as UN GHS Category 1 without further testing. However, as alreadymentioned, positive results obtained with alcohols or ketones should be interpreted cautiously due topotential over-prediction.
7.
The BCOP test method can also be used to identify chemicals that do not require classificationfor eye irritation or serious eye damage under the UN GHS classification system (4). When used for thispurpose, the BCOP test method has an overall accuracy of 69% (135/196), a false positive rate of 69%(61/89), and a false negative rate of 0% (0/107), when compared to in vivo rabbit eye test method dataclassified according to the UN GHS classification system (3) (see Annex 2, Table 2). The false positive
rate obtained (in vivo UN GHS No Category chemicals producing an IVIS > 3, see paragraph 47) is
considerably high, but not critical in this context since all test chemicals that produce an 3 < IVIS ≤ 55
would be subsequently tested with other adequately validated in vitro tests, or as a last option in rabbits,
depending on regulatory requirements, using a sequential testing strategy in a weight-of-evidence approach.
The BCOP test method shows no specific shortcomings for the testing of alcohols, ketones and solids when
the purpose is to identify chemicals that do not require classification for eye irritation or serious eye
3© OECD, (2013)

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damage (UN GHS No Category) (3). Investigators could consider using this test method for all types ofchemicals, whereby a negative result (IVIS ≤ 3) should be accepted as indicative that no classification isrequired (UN GHS No Category). Since the BCOP test method can only identify correctly 31% of thechemicals that do not require classification for eye irritation or serious eye damage, this test method should
not be the first choice to initiate a Bottom-Up approach (5), if other validated and accepted in vitro methods
with similar high sensitivity but higher specificity are available.
8.
The BCOP validation database contained 113 substances and 100 mixtures in total (2)(3). The
BCOP test method is therefore considered applicable to the testing of both substances and mixtures.
9.
The BCOP test method is not recommended for the identification of test chemicals that should beclassified as irritating to eyes (UN GHS Category 2 or Category 2A) or test chemicals that should beclassified as mildly irritating to eyes (UN GHS Category 2B) due to the considerable number of UN GHS
Category 1 chemicals underclassified as UN GHS Category 2, 2A or 2B and UN GHS No Category
chemicals overclassifed as UN GHS Category 2, 2A or 2B (2)(3). For this purpose, further testing withanother suitable method may be required.
10.
All procedures with bovine eyes and bovine corneas should follow the testing facility’s applicableregulations and procedures for handling animal-derived materials, which include, but are not limited to,tissues and tissue fluids. Universal laboratory precautions are recommended (11).
11.
Whilst the BCOP test method does not consider conjunctival and iridal injuries, it addresses
corneal effects, which are the major driver of classification in vivo when considering the UN GHS
classification. The reversibility of corneal lesions cannot be evaluated per se in the BCOP test method. It
has been proposed, based on rabbit eye studies, that an assessment of the initial depth of corneal injury may
be used to identify some types of irreversible effects (12). However, further scientific knowledge isrequired to understand how irreversible effects not linked with initial high level injury occur. Finally, theBCOP test method does not allow for an assessment of the potential for systemic toxicity associated withocular exposure.
12.
This Test Guideline will be updated periodically as new information and data are considered. For
example, histopathology may be potentially useful when a more complete characterization of corneal
damage is needed. As outlined in OECD Guidance Document No. 160 (13), users are encouraged topreserve corneas and prepare histopathology specimens that can be used to develop a database anddecision criteria that may further improve the accuracy of this test method.
13.
For any laboratory initially establishing this test method, the proficiency chemicals provided in
Annex 3 should be used. A laboratory can use these chemicals to demonstrate their technical competencein performing the BCOP test method prior to submitting BCOP test method data for regulatory hazard
classification purposes.

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PRINCIPLE OF THE TEST14.
The BCOP test method is an organotypic model that provides short-term maintenance of normal
physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by thetest c h e m i c a l is assessed by quantitative measurements of changes in corneal opacity andpermeability with an opacitometer and a visible light spectrophotometer, respectively. Bothmeasurements are used to calculate an IVIS, which is used to assign an in vitro irritancy hazard
classification category for prediction of the in vivo ocular irritation potential of a test chemical (seeDecision Criteria in paragraph 47).
15.
The BCOP test method uses isolated corneas from the eyes of freshly slaughtered cattle. Corneal
opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability ismeasured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of thecornea, as detected in the medium in the posterior chamber. Test chemicals are applied to the epithelial
surface of the cornea by addition to the anterior chamber of the corneal holder. Annex 4 provides a
description and a diagram of a corneal holder used in the BCOP test method. Corneal holders can beobtained commercially from different sources or can be constructed.
Source and Age of Bovine Eyes and Selection of Animal Species
16.
Cattle sent to slaughterhouses are typically killed either for human consumption or for othercommercial uses. Only healthy animals considered suitable for entry into the human food chain are used asa source of corneas for use in the BCOP test method. Because cattle have a wide range of weights,
depending on breed, age, and sex, there is no recommended weight for the animal at the time of slaughter.
17.
Variations in corneal dimensions can result when using eyes from animals of different ages.
Corneas with a horizontal diameter >30.5 mm and central corneal thickness (CCT) values ≥ 1100 μm are
generally obtained from cattle older than eight years, while those with a horizontal diameter < 28.5 mm and
CCT <900 μm are generally obtained from cattle less than five years old (14). For this reason, eyes fromcattle greater than 60 months old are not typically used. Eyes from cattle less than 12 months of age havenot traditionally been used since the eyes are still developing and the corneal thickness and corneal
diameter are considerably smaller than that reported for eyes from adult cattle. However, the use of corneas
from young animals (i.e., 6 to 12 months old) is permissible since there are some advantages, such asincreased availability, a narrow age range, and decreased hazards related to potential worker exposure to
Bovine Spongiform Encephalopathy (15). As further evaluation of the effect of corneal size or thickness on
responsiveness to corrosive and irritant substances would be useful, users are encouraged to report theestimated age and/or weight of the animals providing the corneas used in a study.
Collection and Transport of Eyes to the Laboratory
18.
Eyes are collected by slaughterhouse employees. To minimize mechanical and other types ofdamage to the eyes, the eyes should be enucleated as soon as possible after death and cooled immediately
after enucleation and during transport. To prevent exposure of the eyes to potentially irritant substances,
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the slaughterhouse employees should not use detergent when rinsing the head of the animal.
19.
Eyes should be immersed completely in cooled Hanks’ Balanced Salt Solution (HBSS) in asuitably sized container, and transported to the laboratory in such a manner as to minimize deterioration
and/or bacterial contamination. Because the eyes are collected during the slaughter process, they might be
exposed to blood and other biological substances, including bacteria and other microorganisms. Therefore,
it is important to ensure that the risk of contamination is minimized (e.g., by keeping the container
containing the eyes on wet ice during collection and transportation and by adding antibiotics to the HBSS
used to store the eyes during transport [e.g., penicillin at 100 IU/mL and streptomycin at 100 μg/mL]).
20.
The time interval between collection of the eyes and use of corneas in the BCOP test
method should be minimized (typically collected and used on the same day) and should be demonstrated
to not compromise the assay results. These results are based on the selection criteria for the eyes, as well as
the positive and negative control responses. All eyes used in the assay should be from the same group ofeyes collected on a specific day.
Selection Criteria for Eyes Used in the BCOP Test Method
21.
The eyes, once they arrive at the laboratory, are carefully examined for defects including
increased opacity, scratches, and neovascularization. Only corneas from eyes free of such defects are to be
used.
22.
The quality of each cornea is also evaluated at later steps in the assay. Corneas that have
opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after
an initial one hour equilibration period are to be discarded (NOTE: the opacitometer should be calibrated
with opacity standards that are used to establish the opacity units, see Annex 4).
23.
Each treatment group (test chemical, concurrent negative and positive controls) consists of aminimum of three eyes. Three corneas should be used for the negative control corneas in the BCOP test
method. Since all corneas are excised from the whole globe, and mounted in the corneal chambers, there ispotential for artifacts from handling upon individual corneal opacity and permeability values (including
negative control). Furthermore, the opacity and permeability values from the negative control corneas areused to correct the test chemical-treated and positive control-treated corneal opacity and permeability
values in the IVIS calculations.
PROCEDURE
Preparation of the Eyes
24.
Corneas, free of defects, are dissected with a 2 to 3 mm rim of sclera remaining to assistin subsequent handling, with care taken to avoid damage to the corneal epithelium and endothelium.
Isolated corneas are mounted in specially designed corneal holders that consist of anterior and posterior
compartments, which interface with the epithelial and endothelial sides of the cornea, respectively. Both
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chambers are filled to excess with pre-warmed phenol red free Eagle's Minimum Essential Medium(EMEM) (posterior chamber first), ensuring that no bubbles are formed. The device is then equilibrated at
32 ± 1°C for at least one hour to allow the corneas to equilibrate with the medium and to achieve normal
metabolic activity, to the extent possible (the approximate temperature of the corneal surface in vivo is32°C).
25.
Following the equilibration period, fresh pre-warmed phenol red free EMEM is added to bothchambers and baseline opacity readings are taken for each cornea. Any corneas that show macroscopic
tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity greater than seven opacity
units or equivalent for the opacitometer and cornea holders used are discarded. A minimum of threecorneas are selected as negative (or solvent) control corneas. The remaining corneas are thendistributed into treatment and positive control groups.
26.
Because the heat capacity of water is higher than that of air, water provides more stabletemperature conditions for incubation. Therefore, the use a water bath for maintaining the corneal holder
and its contents at 32 ± 1ºC is recommended. However, air incubators might also be used, assuming
precaution to maintain temperature stability (e.g., by pre-warming of holders and media).
Application of the Test Chemical
27.
Two different treatment protocols are used, one for liquids and surfactants (solids or liquids), andone for non-surfactant solids.
28.
Liquids are tested undiluted. Semi-solids, creams, and waxes are typically tested as liquids. Neatsurfactant substances are tested at a concentration of 10% w/v in a 0.9% sodium chloride solution, distilledwater, or other solvent that has been demonstrated to have no adverse effects on the test system.
Appropriate justification should be provided for alternative dilution concentrations. Mixtures containing
surfactants may be tested undiluted or diluted to an appropriate concentration depending on the relevant
exposure scenario in vivo. Appropriate justification should be provided for the concentration tested.
Corneas are exposed to liquids and surfactants for 10 minutes. Use of other exposure times should beaccompanied by adequate scientific rationale. Please see Annex 1 for a definition of surfactant andsurfactant-containing mixture.
29.
Non-surfactant solids are typically tested as solutions or suspensions at 20% w/ v concentration
in a 0.9% sodium chloride solution, distilled water, or other solvent that has been demonstrated to have noadverse effects on the test system. In certain circumstances and with proper scientific justification, solids
may also be tested neat by direct application onto the corneal surface using the open chamber method (seeparagraph 32). Corneas are exposed to solids for four hours, but as with liquids and surfactants, alternative
exposure times may be used with appropriate scientific rationale.
30.
Different treatment methods can be used, depending on the physical nature and chemical
characteristics (e.g., solids, liquids, viscous vs. non-viscous liquids) of the test c h e m i c a l . The
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adequately removed during the rinsing steps. A closed-chamber method is typically used for non-viscous toslightly viscous liquid test c h e m i c a l s, while an open-chamber method is typically used for semiviscous and viscous liquid test chemicals and for neat solids.
31.
In the closed-chamber method, sufficient test chemical (750 μL) to cover the epithelial sideof the cornea is introduced into the anterior chamber through the dosing holes on the top surface of thechamber, and the holes are subsequently sealed with the chamber plugs during the exposure. It is important
to ensure that each cornea is exposed to a test chemical for the appropriate time interval.
32.
In the open-chamber method, the window-locking ring and glass window from the anteriorchamber are removed prior to treatment. The control or test chemical (750 μL, or enough test chemical tocompletely cover the cornea) is applied directly to the epithelial surface of the cornea using a micro-pipet.
If a test chemical is difficult to pipet, the test chemical can be pressure-loaded into a positive displacement
pipet to aid in dosing. The pipet tip of the positive displacement pipet is inserted into the dispensing tip of
the syringe so that the material can be loaded into the displacement tip under pressure. Simultaneously, thesyringe plunger is depressed as the pipet piston is drawn upwards. If air bubbles appear in the pipet tip, thetest article is removed (expelled) and the process repeated until the tip is filled without air bubbles. If
necessary, a normal syringe (without a needle) can be used since it permits measuring an accurate volumeof test chemical and an easier application to the epithelial surface of the cornea. After dosing, the glass
window is replaced on the anterior chamber to recreate a closed system.
Post-Exposure Incubation
33.
After the exposure period, the test chemical, the negative control, or the positive control substanceis removed from the anterior chamber and the epithelium washed at least three times (or until no visualevidence of test chemical can be observed) with EMEM (containing phenol red). Phenol red- containing
medium is used for rinsing since a color change in the phenol red may be monitored to determine
the effectiveness of rinsing acidic or alkaline materials. The corneas are washed more than three times if
the phenol red is still discolored (yellow or purple), or the test chemical is still visible. Once the medium isfree of test chemical, the corneas are given a final rinse with EMEM (without phenol red). The EMEM
(without phenol red) is used as a final rinse to ensure removal of the phenol red from the anterior chamberprior to the opacity measurement. The anterior chamber is then refilled with fresh EMEM without phenol
red.
34.
For liquids or surfactants, after rinsing, the corneas are incubated for an additional two hours at
32 ± 1ºC. Longer post-exposure time may be useful in certain circumstances and could be considered on acase-by-case basis. Corneas treated with solids are rinsed thoroughly at the end of the four-hour exposure
period, but do not require further incubation.
35.
At the end of the post-exposure incubation period for liquids and surfactants and at the end of the
four-hour exposure period for non-surfactant solids, the opacity and permeability of each cornea arerecorded. Also, each cornea is observed visually and pertinent observations recorded (e.g., tissue peeling,residual test chemical, non-uniform opacity patterns). These observations could be important as they may
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be reflected by variations in the opacitometer readings.
Control Substances
36.
Concurrent negative or solvent/vehicle controls and positive controls are included in eachexperiment.
37.
When testing a liquid substance at 100%, a concurrent negative control (e.g., 0.9% sodium
chloride solution or distilled water) is included in the BCOP test method so that nonspecific changes in thetest system can be detected and to provide a baseline for the assay endpoints. It also ensures that the assay
conditions do not inappropriately result in an irritant response.
38.
When testing a diluted liquid, surfactant, or solid, a concurrent solvent/vehicle control group isincluded in the BCOP test method so that nonspecific changes in the test system can be detected and toprovide a baseline for the assay endpoints. Only a solvent/vehicle that has been demonstrated to have noadverse effects on the test system can be used.
39.
A substance known to induce a positive response is included as a concurrent positive control ineach experiment to verify the integrity of the test system and its correct conduct. However, to ensure thatvariability in the positive control response across time can be assessed, the magnitude of irritant response
should not be excessive.
40.
Examples of positive controls for liquid test chemicals are 100% ethanol or 100%
dimethylformamide. An example of a positive control for solid test chemicals is 20% w/v imidazole in
0.9% sodium chloride solution.
41.
Benchmark substances are useful for evaluating the ocular irritancy potential of unknown
chemicals of a specific chemical or product class, or for evaluating the relative irritancy potential of an
ocular irritant within a specific range of irritant responses.
Endpoints Measured
42.
Opacity is determined by the amount of light transmission through the cornea. Corneal opacity ismeasured quantitatively with the aid of an opacitometer, resulting in opacity values measured on acontinuous scale.
43.
Permeability is determined by the amount of sodium fluorescein dye that penetrates all corneal
cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea
surface). O n e mL sodium fluorescein solution (4 or 5 mg/mL when testing liquids and surfactants or
non- surfactant solids, respectively) is added to the anterior chamber of the corneal holder, which interfaces
with the epithelial side of the cornea, while the posterior chamber, which interfaces with the endothelial
side of the cornea, is filled with fresh EMEM. The holder is then incubated in a horizontal position for
90 ± 5 min at 32 ± 1 ºC. The amount of sodium fluorescein that crosses into the posterior chamber is
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quantitatively measured with the aid of UV/VIS spectrophotometry. Spectrophotometric measurements
evaluated at 490 nm are recorded as optical density (OD490) or absorbance values, which aremeasured on a continuous scale. The fluorescein permeability values are determined using OD490
values based upon a visible light spectrophotometer using a standard 1 cm path length.
44.
Alternatively, a 96-well microtiter plate reader may be used provided that; (i) the linear range ofthe plate reader for determining fluorescein OD490 values can be established; and (ii), the correct volumeof fluorescein samples are used in the 96-well plate to result in OD490 values equivalent to the standard
1 cm path length (this could require a completely full well [usually 360μL]).
DATA AND REPORTING
Data Evaluation
45.
Once the opacity and mean permeability (OD490) values have been corrected forbackground opacity and the negative control permeability OD490 values, the mean opacity andpermeability OD490 values for each treatment group should be combined in an empirically-derived
formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:

IVIS = mean opacity value + (15 x mean permeability OD490 value)46.
Sina et al. (16) reported that this formula was derived during in-house and inter-laboratory
studies. The data generated for a series of 36 compounds in a multi-laboratory study were subjected to a
multivariate analysis to determine the equation of best fit between in vivo and in vitro data. Scientists at two
separate companies performed this analysis and derived nearly identical equations.
47.
The opacity and permeability values should also be evaluated independently to determine
whether a test chemical induced corrosivity or severe irritation through only one of the two endpoints (seeDecision Criteria).
Decision Criteria
48.
The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS
Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage(UN GHS No Category) are given hereafter:

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OECD/OCDEIVISUN GHS≤3

No Category> 3; ≤ 55

No prediction can be
made

> 55

Category 1

Study Acceptance Criteria
49.
A test is considered acceptable if the positive control gives an IVIS that falls within two standard
deviations of the current historical mean, which is to be updated at least every three months, or each time
an acceptable test is conducted in laboratories where tests are conducted infrequently (i.e., less than once amonth). The negative or solvent/vehicle control responses should result in opacity and permeability values
that are less than the established upper limits for background opacity and permeability values for bovine
corneas treated with the respective negative or solvent/vehicle control. A single testing run composed of atleast three corneas should be sufficient for a test chemical when the resulting classification is unequivocal.
However, in cases of borderline results in the first testing run, a second testing run should be considered(but not necessarily required), as well as a third one in case of discordant mean IVIS results between thefirst two testing runs. In this context, a result in the first testing run is considered borderline if thepredictions from the 3 corneas were non-concordant, such that:x

2 of the 3 corneas gave discordant predictions from the mean of all 3 corneas, OR,x

1 of the 3 corneas gave a discordant prediction from the mean of all 3 corneas, AND thediscordant result was >10 IVIS units from the cut-off threshold of 55.

x

If the repeat testing run corroborates the prediction of the initial testing run (based upon the mean
IVIS value), then a final decision can be taken without further testing. If the repeat testing run
results in a non-concordant prediction from the initial testing run (based upon the mean IVIS
value), then a third and final testing run should be conducted to resolve equivocal predictions,and to classify the test chemical. It may be permissible to waive further testing for classificationand labeling in the event any testing run results in a UN GHS Category 1 prediction.

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Test Report50.

The test report should include the following information, if relevant to the conduct of the study:
Test and Control Substances Chemical name(s) such as the structural name used by the Chemical Abstracts Service(CAS), followed by other names, if known; The CAS Registry Number (RN), if known; Purity and composition of the test/control substance or preparation (in percentage(s) byweight), to the extent this information is available;
 Physicochemical properties such as physical state, volatility, pH, stability, chemical class,water solubility relevant to the conduct of the study;
 Treatment of the test/control substances prior to testing, if applicable (e.g., warming,
grinding);
 Stability, if known.
Information Concerning the Sponsor and the Test Facility
 Name and address of the sponsor, test facility and study director.
Test Method Conditions
 Opacitometer used (e.g., model and specifications) and instrument settings;
 Calibration information for devices used for measuring opacity and permeability (e.g.,
opacitometer and spectrophotometer) to ensure linearity of measurements;
 Type of corneal holders used (e.g., model and specifications); Description of other equipment used; The procedure used to ensure the integrity (i.e., accuracy and reliability) of the test method
over time (e.g., periodic testing of proficiency chemicals).
Criteria for an Acceptable Test Acceptable concurrent positive and negative control ranges based on historical data; If applicable, acceptable concurrent benchmark control ranges based on historical data.

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Eyes Collection and Preparation
 Identification of the source of the eyes (i.e., the facility from which they were collected); Corneal diameter as a measure of age of the source animal and suitability for the assay;
 Storage and transport conditions of eyes (e.g., date and time of eye collection, time interval
prior to initiating testing, transport media and temperature conditions, any antibiotics used); Preparation & mounting of the bovine corneas including statements regarding their quality,
temperature of corneal holders, and criteria for selection of corneas used for testing.
Test Procedure Number of replicates used; Identity of the negative and positive controls used (if applicable, also the solvent andbenchmark controls);
 Test chemical concentration(s), application, exposure time and post-exposure incubation timeused;
 Description of evaluation and decision criteria used; Description of study acceptance criteria used; Description of any modifications of the test procedure; Description of decision criteria used.
Results
 Tabulation of data from individual test samples (e.g., opacity and OD490 values andcalculated IVIS for the test chemical and the positive, negative, and benchmark controls [ifincluded], reported in tabular form, including data from replicate repeat experiments asappropriate, and means ± the standard deviation for each experiment); Description of other effects observed;
 The derived in vitro UN GHS classification, if applicable.
Discussion of the Results
Conclusion

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LITERATURE
1. ICCVAM (2006). Test Method Evaluation Report - In Vitro Ocular Toxicity Test Methods for
Identifying Ocular Severe Irritants and Corrosives. Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). NIH
Publication
No.:
07-4517.
Available:[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm].
2. ICCVAM (2010). ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test
Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products. NIH
Publication No.10-7553. Research Triangle Park, NC:National Institute of Environmental Health
Sciences. Available: [http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm].
3. OECD (2013). Streamlined Summary Document supporting the Test Guideline 437 for eye
irritation/corrosion. Series on Testing and Assessment, No.189, OECD, Paris.
4. UN (2011). United Nations Globally Harmonized System of Classification and Labelling of Chemicals(GHS), ST/SG/AC.10/30 Rev 4, New York and Geneva: United Nations. Available:[http://www.unece.org/trans/danger/publi/ghs/ghs_rev04/04files_e.html].
5. Scott, L., Eskes, C., Hoffmann, S., Adriaens, E., Alépée, N., Bufo, M., Clothier, R., Facchini, D., Faller,C., Guest, R., Harbell, J., Hartung, T., Kamp, H., Le Varlet, B., Meloni, M., McNamee, P., Osborne, R.,
Pape, W., Pfannenbecker, U., Prinsen, M., Seaman, C., Spielman, H., Stokes, W., Trouba, K., Van den
Berghe, C., Van Goethem, F., Vassallo, M., Vinardell, P., and Zuang, V. (2010). A proposed eye
irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down
approaches. Toxicol. in Vitro 24:1-9.
6. ICCVAM (2006). ICCVAM Recommended BCOP Test Method Protocol. In: ICCVAM Test Method
Evaluation Report - In Vitro Ocular Toxicity Test Methods for Identifying Ocular Severe Irritants andCorrosives. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM)
and the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological
Methods(NICEATM).
NIH
Publication
No.:
07-4517.
Available:[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm].

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7. ICCVAM (2010). ICCVAM Recommended BCOP Test Method Protocol. In: ICCVAM Test Method
Evaluation Report – Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye
Injury Hazard Potential of Chemicals and Products. Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and the National Toxicology Program (NTP)
Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM). NIH
Publication No.: 10-7553A. Available:[http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm].
8. INVITTOX (1999). Protocol 124: Bovine Corneal Opacity and Permeability Assay – SOP of
Microbiological Associates Ltd. Ispra, Italy: European Centre for the Validation of Alternative Methods(ECVAM).
9. Gautheron, P., Dukic, M., Alix, D. and Sina, J.F. (1992). Bovine corneal opacity and permeability
test: An in vitro assay of ocular irritancy. Fundam. Appl. Toxicol. 18:442-449.
10. Prinsen, M.K. (2006). The Draize Eye Test and in vitro alternatives; a left-handed marriage? Toxicol. in
Vitro 20:78-81.
11. Siegel, J.D., Rhinehart, E., Jackson, M., Chiarello, L., and the Healthcare Infection Control Practices
Advisory Committee (2007). Guideline for Isolation Precautions: Preventing Transmission of
Infectious Agents in Healthcare Settings. Available: [http://www.cdc.gov/ncidod/dhqp/pdf].
12. Maurer, J.K., Parker, R.D. and Jester, J.V. (2002). Extent of corneal injury as the mechanistic basis forocular irritation: key findings and recommendations for the development of alternative assays. Reg.
Tox. Pharmacol. 36:106-117.
13. OECD (2011). Guidance Document on The Bovine Corneal Opacity and Permeability (BCOP) and
Isolated Chicken Eye (ICE) Test Methods: Collection of Tissues for Histological Evaluation andCollection of Data on Non-severe Irritants. Series on Testing and Assessment, No. 160. Adopted
October 25, 2011. Paris:Organisation for Economic Co-operation and Development.
14. Doughty, M.J., Petrou, S. and Macmillan, H. (1995). Anatomy and morphology of the cornea ofbovine eyes from a slaughterhouse. Can. J. Zool. 73:2159-2165.
15. Collee, J. and Bradley, R. (1997). BSE: A decade on - Part I. The Lancet 349: 636-641.
16. Sina, J.F., Galer, D.M., Sussman, R.S., Gautheron, P.D., Sargent, E.V., Leong, B., Shah, P.V., Curren,
R.D., and Miller, K. (1995). A collaborative evaluation of seven alternatives to the Draize eye irritation
test using pharmaceutical intermediates. Fundam. Appl. Toxicol. 26:20-31.
17. OECD (2002). Test Guideline 405. OECD Guideline for Testing of Chemicals. Acute eyeirritation/corrosion. Available:
15© OECD, (2013)29

437 OECD/OCDE[http://www.oecd.org/document/40/0,2340,en_2649_34377_37051368_1_1_1_1,00.html].
18. ICCVAM (2006). Current Status of In Vitro Test Methods for Identifying Ocular Corrosives andSevere Irritants: Bovine Corneal Opacity and Permeability Test Method. NIH Publication No.: 064512. Research Triangle Park: National Toxicology Program. Available:[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_bcop.htm].
19. OECD (1998). Series on Good Laboratory Practice and Compliance Monitoring. No. 1 : OECD
Principles on Good Laboratory Practice (revised in 1997). ENV/MC/CHEM(98)17. Available :[http://www.oecd.org/document/63/0,3343,en_2649_34381_2346175_1_1_1_1,00.html]

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ANNEX 1DEFINITIONS
Accuracy: The closeness of agreement between test method results and accepted reference values. It is ameasure of test method performance and one aspect of “relevance.” The term is often used interchangeably
with “concordance”, to mean the proportion of correct outcomes of a test method.
Benchmark substance: A substance used as a standard for comparison to a test chemical. A benchmark
substance should have the following properties; (i) a consistent and reliable source(s); (ii) structural andfunctional similarity to the class of substances being tested; (iii) known physical/chemical characteristics;(iv) supporting data on known effects, and (v) known potency in the range of the desired response.
Bottom-Up Approach: step-wise approach used for a chemical suspected of not requiring classificationfor eye irritation or serious eye damage, which starts with the determination of chemicals not requiringclassification (negative outcome) from other chemicals (positive outcome).
Cornea: The transparent part of the front of the eyeball that covers the iris and pupil and admits light tothe interior.
Corneal opacity: Measurement of the extent of opaqueness of the cornea following exposure to a test
chemical. Increased corneal opacity is indicative of damage to the cornea. Opacity can be evaluatedsubjectively as done in the Draize rabbit eye test, or objectively with an instrument such as an“opacitometer.”Corneal permeability: Quantitative measurement of damage to the corneal epithelium by a determinationof the amount of sodium fluorescein dye that passes through all corneal cell layers.
Eye irritation: Production of changes in the eye following the application of a test chemical to the anterior
surface of the eye, which are fully reversible within 21 days of application. Interchangeable with “Reversible
effects on the eye” and with “UN GHS Category 2” (4).
False negative rate: The proportion of all positive substances falsely identified by a test method as
negative. It is one indicator of test method performance.
False positive rate: The proportion of all negative substances that are falsely identified by a test method as
positive. It is one indicator of test method performance.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects when an
organism, system or (sub) population is exposed to that agent.
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437 OECD/OCDEIn Vitro Irritancy Score (IVIS): An empirically-derived formula used in the BCOP test method whereby
the mean opacity and mean permeability values for each treatment group are combined into a single in vitro
score for each treatment group. The IVIS = mean opacity value + (15 x mean permeability value).
Irreversible effects on the eye: See “Serious eye damage”.
Mixture: A mixture or a solution composed of two or more substances in which they do not react (4)Negative control: An untreated replicate containing all components of a test system. This sample isprocessed with test chemical-treated samples and other control samples to determine whether the solvent
interacts with the test system.
Not Classified: Chemicals that are not classified for Eye irritation (UN GHS Category 2, 2A, or 2B) or
Serious eye damage (UN GHS Category 1). Interchangeable with “UN GHS No Category”.
Opacitometer: An instrument used to measure “corneal opacity” by quantitatively evaluating lighttransmission through the cornea. The typical instrument has two compartments, each with its own lightsource and photocell. One compartment is used for the treated cornea, while the other is used to calibrate
and zero the instrument. Light from a halogen lamp is sent through a control compartment (empty chamber
without windows or liquid) to a photocell and compared to the light sent through the experimental
compartment, which houses the chamber containing the cornea, to a photocell. The difference in lighttransmission from the photocells is compared and a numeric opacity value is presented on a digital display.
Positive control: A replicate containing all components of a test system and treated with ac h e m i c a l known to induce a positive response. To ensure that variability in the positive control response
across time can be assessed, the magnitude of the positive response should not be excessive.
Reversible effects on the eye: See “Eye irritation”.
Reliability: Measures of the extent that a test method can be performed reproducibly within and between
laboratories over time, when performed using the same protocol. It is assessed by calculating intra- andinter-laboratory reproducibility and intra-laboratory repeatability.
Serious eye damage: Production of tissue damage in the eye, or serious physical decay of vision, following
application of a test chemical to the anterior surface of the eye, which is not fully reversible within 21 days ofapplication. Interchangeable with “Irreversible effects on the eye” and with “UN GHS Category 1” (4).
Solvent/vehicle control: An untreated sample containing all components of a test system, including thesolvent or vehicle that is processed with the test chemi cal -treated sampl es and other control samples
to establish the baseline response for the samples treated with the test c h e m i c a l dissolved in the18© OECD, (2013)32

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same solvent or vehicle. When tested with a concurrent negative control, this sample also demonstrates
whether the solvent or vehicle interacts with the test system.
Substance: Chemical elements and their compounds in the natural state or obtained by any production
process, including any additive necessary to preserve the stability of the product and any impurities derivingfrom the process used, but excluding any solvent which may be separated without affecting the stability ofthe substance or changing its composition (4).
Surfactant: Also called surface-active agent, this is a substance, such as a detergent, that can reduce thesurface tension of a liquid and thus allow it to foam or penetrate solids; it is also known as a wetting agent.
Surfactant-containing mixture: In the context of this Test Guideline, it is a mixture containing one ormore surfactants at a final concentration of > 5%.
Top-Down Approach: step-wise approach used for a chemical suspected of causing serious eye damage,which starts with the determination of chemicals inducing serious eye damage (positive outcome) fromother chemicals (negative outcome).
Test chemical: Chemical (substance or mixture) assessed in the test method.
Tiered testing strategy: A stepwise testing strategy where all existing information on a test chemical 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 chemical can be assigned based on the existing information, no additional
testing is required. If the irritancy potential of a test chemical cannot be assigned based on the existing
information, a step-wise sequential animal testing procedure is performed until an unequivocal classification
can be made.
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS):
A system proposing the classification of chemicals (substances and mixtures) according to standardized
types and levels of physical, health and environmental hazards, and addressing corresponding
communication elements, such as pictograms, signal words, hazard statements, precautionary statements
and safety data sheets, so that to convey information on their adverse effects with a view to protect people(including employers, workers, transporters, consumers and emergency responders) and the environment(4).
UN GHS Category 1: See “Serious eye damage”.
UN GHS Category 2: See “Eye irritation”.
UN GHS No Category: Chemicals that do not meet the requirements for classification as UN GHS
Category 1 or 2 (2A or 2B). Interchangeable with “Not Classified”.
Validated test method: A test method for which validation studies have been completed to determine the19© OECD, (2013)33

437 OECD/OCDE
relevance (including accuracy) and reliability for a specific purpose. It is important to note that a validated
test method may not have sufficient performance in terms of accuracy and reliability to be found acceptable
for the proposed purpose.
Weight-of-evidence: The process of considering the strengths and weaknesses of various pieces of
information in reaching and supporting a conclusion concerning the hazard potential of a test chemical.

20© OECD, (2013)34

ENV/JM/WRPR(2013)23

ANNEX 2

PREDICTIVE CAPACITY OF THE BCOP TEST METHOD
Table 1: Predictive Capacity of BCOP for identifying chemicals inducing serious eye damage [UNGHS/ EU CLP Cat 1 vs Not Cat 1 (Cat 2 + No Cat); US EPA Cat I vs Not Cat I (Cat II + Cat III + Cat
IV)]Classification
SystemNo.

Accuracy

Sensitivity

False
Negatives

Specificity

False
Positives

%

%

%

No.

%

%

No.

No.

No.

No.

UN GHS
EU CLP

191

78.53 150/191 86.15 56/65 13.85

9/65

74.60 94/126 25.40 32/126

US EPA190

78.95 150/190 85.71 54/63 14.29

9/63

75.59 96/127 24.41 31/127

Table 2: Predictive Capacity of BCOP for identifying chemicals not requiring classification for eye
irritation or serious eye damage (“non-irritants”) [UN GHS/ EU CLP No Cat vs Not No Cat (Cat 1 +Cat 2); US EPA Cat IV vs Not Cat IV (Cat I + Cat II + Cat III)]Classification
SystemNo.

Accuracy

Sensitivity

False
Negatives

Specificity

False
Positives

%

%

No.

%

No.

%

%

100

31.46 28/89 68.54 61/89

No.

No.

No.

UN GHS
EU CLP

196

68.88 135/196

107/1070

0/107

US EPA

190

82.11 156/190 93.15 136/146

6.85

10/146 45.45 20/44 54.55 24/44

21© OECD, (2013)35

ENV/JM/WRPR(2013)23

ANNEX 3

PROFICIENCY CHEMICALS FOR THE BCOP TEST METHOD
Prior to routine use of a test method that adheres to this Test Guideline, laboratories should demonstratetechnical proficiency by correctly identifying the eye hazard classification of the 13 substances
recommended in Table 1. These substances were selected to represent the range of responses for eye
hazards based on results in the in vivo rabbit eye test ( T G 4 0 5 ) (17) a n d the U N GHS
classification system (i.e., Categories 1, 2A, 2B, or Not Classified) (4). Other selection criteria werethat substances are commercially available, that there are high quality in vivo reference data available, andthat there are high quality in vitro data available from the BCOP test method. Reference data are available
in the Streamlined Summary Document (3) and in the ICCVAM Background Review Document for the
BCOP test method (2)(18).

22© OECD, (2013)

36

437

OECD/OCDE

Table 1: Recommended substances for demonstrating technical proficiency with the BCOP Test
MethodChemicalBenzalkonium
(5%)

CASRN

8001-54-5
55-56-1

Amine,
AmidineSolid

Category 1

2743-38-6

Carboxylicacid, Ester

Solid

Category 1

Solid

Category 1

LiquidCategory 1

chloride

Chlorhexidine
Dibenzoyl-L- tartaric acid
Imidazole
Trichloroacetic acid (30%)
2,6-Dichlorobenzoyl
chloride
Ethyl-2-methylacetoacetate
Ammonium nitrate
EDTA, di-potassium salt
Tween 20In Vivo

ChemicalClass1
Onium
compound288-32-4
76-03-9Heterocyclic
Carboxylic acid

Physical
FormClassification2

LiquidCategory 1

4659-45-4

Acyl halide

LiquidCategory 2A

609-14-3

Ketone, Ester

LiquidCategory 2B

Solid

Category 23

Solid

Not Classified

LiquidNot Classified

6484-52-2

Inorganic salt

Amine,
25102-12-9 Carboxylic acid(salt)
9005-64-5

Ester, Polyether

BCOP
Classification
Category 1
Category 1
Category 1
Category 1
Category 1Noaccurate/reliablepredictionNo
accurate/reliablepredictionNo
accurate/reliable
di i
Not Classified
Not Classified

2-Mercaptopyrimidine

1450-85-7

Acyl halide

Solid

Not Classified

Not Classified

Phenylbutazone

50-33-9Heterocyclic

Solid

Not Classified

Not Classified

AlcoholLiquidNot Classified

Not Classified

Polyoxyethylene 23 lauryl
9002-92-0
ether (BRIJ-35) (10%)

Abbreviations: CASRN = Chemical Abstracts Service Registry Number
1Chemical classes were assigned to each test chemical using a standard classification scheme, basedon the National Library of Medicine Medical Subject Headings (MeSH) classification system(available at http//www.nlm.nih.gov/mesh).
2Based on results from the in vivo rabbit eye test (OECD TG 405) (17) and using the UN GHS (4).
© OECD, (2013)2337

437 OECD/OCDE
3Classification as 2A or 2B depends on the interpretation of the UN GHS criterion for distinguishing
between these two categories, i.e. 1 out of 3 vs 2 out of 3 animals with effects at day 7 necessary to
generate a Category 2A classification. The in vivo study included 3 animals. All endpoints apart fromconjunctiva redness in one animal recovered to a score of zero by day 7 or earlier. The one animal that
did not fully recover by day 7 had a conjunctiva redness score of 1 (at day 7) that fully recovered at day
10.

24© OECD, (2013)38

OECD/OCDE

437

ANNEX 4

THE BCOP CORNEAL HOLDER
1.
The BCOP corneal holders are made of an inert material (e.g., polypropylene). The holders
are comprised of two halves (an anterior and posterior chamber), and have two similar cylindricalinternal chambers. Each chamber is designed to hold a volume of about 5 mL and terminates in a
glass window, through which opacity measurements are recorded. Each of the inner chambers is
1

1.7 cm in diameter and 2.2 cm in depth . An o-ring located on the posterior chamber is used toprevent leaks. The corneas are placed endothelial side down on the o-ring of the posterior chambers
and the anterior chambers are placed on the epithelial side of the corneas. The chambers are maintainedin place by three stainless steel screws located on the outer edges of the chamber. The end of eachchamber houses a glass window, which can be removed for easy access to the cornea. An o-ring is
also located between the glass window and the chamber to prevent leaks. Two holes on the top ofeach chamber permit introduction and removal of medium and test compounds. They are closed withrubber caps during the treatment and incubation periods. The light transmission through corneal
holders can potentially change as the effects of wear and tear or accumulation of specific chemical
residues on the internal chamber bores or on the glass windows may affect light scatter or reflectance.
The consequence could be increases or decreases in baseline light transmission (and conversely thebaseline opacity readings) through the corneal holders, and may be evident as notable changes in the
expected baseline initial corneal opacity measurements in individual chambers (i.e., the initial corneal
opacity values in specific individual corneal holders may routinely differ by more than 2 or 3 opacity
units from the expected baseline values). Each laboratory should consider establishing a program forevaluating for changes in the light transmission through the corneal holders, depending upon the natureof the chemistries tested and the frequency of use of the chambers. To establish baseline values, corneal
holders may be checked before routine use by measuring the baseline opacity values (or lighttransmission) of chambers filled with complete medium, without corneas. The corneal holders are thenperiodically checked for changes in light transmission during periods of use. Each laboratory canestablish the frequency for checking the corneal holders, based upon the chemistries tested, thefrequency of use, and observations of changes in the baseline corneal opacity values. If notable changes
in the light transmission through the corneal holders are observed, appropriate cleaning and/or
polishing procedures of the interior surface of the cornea holders or replacement have to be considered.

© OECD, (2013)25

39

437 OECD/OCDE

1The dimensions provided are based on a corneal holder that is used for cows ranging in age from12 to 60 months old. In the event that animals 6 to 12 months are being used, the holder would
instead need to be designed such that each chamber holds a volume of 4 mL, and each of the innerchambers is 1.5 cm in diameter and 2.2 cm in depth. With any newly designed corneal holder, it isvery important that the ratio of exposed corneal surface area to posterior chamber volume shouldbe the same as the ratio in the traditional corneal holder. This is necessary to assure thatpermeability values are correctly determined for the calculation of the IVIS by the proposed formula.

26© OECD, (2013)40

OECD/OCDE

437

ANNEX 5

THE OPACITOMETER2.
The opacitometer is a light transmission measuring device. For example, for the OP-KIT
equipment from Electro Design (Riom, France) used in the validation of the BCOP test method, lightfrom a halogen lamp is sent through a control compartment (empty chamber without windows or liquid)
to a photocell and compared to the light sent through the experimental compartment, which houses thechamber containing the cornea, to a photocell. The difference in light transmission from the photocells is
compared and a numeric opacity value is presented on a digital display. The opacity units are established.
Other types of opacitometers with a different setup (e.g., not requiring the parallel measurements of thecontrol and experimental compartments) may be used if proven to give similar results to the validated
equipment.
3.
The opacitometer should provide a linear response through a range of opacity readings
covering the cut-offs used for the different classifications described by the Prediction Model (i.e., up to
the cut-off determining corrosiveness/severe irritancy). To ensure linear and accurate readings up to 7580 opacity units, it is necessary to calibrate the opacitometer using a series of calibrators. Calibrators areplaced into the calibration chamber (a corneal chamber designed to hold the calibrators) and read on theopacitometer. The calibration chamber is designed to hold the calibrators at approximately the same
distance between the light and photocell that the corneas would be placed during the opacity
measurements. Reference values and initial set point depend on the type of equipment used. Linearityof opacity measurements should be ensured by appropriate (instrument specific) procedures. For
example, for the OP-KIT equipment from Electro Design (Riom, France), the opacitometer is firstcalibrated to 0 opacity units using the calibration chamber without a calibrator. Three different
calibrators are then placed into the calibration chamber one by one and the opacities are measured.
Calibrators 1, 2 and 3 should result in opacity readings equal to their set values of 75, 150, and225 opacity units, respectively, ±5%.

© OECD, (2013)

27
41

42

Unclassified

ENV/JM/MONO(2013)13

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

21-Jun-2013___________________________________________________________________________________________English - Or. English

ENVIRONMENT DIRECTORATEENV/JM/MONO(2013)13
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYSTREAMLINED SUMMARY DOCUMENT SUPPORTING OECD GUIDELINE 437 ON THE BOVINECORNEAL OPACITY AND PERMEABILITY FOR EYE IRRITATION/CORROSION
Series on Testing and Assessment
No. 189

English - Or. English

JT03342251
Complete document available on OLIS in its original format
This document and any map included herein are without prejudice to the status of or sovereignty over any territory, to the delimitation ofinternational frontiers and boundaries and to the name of any territory, city or area.

43ENV/JM/MONO(2013)13

2

44

ENV/JM/MONO(2013)13
OECD Environment, Health and Safety Publications
Series on Testing and Assessment

No. 189

STREAMLINED SUMMARY DOCUMENT SUPPORTING OECD GUIDELINE 437 ON THE
BOVINE CORNEAL OPACITY AND PERMEABILITY FOR EYE IRRITATION/CORROSION

Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT
Paris 2013

345

ENV/JM/MONO(2013)13
ABOUT THE OECDThe Organisation for Economic Co-operation and Development (OECD) is an intergovernmentalorganisation in which representatives of 34 industrialised countries in North and South America, Europe
and the Asia and Pacific region, as well as the European Commission, meet to co-ordinate and harmonise
policies, discuss issues of mutual concern, and work together to respond to international problems. Most of
the OECD’s work is carried out by more than 200 specialised committees and working groups composedof member country delegates. Observers from several countries with special status at the OECD, and frominterested international organisations, attend many of the OECD’s workshops and other meetings.
Committees and working groups are served by the OECD Secretariat, located in Paris, France, which isorganised into directorates and divisions.
The Environment, Health and Safety Division publishes free-of-charge documents in 11 different series:
Testing and Assessment; Good Laboratory Practice and Compliance Monitoring; Pesticides;
Biocides; Risk Management; Harmonisation of Regulatory Oversight in Biotechnology; Safety ofNovel Foods and Feeds; Chemical Accidents; Pollutant Release and Transfer Registers; Emission
Scenario Documents; and Safety of Manufactured Nanomaterials. More information about theEnvironment, Health and Safety Programme and EHS publications is available on the OECD’s World
Wide Web site (www.oecd.org/ehs/).

This publication was developed in the IOMC context. The contents do not necessarily reflect the views orstated policies of individual IOMC Participating Organisations.
The Inter-Organisation Programme for the Sound Management of Chemicals (IOMC) was established in
1995 following recommendations made by the 1992 UN Conference on Environment and Development tostrengthen co-operation and increase international co-ordination in the field of chemical safety. The
Participating Organisations are FAO, ILO, UNDP, UNEP, UNIDO, UNITAR, WHO, World Bank andOECD. The purpose of the IOMC is to promote co-ordination of the policies and activities pursued by theParticipating Organisations, jointly or separately, to achieve the sound management of chemicals in
relation to human health and the environment.

4

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ENV/JM/MONO(2013)13
This publication is available electronically, at no charge.
Also published in the Series on Testing and Assessment link
For this and many other Environment,
Health and Safety publications, consult the OECD’s
World Wide Web site (www.oecd.org/ehs/)
or contact:
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E-mail: ehscont@oecd.org© OECD 2013
Applications for permission to reproduce or translate all or part of this material
should be made to: Head of Publications Service, RIGHTS@oecd.org, OECD,
2 rue André-Pascal, 75775 Paris Cedex 16, France

547

ENV/JM/MONO(2013)13
FOREWORD

This streamlined summary document (SSD) was developed to provide summary information in
support of OECD Test Guideline 437 on the Bovine Corneal Opacity and Permeability Test Methodaddressing the endpoint eye irritation/corrosion. This SSD was developed by a Secretariat consultant
and submitted to the Working Group of the National Coordinators of the Test Guidelines Programme(WNT) in March 2013, together with the updated version of TG 437 (originally adopted in 2009). The
SSD provides useful and more detailed information than is otherwise available from the Test
Guideline itself on: 1) the scientific basis of the test method, 2) the identified limitations, weaknesses
and strengths, 3) the applicability domain, 4) the sensitivity, specificity and accuracy, and 5) thewithin- and between-laboratory reproducibility of the method.
The SSD was approved by the WNT with a few changes to paragraph 12, including additionalreferences 23, 24, 25 and 26, on 30 April 2013.
The Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides
and Biotechnology agreed to its declassification on 14 June, 2013.
This document is published under the responsibility of the Joint Meeting of the Chemicals
committee and the Working Party on Chemicals, Pesticides and Biotechnology.

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ENV/JM/MONO(2013)13
Description of applicability domain and performance based on the retrospective validation
studies of the Bovine Corneal Opacity and Permeability (BCOP) Test Method (Test Guideline437) for identifying i) chemicals inducing serious eye damage and ii) chemicals not requiringclassification for eye irritation or serious eye damage

Introduction and BackgroundThe 2003-2006 Validation Studies
1.
Between 2003 and 2006, a retrospective evaluation was carried out concerning the validation
status of the Bovine Corneal Opacity and Permeability (BCOP) test method for identifying chemicals(substances and mixtures) inducing serious eye damage (“ocular corrosives and severe irritants”), i.e.,
its usefulness and limitations for initiating a Top-Down approach (1). This evaluation, counting with a
total of 161 chemicals, was performed by the US-Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM) and the US-National Toxicology Program Interagency
Center for the Evaluation of Alternative Toxicological Methods (NICEATM), in collaboration with
the EU-European Centre for the Validation of Alternative Methods (ECVAM) and the JapaneseCenter for the Validation of Alternative Methods (JaCVAM). For a full description, see the
Background Review Document (BRD) (2) and the ICCVAM Test Method Evaluation Report (TMER)(3).
2.
The study aimed at characterising the reproducibility and predictive capacity of the BCOP
for the following classification systems: UN GHS (Category 1) (4), US EPA (Category I) (5) and EU
DSD (R41) (6) (the EU CLP classification system (7) based on UN GHS had not yet been adopted in
the EU at that time). On the basis of all the collected data and information the BCOP was considered
as scientifically valid (reliable and relevant) for identifying chemicals inducing serious eye damage(i.e., to initiate a Top-Down approach (1)) and was recommended for regulatory hazard classificationand labelling purposes. Chemicals inducing serious eye damage are defined as those that produce
tissue damage in the eye, or serious physical decay of vision, following application to the anterior
surface of the eye in the in vivo Draize rabbit eye test (Test Guideline 405) (8), which is not (or notexpected to be) fully reversible within 21 days of application. Following these recommendations, theOECD officially adopted the BCOP as OECD Test Guideline (TG) 437 for identifying chemicals
inducing serious eye damage in September 2009 (9).
The 2006-2009 Validation Studies
3.
Following the 2003-2006 retrospective validation study NICEATM/ICCVAM, incollaboration with ECVAM and JaCVAM, further evaluated between 2006 and 2009 the usefulness
and limitations of the BCOP test method for the additional identification of chemicals (substances andmixtures) not causing sufficient effects on the eye to require hazard classification and labelling
according to the UN GHS (4), US EPA (5), US FHSA and EU DSD (6) classification systems (the EU
CLP classification system (7) based on UN GHS had not been adopted in the EU at that time), i.e., its
usefulness and limitations for initiating a Bottom-Up approach (1).
4.
The updated BCOP validation database comprises 238 studies on 213 chemicals (107substances and 106 mixtures) (UN GHS / EU CLP: 65 Cat 1, 32 Cat 2A, 3 Cat 2B, 1 Cat 2, 6 Cat
1/Cat 2, 1 No Cat/Cat 2A, 89 No Cat, 5 SCNM, 11 without in vivo data; US EPA: 63 Cat I, 23 Cat II,
1 Cat II/Cat III, 59 Cat III, 44 Cat IV, 13 SCNM, 11 without in vivo data), collected from sevenindividual studies (Gautheron et al. 1994 (10), Balls et al. 1995 (11), Swanson et al. 1995 (12),Southee 1998 (13), Swanson and Harbell 2000 (14), Bailey et al. 2004 (15), Antimicrobial Cleaning
Products (AMCP) BRD (16)), which were used to determine the predictive capacity of the BCOP test
method (for a comprehensive list see Annex 1). Another 54 mixtures and 13 surfactant substances
reported in Casterton et al. 1996 (17), Gettings et al. 1996 (18), Swanson and Harbell 2000 (14) and in
749

ENV/JM/MONO(2013)13
the AMCP BRD (16) were considered by NICEATM-ICCVAM in the retrospective validation of
BCOP, but were not used to calculate the predictive capacity of the test method due to incomplete invitro and/or in vivo data (2) (3) (19). The components of each of the mixtures tested in the BCOP test
method can be found in Annexes 2-5 (copied from reference 19).
5.
In May 2009, NICEATM/ICCVAM convened a public meeting of an independentinternational scientific peer review panel (PRP) on alternative ocular safety testing methods,
composed of members from several EU Member States, USA, Japan and Canada. The PRP wascharged with reviewing the data compiled in view of evaluating the validation status of the BCOP test
method for identifying chemicals not classified for eye irritation or serious eye damage (UN GHS No
Category; EU DSD Not Classified; US EPA Category IV). The PRP concluded that the usefulness of
the BCOP for the identification of chemicals not classified for eye irritation or serious eye damage
depended on the intended purpose (i.e., the classification system) and that the BCOP can be used forthe identification of chemicals not classified for eye irritation or serious eye damage (i.e., to initiate a
Bottom-Up approach (1)) under the UN GHS and EU DSD classification systems for which no false
negatives were identified. Since the BCOP showed 6% false negatives for the US EPA classification
system, the PRP did not recommend the BCOP for this classification system (20). The final ICCVAM
conclusions and recommendations on the use BCOP to identify chemicals not requiring classificationfor eye irritation or serious eye damage were published in a Test Method Evaluation Report (TMER),
to which, amongst other documents, a revised BRD and the PRP Report were annexed (19).
BCOP Issue Paper Supporting the Adoption of TG 437 for the Identification of Chemicals Not
Classified for Eye Irritation or Serious Eye Damage
6.
In April 2011, following the conclusions of the 2006-2009 retrospective validation study, anOECD project for updating TG 437 (9) was included in the work plan of the OECD Test Guidelines
Programme. The aim of the project was to address a possible update of TG 437 to allow its use also
for the identification of chemicals not requiring classification for eye irritation or serious eye damage
under UN GHS, EU CLP and EU DSD classification systems. An Issue Paper was prepared by aconsultant to the OECD Secretariat with the aim to review existing BCOP data and make a
recommendation on the use of TG 437 for the purpose described above (Annex 6). This document wastabled for discussion by the eye irritation/corrosion expert group at an OECD expert meeting that washeld on 6-7 December 2012. The BCOP Issue Paper reviewed the BCOP data presented in the
ICCVAM BCOP BRD (19) as well as other newly published data in connection with the use of BCOP
to identify chemicals inducing serious eye damage as well as chemicals not requiring classification foreye irritation or serious eye damage. The BCOP Issue Paper further attempted to analyse the added
value of BCOP to identify chemicals inducing serious eye damage as well as chemicals not requiringclassification for eye irritation or serious eye damage, when used together with other test methods in a
testing strategy. All information regarding the predictive capacity of BCOP included in this SSD isbased on the data reviewed in the BCOP Issue Paper and on the analysis presented in that paper.
7.
In 2013, the OECD officially adopted an updated version of TG 437 allowing the use of
BCOP for identifying chemicals inducing serious eye damage as well as for identifying chemicals not
requiring classification for eye irritation or serious eye damage.
Scientific Basis for the BCOP Test Method
8.
The BCOP test method (TG 437) is an organotypic ex vivo assay that uses living bovine
corneas isolated from the eyes of freshly slaughtered animals killed for human consumption. In thistest method, the irritation potential of a test substance is assessed by quantitative measurements ofchanges in opacity and permeability of the isolated exposed corneas, using an opacitometer and avisible light spectrophotometer, respectively (9). Both permeability and opacity are used to calculate
an in vitro irritancy score (IVIS), which is used to assign an in vitro irritancy hazard classification forprediction of the in vivo ocular irritation potential of a test substance.

50

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ENV/JM/MONO(2013)13
9.
The prediction model used by NICEATM/ICCVAM in their evaluations of BCOP predictive
capacity was based on the prediction model originally proposed by Gautheron et al. (1994) (10) andlater modified by Balls et al. (1995) (11) and Southee (1998) (13). This prediction model allows forthe categorisation of chemicals to four regulatory irritant categories, with cut-offs at IVIS values of 3,
25 and 55. In OECD TG 437 updated in 2013, an IIVS  3 is used to identify a chemical as not
requiring classification for eye irritation or serious eye damage (UN GHS No Category), an IVIS > 55
is used to identify a chemical as inducing serious eye damage (UN GHS Category 1), and no
prediction can be made if an 3 < IVIS  55 is obtained. Finally, it has also been recommended thatchemicals producing permeability values equal or greater than 0.6 be identified as inducing serious
eye damage (UN GHS Category 1). This may be especially important for chemicals that do not
produce appreciable opacity in the isolated bovine cornea, but that can damage the epithelium andincrease permeability, e.g., surfactant-based personal care formulations (2) (3).
Comparison of BCOP Test Method with the In Vivo Rabbit Eye Test Method
10.
In the BCOP test method, damage to the isolated cornea is assessed by measuring corneal
opacity and permeability in a short-term test that typically takes less than 8 hours to perform. The two
endpoints are measured quantitatively with an opacitometer and an ultraviolet/visible (UV/VIS)
spectrophotometer, respectively, after ten minutes exposure plus two hours post-exposure incubation
for liquid and surfactant chemicals or after four hours of exposure to solid chemicals (2).
11.
Depending on the physicochemical properties of the test substance, post-exposure
measurements may be extended to 24 hours (e.g., for chemicals with delayed responses). In contrast,
the in vivo rabbit eye test involves a qualitative visual evaluation of the severity of adverse effects on
the cornea, the iris, and the conjunctiva, as well as the reversibility of any ocular effects detected atselected intervals up to 21 days after exposure. In BCOP, liquids, semi-solids, creams and waxes areusually applied undiluted for 10 minutes, then rinsed off the cornea, followed by a 2-hour postexposure incubation of the cornea in assay medium. Neat surfactant substances are typically tested at aconcentration of 10% w/v, while mixtures containing surfactants may be tested undiluted or diluted toan appropriate concentration depending on the relevant exposure scenario in vivo. The same exposure
and post-exposure regime used for liquids is followed for surfactant substances and mixturescontaining surfactants. Solids are usually applied as a suspension or solution (20%) for four hours,
then rinsed off the cornea before opacity and permeability measurements are performed. Postexposure incubation is not required for solids. Whether the test substance is a liquid or a solid, theentire cornea is exposed for a specified duration. In the in vivo rabbit eye test, liquid and solid test
substances are applied to the conjunctival sac, usually in an undiluted form. Because the rabbit eye canblink and/or tear, exposure of the cornea to the test substance will be affected by these factors in termsof coverage or duration. The neurogenic components that drive tear film production are not present in
the BCOP. When compared with an in vivo rabbit eye study, application of a test substance in theabsence of this protective barrier might be expected to cause an increase in false positive outcomes.
One of the conclusions from a workshop on mechanisms of eye irritation highlighted the need for
additional research on the impact of chemicals on tear film and the consequences of tear film
disruption. Protective mechanisms for the eye (e.g., blinking, tear film) are built into in vivo testing,but are absent in in vitro / ex vivo testing. However, note that for some test substances (e.g., solids),
blinking can also induce mechanical damage in vivo, contributing to a higher degree of irritation.
Thus, the BCOP test method differs from the in vivo rabbit eye test method in the following significant
ways (2):•The BCOP evaluates only corneal effects and does not assess effects on the iris and the
conjunctiva as performed in the in vivo rabbit eye test. Measurements are performed
quantitatively in the BCOP assay, while they are assessed with qualitative observations in
the in vivo rabbit eye test.

•

Corneal exposure conditions, including test substance concentration and exposure duration,
are well controlled in the BCOP assay, but subject to potentially greater variation in vivo,
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ENV/JM/MONO(2013)13
due in part to the blink response and natural tearing of the eye in a live animal. Moreover, it
should be noted that solids may lead to variable and extreme exposure conditions in the invivo Draize eye irritation test, which may result in irrelevant predictions of their true
irritation potential (21).
•The observation period of the BCOP assay is typically less than 24 hours, whereas oculareffects are typically evaluated in the in vivo rabbit eye test for a minimum of 72 hours andcan extend up to 21 days.

•

Reversibility/irreversibility of corneal effects induced by a test substance cannot be observedin the BCOP assay per se, but histological evaluation of the exposed cornea may provide
additional information about the depth and type of injury that could aid predictions, as towhether damage is irreversible. It has been proposed, based on rabbit eye studies, that anassessment of the initial depth of corneal injury may be used to identify some types ofirreversible effects (22). However, further scientific knowledge is required to understandhow irreversible effects not linked with initial high level injury occur.

•

Protective mechanisms of the eye, such as tear production and blinking, are built into in vivo
testing, but are absent in in vitro / ex vivo testing.

•

The BCOP assay does not account for systemic effects following ocular instillation that may
be noted with the in vivo rabbit eye test (e.g., toxicity or lethality as in the case of certain
pesticides). However, these effects are typically predicted from other acute toxicity test
methods, and may not be relevant for the many consumer products that are formulated withwell characterized raw materials of known systemic toxicity.

Identified Limitations, Weaknesses and Strengths
12.
The potential shortcomings of the BCOP test method when used to identify chemicals
inducing serious eye damage (UN GHS Category 1) are based on the high false positive rates foralcohols and ketones and the high false negative rate for solids observed in the validation database (2)(3) (19). When substances within these chemical and physical classes are excluded from the database,the accuracy of the BCOP test method is substantially improved (2) (3) (19) (see Table 7 below).
However, since not all alcohols and ketones are over-predicted by the BCOP test method and some arecorrectly predicted as UN GHS Category 1, these two organic functional groups are not considered tobe out of the applicability domain of the test method. Positive results obtained with alcohols or
ketones should nevertheless be interpreted cautiously due to potential over-prediction. Moreover,given the fact that some solids are also correctly predicted by the BCOP test method as UN GHS
Category 1, and that all solids in the validation database that were underpredicted by BCOP showed an
3 < IVIS  55 (i.e., no prediction can be made) and would therefore need to be subsequently tested
with other suitable test method(s) in a sequential testing strategy (8) (Annex 6), this physical state isalso not considered to be out of the applicability domain of the BCOP test method. Evidence suggeststhat there is a certain probability that Cat 1 are predicted as Cat 2 due to the variability of individual
animal responses within the same test (23). Although not based on the same dataset, the resulting
probability seems to be in the same range as the BCOP/ICE under-prediction rate for identifying UN
GHS Category 1. However, variability between laboratories can further contribute to the variability ofin vivo responses (24)(25)(26). Quantitative estimates for such uncertainties, both for the in vivo tests
and for their in vitro alternatives, should be considered in the future development of testing strategies
for serious eye damage/eye irritation.
13.
When used to identify chemicals not requiring classification for eye irritation or serious eye
damage under the UN GHS classification system, the BCOP test method has a considerably high false
positive rate (in vivo UN GHS No Category chemicals producing an IVIS > 3) (19). This is however
not considered critical since all test substances that produce an 3 < IVIS  55 (i.e., no prediction canbe made) would need to be subsequently tested with other suitable test method(s) in a sequential
52

10ENV/JM/MONO(2013)13
testing strategy. The BCOP test method shows no specific shortcomings for the testing of alcohols,
ketones and solids when the purpose is to identify chemicals that do not require classification for eye
irritation or serious eye damage (UN GHS No Category) (see Table 8 below).
14.
Another limitation of the test method is that, although it takes into account some of theocular effects evaluated in the in vivo test method and to some degree their severity, it does notconsider conjunctival and iridal injuries. Nevertheless, the BCOP directly addresses corneal effects,
which are the major driver of classification in vivo when considering the UN GHS classification
system (Annex 6). Also, although the reversibility of corneal lesions cannot be evaluated per se in the
BCOP test method, it has been proposed, based on rabbit eye studies, that an assessment of the initial
depth of corneal injury (e.g., through histological evaluation) may be used to identify some types ofirreversible effects (22). However, further scientific knowledge is required to understand how
irreversible effects not linked with initial high level injury occur. Finally, the BCOP does not allow for
an assessment of the potential for systemic toxicity associated with ocular exposure.
15.
The BCOP test method is not recommended for the identification of test substances thatshould be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test substances thatshould be classified as mildly irritating to eyes (UN GHS Category 2B) due to the considerable
number of UN GHS Category 1 chemicals underclassified as UN GHS Category 2, 2A or 2B and UN
GHS No Category chemicals overclassifed as UN GHS Category 2, 2A or 2B (19). For this purpose,
further testing with another suitable method may be required.

Table 1: Physicochemical properties and compatibility with the BCOP
Is a material with this property
compatible with the BCOP
assay?

Physicochemical property
Fixative

Unknown

Solvent

Yes

Extreme pH

Yes

Gases

No

Liquids

Yes

Solid materials

Yes

Emulsions

Yes

Granular materials

No

Suspensions

Yes

Coloured materials

Yes

Diluted concentrations of chemicals

Yes

Highly viscous materials

Yes

Volatile materials

Yes

Reactive chemistries

Yes1

Hydrophobic/lipophilic chemicals

Yes

Neat concentrations of chemicals

Yes

1

Reactive chemistries may require the use of extended post-treatment incubations, up to 24 hours,
prior to the opacity and permeability endpoints for expression of effects. Histopathology is highly
recommended for such studies.
1153

ENV/JM/MONO(2013)13
Applicability Domain
16.
The Test Guideline can be used for testing all types of substances and mixtures, including
those outside the chemical classes defined by the chemicals used in the validation study, provided
there is no evidence that the method is not valid for the chemical tested.
Categories of Irritancy
17.
Based on the conclusions of the 2003-2006 and 2006-2009 retrospective validation studies(3) (19), TG 437 was approved for classification of chemicals inducing serious eye damage (UN GHS
Category 1) (in 2009), as well as for the identification of chemicals not requiring classification for eye
irritation or serious eye damage (UN GHS No Category), under the UN GHS classification system, in2013.
Potential Role in an ITS
18.
The BCOP can be used as a validated ex vivo test method in a tiered testing approach asrecommended in the supplement to TG 405 (8) with the purpose of identifying chemicals inducing
serious eye damage (UN GHS Category 1), as well as chemicals not requiring classification for eye
irritation or serious eye damage (UN GHS No Category) (Annex 6). Due to its low specificity whenused to identify chemicals not requiring classification for eye irritation or serious eye damage, theBCOP should not be the first choice to initiate a Bottom-Up approach (1), if other validated andaccepted in vitro methods with similar high sensitivity but higher specificity are available.
19.
Two studies have looked at the combination of BCOP with other in vitro test methods intesting strategies for the prediction of ocular hazards. Kolle et al. (2011) (27) combined BCOP with
EpiOcularTM EIT (the latter can only be used to identify UN GHS No Category) in a two-tier Bottomup/Top-Down test strategy and Hayashi et al. (2012) (28) combined BCOP with EpiOcularTM EIT, theShort Time Exposure (STE)1 test method and the Hen’s Egg Test - Chorioallantoic Membrane (HETCAM)2 in a two-stage Bottom-Up tiered approach. In the data published by Kolle et al. (2011), it canbe seen that chemical 10/0229-1, a UN GHS No Category (“non-irritant”) solid organophosphate, isoverpredicted by EpiOcularTM EIT but correctly predicted by BCOP. By using BCOP to identify both
UN GHS Category 1 and UN GHS No Category chemicals, this chemical can be correctly identified
as “non-irritant” using either a Bottom-Up testing strategy (EpiOcularTM EIT first followed by BCOP)
or a Top-Down testing strategy (BCOP first followed by EpiOcularTM EIT) (Annex 6) (27). Hayashi etal. tested 26 UN GHS No Category chemicals from the BCOP validation database in EpiOcularTM
EIT, STE and HET-CAM. Of these, 13 were overpredicted as irritant by EpiOcularTM EIT (50% false
positives). Two of the 13 EpiOcularTM EIT false positives (EDTA di-potassium salt, CAS # 25102-129; triethanolamine, CAS # 102-71-6) are correctly identified by BCOP as not requiring hazard
classification and labelling (UN GHS No Category). HET-CAM also correctly identifies two other1

The STE has undergone a full validation study in Japan, coordinated by JaCVAM, and an international peerreview of the study is currently being organised by ICCVAM. The STE was also submitted by Japanto the OECD for the development of a new Test Guideline, and was included in the OECD work planfor the Test Guidelines Programme in 2011.

2

The HET-CAM was evaluated by ICCVAM together with BCOP and ICE in the retrospective validation studiesof 2003-2006 (3) and 2006-2009 (19). ICCVAM considered that the HET-CAM might be useful for
identifying chemicals not requiring hazard classification and labeling for eye irritation but did notrecommend the test method due to lack of data in the mild/moderate irritancy range. ICCVAM
recommended that additional data be collected on mild and moderate irritants to more adequately
characterise the usefulness of HET-CAM. Following this recommendation, an international workshop
on HET-CAM was recently held at the BfR in Berlin to help advance regulatory acceptance of the test
method.

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ENV/JM/MONO(2013)13
EpiOcularTM EIT false positives as “non-irritant” (1-nitropropane, CAS # 108-03-2; 1-phenyl-3pyrazolidinone, CAS # 92-43-3), but in this case at the expense of 1 UN GHS Category 2A chemical
being underclassified as No Category (4-carboxybenzaldehyde, CAS # 619-66-9). However, if a TopDown approach would be used to test this chemical, starting with BCOP, it would be overclassified as
UN GHS Category 1 instead of underclassified as UN GHS No Category. Finally, STE correctly
identified 8 of 10 EpiOcularTM EIT false positives tested as “non-irritant” (including both chemicals
correctly identified by BCOP but not those identified by HET-CAM), but at the same it underpredicted
3 UN GHS Category 2A and 1 Category 2B as “non-irritants”. If a Top-Down approach would be used
to test these four chemicals, starting with BCOP, two of them (ethanol, CAS # 64-17-5, andisopropanol, CAS # 67-63-0) would be overclassified as UN GHS Category 1 but the other two(methyl acetate, CAS # 79-20-9; ammonium nitrate, 6484-52-2) would still be underclassified as UN
GHS No Category since for these, BCOP produced an IVIS between 3 and 55 (i.e., no prediction canbe made) (24). Methyl acetate produced a mean IVIS of 54.9 in the EC-HO validation study, which isborderline for the cut-off of 55 used to identify UN GHS Category 1. In fact, the same chemical was
predicted as UN GHS Cat 1 by BCOP at BASF with an IVIS of 58.1 (27). By using BCOP to identify
both UN GHS Category 1 and UN GHS No Category chemicals in combination with EpiOcularTM
EIT, the false positive rate for chemicals not requiring classification for eye irritation or serious eye
damage would be reduced by 7.7%, from 13/26 (50%) to 11/26 (42.3%). If HET-CAM is further
integrated in the strategy, the false positive rate would be further reduced to 34.6% (9/26). If STE
would also be integrated in the test strategy despite its higher false negative rate, the false positive ratewould decrease to only 11.5% (3/26) (Annex 6) (28).
Mode of Action (MoA)20.
An Expert meeting held at EC-ECVAM in 2005 (1) recommended to expand the concept ofdefining the applicability domain as not only chemical classes, but also as a function of the mechanismof eye irritation. The four identified MoA that were discussed included: (i) cell membrane lysis(breakdown of membrane integrity as might occur from exposure to membrane active materials, e.g.,surfactants), (ii) saponification (breakdown of lipids by alkaline action), (iii) coagulation(precipitation/denaturation of macromolecules, particularly protein, characteristic of acid, alkali, ororganic solvent exposure), and (iv) actions on macromolecules (chemicals that react with cellularconstituents/organelles that may or may not lead to overt lysis or coagulation, e.g., alkylation,oxidative attack on macromolecules such as essential proteins or nucleic acids). The BCOP test
method addresses the three first MoAs. In addition, it may also address the fourth MoA (actions on
macromolecules) when histopathological information is available.

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ENV/JM/MONO(2013)13
Table 2: Summary of events involved in chemical-induced eye irritation in vivo (text in italics
represents irreversible responses) with information on whether they are modelled by the BCOP test
method or not.
Modelled by the
Events involved in chemical-induced eye irritation
BCOP assay?
Chemical interaction with tear film

No

Chemical binding to the conjunctival epitheliumNo

Adhesion molecules compromised

Yes

Corneal epithelial damage

Yes

Inhibition of receptor-mediated transport

Unknown

Compromise of cell membrane integrity of upper corneal epithelium

Yes

Cell membrane lysis of all corneal epithelial layers

Yes

Hydration of corneal stroma

Yes

Cross-linking of proteins in corneal stroma

Yes

Erosion of corneal stroma

Yes

Cell damage to corneal epithelium and limbus
Dilation and increased lymphatic leakage from scleral vasculature

Yes (corneal
epithelium);
No (limbus)
No

Stimulation of nerve endings, i.e., enhanced blinking, tearing

No

Erosion of nerve endings in corneal and sclera

No

Duration of the response, i.e., length of time cell responses deteriorate.
Duration of response covers the effects of reactive chemicals, which cancause coagulation, saponification, that are effects, which develop andincrease over time.

No1

Recovery from response, i.e., length of time for cell responses to return tocontrol levels

No

1

In OECD TG 437, a time course is not specified. However, a time course for exposures, as well as atime course for post-exposure expression of changes, up to 24 hours, can be designed into the assay.
Chemical Classes
21.
A total of 213 individual chemicals (107 substances and 106 mixtures) were evaluated in the
2006-2009 test method evaluation (19), including representatives from a number of chemical andproduct classes that have been evaluated using in vivo rabbit eye data. Chemicals with a wide range ofindividual responses have been evaluated. Test data were collected from Gautheron et al. (1994) (10),Balls et al. (1995) (11), Swanson et al. (1995) (12), Southee (1998) (13), Swanson and Harbell (2000)(14), Bailey et al. (2004) (15) and the AMCP BRD (16) (Annex 1). The ICCVAM PRP (20)concluded that “the chemical database appeared adequate, however, additional chemicals in certain
chemical classes will provide a more robust statistical inference as these data become available”,
however, the PRP did not specifically identify any chemicals or product classes as being of special
concern. For a complete list of chemical and product classes see Annex 1. The components of each of
the mixtures tested in the BCOP test method can be found in Annexes 2-5 (copied from reference 19).
22.
Tables 3 and 4 show the chemical and product classes for the chemicals included in the
BCOP validation database. Information, including chemical name, Chemical Abstracts Service

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14ENV/JM/MONO(2013)13
Registry Number (CASRN), chemical and/or product class, concentration(s) tested, purity, supplier orsource, and literature reference using the chemical are provided in Annex 1.
Table 3: Chemical classes tested in the BCOP test method.
Chemical Class
Acyl halide
Alcohol
Aldehyde
Alkali
Aluminum compound
Amide
AmidineAmine
Amino acid
Boron compound
Carboxylic acid
Ester
Ether/Polyether
Formulation
Heterocyclic compound
Hydrocarbon# of Chemicals
3
22
1
3
1
2
6
10
4
1
17
12
9
69
12
18

Chemical Class
Imide
Inorganic salt
KetoneLactoneNitrile compound
Nitro compound
Oil
Onium compound
Organic salt
Organic sulfur compound
Organophosphate
Organosilicon compound
PhenolPolycyclic compound
TerpeneWax# of Chemicals
2
6
12
3
1
2
1
12
3
5
1
1
1
3
1
1

Table 4: Product classes tested in the BCOP test method.
Product Class# of Chemicals

Product Class# of Chemicals

Adhesive
Agricultural chemical
Antifreeze agent
Antimicrobial
cleaning
product
Bactericide/Fungicide/
Disinfectant/Germicide
Beverage
BleachChelating agent

1
2
1
66

Fertilizer
Flame retardant
Flavor ingredient
Food additive

1
1
3
1

11

Herbicide

3

1
3
2

8
6
1

Chemical/synthetic
intermediate
Cleaner
Cleanser (personal care)28

Insect repellant
Lubricant/lubricant additive
Paint,
lacquer,varnish(component)
Pesticide
Petroleum productPhotographic
chemical/developing agent
Plant growth regulator
Plasticizer
Preservative
Reagent
Shampoo (hair)
Soap

16
2

Coupling agent
Cutting fluid
Degreaser
DessicantDetergentDrug/Pharmaceutical/
Therapeutic agent and/or
metabolite

15
13
1
2
1
1
11
17

15

8

2
4
2
5
14
3

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ENV/JM/MONO(2013)13
Dry cleaning preparation
Dye, in manufacture ofEmulsifier
Etching and/or electroplating
Explosive
Fabric softener

1
3
1
2
1
1

Solvent
Surfactant
Anionic surfactant
Cationic surfactant
Nonionic surfactant
Thermometer fluid

34
39
3
6
5
1

Sensitivity, Specificity and Accuracy:
23.
The updated BCOP validation database contains a total of 213 chemical, most of which had
sufficient in vivo data to be assigned an ocular irritancy classification according to the UN GHS
classification system (4), sufficient for the calculation of accuracy to identify UN GHS Category 1(Top-Down) (191 chemicals: 65 Cat 1, 32 Cat 2A, 3 Cat 2B, 1 Cat 2, 1 No Cat/Cat 2A and 89 No Cat)or UN GHS No Category (Bottom-Up) (196 chemicals: 65 Cat 1, 32 Cat 2A, 3 Cat 2B, 1 Cat 2, 6 Cat
1/Cat 2 and 89 No Cat). The difference in the number of chemicals used in the two calculations (191vs. 196) derives from (i) the use of 1 chemical (Tetraaminopyrimidine sulphate) that has two in vivo
studies, one indicating UN GHS No Category (ECETOC) and the other UN GHS Category 2A(Gautheron et al. 1996 (10)), for the Top-Down calculations but not for the Bottom-Up calculations;and (ii) the use of 6 chemicals with in vivo Study Criteria Not Met (SCNM), but for which one can becertain they are either UN GHS Category 2 or UN GHS Category 1, for the Bottom-Up calculations
but not for the Top-Down calculations (see Annex 1). In order to calculate the appropriate ocular
irritancy hazard classification, detailed in vivo data consisting of cornea, iris, and conjunctiva scoresfor each animal at 24, 48, and 72 hours following test substance administration and/or assessment of
the presence or absence of lesions at 7, 14, and 21 days were evaluated. Some of the test substances
had insufficient in vivo data to assign a hazard classification (16 chemicals: 5 with study criteria notmet, 11 without in vivo data). These chemicals were therefore not used to evaluate test method
accuracy.

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ENV/JM/MONO(2013)13
Table 5: False positive and false negative rates of the BCOP test method, by chemical class andproperties of interest, for the UN GHS1 classification system for distinguishing chemicals (substancesand mixtures) inducing serious eye damage (UN GHS Category 1) from all other categories.
False Positive Rate
False Negative Rate
Category
N2
3
%
No.
%
No.3
Overall
191
25
32/12614
9/65
Chemical Class4
Alcohol
17
50
7/14
67
2/3
Amine/Amidine
7
0
0/4
0
0/3
Carboxylic acid
14
37
3/8
33
2/6
Ester
10
12
1/8
0
0/2
Ether/Polyether
6
40
2/5
0
0/1
Heterocycle
13
14
1/7
17
1/6
Hydrocarbon
11
9
1/11
0/0
Inorganic salt
7
0
0/3
0
0/4
Ketone944
4/90/0
Onium compound
11
0
0/3
12
1/8
Properties of Interest
Liquids5
150
26
27/1028
4/48
5
Solids
33
15
3/20
38
5/13
Pesticide
8
66
2/3
40
2/5
6
Surfactant – Total
75
17
6/35
7
3/40
-nonionic
25
21
3/14
9
1/11
-anionic
22
0
0/12
10
1/10
-cationic
7
0
0/1
0
0/6
pH – Total7
34
21
4/19
13
2/15
- acidic (pH < 7.0)
13
27
3/11
0
0/2
- basic (pH > 7.0)
16
0
0/3
15
2/13
- equals 7
3
33
1/3
0/0
8
Category 1 Subgroup 17
9/52
Total
529
25
16
4/25
- 4 (CO=4 at any time)10
0/1- 3 (severity/persistence)
333
1/3- 2 (severity)
29
17
5/29
- 2-4 combined10
23
17
4/23
- 1 (persistence)
1
GHS = Globally Harmonized System (UN 2011) (4).
2N = Number of chemicals.
3
Data used to calculate the percentage.
4
Chemical classes included in this table are represented by at least five substances tested in the BCOP
test method and assignments are based on the MeSH categories (www.nlm.nih.gov/mesh).
5
Physical form (i.e., solid or liquid) not known for some chemicals, and therefore the overall number
does not equal the sum of the solid and liquid chemicals.
6
Combines single substances labelled as surfactants along with surfactant-containing mixtures.
7
Total number of chemicals for which pH information was obtained.
8
NICEATM-defined subgroups assigned based on the lesions that drove classification of a UN GHS
Category 1 chemical. 1: based on lesions that are persistent; 2: based on lesions that are severe (not1759ENV/JM/MONO(2013)13
including Corneal Opacity [CO]=4); 3: based on lesions that are severe (not including CO=4) andpersistent; 4: CO = 4 at any time.
9
The number of chemicals evaluated in the Category 1 subgroup analysis may be less than the total
number of in vivo Category 1 chemicals evaluated, since some chemicals could not be classified into
the subgroups used in the evaluation.
10
Subcategories 2 to 4 combined to allow for a direct comparison of UN GHS Category 1 chemicals
classified in vivo based on some lesion severity component and those classified based on persistentlesions alone.

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ENV/JM/MONO(2013)13
Table 6: False positive and false negative rates of the BCOP test method, by chemical class andproperties of interest, for the UN GHS1 classification system for distinguishing chemicals (substancesand mixtures) not requiring classification for eye irritation or serious eye damage (UN GHS No
Category) from all other irritant categories.
False Positive Rate
False Negative Rate
Category
N2
%
No.3
%
No.3
Overall

196

69

61/89

0

0/107

Alcohol

17

0

3/7

0

0/10

Amine/Amidine

7

25

1/4

0

0/3

Carboxylic acid

14

83

5/6

0

0/8

Ester

10

75

3/4

0

0/6

Ether/Polyether

6

25

1/4

0

0/2

Heterocycle

13

67

4/6

0

0/7

Hydrocarbon

11

73

8/11

-

0/0

Inorganic salt

7

50

1/2

0

0/5

Ketone

9

67

4/6

0

0/3

Onium compound

11

100

2/2

0

0/9

Liquids5

154

75

54/72

0

0/82

Solids5

34

38

6/16

0

0/18

Pesticide

8

100

2/2

0

0/6

Surfactant – Total6

75

80

24/30

0

0/45

-nonionic

25

69

9/13

0

0/12

-anionic

23

82

9/11

0

0/12

-cationic

7

100

1/1

0

0/6

pH – Total7

34

78

7/9

0

0/25

- acidic (pH < 7.0)

13

75

3/4

0

0/9

- basic (pH > 7.0)

16

100

3/3

0

0/13

- equals 7

3

-

0/0

0

0/3

Chemical Class4

Properties of Interest

1

GHS = Globally Harmonized System (UN 2011) (4).
19

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ENV/JM/MONO(2013)13
2

N = Number of chemicals.
Data used to calculate the percentage.
4
Chemical classes included in this table are represented by at least five substances tested in the BCOP
test method and assignments are based on the MeSH categories (www.nlm.nih.gov/mesh).
5
Physical form (i.e., solid or liquid) not known for some chemicals, and therefore the overall number
does not equal the sum of the solid and liquid chemicals.
6
Combines single substances labelled as surfactants along with surfactant-containing mixtures.
7
Total number of chemicals for which pH information was obtained.
3

Overall Predictive Capacity
Table 7: Predictive capacity of the BCOP test method for distinguishing chemicals (substances andmixtures) inducing serious eye damage from all other categories, as defined by the UN GHS
classification system1 (based on Table 2 in the BCOP Issue Paper; Annex 6).
ChemicalsNo.

Accuracy

Sensitivity

False
Negatives

Specificity

False
Positives

%

No.

%

No.

%

No.

%

No.

%

No.

All

191

79

150/191

86

56/65

14

9/65

75

94/12625

32/126

Substance
s

100

73

73/100

77

20/26

23

6/26

72

53/74

28

21/74

Mixtures

91

85

77/91

92

36/39

8

3/39

79

41/52

21

11/52

All
without
alcohols,
ketonesand solids

131

85

111/131

92

46/50

8

4/50

80

65/81

20

16/81

Substances withoutalcohols,
ketonesand solids

42

83

35/42

83

10/12

17

2/12

83

25/30

17

5/30

Mixtureswithout
alcohols,
ketonesand solids

89

85

76/89

95

36/38

5

2/38

78

40/51

22

11/51

Abbreviations: No. = data used to calculate the percentage.
1
UN GHS classification system (UN 2011) (4): Category 1 vs. No Category/Category 2A/2B.

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ENV/JM/MONO(2013)13

Table 8: Predictive capacity of the BCOP test method for distinguishing chemicals (substances andmixtures) not requiring classification for eye irritation or serious eye damage from all other irritantcategories, as defined by the UN GHS classification system1 (based on Table 3 in the BCOP Issue
Paper; Annex 6).
ChemicalsNo.

Accuracy

Sensitivity

False
Negatives

Specificity

False
Positives

%

No.

%

No.

%

No.

%

No.

%

No.

All

196

69

135/196100

107/107

0

0/107

31

28/89

69

61/89

Substance
s

104

72

75/104

100

55/55

0

0/55

41

20/49

59

29/49

Mixtures

92

65

60/92

100

52/52

0

0/52

20

8/40

80

32/40

All
without
alcohols,
ketonesand solids

135

64

87/135

100

75/75

0

0/75

20

12/60

80

48/60

Substances withoutalcohols,
ketonesand solids

45

64

29/45

100

24/24

0

0/24

24

5/21

76

16/21

Mixtureswithout
alcohols,
ketonesand solids

90

64

58/90

100

51/51

0

0/51

18

7/39

82

32/39

Abbreviations: No. = data used to calculate the percentage.
1
UN GHS classification system (UN 2011) (4): No Category vs. Category 1/2A/2B.

Within- and Between-Laboratory Reproducibility:
24.
A thorough evaluation of the BCOP reliability was conducted in the 2003-2006 retrospective
validation study concerning the use of BCOP for identifying chemicals inducing serious eye damage(2) (3). This evaluation showed a median coefficient of variation (CV) for IVIS for replicate corneas(n=3) within individual experiments ranging from 11.8% to 14.2% in one study, and mean and median
CV values for IVIS for replicate corneas (n=4) within individual experiments of 71% and 35%,
respectively, in a second study. The between experiment mean CV values of IVIS for 16 chemicals
tested two or more times in three laboratories ranged from 12.6% to 14.8%, while the median CV
values ranged from 6.7% to 12.4%.
25.
Additional analyses of between-laboratory reproducibility were also conducted in the second
retrospective validation study (2006-2009) to evaluate the extent of agreement of BCOP hazard
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ENV/JM/MONO(2013)13
classifications among the laboratories that participated in three different inter-laboratory validation
studies (10) (11) (13) (19). These analyses showed nearly 100% agreement of classification among thedifferent laboratories in each study, confirming the results that had been obtained in the firstevaluation. The extent of agreement was 100% when distinguishing chemicals inducing serious eye
damage from all other ocular hazard categories for 72% (91/127) of the chemicals according to the UN
GHS classification system. When distinguishing chemicals not requiring classification for eye
irritation or serious eye damage from all other ocular hazard categories, the extent of agreement was
100% for 80% (103/128) of the chemicals according to the UN GHS classification system (19).
References:
1. Scott L., Eskes C., Hoffmann S., Adriaens E., Alépée N., Bufo M., Clothier R., Facchini D.,
Faller C., Guest R., Harbell J., Hartung T., Kamp H., Le Varlet B., Meloni M., McNamee P.,
Osborne R., Pape W., Pfannenbecker U., Prinsen M., Seaman C., Spielman H., Stokes W.,
Trouba K., Van den Berghe C., Van Goethem F., Vassallo M., Vinardell P., Zuang V. (2010).
A proposed eye irritation testing strategy to reduce and replace in vivo studies using BottomUp and Top-Down approaches. Toxicol. In Vitro, 24: 1-9.
2. NICEATM (2006). Background Review Document: Current Status of In Vitro Test Methods
for Identifying Ocular Corrosives and Severe Irritants: Bovine Corneal Opacity andPermeability Test Method. NIH Publication No. 06-4512. National Institute of Environmental
HealthSciences,
Research
Triangle
Park,
NC,
USA.
Available:[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_bcop.htm].
3. ICCVAM (2007). ICCVAM Test Method Evaluation Report: In Vitro Ocular Toxicity Test
Methods for Identifying Ocular Severe Irritants and Corrosives. NIH Publication No. 07-4517.
National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
Available: [http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_tmer.htm].
4. UN (2011). Globally Harmonized System of Classification and Labelling of Chemicals(GHS). ST/SG/AC.10/30 Rev 4, New York & Geneva: United Nations Publications.
Available: [http://www.unece.org/trans/danger/publi/ghs/ghs_rev04/04files_e.html].
5. U.S. EPA (2011). Label Review Manual. Washington, DC: U.S. Environmental Protection
Agency. Available: [http://www.epa.gov/oppfead1/labeling/lrm/].
6. EC (2001). Commission Directive 2001/59/EC of 6 August 2001 adapting to technicalprogress for the 28th time Council Directive 67/548/EEC on the approximation of the laws,
regulations and administrative provisions relating to the classification, packaging and labellingof dangerous substances. Official Journal of the European Communities L255:1-333.
7. EC (2008). REGULATION (EC) No 1272/2008 of the European Parliament and of theCouncil of 16 December 2008 on classification, labelling and packaging of substances andmixtures, 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.
8. OECD (2002). Acute eye irritation/corrosion. OECD Guideline for Testing of Chemicals No.
405. Organisation for Economic Co-operation and Development, Paris. Available:[http://www.oecd.org/document/40/0,2340,en_2649_34377_37051368_1_1_1_1,00.html].
9. OECD (2009). Bovine Corneal Opacity and Permeability (BCOP) Test Method for Identifying
Ocular Corrosives and Severe Irritants. OECD Guideline for Testing of Chemicals No. 437.
Organisation for Economic Co-operation and Development, Paris. Available:[http://www.oecd.org/document/40/0,2340,en_2649_34377_37051368_1_1_1_1,00.html].
10. Gautheron P., Giroux J., Cottin M., Audegond L., Morilla A., Mayordomo-Blanco L.,
Tortajada A., Haynes G., Vericat J.A., Pirovano R., Tos E.G., Hagemann C., Vanparys P.,
Deknudt G., Jacobs G., Prinsen M., Kalweit S., Spielmann H. (1994). Interlaboratory
assessment of the bovine corneal opacity and permeability (BCOP) assay. Toxicol. In Vitro, 8:
381-392.
11. Balls M., Botham P.A., Bruner L. H., Spielmann H. (1995). The EC/HO international
validation study on alternatives to the Draize eye irritation test. Toxicol. In Vitro, 9: 871-929.

64

22

ENV/JM/MONO(2013)13
12. Swanson J.E., Lake L.K., Donnelly T.A., Harbell J.W., Huggins J. (1995). Prediction ofocular irritancy of full-strength cleaners and strippers by tissue equivalent and bovine corneal
assays. J. Toxicol. Cutaneous Ocul. Toxicol., 14: 179–195.
13. Southee J.A. (1998). Evaluation of the prevalidation process, Part 2, Final Report Vol. 2. The
bovine corneal opacity and permeability (BCOP) assay. European Community Contract No.
11279-95-10F 1ED ISP GB.
14. Swanson J.E., Harbell J.W. (2000). Evaluating the eye irritancy potential of ethanolic test
materials with the bovine corneal opacity and permeability assay. The Toxicologist, 54: 188–
189.
15. Bailey P.T., Freeman J.J., Phillips R.D., Merrill J.C. (2004). Validation of the BCOP assay as
a predictor of ocular irritation of various petrochemical products. Poster presentation at theSociety of Toxicology 2004 meeting (Baltimore, MD).
16. ICCVAM (2006). AMCP Background Review Document: An in vitro approach of EPA
toxicity labeling of anti-microbial cleaning products and annexes A–G. Available:[http://iccvam.niehs.nih.gov/methods/ocutox/antimicro/BRD.pdf].
17. Casterton P.L., Potts L.F., Klein B.D. (1996). A novel approach to assessing eye irritation
potential using the bovine corneal opacity and permeability assay. J. Toxicol. – Cut. andOcular Toxicol., 15: 147-163.
18. Gettings S., Lordo R., Hintze K., Bagley D., Casterton P., Chudkowski M., Curren R.D.,
Demetrulias J.L., Dipasquale L.C., Earl L.K., Feder P.I., Galli C.L., Glaza S.M., Gordon V.C.,
Janus J., Kurtz P.J., Marenus K.D., Moral J., Pape W.J.W., Renskers K.J., Rheins L.A., Roddy
M.T., Rozen M.G., Tedeschi J.P., Zyracki J. (1996). The CTFA evaluation of alternatives
program: An evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase
III) surfactant-based formulations. Food Chem. Toxic., 34: 79-117.
19. ICCVAM (2010). ICCVAM Test Method Evaluation Report: Current Validation Status of In
Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals andProducts. NIH Publication No. 10-7553. National Institute of Environmental Health Sciences,
Research
Triangle
Park,
NC,
USA.
Available:[http://iccvam.niehs.nih.gov/methods/ocutox/MildMod-TMER.htm].
20. ICCVAM (2009). Independent Scientific Peer Review Panel Report: Evaluation of the
Validation Status of Alternative Ocular Safety Testing Methods and Approaches. Available:[http://iccvam.niehs.nih.gov].
21. Prinsen M.K. (2006). The Draize Eye Test and in vitro alternatives; a left-handed marriage?
Toxicol. in Vitro, 20: 78-81.
22. Maurer, J.K., Parker, R.D. and Jester, J.V. (2002). Extent of corneal injury as the mechanistic
basis for ocular irritation: key findings and recommendations for the development ofalternative assays. Reg. Tox. Pharmacol., 36: 106-117.
23. Adriaens et al. (under preparation). Draize test for eye irritation: importance of the endpoints
evaluated with regard to UN GHS / EU CLP classification.
24. Weil C.S., Scala A. (1971). Study of intra- and inter- laboratory variability in the results ofrabbit eye and skin irritation tests. Toxicology and Applied Pharmacoogy 19, 276-360.
25. Marzulli FN, Ruggles DI (1973). Rabbit eye irritation test: collaborative study. J. Ass. Off.
Analyt. Chem. 56, 905-914.
26. Cormier EM, Parker RD, Henson C, Cruze LW, Merritt AK, Bruce RD, Osborne R (1996).
Determination of the intra- and inter-laboratory reproducibility of the Low Volume Eye Testand its statistical relationship to the Draize tes. Reg. Tox. Pharmac. 23, 156-161.
27. Kolle S.N., Kandárová H., Wareing B., van Ravenzwaay B., Landsiedel R. (2011). Inhousevalidation of the EpiOcularTM eye irritation test and its combination with the bovine corneal
opacity and permeability test for the assessment of ocular irritation. Altern. Lab. Anim., 39:
365-387.
28. Hayashi K., Mori T., Abo T., Ooshima K., Hayashi T., Komano T., Takahashi Y., Sakaguchi
H., Takatsu A., Nishiyama N. (2012). Two-stage bottom-up tiered approach combining
several alternatives for identification of eye irritation potential of chemicals including
insoluble or volatile substances. Toxicol. in Vitro, 26: 1199-1208.

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