᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ  ᖺ  ᭶  ᪥                                  1R 

2013 ᖺᨵゞ OECD TG 438 ࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂(ICE ἲ:Isolated Chicken Eye Test)࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 26 ᖺ 10 ᭶ 28 ᪥࡟ᮾிࠊᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸JaCVAM ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢᥦ᱌ࡀ࡞ࡉࢀࡓࠋ   ᥦ᱌ෆᐜ㸸

TG 438㸦2013㸧ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ
࡟࠾࠸࡚UN GHS༊ศ㸯≀㉁㸦㔜⠜࡞║ࡢയᐖࢆᘬࡁ㉳ࡇࡍ≀㉁㸧࡞ࡽࡧ࡟࣎ࢺ
࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿUN GHS༊ศእ≀㉁㸦║่⃭ᛶ≀㉁࡜ࡋ࡚ศ㢮ࡉࢀ࡞࠸㸧ࡢ⠊ᅖ࡟࠾࠸࡚⾜ᨻⓗ฼⏝ࡣྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋࡇࡢᥦ᱌᭩ࡣࠊOECD TG 438࠾ࡼࡧStreamed Summary Document Supporting OECD TG 438 on the
Isolated Chicken Eye for Eye Irritation/Corrosion. Series on Testing and Assessment No. 188 (Part 1 andPart 2)ࢆࡶ࡜࡟ࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚JaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚ࠕ2013 ᖺᨵゞ OECD TG 438 ࢽ࣡ࢺࣜࡢ
᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂ࠖࡢ౑⏝ࢆᥦ᱌ࡍࡿࡶࡢ࡛࠶ࡿࠋ ኱㔝Ὀ㞝                      すᕝ⛅ె
JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i

-D&9$0 ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧୍㨣 ຮ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᖹ㈡⚽᫂㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧ቑ⏣ග㍤㸦ᗙ㛗᥎⸀㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷ⏣ ⥳

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

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

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

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

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧ᑠᓥ ⫕㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊᪂つヨ㦂⾲ホ౯ᐊ㸧㸸஦ົᒁ

iii

iv

JaCVAM statement on
Isolated Chicken Eye Test Method for Identifying i) 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 28 October 2014 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:
Isolated Chicken Eye 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 and reliability for prediction of eyeirritating test substances for regulatory use.
Following the review of the results of OECD TG No. 438 and Streamed Summary Document
Supporting OECD TG 438 on the Isolated Chicken Eye for Eye Irritation/Corrosion. Series on Testingand Assessment No. 188 (Part 1 and Part 2), it is concluded that Isolated Chicken Eye Test Method

such as rritation testing are clearly beneficial.
The JaCVAM Regulatory Acceptance Board has been regularly kept informed of the
progress of the study, and this endorsement is based on an assessment of various documents,
including, in particular, the evaluation report prepared by the JaCVAM ad hoc peer review
panel for eye irritation testing.

Yasuo Ohno
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

6 January, 2015v

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

Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Hideaki Hiraga (Pharmaceuticals and Medical Devices Agency)
Mr. Tsutomu Ichiki (Japan Chemical Industry Association)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Mitsuteru Masuda (nominee by Chairperson)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Akiyoshi Nishikawa (NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Koko Tanigawa (Japanese Society for Animal Experimentation)
Mr. Takashi Yamada (National Institute of Technology and Evaluation)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Ms. Midori Yoshida (NIHS)
Mr. Takemi Yoshida (Japanese Society of Toxicology)
Mr. Isao Yoshimura (nominee by Chairperson)
Mr. Kazuto Watanabe (Japan Pharmaceutical Manufacturers Association)
Term: From 1st April 2014 to 31st March 2016vi

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

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

vii

viii

2013 ᖺᨵゞ OECD TG 438 ࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂(ICE ἲ : Isolated Chicken Eye Test)┠ḟホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------- 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------- 7
OECD TG438㸦2013㸧---------------------------------------------------------------------- 17
STREAMLINED SUMMARY DOCUMENT SUPPORTING OECD TESTGUIDELINE 438 ON THE ISOLATED CHICKEN EYE FOR EYE
IRRITATION/CORROSION ------------------------------------------------------------- 37ixx║่⃭ᛶヨ㦂௦᭰ἲࡢホ౯఍㆟ሗ࿌᭩

2013 ᖺᨵゞ OECD TG 438 ࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂(ICE ἲ:Isolated Chicken Eye Test)

JaCVAM ホ౯఍㆟

ᖹᡂ 26 ᖺ㸦2014 ᖺ㸧10 ᭶ 28 ᪥

1

1

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝  㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧୍㨣 ຮ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧㇂ᕝᾈᏊ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧ᖹ㈡⚽᫂㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍㸧ቑ⏣ග㍤㸦ᗙ㛗᥎⸀㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᒣ⏣㝯ᚿ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧ྜྷ⏣Ṋ⨾㸦᪥ᮏẘᛶᏛ఍㸧ྜྷ⏣ ⥳ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧Ώ㒊୍ே㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧                  ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥

22ࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂(Isolated Chicken Eye Testࠊ௨ୗ ICE ἲ)ࡣࠊ࢘ࢧࢠࢆ⏝࠸ࡓ Draize ║่⃭ᛶヨ㦂(௨ୗࠊDraize ἲ)ࡢ௦᭰ヨ㦂ἲ࡛࠶ࡿࠋ2003 ᖺ࠿ࡽ 2006 ᖺ࡟࠿ࡅ࡚ࠊࢺ
ࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ㔜⠜࡞║ࡢയᐖࢆᘬࡁ㉳ࡇࡍ໬Ꮫ≀㉁(UN GHS ༊ศ 1 ≀㉁)ࢆྠᐃࡍࡿࡓ
ࡵࡢヨ㦂ἲ࡜ࡋ࡚ࠊ
ᅇ㢳ⓗࣂࣜࢹ࣮ࢩࣙࣥࡀ NICEATM/ICCVAM ࡟ࡼࡾ࡞ࡉࢀࠊ
2009 ᖺ࡟ OECD TG
438 (௨ୗࠊTG438(2009)1))࡜ࡋ࡚᥇ᢥࡉࢀࡓࠋ
ࡇࢀ࡜ࡣู࡟ NICEATM/ICCVAM ࡣࠊECVAM ࠾ࡼࡧ JaCVAM ࡢ༠ຊࡢୗࠊ2006 ᖺ࠿ࡽ 2010ᖺ࡟࠿ࡅ࡚ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ༊ศእ≀㉁ࡢྠᐃἲ࡜ࡋ࡚ࡢ㐺⏝ᛶ࡟㛵ࡍࡿᅇ㢳ⓗࣂࣜࢹ࣮ࢩࣙࣥࢆ᪂ࡓ࡟⾜ࡗࡓࠋࣂࣜࢹ࣮ࢩࣙࣥ࡟ࡣࠊึᅇ࡟౑⏝ࡉࢀࡓ ICCVAM ࢹ࣮ࢱ(2006)2)ࡀࡑࡢ
ࡲࡲ⏝࠸ࡽࢀ࡚ゎᯒࡀ࡞ࡉࢀࡓࡀࠊICE ἲᣑ኱㐺⏝ࡢᥦゝ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ
ࡑࡢᚋࠊ2012 ᖺࠊOECD ᑓ㛛ጤဨ఍࡟ࡼࡾ UN GHS ༊ศୗ࡟࠾࠸࡚෌ホ౯ࡀᐇ᪋ࡉࢀࡓࠋ
ึᅇ࡟౑⏝ࡉࢀࡓࢹ࣮ࢱ࣮࣋ࢫ࡟࠾ࡅࡿ in vivo ࠾ࡼࡧ in vitro ୧᪉ࡢࢹ࣮ࢱࡀぢ┤ࡉࢀࡓ࡯࠿ࠊinvitro ࡢ༊ศእᇶ‽ࡀṇᘧ࡟タᐃࡉࢀࡓࠋࡑࡇ࡛ᨵࡵ࡚ホ౯ࡀ⾜ࢃࢀࡓ⤖ᯝࠊICE ἲࡣࢺࢵࣉࢲ࢘ࣥ
᪉ᘧ࡟࠾ࡅࡿ UN GHS ༊ศ 1 ≀㉁ࡢྠᐃࡢࡳ࡞ࡽࡎࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ༊ศእ≀㉁ࡢྠᐃ
ἲ࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ࡜ุ᩿ࡉࢀࠊ2013 ᖺ 7 ᭶ 26 ᪥௜ࡅ࡛ 2013 ᖺᨵゞ TG 438(௨ୗࠊTG438(2013)3))࡜ࡋ࡚᥇ᢥࡉࢀࡓࠋ
TG 438(2013)࡟࠾ࡅࡿ୺࡞ᨵゞⅬࡣࠊ1) ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟ࡼࡿ UN GHS ༊ศእ≀㉁ྠᐃ࡬ࡢ㐺⏝࠾ࡼࡧ 2) 㐺⏝㝈⏺ࠊ࡛࠶ࡿࠋJaCVAM ホ౯఍㆟ࡣࠊࡇࡢᨵゞࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 2013 ᖺᨵゞ OECD TG 438㸦TG438㸦2013㸧3㸧㸧ࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂 (ICE ἲ: Isolated Chicken Eye Test)௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 Draize ἲヨ㦂ἲࡢᴫ␎㸸 ICE ἲࡣࠊࢽ࣡ࢺࣜ࠿ࡽ᦬ฟࡋࡓ║⌫࡟⿕㦂≀㉁ࢆ᭚㟢ࡋࠊࡑࡢ⤖ᯝࠊ║⌫࡟⏕
ࡌࡿゅ⭷ࡢኚᛶࢆࠊゅ⭷ࡢ⭘⬽ࠊΰ⃮ᗘ࠾ࡼࡧࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶࡢኚ໬࡜ࡋ࡚࡜ࡽ࠼ࡿࠋࡇ
ࢀࡽ 3 ホ౯㡯┠ࡢኚ໬ࢆࡑࢀࡒࢀಶูࡢࢫࢥ࢔࡟ኚ᥮ࡋ࡚ᚓࡽࢀࡿ⥲ྜホ౯ࢆࡶ࡜࡟ in vivo ࡛ࡢ║่⃭ᛶࢆணࡍࡿࠋྛホ౯㡯┠ࡢኚ໬ࡣࠊA)ゅ⭷ࡢ⭘⬽㸸ගᏛⓗཌᗘィࢆ⿦╔ࡋࡓ⣽㝽ⅉ㢧ᚤ㙾࡟࡚ゅ⭷ࡢཌࡉࢆᐃࡋࠊ᭚㟢 240 ศᚋࡲ࡛ࡢ⤒᫬ⓗ࡞ኚ໬⋡ࢆᐃ㔞ⓗ࡟ồࡵࡿࠋB)ゅ⭷ࡢΰ⃮ᗘ㸸⣽㝽ⅉ㢧ᚤ㙾࡟࡚ゅ⭷ΰ⃮ᗘࡢ⤒᫬ⓗ࡞ኚ໬ࢆ᭚㟢 240 ศᚋࡲ࡛ほᐹࡍࡿࠋC)ࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶ㸸⣽㝽ⅉ㢧ᚤ㙾࡟࡚᭚㟢 30 ศᚋࡢゅ⭷⾲㠃ࡢࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶࢆㄪ࡭ࡿࠋྛ㡯┠ࡢ⤖ᯝࢆയᐖࡢ⛬3

3

ᗘ࡟ࡼࡾࠊ║่⃭ᛶࡢ᭱ࡶᙅ࠸ࢡࣛࢫ I ࠿ࡽ᭱ࡶᙉ࠸ࢡࣛࢫ IV ࡢ 4 ẁ㝵࡟ศ㢮ࡋࠊࡑࢀࡽࡢศ
㢮⤖ᯝࢆ⥲ྜࡋ࡚ࠊ⿕㦂≀㉁ࡢ║่⃭ᛶࢆุᐃࡍࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ
NICEATM/ICCVAM ࡟ࡼࡿᅇ㢳ⓗࣂࣜࢹ࣮ࢩࣙࣥࢆ⤒࡚ OECD ࡟࡚ TG 438(2009)࡜ࡋ࡚᥇ᢥࡉ
ࢀࡓᚋࠊ2012 ᖺ࡟ OECD ᑓ㛛ጤဨ఍࡟ࡼࡾ UN GHS ༊ศୗ࡟࠾࠸࡚෌ホ౯ࡀᐇ᪋ࡉࢀࠊ2013 ᖺ࡟
ࡣ TG 438(2013)࡜ࡋ࡚᥇ᢥࡉࢀ࡚࠾ࡾࠊࡑࡢホ౯࡟⏝࠸ࡓ㈨ᩱ 1-5)࡜ホ౯ෆᐜ࡟⛉Ꮫⓗጇᙜᛶࡀ࠶
ࡿࠋࡲࡓࠊ௒ᅇࡢᨵゞ࡟ඛ❧ࡕ OECD ᑓ㛛ጤဨ఍࡟ࡼࡾࠊ๓ᅇホ౯ࢆ⾜ࡗࡓ ICCVAM(2006)ࣂࣜࢹ
࣮ࢩࣙࣥࢹ࣮ࢱ࣮࣋ࢫࡢ 175 ≀㉁࡟ࡘ࠸࡚ࠊin vivo ࠾ࡼࡧ in vitro ࡢಶࠎࡢࢹ࣮ࢱ࡟ࡘ࠸࡚෌ホ౯ࡉࢀࡓࡀࠊ᭱⤊ⓗ࡟࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢホ౯࡟ࡣ 175 ≀㉁୰ 152 ≀㉁㸦༢୍≀㉁㸸72ࠊΰྜ≀㉁㸸
80㸧ࡀࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧࡢホ౯࡟ࡣ 140 ≀㉁㸦༢୍≀㉁㸸65ࠊΰྜ≀㉁㸸75㸧ࡀ⏝࠸ࡽࢀࠊ༑ศ
࡞ᩘࢆ⏝࠸࡚ホ౯ࡉࢀ࡚࠸ࡿࠋ

㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
➨୕⪅ホ౯ሗ࿌᭩(ICCVAM 20064)ࠊ➨୕⪅ホ౯ሗ࿌᭩ 20126))࡛ሗ࿌ࡉࢀ࡚࠸ࡿ࡜࠾ࡾࠊࢺࢵࣉࢲ
࢘ࣥ᪉ᘧ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ᪋タෆ࣭᪋タ㛫෌⌧ᛶࡣ㧗࠸ࠋࡲࡓࠊ㐺⏝㝈⏺࡟㛵ࡋ࡚ࡣࠊ㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱࠊᅛయ≀㉁ࠊ⏺㠃άᛶ๣࠾ࡼࡧ࢔ࣝࢥ࣮ࣝ㢮࡟ࡘ࠸࡚ࡣࠊ㧗࠸ഇ㝜ᛶ⋡࠶ࡿ࠸ࡣഇ㝧ᛶ⋡ࡀ♧ࡉࢀࡓࡀࠊ௨ୗࡢ⌮⏤࠿ࡽࡇࢀࡽࡢ໬Ꮫ≀㉁࡟ࡶ ICE ἲࢆ㐺⏝ࡍࡿࡇ࡜ࡀ࡛
ࡁࡿ࡜ሗ࿌ࡉࢀࡓࠋ
࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ㸸࣭㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱ࡛㝧ᛶ 2 ≀㉁ࡢ࠺ࡕࠊ1 ≀㉁㸦TNO-94㸧ࡀഇ㝜ᛶ࡛࠶ࡗࡓࡀࠊ⏝㏵ࡢ㢮ఝࡋࡓ໬ྜ≀㸦TNO-93㸧ࡣ㝧ᛶ࡛࠶ࡗࡓࠋᚑࡗ࡚ࠊICE ἲ࡬ࡢ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿ࡟ࡣ᰿ᣐࡀ
୙༑ศ࡛࠶ࡾࠊ㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱࢆ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸ࠋ࣭༢୍≀㉁ࠊΰྜ≀㉁ࠊᾮయࠊᅛయ➼࡟࠾ࡅࡿഇ㝜ᛶ⋡ࡣ 5㸣௨ୗ࡜ప࠸ࠋഇ㝧ᛶ⋡࡟ࡘ࠸࡚ࡣࠊ
ICE ἲ࡛ࡣ 33%࡜௚ࡢヨ㦂ἲ࡟ẚ࡭ప࠸ࡇ࡜ࡀ♧ࡉࢀࡓ㸦BCOP㸦Bovine Corneal Opacity andPermeability㸧ἲ; 69㸣ࠊCM (Cytosensor Microphysiometer)ἲ㸸68%5㸧㸧ࠋ༢୍≀㉁࠾ࡼࡧ⏺㠃άᛶ๣࡟ࡘ࠸࡚ࡢഇ㝧ᛶ⋡ࡣ㧗࠸ࡀࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚㝧ᛶ࡜ุ᩿ࡉࢀࡓሙྜ࡟ࡣࠊ௚ࡢ㐺ษ࡞ヨ㦂ἲ࡟ࡼࡿ☜ㄆࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊࡇࢀࡽࡢ໬Ꮫ≀㉁ࢆ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸ࠋࢺࢵࣉࢲ࢘ࣥ᪉ᘧ㸸44࣭ഇ㝜ᛶ⋡ࡣᅛయ≀㉁࠾ࡼࡧ⏺㠃άᛶ๣࡟࠾࠸࡚㧗ࡃࠊഇ㝧ᛶ⋡ࡣ࢔ࣝࢥ࣮ࣝ㢮࡟࠾࠸࡚㧗࠿ࡗࡓࠋࡋ࠿ࡋࠊࡇࢀࡽࡢศ㢮࡟࠾࠸࡚ࡶ║่⃭ᛶࡀṇ☜࡟ணࡉࢀࡓ≀㉁ࡀ࠶ࡿࡇ࡜ࠊࡉࡽ࡟ࢺࢵࣉࢲ

࢘ࣥ᪉ᘧ࡟࠾࠸࡚㝜ᛶ࡜ุ᩿ࡉࢀࡓሙྜ࡟ࡣ௚ࡢ㐺ษ࡞ヨ㦂ἲ࡟ࡼࡿ☜ㄆࡀᚲせ࡜ࡉࢀ࡚࠸ࡿ
ࡇ࡜࠿ࡽࠊᅛయ≀㉁࠾ࡼࡧ⏺㠃άᛶ๣ࢆ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸ࠋࡓࡔࡋࠊ࢔ࣝࢥ࣮ࣝ
㢮࡛㝧ᛶ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊ⤖ᯝࡢゎ㔘ࡣៅ㔜࡟ࡍ࡭ࡁ࡛࠶ࡿࠋ ௨ୖ࠿ࡽࠊICE ἲࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡢ༊ศእ≀㉁࠾ࡼࡧࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢ UN GHS ༊ศ
1 ≀㉁ࡢྠᐃἲ࡜ࡋ࡚ࠊࡍ࡭࡚ࡢ✀㢮ࡢ໬Ꮫ≀㉁࡟㐺⏝࡛ࡁࡿ࡜ࡉࢀࡓࠋ

㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ⾜ᨻୖ฼⏝ཬࡧ♫఍ⓗཷࡅධࢀࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸
ICE ἲ࡛ࡣࠊ㣗⏝࡜ࡋ࡚ᒕẅࡉࢀࡓࢽ࣡ࢺࣜࡢ║⌫ࢆ⏝࠸ࡿࡓࡵ Draize ἲࡼࡾࡶ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ௒ᅇࡢᨵゞ࡟࠾࠸࡚ࡣࠊື≀⚟♴ࡢほⅬ࠿ࡽࡢኚ᭦ࡀ࡞࠸ࡇ࡜࠿ࡽࠊᮏᨵゞἲࡢ♫఍ⓗཷࡅධࢀᛶࡣࠊᨵゞ๓࡜ኚࢃࡽ࡞࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸  TG 438㸦2013㸧ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚UN GHS༊ศ㸯≀㉁㸦㔜⠜࡞║ࡢയᐖࢆᘬࡁ㉳ࡇࡍ≀㉁㸧࡞ࡽࡧ࡟࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿUN GHS༊ศእ≀㉁㸦║่⃭ᛶ≀㉁࡜ࡋ࡚ศ㢮ࡉࢀ࡞࠸㸧ࡢ⠊ᅖ࡟࠾࠸࡚⾜ᨻⓗ฼⏝ࡣྍ⬟࡛࠶ࡿ࡜⪃࠼ࡿࠋཧ⪃ᩥ⊩1㸧 OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test for
Identifying Ocular Corrosives and Severe Irritants, TG438 (Adopted 7 September 2009)2㸧 ICCVAM, 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. Volume 1 and Volume 2 (2010).
3)

OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test for
Identifying Ocular Corrosives and Severe Irritants, TG438 (Adopted 26 July 2013)4㸧 Streamlined Summary Document Supporting OECD Test Guideline 438 on the Isolated Chicken
Eye for Eye Irritation/Corrosion. Series on Testing and Assessment No. 188 (Part 1 andPart 2), OECD, Paris. (21 June 2013)5㸧 ICCVAM, Background Review Document: Current Status of In Vitro Test Methods for Identifying
Ocular Corrosives and Severe Irritants: Isolated Chicken Eye Test Methods.

NIH Publication No.

06-4513 (2006).
5

5

6㸧 JaCVAM, ║่⃭ᛶヨ㦂௦᭰ἲࡢ➨୕⪅ホ౯ሗ࿌᭩̺ホ౯ᑐ㇟ヨ㦂㸸║࡟ᑐࡍࡿ⭉㣗ᛶ࠾ࡼࡧᙉ่⃭ᛶホ౯ࡢࡓࡵࡢࢽ࣡ࢺࣜ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂ἲ, AATEX-JaCVAM J1,
16-29 (2012)

6

6



║่⃭ᛶヨ㦂௦᭰ἲホ౯ሗ࿌᭩2013 ᖺᨵゞ OECD TG 438 ࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂㸦ICE ἲ㸸Isolated Chicken Eye Test㸧               

ᖹᡂ 26 ᖺ 9 ᭶ 12 ᪥

JaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍


7

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JaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍
ጤဨ㛗      ᑠᆏ ᛅྖ 㸦ṧ␃㎰⸆◊✲ᡤ㸧ጤ ဨ      ⡘ෆ ᱈Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ㸧         ➉ෆ ᑠⱑ 㸦P&G ࢖ࣀ࣮࣋ࢩࣙࣥྜྠ఍♫㸧         ቑ⏣ ග㍤
         ຍ⸨ 㞞୍ 㸦ᰴᘧ఍♫ࢪࣕࣃࣥ㺃ࢸ࢕ࢵࢩ࣭࢚ࣗࣥࢪࢽ࢔ࣜࣥࢢ㸧         ⣽஭ ୍ᘯ 㸦ཧኳ〇⸆ᰴᘧ఍♫㸧         ᒣᮏ ┤ᶞ 㸦⸨⏣ಖ೺⾨⏕኱Ꮫ㸧         ྜྷᮧ ຌ 㸦ᮾி⌮⛉኱Ꮫྡ㄃ᩍᤵ㸧

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␎ㄒ
BCOP:

Bovine Corneal Opacity and Permeability

BRD:

Background Review Document

CM:

Cytosensor Microphysiometer

CV:

Coefficient of Variation

EURL ECVAM: European Union Reference Laboratory for Alternatives to Animal Testing
GHS:

Globally Harmonized System of Classification and Labeling of Chemicals

ICCVAM:

Interagency Coordinating Committee on the Validation of Alternative Methods

ICE:

Isolated Chicken Eye Test

JaCVAM:

Japanese Centre for the Validation of Alternative Methods

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

OECD:

Organization for Economic Co-operation and Development

SSD:

Streamlined Summary Document

TG:

Test GuidelineUN:

United Nations⏝ ㄒࢺࢵࣉࢲ࢘ࣥ᪉ᘧ㸸㔜ᗘࡢ่⃭ᛶࢆṇ☜࡟㆑ู࡛ࡁࡿヨ㦂ἲ࠿ࡽጞࡵࡿホ౯᪉ᘧࠋᮏ࢞࢖ࢻࣛ࢖࡛ࣥ
ࡣࠊ㔜⠜࡞║ࡢᦆയࢆᘬࡁ㉳ࡇࡍ࡜␲ࢃࢀࡿ໬Ꮫ≀㉁࡟㐺⏝ࡉࢀࡿẁ㝵ⓗ࡞ホ౯᪉ᘧࢆᣦࡋࠊࡲࡎࠊ㔜⠜࡞║ࡢᦆയࢆ㉳ࡇࡍ≀㉁ࢆࠊࡑࢀ௨እࡢ≀㉁࠿ࡽṇ☜࡟㆑ู࡛ࡁࡿヨ㦂ἲุู࡛ࡍࡿࡇ࡜࠿ࡽጞࡵࡿ㸦TG438 ANNEX 1,
DEFINITIONS㸧ࠋ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ㸸㠀่⃭ᛶࢆṇ☜࡟㆑ู࡛ࡁࡿヨ㦂ἲ࠿ࡽጞࡵࡿホ౯᪉ᘧࠋᮏ࢞࢖ࢻࣛ࢖࡛ࣥࡣࠊ
║่⃭ᛶ࠶ࡿ࠸ࡣ㔜⠜࡞║ࡢയᐖᛶ࡛ࡣ࡞࠸࡜ண

ࡉࢀࡿ໬Ꮫ≀㉁࡟㐺⏝ࡉࢀࡿẁ㝵ⓗ࡞ホ౯᪉ᘧࢆᣦࡋࠊࡲࡎࠊ║่⃭ᛶ࠶ࡿ࠸ࡣ㔜⠜࡞║ࡢയᐖᛶ࡛ࡣ࡞࠸≀㉁ࢆࠊࡑࢀ௨እࡢ≀㉁࠿ࡽṇ☜࡟㆑ู࡛ࡁࡿヨ㦂ἲุู࡛ࡍࡿࡇ࡜࠿ࡽጞࡵࡿ
㸦TG438 ANNEX 1, DEFINITIONS㸧ࠋ༊ศ1≀㉁:UN (United Nations) GHS (Globally Harmonized Systems of Classification and LabelingofChemicals) ༊ศయ⣔ୗࠊ㔜⠜࡞║ࡢയᐖࢆᘬࡁ㉳ࡇࡍ≀㉁࡜ࡋ࡚༊ศࡉࢀࡿ໬Ꮫ≀㉁༊ศእ≀㉁:UN GHS༊ศయ⣔ୗࠊ║่⃭ᛶ≀㉁࡜ࡋ࡚༊ศࡉࢀ࡞࠸໬Ꮫ≀㉁


9

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㸯㸬ᨵゞࡢ⫼ᬒࢽ࣡ࢺࣜࡢ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂㸦Isolated Chicken Eye Testࠊ௨ୗ ICE ἲ㸧ࡣࠊ࢘ࢧࢠࢆ⏝࠸ࡓ Draize ║่⃭ᛶヨ㦂ἲ㸦௨ୗࠊDraize ἲ㸧ࡢ௦᭰ヨ㦂ἲ࡛࠶ࡿࠋ2003 ᖺ࠿ࡽ 2006 ᖺ࡟࠿ࡅ࡚ࠊࢺࢵ
ࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ㔜⠜࡞║ࡢയᐖࢆᘬࡁ㉳ࡇࡍ໬Ꮫ≀㉁㸦༊ศ 1 ≀㉁㸧ࢆྠᐃࡍࡿࡓࡵࡢヨ㦂ἲ࡜ࡋ࡚ࠊᅇ㢳ⓗࣂࣜࢹ࣮ࢩࣙࣥࡀ NICEATM (NTP Interagency Center for the Evaluation of Alternative
Toxicological Methods) / ICCVAM (Interagency Coordinating Committee on the Validation of Alternative
Methods) ࡟ࡼࡾ࡞ࡉࢀࠊ2009 ᖺ࡟ OECD (Organization for Economic Co-operation and Development) ࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ㸦TG: Test Guideline㸧438㸦TG 438, 2009㸧࡜ࡋ࡚᥇ᢥࡉࢀࡓࠋ
ࡇࢀ࡜ࡣู࡟ NICEATM/ICCVAM ࡣ EURL ECVAM (European Union Reference Laboratory for
Alternatives to Animal Testing) ࠾ࡼࡧ JaCVAM (Japanese Centre for the Validation of Alternative Methods)ࡢ༠ຊࡢୗࠊ2006 ᖺ࠿ࡽ 2010 ᖺ࡟࠿ࡅ࡚ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ༊ศእ≀㉁ࡢྠᐃἲ࡜ࡋ࡚ࡢ㐺⏝ᛶ࡟㛵ࡍࡿᅇ㢳ⓗࣂࣜࢹ࣮ࢩࣙࣥࡀ᪂ࡓ࡟⾜ࢃࢀࡓࠋࣂࣜࢹ࣮ࢩࣙࣥ࡟ࡣࠊึᅇ࡟౑⏝ࡉࢀࡓ ICCVAM
ࢹ࣮ࢱ㸦2006㸧ࡀࡑࡢࡲࡲ⏝࠸ࡽࢀ࡚ゎᯒࡀ࡞ࡉࢀࡓࡀࠊICE ἲࡢᣑ኱㐺⏝ࡢᥦゝ࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ
ࡑࡢᚋࠊ2012 ᖺࠊOECD ᑓ㛛ጤဨ఍࡟ࡼࡾ UN (United Nations) GHS (Globally Harmonized System ofClassification and Labeling of Chemicals) ༊ศୗ࡟࠾࠸࡚෌ホ౯ࡀᐇ᪋ࡉࢀࡓࠋึᅇ࡟౑⏝ࡉࢀࡓࢹ࣮ࢱ࣋
࣮ࢫ࡟࠾ࡅࡿ in vivo ࠾ࡼࡧ in vitro ୧᪉ࡢࢹ࣮ࢱࡀぢ┤ࡉࢀࡓ࡯࠿ࠊin vitro ࡢ༊ศእᇶ‽ࡀṇᘧ࡟タᐃࡉࢀࡓࠋࡑࡇ࡛ᨵࡵ࡚ホ౯ࡀ⾜ࢃࢀࡓ⤖ᯝࠊICE ἲࡣࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ༊ศ 1 ≀㉁ࡢྠᐃࡢࡳ࡞
ࡽࡎࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ༊ศእ≀㉁ࡢྠᐃἲ࡜ࡋ࡚౑⏝ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿ࡜ุ᩿ࡉࢀࠊ2013ᖺ 7 ᭶ 26 ᪥௜ࡅ࡛ᨵゞ TG

438 (TG 438, 2013) ࡜ࡋ࡚᥇ᢥࡉࢀࡓࠋ

TG 438㸦2013㸧ࡢ୺࡞ᨵゞⅬࡣࠊ1) ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟ࡼࡿ UN GHS ༊ศእ≀㉁ྠᐃ࡬ࡢ㐺⏝࠾ࡼࡧ2) 㐺⏝㝈⏺࡛࠶ࡿࠋᮏ㈨ᩱ⦅⧩ጤဨ఍ࡣࠊࡇࡢ TG 438㸦2013㸧ࡢᨵゞⅬ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ௨ୗ࡟ࡑࡢ
⤖ᯝࢆሗ࿌ࡍࡿࠋ
㸰㸬ヨ㦂ࡢᴫせICE ἲ࡛ࡣࠊࢽ࣡ࢺࣜ࠿ࡽ᦬ฟࡋࡓ║⌫࡟⿕㦂≀㉁ࢆᭀ㟢ࡋࠊࡑࡢ⤖ᯝࠊ║⌫࡟⏕ࡌࡿゅ⭷ࡢኚᛶࢆࠊ
ゅ⭷ࡢ⭘⬽ࠊΰ⃮ᗘ࠾ࡼࡧࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶࡢኚ໬࡜ࡋ࡚࡜ࡽ࠼ࡿࠋࡇࢀࡽ 3 ホ౯㡯┠ࡢኚ໬ࢆࡑ
ࢀࡒࢀಶูࡢࢫࢥ࢔࡟ኚ᥮ࡋ࡚ᚓࡽࢀࡿ⥲ྜホ౯ࢆࡶ࡜࡟ in vivo ࡛ࡢ║่⃭ᛶࢆணࡍࡿࠋྛホ౯㡯┠ࡢኚ໬࡜ࡋ࡚ࡣࠊA) ゅ⭷ࡢ⭘⬽㸦ගᏛⓗཌᗘィࢆ⿦╔ࡋࡓ⣽㝽ⅉ㢧ᚤ㙾࡟࡚ゅ⭷ࡢཌࡉࢆᐃࡋ࡚ᭀ㟢 240 ศᚋࡲ࡛ࡢ⤒᫬ⓗ࡞ኚ໬⋡ࢆᐃ㔞ⓗ࡟ồࡵࡿ㸧ࠊB) ゅ⭷ࡢΰ⃮ᗘ㸦⣽㝽ⅉ㢧ᚤ㙾࡟࡚
ゅ⭷ΰ⃮ᗘࡢ⤒᫬ⓗ࡞ኚ໬ࢆᭀ㟢 240 ศᚋࡲ࡛ほᐹࡍࡿ㸧ࠊC) ࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶ㸦⣽㝽ⅉ㢧ᚤ㙾࡟࡚ᭀ㟢 30 ศᚋࡢゅ⭷⾲㠃ࡢࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶ㸧ࢆᐃࡍࡿࡇ࡜࡛࠶ࡿࠋྛ㡯┠ࡢ⤖ᯝࢆയᐖࡢ⛬ᗘ࡟ࡼࡾࠊ║่⃭ᛶࡢ᭱ࡶᙅ࠸ࢡࣛࢫ I ࠿ࡽ᭱ࡶᙉ࠸ࢡࣛࢫ IV ࡢ 4 ẁ㝵࡟ศ㢮ࡋࠊࡑࢀࡽࡢศ㢮⤖ᯝࢆ⥲ྜࡋ࡚ࠊ⿕㦂≀㉁ࡢ║่⃭ᛶࢆุᐃࡍࡿࠋ
㸱㸬ᨵゞⅬ
㸱Ѹ㸯㸬In vivo – in vitro ࢹ࣮ࢱ࣮࣋ࢫࡢぢ┤ࡋ TG 438 㸦2013㸧ࡢᨵゞ࡟ඛ❧ࡕ OECD ᑓ㛛ጤဨ఍࡟ࡼࡾࠊ๓ᅇホ౯ࢆ⾜ࡗࡓ ICCVAM (2006)ࣂࣜࢹ
࣮ࢩࣙࣥࢹ࣮ࢱ࣮࣋ࢫࢆࡶ࡜࡟ࡲ࡜ࡵࡽࢀࡓ SSD (Streamlined Summary Document), Appendix 1 (OECD,


10

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2013) ࠿ࡽ㔜」ࡍࡿ 3 ≀㉁ࢆ㝖እࡋࡓ 175 ≀㉁࡟ࡘ࠸࡚ࠊin vivo ࠾ࡼࡧ in vitro ࡢಶࠎࡢࢹ࣮ࢱ࡟ࡘ࠸࡚෌ホ౯ࡉࢀࡓࠋIn vivo ࢹ࣮ࢱ࡟ࡘ࠸࡚ࡣࠊ⏕ࢹ࣮ࢱࡀḞዴࡋ࡚࠸ࡿ 15 ≀㉁ࢆᑐ㇟࠿ࡽ㝖እࡋࠊࡉࡽ࡟ಶࠎ
ࡢࢹ࣮ࢱࢆぢ┤ࡍࡇ࡜࡟ࡼࡾ UN GHS ༊ศ࡛ࡢ෌ุᐃࡀ࡞ࡉࢀࡓࠋࡲࡓࠊin vitro ࢹ࣮ࢱ࡛ࡣࢹ࣮ࢱࡢぢ┤ࡋ࡜᪂ࡓ࡟タᐃࡋࡓ UN GHS ༊ศ㸦㸱í㸰í㸯㡯㸧࡟ᇶ࡙ࡃ෌ุᐃࡀ࡞ࡉࢀࡓࠋ௨ୖࡢ෌ホ౯࡟࠾࠸࡚ࠊ
᭱⤊ⓗ࡟࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢホ౯࡟ࡣ 175 ≀㉁୰ 152 ≀㉁㸦༢୍≀㉁㸸72ࠊΰྜ≀㉁㸸80㸧ࡀࠊࢺࢵࣉ
ࢲ࢘ࣥ᪉ᘧࡢホ౯࡟ࡣ 140 ≀㉁㸦༢୍≀㉁㸸65ࠊΰྜ≀㉁㸸75㸧ࡀ⏝࠸ࡽࢀࡓࠋ
㸱í㸰㸬࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡢ UN GHS ༊ศእ≀㉁ྠᐃ࡬ࡢ㐺⏝㸱í㸰í㸯㸬 UN GHS ༊ศእ≀㉁ホ౯ࡢ᪂ࡓ࡞ุᐃᇶ‽ࡢタᐃ
 TG 438 (2013)࡛ࡣࠊ᪂ࡓ࡞࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟ࡼࡿ UN GHS ༊ศእ≀㉁ࡢྠᐃ࡟ᑐᛂࡍࡿࡓࡵࠊ༊ศእ≀㉁ホ౯ࡢࡓࡵࡢุᐃᇶ‽ࡀ᪂ࡓ࡟タᐃࡉࢀࡓࠋ㏣ຍࡉࢀࡓุᐃᇶ‽ࢆ⾲ 1 ࡟♧ࡍࠋ    ⾲ 1㸬 UN GHS ༊ศእ≀㉁ホ౯ࡢࡓࡵࡢุᐃᇶ‽UN GHS ༊ศ

3 ホ౯㡯┠ࡢ⤌ࡳྜࢃࡏ㸨༊ศእ

3 㡯┠࡜ࡶࢡࣛࢫ I ࡟ศ㢮ࡉࢀࡿ
2 㡯┠ࡀࢡࣛࢫ I ࡟ศ㢮ࡉࢀࠊ1 㡯┠ࡀࢡࣛࢫ II ࡟ศ㢮ࡉࢀࡿ   㸨㸧3 ホ౯㡯┠㸸ゅ⭷ࡢ⭘⬽ࠊゅ⭷ΰ⃮ࠊゅ⭷ࡢࣇࣝ࢜ࣞࢭ࢖ࣥᰁⰍᛶ      ࢡࣛࢫ I㸸᭱ࡶᙅ࠸║่⃭ᛶ㸱í㸰í㸰㸬࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡢṇ☜ᛶ ୖグࡢࡼ࠺࡟ホ౯ࡋࡓ᪂ࡓ࡞ࢹ࣮ࢱ࣮࣋ࢫࢆ⏝࠸ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ༊ศእ≀㉁ࢆྠᐃࡍࡿ
ሙྜࡢṇ☜ᛶ࡟ࡘ࠸࡚ホ౯ࡉࢀࡓࠋ⤖ᯝࢆ⾲ 2 ࡟♧ࡍࠋ ༊ศእ≀㉁ࡢྠᐃ࡟࠾࠸࡚ࠊDraize ἲࡢ⤖ᯝ࡜ẚ㍑ࡍࡿ࡜ࠊICE ἲࡢṇ☜ᗘࡣ 82㸣㸦125/152㸧ࠊឤᗘ99㸣 㸦72/73㸧ࠊ≉␗ᗘࡣ 67㸣㸦53/79㸧ࠊഇ㝜ᛶ⋡ࡣ 1㸣㸦1/73㸧࠾ࡼࡧഇ㝧ᛶ⋡ࡣ 33㸣㸦26/79㸧ࢆ♧ࡋࡓࠋഇ㝜ᛶ⋡ࡣపࡃࠊഇ㝜ᛶࢆ♧ࡋࡓ≀㉁ࡣ㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱ 3 ≀㉁୰ 1 ≀㉁㸦TNO-94㸧ࡢࡳ࡛࠶ࡗࡓࠋ୍᪉ࠊഇ㝧ᛶ⋡ࡣ㧗࠸ࡀࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡣ㝧ᛶ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊ௚ࡢ㐺ษ࡞ヨ㦂ἲ࡟ࡼࡿ☜ㄆࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࠋ
⾲ 2㸬࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ ICE ἲࡢṇ☜ᛶ㸫UN GHS ༊ศἲ࡛ࡢ༊ศእ≀㉁ࡢྠᐃ 1࣎ࢺ࣒࢔ࢵࣉ
᪉ᘧ

No.

୍⮴ᗘ
㸣
No.

ឤᗘ
㸣
No.

ഇ㝜ᛶ⋡
㸣 No.

≉␗ᗘ
㸣
No.

ഇ㝧ᛶ⋡
㸣
No.

⿕㦂≀㉁

152

82

125/152

99

72/73

1

1/73

67

53/79

33

26/79

㜵ở᭷ᶵ⁐፹
ྵ᭷ሬᩱࢆ㝖እ
ࡋࡓሙྜ

149

83

123/149

100

71/71

0

0/71

67

52/78

33

26/78

No. = ⿕㦂≀㉁ᩘ 
1

UN GHS ༊ศ: (༊ศእ≀㉁) ᑐ (༊ศእ≀㉁௨እ)


11



㸱í㸰í㸱㸬 ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ᪋タෆ࣭᪋タ㛫෌⌧ᛶ➨୕⪅ホ౯ሗ࿌᭩㸦➨୕⪅ホ౯ሗ࿌᭩ 2012ࠊICCVAM BRD : Background Review Document 2006㸧࡛ሗ࿌ࡉࢀ࡚࠸ࡿ࡜࠾ࡾࠊICE ἲࡢゅ⭷⭘⬽ࢆᣦᶆ࡜ࡋࡓሙྜࡢ᪋タෆ CV ್ࡣ 1.8㸣ࠥ6.3㸣࡛࠶ࡗࡓࠋ ⿕㦂≀㉁ࡢ࠺ࡕࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࠾ࡼࡧࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡜ࡶࠊ඲࡚ࡢ᪋タ࡟࠾࠸࡚ UN GHS ༊ศࡀ୍⮴ࡋࡓ⿕㦂≀㉁ࡣ 75㸣ࢆ༨ࡵࡓࠋࡲࡓࠊUN GHS ༊ศ࡜ࡢ୍⮴ࢆᣦᶆ࡜ࡋࡓሙྜࠊ୺ᑟ᪋タ࡜௚ࡢཧຍ᪋タ࡜ࡢ㛫ࡢ┦㛵ಀᩘࡣ 0.829ࠥ0.849 ࡛࠶ࡗࡓࠋ
㸱í㸱㸬ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢ UN GHS ༊ศ㸯≀㉁ྠᐃ࡬ࡢ㐺⏝㸱í㸱í㸯㸬ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢṇ☜ᛶ ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ༊ศ 1 ≀㉁ࢆྠᐃࡍࡿሙྜࡢṇ☜ᛶ࡟ࡘ࠸࡚ࠊ᪂ࡓ࡞ࢹ࣮ࢱ࣮࣋ࢫࢆ⏝࠸࡚ホ౯ࡉࢀࡓࠋ⤖ᯝࢆ⾲ 3 ࡟♧ࡍࠋ ༊ศ 1 ≀㉁ࡢྠᐃ࡟࠾࠸࡚ Draize ἲࡢ⤖ᯝ࡜ẚ㍑ࡍࡿ࡜୍⮴ᗘࡣ 86㸣㸦120/140㸧ࠊഇ㝜ᛶ⋡ࡣ 48㸣
㸦13/27㸧ࠊഇ㝧ᛶ⋡ࡣ 6㸣㸦7/113㸧࡛࠶ࡗࡓࠋ࢔ࣝࢥ࣮ࣝࠊᅛయ࠾ࡼࡧ⏺㠃άᛶ๣ࢆ㝖እࡋࡓሙྜࠊ୍
⮴ᗘࡣୖࡀࡾഇ㝜ᛶ⋡࡜ഇ㝧ᛶ⋡ࡣపୗࡋࡓࠋ
⾲ 3㸬ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ ICE ἲࡢṇ☜ᛶ㸫UN GHS ༊ศ 1 ≀㉁ࡢྠᐃ 1ࢺࢵࣉࢲ࢘ࣥ
᪉ᘧ

No.

⿕㦂≀㉁
࢔ࣝࢥ࣮ࣝࠊᅛ
య࠾ࡼࡧ⏺㠃
άᛶ๣ࢆ㝖እ
ࡋࡓሙྜ

୍⮴ᗘ

ឤᗘ

ഇ㝜ᛶ⋡

≉␗ᗘ

㸣

No.

㸣

No.

㸣

No.

㸣

140

86

120/140

52

14/27

48

13/27

82

94

77/82

71

5/7

29

2/7

No.

ഇ㝧ᛶ⋡
㸣

No.

94 106/113

6

7/113

96

4

3/75

72/75

No. = ⿕㦂≀㉁ᩘ
1

UN GHS ༊ศ: (༊ศ 1 ≀㉁) ᑐ (༊ศ 1 ≀㉁௨እ)㸱í㸱í㸰㸬 ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ᪋タෆ࣭᪋タ㛫෌⌧ᛶ ➨୕⪅ホ౯ሗ࿌᭩㸦➨୕⪅ホ౯ሗ࿌᭩ 2012ࠊICCVAM BRD 2006㸧࡛ሗ࿌ࡉࢀ࡚࠸ࡿ࡜࠾ࡾࠊࢺࢵࣉࢲ
࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ᪋タෆ࣭᪋タ㛫෌⌧ᛶࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡜ྠᵝ࡞⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿࠋ
㸱í㸲㸬㐺⏝㝈⏺
 㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱࠊᅛయ≀㉁ࠊ⏺㠃άᛶ๣࠾ࡼࡧ࢔ࣝࢥ࣮ࣝ㢮࡟ࡘ࠸࡚ࡣࠊ㧗࠸ഇ㝜ᛶ⋡࠶ࡿ࠸ࡣഇ㝧ᛶ⋡ࡀ♧ࡉࢀࡓࡀࠊ௨ୗࡢ⌮⏤࠿ࡽࡇࢀࡽࡢ໬Ꮫ≀㉁࡟ࡶ ICE ἲࢆ㐺⏝ࡍࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ሗ࿌
ࡉࢀࡓࠋ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ㸸y㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱ࡛㝧ᛶ 2 ≀㉁ࡢ࠺ࡕࠊ1 ≀㉁㸦TNO-94㸧ࡀഇ㝜ᛶ࡛࠶ࡾࠊ⏝㏵ࡢ㢮ఝࡋࡓ໬ྜ≀㸦TNO-93㸧࡟ࡣㄆࡵࡽࢀ࡞࠿ࡗࡓࠋᚑࡗ࡚ࠊICE ἲ࡬ࡢ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿ࡟ࡣ᰿ᣐࡀ୙


12



༑ศ࡛࠶ࡾࠊ㜵ở᭷ᶵ⁐፹ྵ᭷ሬᩱࢆ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸࡜ࡉࢀࡓࠋy༢୍≀㉁ࠊΰྜ≀㉁ࠊᾮయࠊᅛయ➼࡟࠾ࡅࡿഇ㝜ᛶ⋡ࡣ 5㸣௨ୗ࡜పࡃࠊഇ㝧ᛶ⋡࡟ࡘ࠸࡚ࡣ 33㸣࡜௚ࡢヨ㦂ἲ㸦BCOP: Bovine Corneal Opacity and Permeability㸹 69㸣ࠊCM : Cytosensor
Microphysiometer㸹68㸣㸧࡟ẚ࡭ప࠸ࡇ࡜ࡀ♧ࡉࢀࡓࠋ༢୍≀㉁࠾ࡼࡧ⏺㠃άᛶ๣࡟ࡘ࠸࡚ࡢഇ㝧ᛶ⋡ࡣ㧗࠸ࡀࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ࡣ㝧ᛶ࡜ุ᩿ࡉࢀࡓሙྜࠊ௚ࡢ㐺ษ࡞ヨ㦂ἲ࡟ࡼࡿ☜ㄆࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊࡇࢀࡽࡢ໬Ꮫ≀㉁ࢆ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸࡜ࡉࢀࡓࠋ

ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ㸸yഇ㝜ᛶ⋡ࡣᅛయ≀㉁㸦n=34, 55㸣=6/11㸧࠾ࡼࡧ⏺㠃άᛶ๣㸦n=21, 67㸣=6/9㸧࡟࠾࠸࡚㧗ࡃࠊഇ㝧ᛶ⋡ࡣ࢔ࣝࢥ࣮ࣝ㢮㸦n=12, 0㸣=4/10㸧࡟࠾࠸࡚㧗࠿ࡗࡓࠋࡋ࠿ࡋࠊࡇࢀࡽࡢศ㢮࡟࠾࠸࡚ࡶ║่⃭ᛶࡀṇ☜࡟ண

ࡉࢀࡓ≀㉁ࡀ࠶ࡿࡇ࡜ࠊࡉࡽ࡟ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㝜ᛶ࡜ุ᩿ࡉࢀࡓሙྜ

ࡣ௚ࡢ㐺ษ࡞ヨ㦂ἲ࡟ࡼࡿ☜ㄆࡀᚲせ࡜ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊᅛయ≀㉁࠾ࡼࡧ⏺㠃άᛶ๣ࢆ㐺⏝㡿ᇦ࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸࡜ࡉࢀࡓࠋࡓࡔࡋࠊ࢔ࣝࢥ࣮ࣝ㢮࡛㝧ᛶ⤖ᯝࡀᚓࡽࢀࡓሙྜࡣࠊ⤖ᯝࡢゎ㔘ࡣៅ㔜࡟ࡍ࡭ࡁ࡜ࡉࢀࡓࠋ ௨ୖ࠿ࡽࠊICE ἲࡣ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡢ༊ศእ≀㉁࠾ࡼࡧࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢ༊ศ 1 ≀㉁ࡢྠᐃἲ
࡜ࡋ࡚ࠊࡍ࡭࡚ࡢ✀㢮ࡢ໬Ꮫ≀㉁࡟㐺⏝࡛ࡁࡿ࡜ࡉࢀࡓࠋ
㸱í㸳㸬ࡑࡢ௚y⩦⇍ᗘ☜ㄆ≀㉁ࡢ㏣ຍ㸸  TG 438㸦2009㸧࡛ࡣࠊICE ἲࡢ⩦⇍ᗘࢳ࢙ࢵࢡ⏝≀㉁࡜ࡋ࡚ 10 ≀㉁ࡀ᥎ዡࡉࢀࡓࠋࡋ࠿ࡋࠊ10 ᥎ዡ≀㉁ࡢ୰࡟ࠊ༊ศእ≀㉁ࡀྵࡲࢀ࡚࠸࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊᮏᨵゞ∧࡛ࡣࠊ3 ✀㢮ࡢ༊ศእ≀㉁ࡀ
㏣ຍࡉࢀࠊ௨ୗࡢࡼ࠺࡞⩦⇍ᗘ☜ㄆ≀㉁࡜ࡋ࡚ࡲ࡜ࡵࡽࢀࡓ㸦⾲ 4㸧ࠋᗘ㸧࡜ࡋ࡚“18Υ࠿ࡽ 25Υ”ࡢ

y

║⌫ࡢ㐠ᦙ

y

⿕㦂≀㉁ᭀ㟢ᚋࡢ║⌫Ὑί࡟࠾࠸࡚ࠊ“㏣ຍࡢὙί”ࡀㄆࡵࡽࢀࡓࠋyሗ࿌᭩࡬ࡢグ㍕㡯┠ࡀ㏣ຍࡉࢀࡓࠋyㄒྃࡢᐃ⩏࡟ࡘ࠸࡚㏣ຍ࣭ゞṇࡀ࡞ࡉࢀࡓࠋy

ICE ἲุᐃ࡟㛵ࡍࡿ⏝ㄒ࡜ࡋ࡚ࠊ࢝ࢸࢦ࣮ࣜࡀࢡࣛࢫ࡜࠸࠺⏝ㄒ࡟⨨ࡁ᥮ࢃࡗࡓࠋᗘ㸦እ⏺

ᗘ⠊ᅖࡀ♧ࡉࢀࡓࠋ


13



⾲ 4㸬ICE ἲࡢ⩦⇍ᗘ☜ㄆ≀㉁
໬Ꮫ≀㉁

CAS␒ྕ

ሷ໬࣋ࣥࢨࣝࢥࢽ࢘㸦5㸣㸧
8001-54-5
Benzalkonium chloride (5㸣)ࢡࣟࣝ࣊࢟ࢩࢪࣥ
Chlorhexidine

55-56-1ࢪ࣋ࣥࢰ࢖ࣝ-L-㓇▼㓟
Dibenzoyl-L- tartaric acid2743-38-6

࢖࣑ࢲࢰ࣮ࣝ
Imidazole288-32-4

ࢺࣜࢡࣟࣟ㓑㓟㸦30㸣㸧

ศ㢮
࢜ࢽ࣒࢘
໬ྜ≀㢮
Onium
compound࢔࣑ࣥ㢮࢔࣑ࢪࣥ㢮
Amine, Amidine࢝ࣝ࣎ࣥ㓟㢮
࢚ࢫࢸࣝ㢮
Carboxylic acid,
Ester
࣊ࢸࣟ
ࢧ࢖ࢡࣜࢵࢡ໬ྜ≀㢮Heterocyclic≀⌮ⓗ≧ែ

In vivo࡛ࡢ༊ศ

ICE࡛ࡢ༊ศ

ᾮయLiquid༊ศ1Category 1

༊ศ1Category 1

ᅛయ
Solid

༊ศ1
Category 1

༊ศ1Category 1

ᅛయ
Solid

༊ศ1
Category 1

༊ศ1Category 1

ᅛయ
Solid

༊ศ1
Category 1

༊ศ1Category 1

76-03-9࢝ࣝ࣎ࣥ㓟㢮
Carboxylic acidᾮయLiquid༊ศ1Category 1

༊ศ1Category 1

2,6-ࢪࢡࣟࣟ࣋ࣥࢰ࢖ࣝ
ࢡࣟࣜࢻDichlorobenzoyl chloride

4659-45-4

࢔ࢩࣝ
ࣁࣛ࢖ࢻ㢮
Acyl halideᾮయLiquid༊ศ2ACategory 2A༊ศ୙ྍ◪㓟࢔ࣥࣔࢽ࣒࢘
Ammonium nitrate

6484-52-2

ᅛయ
Solid

༊ศ2B
Category 2B༊ศ୙ྍ࢚ࢳࣝ-2-࣓ࢳࣝ࢔ࢭࢺ㓑㓟
Ethyl-2-methylacetoacetate609-14-3

ᾮయLiquid༊ศ2B
Category 2B༊ศ୙ྍࢪ࣓ࢳࣝࢫࣝ࣍࢟ࢩࢻ
Dimethyl sulfoxide

67-68-5↓ᶵሷ
Inorganic salt
ࢣࢺࣥ㢮
࢚ࢫࢸࣝ㢮
Ketone, Ester᭷ᶵ◲㯤໬ྜ≀
Organic sulphur
compoundᾮయ
Liquid༊ศእ
Not Classified༊ศእ
Not Classifiedࢢࣜࢭ࣮ࣟࣝ
Glycerol

56-81-5࢔ࣝࢥ࣮ࣝ㢮
AlcoholᾮయLiquid༊ศእ
Not Classified༊ศእ
Not Classifiedᾮయ
Liquid༊ศእ
Not Classified༊ศእ
Not Classifiedᾮయ
Liquid༊ศእ
Not Classified༊ศእ
Not Classifiedᾮయ
Liquid༊ศእ
Not Classified༊ศእ
Not Classified

Trichloroacetic acid (30㸣)࣓ࢳࣝࢩࢡࣟ࣌ࣥࢱࣥ
Methylcyclopentane

96-37-7n-࣊࢟ࢧࣥ
n-Hexane

110-54-3

ࢺࣜ࢔ࢭࢳࣥ
Triacetin102-76-1

Ⅳ໬Ỉ⣲㸦⎔≧㸧Hydrocarbon(cyclo)Ⅳ໬Ỉ⣲㸦㙐≧㸧Hydrocarbon(acylic)
⬡㉁
Lipid


14



㸲㸬⤖ㄽ
 TG 438㸦2013㸧࡛ࡣࠊࡑࡢṇ☜ᛶ࡜෌⌧ᛶࡢ⤖ᯝ࠿ࡽࠊUN GHS ࡢ║่⃭ᛶ༊ศ࡟࠾࠸࡚ࢺࢵࣉࢲ࢘
ࣥ᪉ᘧ࡟࠾ࡅࡿ༊ศ 1 ≀㉁ࡢྠᐃࡢࡳ࡞ࡽࡎࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ༊ศእ≀㉁ࡢྠᐃ࡟ ICE ἲࢆ㐺⏝ࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡾࠊ࡝ࡕࡽࡢ᪉ᘧ࡟࠾࠸࡚ࡶ㐺⏝≀㉁࡟ไ㝈ࢆタࡅࡿᚲせࡣ࡞࠸࡜ࡉࢀ࡚࠸ࡿࠋ
║่⃭ᛶ㈨ᩱ⦅⧩ጤဨ఍࡜ࡋ࡚ࡶࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾ࡅࡿ UN GHS ༊ศ 1 ≀㉁࡞ࡽࡧ࡟࣎ࢺ࣒࢔
ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ UN GHS ༊ศእ≀㉁ࡢྠᐃ࡟ࠊDraize ἲࡢ௦᭰ヨ㦂ἲ࡜ࡋ࡚ TG 438㸦2013㸧ࢆ㐺⏝ࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿ࡜⪃࠼ࡓࠋཧ⪃ᩥ⊩
㸯㸧OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test Method for     
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring     Classification for EyeIrritation or Serious Eye Damage, TG438 (Adopted 26 July 2013)
㸰㸧OECD Guidelines for The Testing of Chemicals, Isolated Chicken Eye Test for Identifying OcularCorrosives and Severe Irritants, TG438 (Adopted 7 September 2009)
㸱㸧Streamlined Summary Document Supporting OECD Test Guideline 438 on the Isolated Chicken Eye for Eye
Irritation/Corrosion. Series on Testing and Assessment No. 188 (Part 1 and Part 2), OECD, Paris. (21 June
2013)
㸲㸧ICCVAM, Background Review Document: Current Status of In Vitro Test Methods for Identifying OcularCorrosives and Severe Irritants: Isolated Chicken Eye Test Methods. NIH Publication No. 06-4513 (2006).
㸳㸧ICCVAM, 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. Volume 1 and Volume 2 (2010).
㸴㸧JaCVAM, ║่⃭ᛶヨ㦂௦᭰ἲࡢ➨୕⪅ホ౯ሗ࿌᭩̺ホ౯ᑐ㇟ヨ㦂㸸║࡟ᑐࡍࡿ⭉㣗ᛶ࠾ࡼࡧᙉ่⃭ᛶホ౯ࡢࡓࡵࡢࢽ࣡ࢺࣜ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂ἲ, AATEX-JaCVAM J1, 16-29 (2012)


15

16

OECD/OCDE

438
Adopted:26 July 2013

OECD GUIDELINES FOR THE TESTING OF CHEMICALSIsolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage
and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage

INTRODUCTION
1.
The Isolated Chicken Eye (ICE) test method was evaluated by the Interagency Coordinating
Committee on the Validation of Alternative Methods (ICCVAM), in conjunction with the European
Centre for the Validation of Alternative Methods (ECVAM) and the Japanese Centre for the
Validation of Alternative Methods (JaCVAM), in 2006 and 2010 (1) (2) (3). In the first evaluation, theICE was endorsed as a scientifically valid test method for use as a screening test to identify chemicals(substances and mixtures) inducing serious eye damage (Category 1) as defined by the United Nations(UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) (1) (2) (4). In
the second evaluation, the ICE test method was evaluated for use as a screening test to identify
chemicals not classified for eye irritation or serious eye damage as defined by UN GHS (3) (4). The
results from the validation study and the peer review panel recommendations maintained the original
recommendation for using the ICE for classification of chemicals inducing serious eye damage (UNGHS Category 1), as the available database remained unchanged since the original ICCVAM
validation. At that stage, no further recommendations for an expansion of the ICE applicability domain
to also include other categories were suggested. A re-evaluation of the in vitro and in vivo dataset usedin the validation study was made with the focus of evaluating the usefulness of the ICE to identify
chemicals not requiring classification for eye irritation or serious eye damage (5). This re-evaluation
concluded that the ICE test method can also be used to identify chemicals not requiring classificationfor eye irritation and serious eye damage as defined by the UN GHS (4) (5). This Test Guideline(adopted in 2009 and updated in 2013) includes the recommended uses and limitations of the ICE test
method based on these evaluations. The main differences between the original 2009 version and the2013 updated version include, but are not limited to, the use of the ICE test method to identify
chemicals not requiring classification according to the UN GHS Classification System, an update to
the test report elements, an update of Annex 1 on definitions, and an update to Annex 2 on the
proficiency chemicals.
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 for different chemical classes. However, strategic combinations of several alternative test
methods within a (tiered) testing strategy may be able to replace the Draize eye test (6). The TopDown approach (7) is designed to be used when, based on existing information, a chemical is expected
to have high irritancy potential, while the Bottom-Up approach (7) is designed to be used when, basedon existing information, a chemical is expected not to cause sufficient eye irritation to require aclassification. The ICE test method is an in vitro test method that can be used, under certain
circumstances and with specific limitations as described in paragraphs 8 to 10 for eye hazard
classification and labelling of chemicals. While it is not considered valid as a stand-alone replacement
1© OECD, (2013)
You are free to use this material for personal, non-commercial purposes without seeking prior consent
from the OECD, provided the source is duly mentioned. Any commercial use of this material is subject
to written permission from the OECD.

17

438

OECD/OCDE

for the in vivo rabbit eye test, the ICE test method is recommended as an initial step within a testing
strategy such as the Top-Down approach suggested by Scott et al. (7) to identify chemicals inducing
serious eye damage, i.e., chemicals to be classified as UN GHS Category 1 without further testing (4).
The ICE test method is also recommended to identify chemicals that do not require classification foreye irritation or serious eye damage as defined by the UN GHS (No Category, NC) (4), and may
therefore be used as an initial step within a Bottom-Up testing strategy approach (7). However, achemical that is not predicted as causing serious eye damage or as not classified for eye
irritation/serious eye damage with the ICE test method would require additional testing (in vitro and/or
in vivo) to establish a definitive classification. Furthermore, the appropriate regulatory authorities
should be consulted before using the ICE in a bottom up approach under other classification schemes
than the UN GHS.
3.
The purpose of this Test Guideline is to describe the procedures used to evaluate the eyehazard potential of a test chemical as measured by its ability to induce or not toxicity in an enucleatedchicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity, (ii) a
qualitative assessment of damage to epithelium based on application of fluorescein to the eye(fluorescein retention), (iii) a quantitative measurement of increased thickness (swelling), and (iv) a
qualitative evaluation of macroscopic morphological damage to the surface. The corneal opacity,swelling, and damage assessments following exposure to a test chemical are assessed individually andthen combined to derive an Eye Irritancy Classification.
4.

Definitions are provided in Annex 1.

INITIAL CONSIDERATIONS AND LIMITATIONS
5.
This Test Guideline is based on the protocol suggested in the OECD Guidance Document
160 (8), which was developed following the ICCVAM international validation study (1) (3) (9), with
contributions from the European Centre for the Validation of Alternative Methods, the JapaneseCenter for the Validation of Alternative Methods, and TNO Quality of Life Department of Toxicology
and Applied Pharmacology (Netherlands). The protocol is based on information obtained frompublished protocols, as well as the current protocol used by TNO (10) (11) (12) (13) (14).
6.
A wide range of chemicals has been tested in the validation underlying this Test Guideline
and the empirical database of the validation study amounted to 152 chemicals including 72 substancesand 80 mixtures (5). The Test Guideline is applicable to solids, liquids, emulsions and gels. The
liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Gases andaerosols have not been assessed yet in a validation study.
7.
The ICE test method can be used to identify chemicals inducing serious eye damage, i.e.,chemicals to be classified as UN GHS Category 1 (4). When used for this purpose, the identified
limitations for the ICE test method are based on the high false positive rates for alcohols and thehigh false negative rates for solids and surfactants (1) (3) (9). However, false negative rates in thiscontext (UN GHS Category 1 identified as not being UN GHS Category 1) are not critical since all
test chemicals that come out negative would be subsequently tested with other adequately validated
in vitro test(s), or as a last option in rabbits, depending on regulatory requirements, using a sequential
testing strategy in a weight-of-evidence approach. 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 (15). Investigators could consider using this test method for
all types of chemicals, whereby a positive result should be accepted as indicative of serious eye
2© OECD, (2013).

18

OECD/OCDE

438

damage, i.e., UN GHS Category 1 classification without further testing. However, positive results
obtained with alcohols should be interpreted cautiously due to risk of over-prediction.
8.
When used to identify chemicals inducing serious eye damage (UN GHS Category 1), theICE test method has an overall accuracy of 86% (120/140), a false positive rate of 6% (7/113) and afalse negative rate of 48% (13/27) when compared to in vivo rabbit eye test method data classified
according to the UN GHS classification system (4) (5).
9.
The ICE 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). The appropriate
regulatory authorities should be consulted before using the ICE in a bottom up approach under otherclassification schemes. This test method can be used for all types of chemicals, whereby a negative
result could be accepted for not classifying a chemical for eye irritation and serious eye damage.
However, on the basis of one result from the validation database, anti-fouling organic solventcontaining paints may be under-predicted (5).
10.
When used to identify chemicals that do not require classification for eye irritation andserious eye damage, the ICE test method has an overall accuracy of 82% (125/152), a false positive
rate of 33% (26/79), and a false negative rate of 1% (1/73), when compared to in vivo rabbit eye test
method data classified according to the UN GHS (4) (5). When test chemicals within certain classes(i.e., anti-fouling organic solvent containing paints) are excluded from the database, the accuracy of
the ICE test method is 83% (123/149), the false positive rate 33% (26/78), and the false negative
rate of 0% (0/71) for the UN GHS classification system (4) (5).
11.
The ICE test method is not recommended for the identification of test chemicals that should
be classified as irritating to eyes (i.e., UN GHS Category 2 or Category 2A) or test chemicals 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. For this purpose,
further testing with another suitable method may be required.
12.
All procedures with chicken eyes should follow the test facility’s applicable regulations andprocedures for handling of human or animal-derived materials, which include, but are not limited to,tissues and tissue fluids. Universal laboratory precautions are recommended (16).
13.
Whilst the ICE test method does not consider conjunctival and iridal injuries as evaluated in
the rabbit ocular irritancy test method, it addresses corneal effects which are the major driver ofclassification in vivo when considering the UN GHS Classification. Also, although the reversibility ofcorneal lesions cannot be evaluated per se in the ICE 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 (17). In particular, further scientific knowledge is required tounderstand how irreversible effects not linked with initial high level injury occur. Finally, the ICE test
method does not allow for an assessment of the potential for systemic toxicity associated with ocular
exposure.
14.
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. To evaluate this possibility, users are encouraged to preserve eyes andprepare histopathology specimens that can be used to develop a database and decision criteria that may
further improve the accuracy of this test method. The OECD has developed a Guidance Document on
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the use of in vitro ocular toxicity test methods, which includes detailed procedures on the collection ofhistopathology specimens and information on where to submit specimens and/or histopathology data
(8).
15.
For any laboratory initially establishing this assay, the proficiency chemicals provided in
Annex 2 should be used. A laboratory can use these chemicals to demonstrate their technicalcompetence in performing the ICE test method prior to submitting ICE data for regulatory hazard
classification purposes.
PRINCIPLE OF THE TEST16.
The ICE test method is an organotypic model that provides short-term maintenance of thechicken eye in vitro. In this test method, damage by the test chemical is assessed by determination of
corneal swelling, opacity, and fluorescein retention. While the latter two parameters involve a
qualitative assessment, analysis of corneal swelling provides for a quantitative assessment. Eachmeasurement is either converted into a quantitative score used to calculate an overall Irritation Index,
or assigned a qualitative categorization that is used to assign an in vitro ocular hazard classification,either as UN GHS Category 1 or as UN GHS non-classified. Either of these outcomes can then be used
to predict the potential in vivo serious eye damage or no requirement for eye hazard classification of atest chemical (see Decision Criteria). However, no classification can be given for chemicals notpredicted as causing serious eye damage or as not classified with the ICE test method (seeparagraph 11).
Source and Age of Chicken Eyes
17.
Historically, eyes collected from chickens obtained from a slaughterhouse where they are
killed for human consumption have been used for this assay, eliminating the need for laboratory
animals. Only the eyes of healthy animals considered suitable for entry into the human food chain are
used.
18.
Although a controlled study to evaluate the optimum chicken age has not been conducted,
the age and weight of the chickens used historically in this test method are that of spring chickens
traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
Collection and Transport of Eyes to the Laboratory
19.
Heads should be removed immediately after sedation of the chickens, usually by electric
shock, and incision of the neck for bleeding. A local source of chickens close to the laboratory should
be located so that their heads can be transferred from the slaughterhouse to the laboratory quickly
enough to minimize deterioration and/or bacterial contamination. The time interval between collectionof the chicken heads and placing the eyes in the superfusion chamber following enucleation should be
minimized (typically within two hours) to assure meeting assay acceptance criteria. All eyes used in
the assay should be from the same group of eyes collected on a specific day.
20.
Because eyes are dissected in the laboratory, the intact heads are transported from theslaughterhouse at ambient temperature (typically between 18oC and 25oC) in plastic boxes humidified
with tissues moistened with isotonic saline.

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Selection Criteria and Number of Eyes Used in the ICE
21.
Eyes that have high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score(i.e., > 0.5) after they are enucleated are rejected.
22.
Each treatment group and concurrent positive control consists of at least three eyes. The
negative control group or the solvent control (if using a solvent other than saline) consists of at least
one eye.
23.
In the case of solid materials leading to a GHS NC outcome, a second run of three eyes isrecommended to confirm or discard the negative outcome.
PROCEDURE
Preparation of the Eyes
24.
The eyelids are carefully excised, taking care not to damage the cornea. Corneal integrity isquickly assessed with a drop of 2% (w/v) sodium fluorescein applied to the corneal surface for a few
seconds, and then rinsed with isotonic saline. Fluorescein-treated eyes are then examined with a slitlamp microscope to ensure that the cornea is undamaged (i.e., fluorescein retention and corneal
opacity scores ≤ 0.5).
25.
If undamaged, the eye is further dissected from the skull, taking care not to damage thecornea. The eyeball is pulled from the orbit by holding the nictitating membrane firmly with surgicalforceps, and the eye muscles are cut with a bent, blunt-tipped scissor. It is important to avoid causing
corneal damage due to excessive pressure (i.e., compression artifacts).
26.
When the eye is removed from the orbit, a visible portion of the optic nerve should be left
attached. Once removed from the orbit, the eye is placed on an absorbent pad and the nictitating
membrane and other connective tissue are cut away.
27.
The enucleated eye is mounted in a stainless steel clamp with the cornea positioned
vertically. The clamp is then transferred to a chamber of the superfusion apparatus (18). The clamps
should be positioned in the superfusion apparatus such that the entire cornea is supplied with theisotonic saline drip (3-4 drops per minute or 0.1 to 0.15 mL/min). The chambers of the superfusion
apparatus should be temperature controlled at 32 ± 1.5°C. Annex 3 provides a diagram of a typicalsuperfusion apparatus and the eye clamps, which can be obtained commercially or constructed. The
apparatus can be modified to meet the needs of an individual laboratory (e.g., to accommodate a
different number of eyes).
28.
After being placed in the superfusion apparatus, the eyes are again examined with a slit-lamp
microscope to ensure that they have not been damaged during the dissection procedure. Corneal
thickness should also be measured at this time at the corneal apex using the depth measuring device on
the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal
opacity > 0.5; or, (iii), any additional signs of damage should be replaced. For eyes that are notrejected based on any of these criteria, individual eyes with a corneal thickness deviating more than
10% from the mean value for all eyes are to be rejected. Users should be aware that slit-lamp
microscopes could yield different corneal thickness measurements if the slit-width setting is different.
The slit-width should be set at 0.095 mm.

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29.
Once all eyes have been examined and approved, the eyes are incubated for approximately
45 to 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration
period, a zero reference measurement is recorded for corneal thickness and opacity to serve as abaseline (i.e., time = 0). The fluorescein score determined at dissection is used as the baseline
measurement for that endpoint.
Application of the Test Chemical
30.
Immediately following the zero reference measurements, the eye (in its holder) is removed
from the superfusion apparatus, placed in a horizontal position, and the test chemical is applied to thecornea.
31.
Liquid test chemicals are typically tested undiluted, but may be diluted if deemed necessary(e.g., as part of the study design). The preferred solvent for diluted test chemicals is physiologicalsaline. However, alternative solvents may also be used under controlled conditions, but theappropriateness of solvents other than physiological saline should be demonstrated.
32.
Liquid test chemicals are applied to the cornea such that the entire surface of the cornea isevenly covered with the test chemical; the standard volume is 0.03 mL.
33.
If possible, solid test chemicals should be ground as finely as possible in a mortar and pestle,
or comparable grinding tool. The powder is applied to the cornea such that the surface is uniformly
covered with the test chemical; the standard amount is 0.03 g.
34.
The test chemical (liquid or solid) is applied for 10 seconds and then rinsed from the eyewith isotonic saline (approximately 20 mL) at ambient temperature. The eye (in its holder) is
subsequently returned to the superfusion apparatus in the original upright position. In case of need,additional rinsing may be used after the 10-sec application and at subsequent time points (e.g., upon
discovery of residues of test chemical on the cornea). In general the amount of saline additionally used
for rinsing is not critical, but the observation of adherence of chemical to the cornea is important.
Control Substances
35.
Concurrent negative or solvent/vehicle controls and positive controls should be included ineach experiment.
36.
When testing liquids at 100% or solids, physiological saline is used as the concurrent
negative control in the ICE test method to detect non-specific changes in the test system, and to ensurethat the assay conditions do not inappropriately result in an irritant response.
37.
When testing diluted liquids, a concurrent solvent/vehicle control group is included in thetest method to detect non-specific changes in the test system, and to ensure that the assay conditions
do not inappropriately result in an irritant response. As stated in paragraph 31, only a solvent/vehicle
that has been demonstrated to have no adverse effects on the test system can be used.
38.
A known ocular irritant is included as a concurrent positive control in each experiment toverify that an appropriate response is induced. As the ICE assay is being used in this Test Guideline toidentify corrosive or severe irritants, the positive control should be a reference substance that induces asevere response in this test method. However, to ensure that variability in the positive control response
across time can be assessed, the magnitude of the severe response should not be excessive. Sufficient
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in vitro data for the positive control should be generated such that a statistically defined acceptable
range for the positive control can be calculated. If adequate historical ICE test method data are notavailable for a particular positive control, studies may need to be conducted to provide thisinformation.
39.
Examples of positive controls for liquid test chemicals are 10% acetic acid or 5%
benzalkonium chloride, while examples of positive controls for solid test chemicals are sodiumhydroxide or imidazole.
40.
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
41.
Treated corneas are evaluated prior to treatment and at 30, 75, 120, 180, and 240 minutes (±5 minutes) after the post-treatment rinse. These time points provide an adequate number ofmeasurements over the four-hour treatment period, while leaving sufficient time between
measurements for the requisite observations to be made for all eyes.
42.
The endpoints evaluated are corneal opacity, swelling, fluorescein retention, andmorphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the
exception of fluorescein retention (which is determined only prior to treatment and 30 minutes after
test chemical exposure) are determined at each of the above time points.
43.
Photographs are advisable to document corneal opacity, fluorescein retention, morphologicaleffects and, if conducted, histopathology.
44.
After the final examination at four hours, users are encouraged to preserve eyes in an
appropriate fixative (e.g., neutral buffered formalin) for possible histopathological examination (seeparagraph 14 and reference (8) for details).
45.
Corneal swelling is determined from corneal thickness measurements made with an opticalpachymeter on a slit-lamp microscope. It is expressed as a percentage and is calculated from corneal
thickness measurements according to the following formula:§corneal thickness at time t  corneal thickness at time 0 ·¨¸ u 100
corneal thickness at time 0
©
¹
46.
The mean percentage of corneal swelling for all test eyes is calculated for all observation
time points. Based on the highest mean score for corneal swelling, as observed at any time point, an
overall category score is then given for each test chemical (see paragraph 51).
47.
Corneal opacity is evaluated by using the area of the cornea that is most densely opacified
for scoring as shown in Table 1. The mean corneal opacity value for all test eyes is calculated for allobservation time points. Based on the highest mean score for corneal opacity, as observed at any time
point, an overall category score is then given for each test chemical (see paragraph 51).

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Table 1. Corneal opacity scores.
Score

Observation0

No opacity

0.5

Very faint opacity

1

Scattered or diffuse areas; details of the iris are clearly visible

2

Easily discernible translucent area; details of the iris are slightly
obscured3

Severe corneal opacity; no specific details of the iris are visible; sizeof the pupil is barely discernible

4Complete corneal opacity; iris invisible

48.
Fluorescein retention is evaluated at the 30 minute observation time point only as shown in
Table 2. The mean fluorescein retention value of all test eyes is then calculated for the 30-minute
observation time point, and used for the overall category score given for each test chemical (seeparagraph 51).
Table 2. Fluorescein retention scores.
Score

Observation0

No fluorescein retention

0.5

Very minor single cell staining

1

Single cell staining scattered throughout the treated area of thecornea2

Focal or confluent dense single cell staining

3

Confluent large areas of the cornea retaining fluorescein

49.
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium,“roughening” of the corneal surface and “sticking” of the test chemical to the cornea. These findings
can vary in severity and may occur simultaneously. The classification of these findings is subjective
according to the interpretation of the investigator.
DATA AND REPORTING
Data Evaluation
50.
Results from corneal opacity, swelling, and fluorescein retention should be evaluatedseparately to generate an ICE class for each endpoint. The ICE classes for each endpoint are then
combined to generate an Irritancy Classification for each test chemical.
Decision Criteria
51.
Once each endpoint has been evaluated, ICE classes can be assigned based on apredetermined range. Interpretation of corneal swelling (Table 3), opacity (Table 4), and fluorescein
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retention (Table 5) using four ICE classes is done according to the scales shown below. It is important
to note that the corneal swelling scores shown in Table 3 are only applicable if thickness is measured
with a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. 1 and slit-width
setting at 9½, equalling 0.095 mm. Users should be aware that slit-lamp microscopes could yield
different corneal thickness measurements if the slit-width setting is different.
Table 3. ICE classification criteria for corneal swelling.
Mean Corneal Swelling (%)

ICE Class0 to 5I>5 to 12II

>12 to 18 (>75 min after treatment)II>12 to 18 (≤75 min after treatment)III>18 to 26III>26 to 32 (>75 min after treatment)III>26 to 32 (≤75 min after treatment)

IV>32
Highest mean score observed at any time point

IV*

Table 4. ICE classification criteria for opacity.
Maximum Mean Opacity Score*

ICE Class

0.0-0.5I

0.6-1.5II

1.6-2.5III

2.6-4.0
IV
Maximum mean score observed at any time point (based on opacity scores as
defined in Table 1).
*

Table 5. ICE classification criteria for mean fluorescein retention.
Mean Fluorescein Retention Score
at 30 minutes post-treatment*

ICE Class

0.0-0.5I

0.6-1.5II

1.6-2.5III

2.6-3.0
Based on scores as defined in Table 2.

IV

*

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The in vitro classification for a test chemical is assessed by reading the GHS classificationthat corresponds to the combination of categories obtained for corneal swelling, corneal opacity, andfluorescein retention as described in Table 6.
Table 6. Overall in vitro classifications.
UN GHSClassification

Combinations of the 3 Endpoints

No Category

3xI
2 x I, 1 x II

No prediction
can be madeOther combinations

Category 1

3 x IV
2 x IV, 1 x III
2 x IV, 1 x II*
2 x IV, 1 x I*
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of the epithelium (in at least 1 eye)*Combinations less likely to occur.
Study Acceptance Criteria
53.
A test is considered acceptable if the concurrent negative or vehicle/solvent controls and theconcurrent positive controls are identified as GHS Non-Classified and GHS Category 1, respectively.
Test Report54.
study:The test report should include the following information, if relevant to the conduct of the
Test Chemical 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 chemical/control substance or preparation (inpercentage(s) by weight), to the extent this information is available;
 Physicochemical properties such as physical state, volatility, pH, stability, chemical classwater solubility relevant to the conduct of the study;
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 Treatment of the test chemical/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;
 Identification on the source of the eyes (e.g., the facility from which they werecollected);
Test Method Conditions
 Description of test system used; Slit-lamp microscope used (e.g., model) and instrument settings for the slit-lamp
microscope used; Reference to historical negative and positive control results and, if applicable, historical
data demonstrating acceptable concurrent benchmark control ranges;
 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)).
Eyes Collection and Preparation
 Age and weight of the donor animal and if available, other specific characteristics of theanimals from which the eyes were collected (e.g., sex, strain); Storage and transport conditions of eyes (e.g., date and time of eye collection, timeinterval between collection of chicken heads and placing the enucleated eyes insuperfusion chamber); Preparation & mounting of the eyes including statements regarding their quality,
temperature of eye chambers, and criteria for selection of eyes 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 dose, application and exposure time used; Observation time points (pre- and post- treatment); Description of evaluation and decision criteria used;
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OECD/OCDE Description of study acceptance criteria used; Description of any modifications of the test procedure.
Results
 Tabulation of corneal swelling, opacity and fluorescein retention scores obtained for eachindividual eye and at each observation time point, including the mean scores at eachobservation time of all tested eyes; The highest mean corneal swelling, opacity and fluorescein retention scores observed(from any time point), and its relating ICE class.
 Description of any other effects observed;
 The derived in vitro GHS classification;
 If appropriate, photographs of the eye;
Discussion of the Results
Conclusion

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LITERATURE(1) ICCVAM (2007). 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) ESAC (2007). Statement on the conclusion of the ICCVAM retrospective study on organotypic invitro assays as screening tests to identify potential ocular corrosives and severe eye irritants.
Available: [http://ecvam.jrc.it/index.htm].
(3) ICCVAM (2010). ICCVAM Test Method Evaluation Report – Current Status of In Vitro Test
Methods for Identifying Mild/Moderate Ocular Irritants: The Isolated Chicken Eye (ICE) Test
Method. 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](4) United nations (UN) (2011). Globally Harmonized System of Classification and Labelling ofChemicals (GHS), Fourth revised edition, UN New York and Geneva, 2011. Available at:[http://www.unece.org/trans/danger/publi/ghs/ghs_rev04/04files_e.html](5) Streamlined Summary Document Supporting OECD Test Guideline 438 on the Isolated Chicken
Eye for Eye Irritation/Corrosion. Series on Testing and Assessment no. 188 (Part 1 and Part 2),
OECD, Paris.
(6) OECD (2012). Test Guideline 405. OECD Guidelines for Testing of Chemicals. Acute eyeirritation/corrosion. DOI : 10.1787/9789264185333-en.
(7) Scott L, Eskes C, Hoffman S, Adriaens E, Alepee N, Bufo M, Clothier R, Facchini D, Faller C,
Guest R, Hamernik K, Harbell J, Hartung T, Kamp H, Le Varlet B, Meloni M, Mcnamee P, Osborn R,
Pape W, Pfannenbecker U, Prinsen M, Seaman C, Spielmann H, Stokes W, Trouba K, Vassallo M,
Van den Berghe C, Van Goethem F, Vinardell P, Zuang V (2010). A proposed Eye Irritation Testing
Strategy to Reduce and Replace in vivo Studies Using Bottom-up and Top-down Approaches.
Toxicology In Vitro 24, 1-9.
(8) 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, OECD, Paris.
(9) ICCVAM. (2006). Background review document: Current Status of In Vitro Test Methods for
Identifying Ocular Corrosives and Severe Irritants: Isolated Chicken Eye Test Method. NIH
Publication No.: 06-4513. Research Triangle Park: National Toxicology Program. Available at:[http://iccvam.niehs.nih.gov/methods/ocutox/ivocutox/ocu_brd_ice.htm](10) Prinsen, M.K. and Koëter, B.W.M. (1993). Justification of the enucleated eye test with eyes ofslaughterhouse animals as an alternative to the Draize eye irritation test with rabbits. Fd. Chem.
Toxicol. 31:69-76.
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OECD/OCDE(11) DB-ALM (INVITTOX) (2009). Protocol 80: Chicken enucleated eye test (CEET) / Isolated
Chicken Eye Test, 13pp. Available: [http://ecvam-dbalm.jrc.ec.europa.eu/].
(12) Balls, M., Botham, P.A., Bruner, L.H. and Spielmann H. (1995). The EC/HO international
validation study on alternatives to the Draize eye irritation test. Toxicol. In Vitro 9:871-929.
(13) Prinsen, M.K. (1996). The chicken enucleated eye test (CEET): A practical (pre)screen for theassessment of eye irritation/corrosion potential of test materials. Food Chem. Toxicol. 34:291-296.
(14) Chamberlain, M., Gad, S.C., Gautheron, P. and Prinsen, M.K. (1997). IRAG Working Group I:
Organotypic models for the assessment/prediction of ocular irritation. Food Chem. Toxicol. 35:23-37.
(15) Prinsen, M.K. (2006). The Draize Eye Test and in vitro alternatives; a left-handed marriage?
Toxicology in Vitro 20,78-81.
(16) Siegel, J.D., Rhinehart, E., Jackson, M., Chiarello, L., and the Healthcare Infection Control
Practices Advisory Committee (2007). Guideline for Isolation Precautions: Preventing TransmissionofInfectious
Agentsin
Healthcare
Settings.
Available:[http://www.cdc.gov/ncidod/dhqp/pdf/isolation2007.pdf].
(17) 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 of alternative
assays. Reg. Tox. Pharmacol. 36:106-117.
(18) Burton, A.B.G., M. York and R.S. Lawrence (1981). The in vitro assessment of severe irritants.
Fd. Cosmet.- Toxicol.- 19, 471-480.

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ANNEX 1DEFINITIONS
Accuracy: The closeness of agreement between test method results and accepted reference values. It
is a measure of test method performance and one aspect of “relevance.” The term is often usedinterchangeably 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 and functional similarity to the class of substances being tested; (iii), known
physical/chemical characteristics; (iv) supporting data on known effects; and (v), known potency inthe range of the desired response
Bottom-Up Approach: step-wise approach used for a chemical suspected of not requiringclassification for eye irritation or serious eye damage, which starts with the determination of chemicals
not requiring classification (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 lightto the 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.
Corneal swelling: An objective measurement in the ICE test of the extent of distension of the cornea
following exposure to a test chemical. It is expressed as a percentage and is calculated from baseline(pre-dose) corneal thickness measurements and the thickness recorded at regular intervals after
exposure to the test material in the ICE test. The degree of corneal swelling is indicative of damage tothe cornea.
Eye Irritation: Production of changes in the eye following the application of test chemical to theanterior surface of the eye, which are fully reversible within 21 days of application. Interchangeablewith "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.
Fluorescein retention: A subjective measurement in the ICE test of the extent of fluorescein sodiumthat is retained by epithelial cells in the cornea following exposure to a test substance. The degree offluorescein retention is indicative of damage to the corneal epithelium.
Hazard: Inherent property of an agent or situation having the potential to cause adverse effects whenan organism, system or (sub) population is exposed to that agent.
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Irreversible effects on the eye: see "Serious eye damage" and "UN GHS Category 1".
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 thesolvent interacts with the test system.
Not Classified: Substances that are not classified for eye irritation (UN GHS Category 2) or serious
damage to eye (UN GHS Category 1). Interchangeable with “UN GHS No Category”.
Positive control: A replicate containing all components of a test system and treated with a chemical
known to induce a positive response. To ensure that variability in the positive control response acrosstime can be assessed, the magnitude of the severe response should not be excessive.
Reliability: Measures of the extent that a test method can be performed reproducibly within andbetween laboratories over time, when performed using the same protocol. It is assessed by calculating
intra- and inter-laboratory reproducibility and intra-laboratory repeatability.
Reversible effects on the Eye: see "Eye Irritation" and "UN GHS Category 2".
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 of application. Interchangeable with "Irreversible effects on the eye" and with "UN
GHS Category 1" (4).
Slit-lamp microscope: An instrument used to directly examine the eye under the magnification of abinocular microscope by creating a stereoscopic, erect image. In the ICE test method, this instrument
is used to view the anterior structures of the chicken eye as well as to objectively measure corneal
thickness with a depth-measuring device attachment.
Solvent/vehicle control: An untreated sample containing all components of a test system, including
the solvent or vehicle that is processed with the test chemical-treated and other control samples toestablish the baseline response for the samples treated with the test chemical dissolved in the 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
deriving from the process used, but excluding any solvent which may be separated without affecting
the stability of the substance or changing its composition (4).
Surfactant: Also called surface-active agent, this is a substance, such as a detergent, that can reduce
the surface tension of a liquid and thus allow it to foam or penetrate solids; it is also known as awetting agent.
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) from other chemicals (negative outcome).
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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
is reviewed, 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 nexttier. If the irritancy 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 anunequivocal classification can be made.
United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UNGHS): A system proposing the classification of chemicals (substances and mixtures) according tostandardized 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 damage to eyes" and/or "Irreversible effects on the eye".
UN GHS Category 2: see "Eye Irritation" and/or "Reversible effects to the eye".
UN No Category: Substances 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
the relevance (including accuracy) and reliability for a specific purpose. It is important to note that avalidated test method may not have sufficient performance in terms of accuracy and reliability to befound 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 chemical.

17© OECD, (2013).

33

438

OECD/OCDEANNEX 2PROFICIENCY CHEMICALS FOR THE ICE TEST METHOD

Prior to routine use of a test method that adheres to this Test Guideline, laboratories should
demonstrate technical 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 from the in vivo rabbit eye test (TG 405) and the UN GHS
classification system (i.e., UN GHS Categories 1, 2A, 2B, or No Category) (4)(6). Other selection
criteria were that substances are commercially available, there are high quality in vivo reference data
available, and there are high quality data from the ICE in vitro method. Reference data are available in
the SSD (5) and in the ICCVAM Background Review Documents for the ICE test method (9).

18
© OECD, (2013).

34

438

OECD/OCDE

Table 1: Recommended substances for demonstrating technical proficiency with ICE
Chemical

CASRN

ChemicalClass1

PhysicalForm

In Vivo
Classification2

In Vitro
Classification3

Benzalkonium
chloride (5%)

8001-545

Onium
compoundLiquidCategory 1

Category 1

Chlorhexidine

55-56-1

Amine,
AmidineSolid

Category 1

Category 1

Dibenzoyl-Ltartaric acid2743-386

Carboxylicacid, Ester

Solid

Category 1

Category 1

Imidazole288-32-4

Heterocyclic

Solid

Category 1

Category 1

Trichloroaceti
c acid (30%)

76-03-9

Carboxylic
Acid

LiquidCategory 1

Category 1

2,6Dichlorobenzoyl chloride

4659-454

Acyl halide

LiquidCategory 2A

No predictions
can be made 4

Ammonium
nitrate6484-522

Inorganic salt

Solid

Category 2A5

No predictions
can be made 4

Ethyl-2methylacetoacetate609-14-3

Ketone, Ester

LiquidCategory 2B

No predictions
can be made 4

Dimethylsulfoxide

67-68-5

Organic
sulphurcompoundLiquidNo Category

No Category

Glycerol

56-81-5

AlcoholLiquidNo Category

No Category(borderline)

Methylcyclopentane

96-37-7

Hydrocarbon(cyclic)

LiquidNo Category

No Categoryn-Hexane

110-54-3

Hydrocarbon(acyclic)

LiquidNo Category

No Category

Triacetin102-76-1

Lipid

LiquidNot classified

No Category

Abbreviations: CASRN = Chemical Abstracts Service Registry Number
1
Chemical classes were assigned to each test substance using a standard classification scheme, based on the National Libraryof Medicine Medical Subject Headings (MeSH) classification system (available at http//www.nlm.nih.gov/mesh)
2
Based on results from the in vivo rabbit eye test (OECD TG 405) and using the UN GHS (4)(6).
3
Based on results in ICE as described in table 6.
4
Combination of ICE scores other than the ones described in table 6 for the identification of GHS no-category and GHS
Category 1 (see table 6)
5Classification as 2A or 2B depends on the interpretation of the UN GHS criterion for distinguishing between these twocategories, 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 invivo study included 3 animals. All endpoints apart from conjunctiva 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.

19© OECD, (2013).

35

438

OECD/OCDEANNEX 3

DIAGRAMS OF THE ICE SUPERFUSION APPARATUS AND EYE CLAMPS(See Burton et al. (18) for additional generic descriptions of the superfusion apparatus and eye clamp)© OECD, (2013).

36

20

Unclassified

ENV/JM/MONO(2013)12/PART1

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)12/PART1
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYSTREAMLINED SUMMARY DOCUMENT SUPPORTING OECD TEST GUIDELINE 438 ON THE
ISOLATED CHICKEN EYE FOR EYE IRRITATION/CORROSION
Series on Testing and Assessment
No. 188

English - Or. English

JT03342244
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.

37

ENV/JM/MONO(2013)12/PART1

2

38

ENV/JM/MONO(2013)12PART1OECD Environment, Health and Safety Publications
Series on Testing and Assessment

No. 188

STREAMLINED SUMMARY DOCUMENT SUPPORTING OECD TEST GUIDELINE 438 ON
THE ISOLATED CHICKEN EYE FOR EYE IRRITATION/CORROSION

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

3

39

ENV/JM/MONO(2013)12/PART1ABOUT 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 ten 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.

440

ENV/JM/MONO(2013)12PART1
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:
OECD Environment Directorate,
Environment, Health and Safety Division
2 rue André-Pascal
75775 Paris Cedex 16
FranceFax: (33-1) 44 30 61 80
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 AndréPascal, 75775 Paris Cedex 16, France

541

ENV/JM/MONO(2013)12/PART1
FOREWORD

This streamlined summary document (SSD) was developed to provide summary information in
support of OECD Test Guideline 438 on the Isolated Chicken Eye Test Method addressing 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 438 (originally adopted in 2009). The SSD provides useful and moredetailed 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) the within-laboratory and between-laboratory reproducibility ofthe method.
The SSD was approved by the WNT with a few changes to paragraph 11, including additionalreferences 22, 23, 24 and 25, on 30 April 2013.
The Joint Meeting of the Chemicals Committee and the Working Party on Chemicals, Pesticides andBiotechnology 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)12PART1
STREAMLINED SUMMARY DOCUMENT

Description of applicability domain and performance, based on the retrospective validation studies
and their revisions, of the Isolated Chicken Eye (ICE) Test Method (Test Guideline 438) for
identifying i) chemicals inducing serious eye damage and ii) chemicals not requiring classification foreye irritation or serious eye damage

INTRODUCTION AND BACKGROUND
The 2003-2006 Validation Studies
1. Between 2003 and 2006, a retrospective evaluation was carried out concerning the validation status of
the Isolated Chicken Eye (ICE) 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 175 chemicals, wasperformed 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 Japanese Center 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 ICE for thefollowing 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 at that time). On thebasis of all collected data and information the ICE was considered as scientifically valid (reliable andrelevant) for identifying chemicals inducing serious eye damage (i.e., to initiate a Top-Down approach (1))
and was recommended for regulatory hazard classification and labelling purposes. Chemicals inducing
serious eye damage are defined as those that produce tissue damage in the eye, or serious physical decay ofvision, following application to the anterior surface of the eye in the in vivo Draize rabbit eye test (8),which is not (or not expected to be) fully reversible within 21 days of application. Following theserecommendations, the OECD officially adopted the ICE test method as OECD Test Guideline (TG) 438 for
identifying chemicals inducing serious eye damage in September 2009 (9).
The 2006-2009 Validation Studies
3. NICEATM/ICCVAM, in collaboration with ECVAM and JaCVAM, further evaluated between 2006
and 2009 the usefulness and limitations of the ICE test method for the additional identification ofchemicals 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 at that time), i.e., its usefulness andlimitations for initiating a Bottom-Up approach (1). The ICE validation database remained unchanged
and comprised 175 chemicals (90 substances and 85 mixtures) collected from five individual studies(Prinsen and Koëter 1993 (10), Balls et al. 1995 (11), Prinsen 1996 (12), Prinsen 2000 (13) and Prinsen
2005 (14)), which were used to determine the predictive capacity of the ICE test method (ICCVAM BRD
from April 2009 (15)).
4. In May 2009, NICEATM/ICCVAM convened an independent international scientific peer review panel(PRP) on alternative ocular safety testing methods, composed of members from EU, USA, Japan and7

43ENV/JM/MONO(2013)12/PART1
Canada. The PRP maintained the original recommendation for using the ICE for classification of serious
eye damage (16). At that stage however, no further recommendations were made for an expansion of the
ICE applicability domain to also include other classification categories, and in particular the identificationof non-classified chemicals (16). This was due to the false negatives rates (6% or 4/62 for the UN GHS
classification system) and the fact that amongst the false negatives there was one substance was classified
as GHS Cat 1 based on Draize rabbit eye test data (16).
Revisions of the Validation Dataset
5. On the basis of revisions of the validation dataset carried out in 2012 it was found that the individual in
vitro and in vivo classifications of a number of chemicals deserved further considerations. In particular
discrepancies were found in the final in vivo and in vitro classifications for a number of chemicals in the
ICCVAM BRD from April 2009 (15) which had an impact on the number of false negative chemicals (1out of 73 false negative instead of 4 out of 62; see Appendixes 1, 2 and 3). In addition, it was felt importantto recognize the limitations of the in vivo Draize rabbit eye irritation test and their implications forvalidation purposes (17). Some of the drawbacks of the Draize rabbit eye test referred in literature include(see extract from Eskes (18) in Appendix 3 for details):- The fact that the in vivo rabbit eye irritation/corrosion test has no standardized exposure regimen, sothat the duration of exposure of the test substance with the rabbit eyes remains unknown and can varyfrom a few minutes to several hours. In addition, for solids and sticky chemicals it is unclear how muchof the compound (solid, paste or liquid) stays in contact with the eye (19);- The limited reproducibility of the Draize rabbit eye test method;- The subjectivity in the allocation of the rabbit ocular tissue scores;- The type of exposure which does not reflect a potential human accidental exposure;- The differences in physiology and sensitivity to tested chemicals between rabbit and human eyes;- Ethical issues and the fact that the Draize test can be very painful to the rabbits.
Adoption of TG 438 for the Identification of Chemicals Not Classified for Eye Irritation or Serious Eye
Damage6. In April 2012, following a proposal from the Netherlands and the European Commission, a project for
updating TG 438 (9) was included in the work plan of the OECD Test Guidelines Programme. The aim of
the project was, taking into account the review of the individual in vivo and in vitro data, to reassess theperformances of ICE and address a possible update of TG 438 to allow its use also for the identification ofchemicals not requiring classification for eye irritation or serious eye damage under the UN GHS
classification system. An initial re-evaluation of the in vitro and in vivo ICE dataset was carried out by the
Netherlands, followed by the preparation of an Issue Paper and its addendum by a consultant to the OECD
Secretariat with the aim to review existing ICE data and make a recommendation on the use of TG 438 forthe identification of chemicals not requiring classification for eye irritation or serious eye damage(Appendixes 3 and 4). The ICE Issue Paper and its addendum reviewed both the ICE data presented in the
ICCVAM-NICEATM ICE BRDs (2) (15) as well as the evaluation carried out by The Netherlands in thefirst draft version of this SSD (17). The Issue Paper and its addendum were discussed by the eyeirritation/corrosion expert group at an OECD expert meeting held on 6-7 December 2012. The present SSD
represents a compilation of all relevant data on the ICE test method evaluation and takes into account allcomments received at and after the eye irritation expert meeting, as well as the comments received fromthe two commenting rounds on the proposed revised TG 438.
7. In 2013, the OECD approved the updated version of TG 438 allowing the use of ICE for identifying
chemicals inducing serious eye damage as well as for identifying chemicals not requiring classification foreye irritation or serious eye damage.

8

44

ENV/JM/MONO(2013)12PART1SCIENTIFIC BASIS FOR THE ICE TEST METHOD
8. The ICE test method (TG 438) is an organotypic model that provides short-term maintenance of thechicken eye in vitro. Damage to the cornea is assessed by determination of corneal swelling, corneal
opacity, and fluorescein retention in a test that typically takes less than 8 hours to perform. Analysis ofcorneal swelling provides for a quantitative assessment, whereas corneal opacity and fluorescein retention
involve a qualitative assessment based nevertheless on slit-lamp observations. Each endpoint is then either
converted into a quantitative score or assigned a qualitative categorization (ICE Classes) which are then
combined together and used to assign an in vitro ocular hazard classification (9) (20).
Data Interpretation Procedures
9. If the criteria used to attribute the ICE classes (I to IV) for the three endpoints (corneal swelling,corneal opacity and fluorescein retention) remained unchanged, the Data Interpretation Procedure (DIP)
proposed by ICCVAM and in the OECD Guidance Document 160 (15) (20) differed slightly from the one
proposed by The Netherlands (see Appendix 3). Following consultation of the OECD Expert Group on Eye
Irritation, it was recommended to make use of the DIP proposed by ICCVAM and by Guidance Document
160 (15) (20) complemented with some effects proposed by The Netherlands as described in Table 1.
Table 1. Data Interpretation Procedures applied for the ICE test method for identifying i) chemicals
inducing serious eye damage and ii) chemicals not requiring classification for eye irritation or serious eye
damage.
UN GHS Classification

Combinations of the 3 Endpoints*

No Category

3xI
2 x I, 1 x II

No prediction
can be madeOther combinations

Category 1

3 x IV
2 x IV, 1 x III
2 x IV, 1 x II**
2 x IV, 1 x I**
Corneal opacity  3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of the epithelium (in at least 1 eye)* Based on the criteria proposed in the original TG 438 (9) and in the Guidance Document 160 (20)**Combinations less likely to occur.

Comparison of the ICE Test Method with the In Vivo Rabbit Eye Test Method
10. In contrast to ICE, the in vivo rabbit eye test involves only qualitative evaluations based mainly on
visual observations of the severity of adverse effects on the cornea, the iris, and the conjunctiva, as well as
the reversibility of any ocular effects detected at selected intervals up to 21 days after exposure. In ICE,
liquids and solids are typically tested undiluted and are applied to evenly cover the entire surface of thecornea. In the in vivo rabbit eye test, liquid and solid test substances are also tested usually undiluted,
however they are applied to the conjunctival sac of the rabbit eyes. Because rabbits blink and/or tear,
exposure of the cornea to the test substance will be affected by these factors in terms of coverage or
945

ENV/JM/MONO(2013)12/PART1
duration. The neurogenic components that drive tear film production are not present in the ICE. When
compared with an in vivo rabbit eye study, application of a test substance in the absence of this protective
barrier might be expected to cause an increase in false positive outcomes. One of the conclusions from aworkshop on mechanisms of eye irritation highlighted the need for additional research on the impact ofchemicals on tear film and the consequences of tear film disruption. However, for some test substances(e.g., solids), blinking can also induce mechanical damage in vivo, contributing to a higher degree of
irritation. Thus, the ICE test method differs from the in vivo rabbit eye test method in the following
significant ways:• The ICE 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 and qualitatively with the help of a slit-lamp in the ICE assay, while they areassessed only qualitatively based mainly on visual observations in the in vivo rabbit eye test.
• Corneal exposure conditions, including test substance concentration and exposure duration,
are well defined in the ICE assay, whereas subject to potentially large variations in vivo due tothe ill-defined exposure conditions, blink response and natural tearing of the eye in a live
animal. Moreover, based on the unrealistic accidental in vivo exposure conditions, solids maylead to variable and extreme responses in the in vivo Draize eye irritation test, which may notreflect their true irritation potential in humans (19).
• The observation period of the ICE assay is typically of 4 hours, whereas ocular effects are
typically evaluated in the in vivo rabbit eye test for a minimum of 72 hours and can extended
up to 21 days.
• Reversibility/irreversibility of corneal effects induced by a test substance cannot be observedin the ICE assay per se, but histological evaluation of the exposed eyes 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 (21) although further scientific knowledge is required to understand how
irreversible effects not linked with initial high level injury occur.
• Protective mechanisms of the eye, such as tear production and blinking (e.g., against drying
and infection), are built into in vivo testing, but are absent in in vitro / ex vivo testing.
However, if regeneration of the tear film might be important for the in vivo healing process, it
may play a minor role in the ICE test method where the initial damage is measured rather thanrecovery.
• The ICE 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
11. The potential shortcomings of the ICE test method when used to identify chemicals inducing serious
eye damage (UN GHS Category 1) in e.g., a Top-Down approach, are based on the high false positive
rate for alcohols and the high false negative rates for solids and surfactants, as observed in the 2003-2006
retrospective validation study (2) (3). When substances within these chemical and physical classes are
excluded from the database, the accuracy of the ICE test method is substantially improved (2) (3) (seeTable 6 below). However, since not all alcohols are over-predicted (4 out of 10) and some are correctlypredicted as UN GHS Category 1, this organic functional group is not considered to be out of theapplicability domain of the test method. Positive results obtained with alcohols should nevertheless be
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ENV/JM/MONO(2013)12PART1
interpreted cautiously due to potential over-prediction. Similarly, given the fact that i) some solids andsurfactants are correctly predicted by the ICE test method as UN GHS Category 1, ii) that not all solids andsurfactants are underpredicted (6 underpredictions out of 11 solids, and 6 out of 9 surfactants), and that iii)
the underpredicted solids and surfactants would need to be subsequently tested with other suitable test
method(s) in a sequential testing strategy (as none of the GHS Cat 1 solid and surfactant materials in thevalidation database are underpredicted as GHS non-classified), solids and surfactants are also notconsidered to be out of the applicability domain of the ICE test method. Evidence suggests that there is acertain probability that Cat 1 are predicted as Cat 2 due to the variability of individual animal responses
within the same test (22). 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 of in vivo responses (23)(24)(25).
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.
12. When used to identify chemicals not requiring classification for eye irritation or serious eye damage
under the UN GHS classification system, in e.g., a Bottom-Up approach, anti-fouling organic solvent
containing paint were found to risk under-prediction (1 out of 2 classified anti-fouling solvent containing
paint was found to be under-predicted as non-classified). When chemicals within this product classes are
excluded from the database, the accuracy of the ICE test method is only slightly improved (see Table 7
below). In addition, the only underpredicted material (TNO-94) was classified in vivo as GHS Cat 1 due tounusual effects, i.e., a residue of paint got attached to the cornea most probably caused bygrooming/scratching of the eye by the rabbit and the type of exposure of the rabbit eyes (i.e., adding thepaint in the conjunctival sac of the eye and holding the eye lids together). This was observed in only one
animal, whereas the two other animals only showed GHS Cat 2-type effects (see Appendix 5). The OECD
Expert Group decided to add a warning sentence for this category of materials, but not to exclude themfrom the applicability domain of the ICE test method for the following reasons: i) there was insufficientevidence to exclude those types of formulations (only two classified chemicals from this category), ii) thetype of exposure to this material is unlikely to occur in humans, and iii) sticky materials present similardifficulties to test either in vitro and in vivo. Regarding the false positive rates, surfactants (5 out of 8) may
risk overprediction. However due to i) the fact that not all surfactants were overpredicted, ii) that somesurfactants were correctly predicted, and that iii) surfactants not predicted as GHS non-classified would
need to be subsequently tested with other suitable test method(s) in a sequential testing strategy (2) (asnone of the in vivo GHS non-classified chemicals (n=73) was overpredicted as GHS Cat. 1), surfactants are
not considered to be out of the applicability domain of the ICE test method.
13. Although the ICE test takes into account some of the ocular effects evaluated in the in vivo test method
and to some degree their severity, it does not consider conjunctival and iridal injuries. Nevertheless, theICE directly addresses corneal effects, which are the major driver of classification in vivo when
considering the UN GHS classification system (see Annex 6 of SSD for TG 437). In addition, Burton (26)found a direct relation between corneal swelling and the conjunctival reactions in a study with 600 rabbits
and approximately 100 test substances. Prinsen (12) also reported a high correlation between conjunctival
reactions and the endpoints assessed in the ICE test method after parallel testing of test substances in vivo
and in vitro. Furthermore, although the reversibility of corneal lesions cannot be evaluated per se in the
ICE test method it has been proposed, based on rabbit eye studies, that an assessment of the initial depth ofcorneal injury (e.g., through histological evaluation) may be used to identify some types of irreversible
effects (21). Finally, the ICE does not allow for an assessment of the potential for systemic toxicity
associated with ocular exposure.
14. The ICE test method is not recommended for the identification of test substances classified as irritating
to eyes (UN GHS Category 2 or Category 2A) or test substances classified as mildly irritating to eyes (UNGHS 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. For this purpose, further testing with another suitable method may be required.
11

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ENV/JM/MONO(2013)12/PART1
Table 2: Physicochemical properties and compatibility with the ICE
Is a material with this property
compatible with the ICE assay?

Physicochemical property
Fixative
Solvent
Extreme pH
Gases
Liquids
Solid materials
Emulsions
Granular materials
Suspensions
Coloured materials
Diluted concentrations of chemicals
Highly viscous materials
Volatile materials
Reactive chemistries
Hydrophobic/lipophilic chemicals
Neat concentrations of chemicals

Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes

APPLICABILITY DOMAIN
15. The Test Guideline can be used for testing all types of substances and mixtures, provided there is noevidence that the method is not valid for the chemical tested.
Categories of Irritancy
16. Based on the conclusions of the 2003-2006 and 2006-2009 retrospective validation studies (3) (16), TG
438 was adopted in 2009 for classification of chemicals inducing serious eye damage (UN GHS Category1). In addition, following the re-evaluations carried out in 2012 and their review by the OECD Expert
Group on Eye Irritation, TG 438 was also approved in 2013 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.
Potential Role in an ITS
17. The ICE can be used as a validated ex vivo test method in a tiered testing approach as described in theaddendum 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). No published studies have been found at the current date, on the combination of
ICE with other in vitro test methods in testing strategies for the prediction of ocular hazards.
Mode of Action (MoA)
18. An expert meeting held at EC-ECVAM in 2005 (1) recommended to expand the concept of defining
the applicability domain as not only chemical classes, but also as a function of the mechanism of eye
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ENV/JM/MONO(2013)12PART1
irritation. The four identified MoA that were discussed included: (i) cell membrane lysis (breakdown ofmembrane 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 ofmacromolecules, particularly protein, characteristic of acid, alkali, or organic solvent exposure), and (iv)actions on macromolecules (chemicals that react with cellular constituents/organelles that may or may notlead to overt lysis or coagulation, e.g., peroxides, mustards and bleaches). The ICE test method addresses
the three first MoAs. In addition, it may also address the fourth MoA (actions on macromolecules) when
histopathological information is available.
Table 3: Summary of events involved in chemical-induced eye irritation in vivo
Events involved in chemical-induced eye irritation
Chemical interaction with tear film
Chemical binding to the conjunctival epithelium
Adhesion molecules compromised
Corneal epithelial damage
Inhibition of receptor-mediated transportCompromise of cell membrane integrity of upper corneal epithelium
Cell membrane lysis of all corneal epithelial layers
Hydration of corneal stroma
Cross-linking of proteins in corneal stroma
Erosion of corneal stroma
Cell damage to corneal epithelium and limbus
Dilation and increased lymphatic leakage from scleral vasculature
Stimulation of nerve endings, i.e., enhanced blinking, tearing
Erosion of nerve endings in corneal and sclera
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.
Recovery from response, i.e., length of time for cell responses to return tocontrol levels

Modelled by the
ICE assay?
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
No
Yes

No

Chemical Classes
19. The ICE validation dataset comprised a total of 175 individual chemicals (90 substances and 85
mixtures) collected from Prinsen and Koëter 1993 (10), Balls et al. 1995 (11), Prinsen 1996 (12), Prinsen
2000 (13) and Prinsen 2005 (14) as described in the retrospective validation studies (2) (15). Tables 4 and5 show the chemical and product classes representation within the ICE validation database. Although thesingle components from the mixtures used in the validation dataset could not be disclosed due toproprietary reasons they represented relevant to current commerce mixtures and formulations. In addition,
details on the product categories and chemical classes of most of the tested mixtures and substances wereavailable as described in the ICCVAM BRD from April 2009 (15). Out of the 175 chemicals, 85 (including
mixtures) could not be assigned a specific chemical class and 23 (including substances) could not be
assigned a specific product category. Detailed information, including chemical name, Chemical Abstracts
Service Registry Number (CASRN), chemical class, product category, physical state, purity,
concentration(s) tested, in vivo GHS classification, in vitro GHS classification, in vitro raw data, in vitro
categories and literature reference using the chemical are provided in Appendix 1.
13

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ENV/JM/MONO(2013)12/PART1

Table 4: Chemical classes tested in the ICE test method*.
Chemical Class# of Chemicals

Chemical Class# of Chemicals

Acetate1

Inorganic Chloride Compound

1

Acid

5

Inorganic Salt

3

Acyl halide

1

Inorganic Silver /Nitrogen Compound

1

Alcohol

15

Ketone4

Aldehyde2

Lactone1

Alkali

3

Lipid

1

Amide /Amidine

7

Nitrile1

Amino acid1

Nitro Compound

1

Boron compound1

Not Classified

85

Carbohydrate

2Onium Compound

8

Carboxylic acid

12

Organic Silicon Compound

2

Ester

10

Organic Sulfur Compound

3

Ether

1

Organometallic

2

Heterocyclic

9Organophosphrous Compound1

Hydrocarbon

5

Polycyclic

4

Imide

2

Polyether5

Inorganic Chemical

1

Urea Compound1* Revised from (15) based on information presented in Appendix 1.

Table 5: Product classes tested in the ICE test method*.
Product Class# of Chemicals

Product Class

Adhesive

2

Fertilizer

1

Antifungal

3

Food additive1

Antihistamine

1

Fungicide / Germicide /Bactericide8

Anti-infective

3

Industrial Chemical,
Intermediate or Formulation

19

Antiseptic

2

Not Classified

23

Caustic Agent

4

Optical Resolution Agent

1

14

50

# of Chemicals

ENV/JM/MONO(2013)12PART1
Chlorination by-product

1

Paint

4

Cleaner

8

Pesticide / Herbicide

17

Copolymer

8

Preservative

6

Cosmetic Ingredient

1

Pharmaceutical Compounds /Intermediates

6

Detergent

8

Raw Material

9

Developer

1

Reagent

4

Disinfectant

5

Resin

2

Dyes & Stains

10

Silicon Resin

1

Elastomer

2

Soap

9

Enzyme Inhibitor

1

Surfactant

25

Enzyme Solution

3

Solvent

37* Revised from (15) based on information presented in Appendix 1.

SENSITIVITY, SPECIFICITY AND ACCURACY
20. Within the ICE validation database a total of 152 chemicals (72 substances and 80 mixtures) had
sufficient in vivo and in vitro data to assess the ICE predictive capacity (Appendix 1). Their distribution
according to the UN GHS classification categories are described below.
- Identification of GHS NC (Bottom-Up approach):
152 chemicals (72 substances + 80 mixtures)
79 NC + 73 classified chemicals (30 Cat 1 + 6 Cat 1/2 + 27 Cat 2A + 8 Cat 2B + 2 Cat 2A/2B)- Identification of GHS Cat 1 (Top-Down approach):
139 chemicals (65 substances + 75 mixtures)
27 Cat 1 + 113 non-Cat 1 (26 Cat 2A + 8 Cat 2B + 79 Non-Classified (NC))
Out of the 175 chemicals from the validation dataset, a number of chemicals (n=15) had no raw in vivo
data to allocate a UN GHS Classification. In addition, a number of chemicals (n=13) had Study Criteria
Not Met (SCNM) to assign an in vivo classification (e.g., incomplete dataset to assess reversibility /irreversibility of effects at day 21). For a large number of them (n=11), the in vivo scores suggested theneed for classification even if not possible to allocate a specific classification category (i.e., GHS Cat 2B
versus 2A versus 1). These chemicals were used for the evaluation of the predictive capacity of the ICE
test method in a bottom-up approach, but not for the top-down approach due to uncertainty as to whichclassification category to assign (i.e., GHS Cat 1 versus GHS Cat 2). Chemicals that had a SCNM andwere estimated to be non-classified based on expert judgement (n=2), were not included in any of theanalyses for precautionary reasons (although in the original evaluation they were considered as NC). A
total of 5 chemicals that were classified as Eye GHS Cat 1 based on data from skin corrosion studies were
not included for the purposes of the Test Guideline, in order to consider only chemicals for which highquality in vivo ocular data was available. Finally, two chemicals had a borderline GHS Cat 1 / Cat 2classification so that they could only be used for the evaluation of the predictive capacity of the ICE test
method in a bottom-up approach, and not in a top-down approach.
Overall Predictive Capacity
1551

ENV/JM/MONO(2013)12/PART1
21. Due to discrepancies found in a number of in vitro and in vivo classifications from previous
validation studies (for details see Appendixes 1, 2 and 3), the predictive capacities of the ICE test
method were re-calculated for i) the identification of GHS Category 1 chemicals (Top-Down
approach) and ii) the identification of non-classified chemicals (Bottom-up approach) as shown in
Tables 6 and 7. The analyses were based on the outcome of individual test substances (and not on
individual laboratory outcome), as recommended by the Expert Group on Eye Irritation, in order to be
in alignment with previous ICCVAM evaluations and with the analyses carried out in the context of
the revisions of the BCOP Test Guideline.

Table 6: Predictive capacity of the ICE test method for distinguishing chemicals (substances andmixtures) inducing serious eye damage (UN GHS1 Category 1) from all other categories.
Top-.Down
Approach

No.

OverallWithout
alcohols, solids
and surfactants

Accuracy

Sensitivity

False
Negatives

Specificity

False
Positives

%

No.

%

No.

%

No.

%

No.

%

No.

140

86

120/140

52

14/27

48

13/27

94

106/113

6

7/113

82

94

77/82

71

5/7

29

2/7

96

72/75

4

3/75

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

Table 7: Predictive capacity of the ICE test method for distinguishing chemicals (substances andmixtures) not requiring classification for eye irritation or serious eye damage (UN GHS1 NonClassified) from all other irritant categories.
Bottom-Up
Approach

No.

Overall

Accuracy

Sensitivity

False
Negatives

Specificity

False
Positives

%

No.

%

No.

%

No.

%

No.

%

No.

152

82

125/152

99

72/73

1

1/73

67

53/79

33

26/79

Without anti-fouling
149
organic-solvent
containing paints

83

123/149

100

71/71

0

0/71

67

52/78

33

26/78

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

22. For the Top-Down approach, alcohols were found to risk over-prediction (4 alcohols out of 10 nonCategory 1 were over-predicted as Category 1) whereas solids and surfactants were found to risk underprediction (6 out of 11 Category 1 solids were found to be under-predicted, and 6 out of 9 Category 1
surfactants were found to be under-predicted). Table 8 shows the false positive and false negative rates
16

52

ENV/JM/MONO(2013)12PART1
obtained for specific chemical classes and properties of interest, including mixtures and substances basedon the revised dataset (Appendix 1). Substances, mixtures, liquids and solids all showed false positive rates
below or equal to 15% suggesting an appropriate identification of GHS Category 1. The rate of false
negatives was found to be particularly high (i.e., higher than 50% for 5 chemicals or more), for solids andsurfactants. However due to the fact that the underpredicted solids and surfactants would need to be
subsequently tested with other suitable test method(s) in a sequential testing strategy (as none of the GHS
Cat 1 solid and surfactant materials in the validation database are underpredicted as GHS non-classified),solids and surfactants are not considered to be out of the applicability domain of the ICE test method.
23. For the Bottom-up approach, anti-fouling organic solvent containing paint were found to risk underprediction (1 out of 2 classified anti-fouling solvent containing paint was found to be under-predicted as
non-classified). As explained in paragraph 12, a warning sentence was included in the Test Guideline forthis category of materials but they were not to excluded from the applicability domain of the ICE test
method for the following reasons: i) there was insufficient evidence (only two classified chemicals fromthis category), ii) the type of exposure is unlikely to occur in humans, and iii) sticky materials present
similar difficulties to test either in vitro and in vivo. Table 9 shows the false positive and false negative rates
obtained for specific chemical classes and properties of interest, including mixtures and substances basedon the revised dataset (Appendix 1). Substances, mixtures, liquids and solids all showed false negative
rates below or equal to 5% suggesting an appropriate identification of GHS Non-classified chemicals basedon the criteria discussed by the OECD Expert Group on Eye Irritation. Regarding the false positive rates,
the ICE test method was found to have a lower overall false positive rate as compared to other test methods
accepted for this purpose (i.e., 33% for ICE versus 69% for BCOP and 68% for CM). The false positive
rates was found nevertheless to be particularly high (i.e., higher than 50% for 5 chemicals or more) for
surfactants. However due to the fact that the overpredicted surfactants would need to be subsequently
tested with other suitable test method(s) in a sequential testing strategy (2) (as none of the in vivo GHS
non-classified chemicals (n=73) was overpredicted as GHS Cat. 1), surfactants are not considered to be outof the applicability domain of the ICE test method.
Table 8: False positive and false negative rates of the ICE test method, by properties of interest, chemicalclass and product categories, for distinguishing chemicals (substances and mixtures) inducing serious eye
damage (UN GHS1 Category 1) from all other categories.
Top-Down Approach

N2

Overall

False Positive Rate

False Negative Rate

%

No.

3

%

No.3

140

6

7/113

48

13/27

Properties of interest
Substances
Mixtures
Liquids5
Solids5
Emulsions and gels5

65
75
95
34
7

15
1
8
0
14

6/40
1/73
6/80
0/23
1/7

44
100*
40
55
n.a.

11/25
2/24,*
6/15
6/11
n.a.

Chemical Classes6
Alcohol
Amine/Amidine
Carboxylic acid
Ester
Heterocyclic
Onium compound
Polyether

12
5
10
9
9
8
5

40
0*
0*
13
0*
0*
25*

4/10
0/1*
0/3*
1/8
0/3*
0/2*
1/4*

50*
50*
43
0*
50
50
100*

1/2*
2/4*
3/7
0/1*
3/6
3/6
1/1*

17

53

ENV/JM/MONO(2013)12/PART1
Product categories
Cleaners
Copolymer
Detergent
Dyes & stains
Fungicide / Germicide / Bactericide
Industrial Chemical, Intermediate or
Formulation
Pesticide / Herbicide
Preservative
Pharmaceutical compound or intermediate
Raw material
Soap
Solvent
Surfactant – Total7-cationic-nonionic-anionic

4
8
7
8
7

0*
13
0*
0
0*

0/3*
1/8
0/4*
0/7
0/1*

100*
n.a.
100*
0*
50

1/1*
n.a.
3/3*
0/1*
3/6

16

0

0/14

50*

1/2*

12
5
4
8
7
34
21
7
5
2

0
0*
50*
0
0
19
0
0*
0*
0*

0/6
0/1*
1/2*
0/8
0/6
6/31
0/12
0/1*
0/4*
0/1*

50
25*
50*
n.a.
100*
0*
67
50
100*
100*

3/6
1/4*
1/2*
n.a.
1/1*
0/3*
6/9
3/6
1/1*
1/1*

*

Too small dataset to make definitive conclusions; n.a.: not applicable.
GHS = Globally Harmonized System (UN 2011) (4).
2N = Number of chemicals.
3
Data used to calculate the percentage.
4
Only few formulations having severe effects are available.
5
Physical form (i.e., solid or liquid) not known for 4 chemicals.
6
Chemical classes included in this Table are represented by at least five chemicals tested in the ICE test method andassignments are based on the MeSH categories (www.nlm.nih.gov/mesh) as described in (2) and (15).
7Combines single substances labelled as surfactants along with surfactant-containing mixtures.

1

Table 9: False positive and false negative rates of the ICE test method, by properties of interest, chemicalclass and product categories, for distinguishing chemicals (substances and mixtures) not requiringclassification for eye irritation or serious eye damage (UN GHS1 No Category) from all other irritantcategories.
Bottom-Up Approach

N2

Overall

False Positive Rate
%

No.3

%

No.3

152

33

26/79

1

1/73

Properties of interest
Substances
Mixtures
Liquids4
Solids4
Emulsions, gels and paste4

72
80
101
38
9

70
20
36
18
50*

14/20
12/59
20/55
3/17
2/4*

0
5
2
0
0

0/52
1/21
1/46
0/21
0/5

Chemical Classes5
Alcohol
Amine/Amidine
Carboxylic acid

13
6
11

100*
100*
100*

4/4*
1/1*
2/2*

0
0
0

0/9
0/5
0/9

18

54

False Negative Rate

ENV/JM/MONO(2013)12PART1
Ester
Heterocyclic
Onium compound
Polyether
Product categories
Cleaners
Copolymer
Detergent
Dyes & stains
Fungicide / Germicide / Bactericide
Industrial Chemical, Intermediate or
Formulation
PaintsPesticide / Herbicide
Preservative
Pharmaceutical compound or intermediate
Raw material
Soap
Solvent
Surfactant – Total6-cationic-nonionic-anionic

10
9
8
5

100*
100*
100*
100*

4/4*
2/2*
1/1*
2/2*

0
0
0
0*

0/6
0/7
0/7
0/3*

5
8
7
9
8

100*
33
100*
43
100*

1/1*
2/6
2/2*
3/7
1/1*

0*
0*
0
0*
0

0/4*
0/2*
0/5
0/2*
0/7

18

50*

2/4*

0

0/14

4
14
5
6
9
8
35
22
7
5
2

0*
50*
n.a.
n.a.
20
25*
38
63
100*
100*
100*

0/2*
2/4*
n.a.
n.a.
1/5
1/4*
8/21
5/8
1/1*
2/2*
1/1*

50*
0
0
0
0*
0*
0
0
0
0*
0*

1/2*
0/10
0/5
0/6
0/4*
0/4*
0/14
0/14
0/6
0/3*
0/1*

*

Too small dataset to make definitive conclusions; n.a.: not applicable.
GHS = Globally Harmonized System (UN 2011) (4).
2N = Number of chemicals.
3
Data used to calculate the percentage.
4
Physical form (i.e., solid or liquid) not known for 4 chemicals.
5
Chemical classes included in this Table are represented by at least five chemicals tested in the ICE test method andassignments are based on the MeSH categories (www.nlm.nih.gov/mesh) as described in (2) and (15).
6Combines single substances labelled as surfactants along with surfactant-containing mixtures.

1

WITHIN- AND BETWEEN-LABORATORY REPRODUCIBILITY
24. A thorough evaluation of the ICE reproducibility was conducted in the 2003-2006 retrospective
validation study (2). Based on a quantitative analysis of within-laboratory reproducibility of the ICE test
method endpoints, the evaluation showed CV values for the corneal thickness measurement, when results
were compared within experiments, varying from 1.8% to 6.3% (2) (3). The other endpoints evaluatedproduced ranges of CV values that were larger, with variability most prominent with the non-irritating
substance. However, this can be explained by an exaggeration of variability given the relatively smallvalues that were produced by chemicals not requiring classification relative to chemicals inducing eye
irritation and serious eye damage (i.e., corneal swelling values of 2, 0, and 3 yield a higher CV than valuesof 11, 14, and 18). A similar discussion also can be applied to the variability among the qualitative
endpoints (i.e., corneal opacity and fluorescein retention) given the small dynamic range of their scores (04 or 0-3, respectively).

19

55

ENV/JM/MONO(2013)12/PART1
25. Regarding the between-laboratory reproducibility, the retrospective studies showed median/mean %CV values to be 32%/35% for the Irritation Index, 36%/39% for fluorescein retention, 37/47% for corneal
opacity, and 75%/77% for corneal swelling (2). All laboratories were in 100% agreement on theclassification of 75% (44/59) of the substances according to the UN GHS classification system for both the
top-down (2) and bottom-up approaches (15) according to the UN GHS classification. Finally, the EC/HO
study showed the following inter-laboratory correlations between the ICE classification at TNO (leadlaboratory) and the classifications obtained in three other laboratories: 0.829, 0.849 and 0.844 (11).
26. Specific issues were raised on the between-laboratory variability of the corneal swelling endpoint. Thiswas due to the use of different slit-lamp measuring devices by the participating laboratories of the EC/HO
study which, unless normalized, can contribute to the increased variability and/or the excessive values
calculated for this endpoint (2). In particular, out of the four participating laboratories, two (that are nolonger active in the area of toxicity testing) were reported to use different slit-lamps and different slit width
settings resulting in different ranges of values for corneal swelling (see Appendix 4). In order to avoid
potential variability issues linked to this endpoint, the use of a specific pachymeter and appropriate slit
width, together with the use of proficiency chemicals are requested in both the adopted TG 438 (9) and therevised TG 438 (i.e., old paragraph 45, new paragraph 50: “Corneal swelling scores are only applicable if
thickness is measured with a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I
and slit-width setting at 9½, equalling 0.095 mm. Users should be aware that slit-lamp microscopes could
yield different corneal thickness measurements if the slit-width setting is different.”).
Considerations on variability for the Bottom-Up approach
27. As shown in Table 7 only one chemical was identified as a false negative in the ICE test method for theidentification of chemicals not requiring classification for eye irritation or serious eye damage in a BottomUp approach (i.e., TNO-94, a anti-fouling solvent containing paint). However, a total of eight chemicals
that were correctly predicted as causing ocular effects that require a UN GHS classification, were found tobe false negatives in some of the participating laboratories (Table 10).

Table 10. Chemicals showing one or more under-classification in the various participating laboratories.
In vivo
GHS Cat.

Physical
state

Lab
22

Lab
25

Lab
24

Lab
27

Overall in
vitro class

Captan 90

Cat 1

Solid

NC

2

2B

2B

2B

16

4- Carboxybenzaldehyde

Cat 2A

Solid

2B

1

NC

2

2A

36

Ethyl-2-methylacetoacetate

Cat 2B

Liquid

NC

2B

2B

NC

2B

46

Maneb

Cat 2A (EJ)

Solid

NC

2A

NC

2B

2B

50

Methyl cyanoacetate

Cat 2A

Liquid

NC

2A

NC

2B

2B

62

Quinacrine

Cat 1

Solid

2B

NC

2A

2B

2B

71

Sodium oxalate

Cat 1

Solid

2B

2B

NC

NC

2B

72

Sodium perborate

Cat 1

Solid

NC

2B

2B

2B

2B

N.

Chemical name15

EJ : classification based on expert judgment

Over the entire dataset, these chemicals represent 6 solids out of the 21 GHS classified solids present in
the ICE validation dataset (i.e., 29%), and 2 liquids out of the 46 GHS classified liquids present in the ICE
validation dataset (i.e., 4%). Due to higher probability of solids to have discordant classifications and in a
20

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ENV/JM/MONO(2013)12PART1
precautionary approach, the revised Test Guideline requires that “In the case of solid materials leading to a
GHS NC outcome, a second run of three eyes is recommended to confirm or discard the negative outcome”(revised paragraph 22).

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