᪂つヨ㦂ἲᥦ᱌᭩             ᖹᡂ 28 ᖺ 3 ᭶ 10 ᪥                                 No. 2015-03║่⃭ᛶヨ㦂௦᭰ἲ In vitro ▷᫬㛫᭚㟢ἲ (STE ἲ)࡟㛵ࡍࡿᥦ᱌ ᖹᡂ 28 ᖺ 3 ᭶ 3 ᪥䛻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 In vitro ▷᫬㛫᭚㟢ἲ㸦The Short Time Exposure In Vitro Test Method㸸STE ἲ㸧ࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢയᐖࢆ㉳ࡇࡍ≀㉁ (United Nations Globally Harmonized System of Classification and Labeling ofChemicals(UN GHS) ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࡞ࡽࡧ࡟࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸
࡚║่⃭ᛶ≀㉁࡜ࡣศ㢮ࡉࢀ࡞࠸≀㉁㸦UN GHS ༊ศእ㸧ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿࠋࡇࡢᥦ᱌᭩ࡣࠊ᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍ (JSAAE)࡜JaCVAMࡢࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩ࠊInteragency
Coordinating Committee on the Validation of Alternative Methods(ICCVAM)/ National Toxicology
Program (NTP) Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM)࡟ࡼࡿ➨୕⪅ホ౯ሗ࿌᭩࠾ࡼࡧ Organisation for Economic Co-operation and Development (OECD)
Test Guideline (TG)491ࢆࡶ࡜࡟ࠊ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾࡲ࡜ࡵࡽࢀࡓᩥ᭩ࢆ⏝࠸࡚ࠊ
JaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨ウࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ ௨ୖࡢ⌮⏤࡟ࡼࡾࠊ⾜ᨻᙜᒁࡢᏳ඲ᛶホ౯᪉ἲ࡜ࡋ࡚║่⃭ᛶヨ㦂௦᭰ἲ STE ἲࡢ౑⏝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㛗㣤ሯᑦᩥ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼஭㞝஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ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∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍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఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀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ᑠᕝஂ⨾Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ຍ⸨ ⠜

㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧Ⳣ㔝 ⣧

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

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

㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧⨾ୖ᠇୍㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧୕⃝ 㤾㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁᑂᰝ⟶⌮ㄢ㸧ᑠᓥ ⫕㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊 ➨஧ᐊ㸧㸸஦ົᒁ

iii

iv

JaCVAM statement on the
Short Time Exposure In Vitro Test Method for assessing ocular irritation
At a meeting held on 3 March 2016 at the National Institute of Health Sciences (NIHS) in
Tokyo, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: The Short Time Exposure (STE) In Vitro Test Method is a suitable means forassessing ocular irritation potency in a regulatory context as part of either a topdown approach to screening test chemicals that potentially induce serious eye
damage (Category 1) or a bottom-up approach to screening test chemicals that
potentially induce neither eye irritation nor serious eye damage and therefore do
not require classification (No Category) under the United Nations Globally
Harmonized System of Classification and Labeling of Chemicals (GHS).
This statement was prepared following a review of a validation report prepared by theJapanese Society for Alternatives to Animal Experiments (JSAAE) and JaCVAM, a peer
review report prepared by the 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), andOrganisation for Economic Co-operation and Development (OECD) Test Guideline (TG)491 “Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing
Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or
Serious Eye Damage,” as well as other materials prepared by the Ocular Irritation Testing
JaCVAM Editorial Committee to acknowledge that the results of a review and study by the
JaCVAM Regulatory Acceptance Board have confirmed the usefulness of this assay.
Based on the above, we propose the Short Time Exposure In Vitro Test Method as a usefulmeans for assessing ocular irritation potency during safety assessments by regulatory
agencies.

suo Ohno
Yasuo
Chairperson
JaCVAM Regulatory Acceptance Board

Akiyoshi Nishikawa
Chairperson
JaCVAM Steering Committee

10 March 2016v

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

Mr. Yasuo Ohno (nominee by JaCVAM Steering Committee) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Yuji Ishii (Biological Safety Research Center: BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Kazuhiro Kaneko (Japan Chemical Industry Association)
Mr. Eiji Maki (Japanese Society of Immunotoxicology)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Ms. Koko Tanigawa (Japanese Society for Alternatives to Animal Experiments)
Mr. Takashi Yamada (National Institute of Technology and Evaluation)
Mr. Hiroo Yokozeki (Japanese Society for Dermatoallergology and Contact Dermatitis)
Mr. Takemi Yoshida (Japanese Society of Toxicology)
Mr. Isao Yoshimura (nominee by Chairperson)
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. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Kenichi Mikami (Ministry of Health, Labour and Welfare)
Mr. Kaoru Misawa (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. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
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. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretary

vii

viii

║่⃭ᛶヨ㦂௦᭰ἲ In vitro ▷᫬㛫᭚㟢ἲ (STE ἲ)┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------- 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------- 7
OECD GUIDELINE FOR THE TESTING OF CHEMICALS 491-------------23
Short Time Exposure (STE) Test Method Summary Review Document ----37ixx║่⃭ᛶヨ㦂௦᭰ἲࡢホ౯఍㆟ሗ࿌᭩

In vitro ▷᫬㛫᭚㟢ἲ(The Short Time Exposure In Vitro Test Method: STE ἲ)

JaCVAM ホ౯఍㆟

ᖹᡂ 27 ᖺ㸦2015 ᖺ㸧12 ᭶ 18 ᪥

1
1

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤ሯᑦᩥ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼஭㞝஧㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧㔠Ꮚ࿴ᘯ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∾ ᰤ஧㸦᪥ᮏච␿ẘᛶᏛ఍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఍㸧ྜྷᮧ ຌ㸦ᗙ㛗᥎⸀㸧                ௵ᮇ㸸ᖹᡂ 26 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 28 ᖺ 3 ᭶ 31 ᪥

22

In vitro ▷᫬㛫᭚㟢ἲ㸦The Short Time Exposure In Vitro Test Method㸸STE ἲ㸧ࡣࠊ࢘ࢧࢠࢆ⏝࠸ࡓ
Draize ║่⃭ᛶヨ㦂ἲࡢ௦᭰ヨ㦂ἲ࡛࠶ࡾࠊ࢘ࢧࢠゅ⭷⏤᮶ࡢୖ⓶⣽⬊ᰴ㸦SIRC ⣽⬊㸧࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢᦆയࢆ㉳ࡇࡍ≀㉁㸦United Nations Globally Harmonized System of Classification and Labeling of Chemicals:
UN GHS ༊ศ 1㸧ࢆ᳨ฟࡍࡿ᪉ἲࠊ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║่⃭ᛶ≀㉁࡜ࡣศ㢮ࡉࢀ࡞࠸≀㉁㸦UN GHS ༊ศእ㸧ࢆ᳨ฟࡍࡿ᪉ἲ 1) ࡜ࡋ࡚ࣂࣜࢹ࣮ࢩࣙࣥࡀ⾜ࢃࢀࠊ2015 ᖺ࡟ OECD TG491 ࡜ࡋ࡚᥇ᢥࡉࢀࡓ 2㸧ࠋJaCVAM ホ౯఍㆟ࡣࠊࡇࡢ TG491 ࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 In vitro ▷᫬㛫᭚㟢ἲ㸦The Short Time Exposure In Vitro Test Method㸸STE ἲ㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 Draize ║่⃭ᛶヨ㦂ヨ㦂ἲࡢᴫ␎㸸ࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡿࡼ࠺࡟༢ᒙᇵ㣴ࡋࡓ SIRC ⣽⬊࡟⿕㦂≀㉁ᕼ㔘ᾮࢆ 5 ศ㛫᭚㟢ࡋࡓᚋࠊ⣽⬊ࢆὙίࡋࠊࡑࡢᚋࠊMTT㸦3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide㸧ࡢྲྀࡾ㎸ࡳ㔞࡛ᐃࡋࡓ⣽⬊⏕Ꮡ⋡ࢆᣦᶆ࡟⏝࠸ࡿࠋࡇࢀࡣ MTT ࡀ࣑ࢺࢥࣥࢻࣜ࢔ෆࡢ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿᛶ㉁ࢆ฼
⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡀ㑏ඖࡉࢀࠊ⏕ᡂࡍࡿ࣐࣍ࣝࢨࣥ㔞ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆ
฼⏝ࡋ࡚࠸ࡿ㸦MTT 㑏ඖἲ㸧ࠋ      㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶ║࡟␗≀ࡀධࡗࡓሙྜࠊ║ࡢ่⃭ᛶࡣゅ⭷ࡸ⤖⭷ࡢ⣽⬊യᐖ࠿ࡽጞࡲࡿࠋᮏヨ㦂ἲࡣࡇࢀ࡟ὀ┠ࡋ࡚ࠊ
ゅ⭷ୖ⓶⣽⬊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ║࡟⁐ᾮࡀධࡗࡓሙྜࠊ
ࣄࢺ࡛ࡣࡑࡢ኱㒊ศࡀ 1㹼2 ศ࡛║ෆ࠿ࡽ᤼ฟࡉࢀࠊ࢘ࢧࢠ࡛ࡣ 3㹼4 ศ࡛ 80%ࡀ᤼ฟࡉࢀࡿ࡜ሗ࿌ࡉࢀ
࡚࠸ࡿ 3㸧ࠋࡇࡢࡼ࠺࡞ᐇ㝿ࡢ᭚㟢≧ἣࢆ⪃៖ࡋ࡚ࠊᮏヨ㦂ἲࡣ㏻ᖖࡢ⣽⬊ẘᛶヨ㦂࡜ẚ࡭࡚▷᫬㛫᭚㟢ࡢヨ㦂࡜ࡋ࡚タィࡉࢀ࡚࠸ࡿࠋࡇࢀࡽࡢࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ࢘ࢧࢠࢆ⏝࠸ࡿ║่⃭ᛶヨ㦂ࡢ௦᭰ἲ࡜
ࡋ࡚⛉Ꮫⓗጇᙜᛶࡀ࠶ࡿࠋ
ᮏヨ㦂ἲࡣ᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍ 4㸧࡜ JaCVAM5㸧ࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࢆ⤒࡚ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ
࡟࠾࠸࡚ UN GHS ༊ศ㸯≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ≀㉁ࢆ᳨
ฟࡍࡿ᪉ἲ࡜ࡋ࡚ ICCVAM(Interagency Coordinating Committee on the Validation of Alternative
Methods)/NICEATM(National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods)࡟ࡼࡾ➨୕⪅ホ౯ࢆཷࡅࠊࡑࡢሗ࿌᭩ࡀ 2013 ᖺ 6 ᭶࡟බ⾲ࡉࢀࡓ 6㸧ࠋࡑࡢᚋࠊ2015ᖺ 7 ᭶࡟ OECD ࡟ࡼࡾࢺࢵࣉࢲ࣭࢘ࣥ࣎ࢺ࣒࢔ࢵࣉ୧᪉ᘧ࡛฼⏝࡛ࡁࡿ║่⃭ᛶヨ㦂௦᭰ἲ࡜ࡋ࡚
TG491 ࡀ᥇ᢥࡉࢀࡓ 2㸧ࠋ

33

ICCVAM/NICEATM ࡟ࡼࡿࢺࢵࣉࢲ࢘ࣥ᪉ᘧࡢṇ☜ᛶࡢ᳨ウ࡟ࡣ 125 ≀㉁ࡀࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢṇ☜ᛶࡢ᳨ウ࡟ࡣ 130 ≀㉁ࡀ⏝࠸ࡽࢀࠊ༑ศ࡞ᩘࡀ ICCVAM/NICEATM ࡟ࡼࡾ➨୕⪅ホ౯ጤဨ఍࡟ࡼࡾホ౯ࡉࢀ࡚࠸ࡿࠋJaCVAM ║่⃭ᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡣࠊࡑࢀࡽࡢ㈨ᩱࢆ⏝࠸࡚ᮏヨ㦂ἲࢆホ౯ࡋ࡚࠾ࡾࠊጇᙜ࡛࠶ࡿࠋ
㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡢࣂࣜࢹ࣮ࢩࣙࣥࡢ⤖ᯝࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧࡢഇ㝧ᛶ⋡ࡣ 1.2%ࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢഇ㝜ᛶ⋡ࡣࠊ㐺⏝㝈⏺ࢆ⪃៖ࡋ࡞࠸ሙྜ 12.3% ⪃៖ࡍࡿሙྜ 1.9%࡜࠸࠺ඃࢀࡓ್࡛࠶ࡿࠋ
ᮏヨ㦂ἲࡣࠊ▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ౑⏝⣽⬊ࡣබⓗ࡞⣽⬊ࣂࣥࢡ࠿ࡽධᡭྍ⬟࡛࠶ࡾࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡋ࡞࠸ࡇ࡜࠿ࡽࠊᇶᮏⓗ࡞⣽⬊ᇵ㣴ࡢᢏ⾡࡜タഛࢆᣢࡗ࡚࠸ࡿ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡛࠶ࡾࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸ࠋ2ࡘࡢࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂7,8㸧࡟࠾࠸࡚ࡶᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ࡢၥ㢟ࡣᣦ᦬ࡉࢀ࡚࠸࡞࠸ࡀࠊOECDࡢ࢞࢖ࢻࣛ࢖ࣥ࡟♧ࡉࢀࡓ⇍㐩ᗘ☜ㄆ≀㉁2, 9)ࢆ⏝࠸࡚ࠊᐇ᪋ࡍࡿヨ㦂᪋タ࡬㐺ษ࡞ᢏ⾡⛣㌿ࡀ㐩ᡂ࡛ࡁࡓࡇ࡜ࢆ☜࠿ࡵࡿᚲせࡀ࠶ࡿࠋᮏヨ㦂ἲ࡟ࡣヨ㦂ࡢᛶ㉁࡜ṇ☜ᛶࡢほⅬ࠿ࡽ௨ୗࡢไ㝈ࡀタࡅࡽࢀ࡚࠸ࡿࠋ
1) ⏕⌮㣗ሷỈࠊ5% (w/w) DMSOࢆྵࡴ⏕⌮㣗ሷỈࠊ࠾ࡼࡧ࣑ࢿࣛࣝ࢜࢖ࣝࡢ࠸ࡎࢀ࡟ࡶ⁐ゎࡋ࡞࠸ࠊ࠶ࡿ࠸ࡣࡑࢀࡽ⁐፹୰࡛ᆒ୍ศᩓࡋ࡚࠸ࡿ≧ែࡀ5ศ௨ୖಖࡓࢀ࡞࠸≀㉁ࡣ㐺⏝୙ྍ⬟࡛࠶ࡿࠋ2) ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛༢୍≀㉁࡟㐺⏝ࡍࡿሙྜࠊ⵨Ẽᅽࡀ6 kPaࢆ㉸࠼ࡿ≀㉁࠾ࡼࡧ⏺㠃άᛶ๣࡛ࡣ
࡞࠸ᅛయࡣࠊ㧗࠸ഇ㝜ᛶ⋡ࢆ♧ࡍࡇ࡜࠿ࡽࠊ㐺⏝ྍ⬟≀㉁࠿ࡽ㝖እࡉࢀࡿࠋᅛయΰྜ≀࡟㐺⏝ࡍࡿሙྜࡣࠊ⏺㠃άᛶ๣ࡢࡳ࠿ࡽᵓᡂࡉࢀࡿ≀㉁ࡣᑐ㇟࡟ࡍࡿࡀࠊࡑࢀ௨እࡢ≀㉁ࡣ㝖እࡍࡿࠋࡲ
ࡓࠊ⵨Ẽᅽࡀ6 kPaࢆ㉸࠼ࡿ≀㉁ࢆྵࡴΰྜ≀࡟㐺⏝ࡍࡿሙྜࡣࠊ║่⃭ᛶࡀ㐣ᑠホ౯ࡉࢀࡿྍ
⬟ᛶࡀ࠶ࡿࡢ࡛ὀពࡍࡿᚲせࡀ࠶ࡿࠋ3) ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛༢୍≀㉁࡟㐺⏝ࡍࡿࡇ࡜࡟ࡣไ㝈ࡀ࡞ࡃࠊࣂࣜࢹ࣮ࢩࣙࣥࢹ࣮ࢱࡣ㝈ࡽࢀ࡚ࡣ࠸ࡿࡶࡢࡢࠊΰྜ≀㉁࡬ࡢ㐺⏝࡟ࡶไ㝈ࡀ࡞࠸ࠋ
௨ୖࡢⅬ࠿ࡽࠊTG491 ࡟‽ᣐࡋ࡚ᐇ᪋ࡋࡓሙྜࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢᦆയࢆ㉳ࡇࡍ㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║่⃭ᛶ≀㉁࡜
ࡣศ㢮ࡉࢀ࡞࠸㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศእ㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚᭷⏝࡛࠶ࡿࠋ

㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣ࢘ࢧࢠゅ⭷⏤᮶ࡢୖ⓶⣽⬊ᰴ㸦SIRC ⣽⬊㸧࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡾࠊ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲ
ࡓࠊ▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ౑⏝⣽⬊ࡢධᡭࡣᐜ࡛᫆࠶ࡾࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡋ࡞࠸ࡇ࡜࠿

44ࡽࠊᇶᮏⓗ࡞⣽⬊ᇵ㣴ࡢᢏ⾡࡜タഛࢆᣢࡗ࡚࠸ࡿ᪋タ࡛࠶ࢀࡤᐇ᪋ྍ⬟࡛࠶ࡾࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸ࠋ௨ୖࡼࡾࠊᮏヨ㦂ἲࡣ♫఍ⓗཷࡅධࢀᛶࡀ㧗࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲࡣࠊ໬Ꮫ≀㉁࡟ࡼࡿ║่⃭ᛶࢆホ౯࡛ࡁࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢയᐖࢆ㉳ࡇࡍ≀㉁㸦UN GHS ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࡞ࡽࡧ࡟࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║่⃭ᛶ≀㉁࡜ࡣ
ศ㢮ࡉࢀ࡞࠸≀㉁㸦UN GHS ༊ศእ㸧ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊ⾜ᨻⓗ฼⏝ࡀྍ⬟࡛࠶ࡿࠋ

ཧ⪃ᩥ⊩

1)

Scott, L., et al. (2010). A proposed eye irritation testing strategy to reduce and replace in vivo studies using
Bottom-Up and Top-Down approaches. Toxicol in Vitro, 24(1): 1-9.

2)

OECD (2015) Test Guideline 491. Short Time Exposure in vitro test method for identifying 1) Chemicals
inducing serious eye damage and ii) Chemicals not requiring classification for eye irritation or serious eye
damage.

3)

Mikkelson TJ, et al. (1973). Altered bioavailability of drugs in the eye due to 34 drug-protein interaction. J.
Pharm. Sci.1648-1653.

4)

Sakaguchi et al. (2011) Validation study of the Short Time Exposure (STE) test to assess the eye irritation
potential of chemicals. Toxicol. In Vitro, 25(4): 796-809.

5)

Kojima et al. (2013) Second-phase validation study of the short time exposure test for assessment of eyeirritation potency of chemicals. Toxicol. In Vitro, 27(6): 1855-1869

6)

ICCVAM (2013) Short Time Exposure (STE) test method, Summary review document.

7)

Takahashi et al. (2009) Inter-laboratory study of short time exposure (STE) test for predicting eye irritation
potential of chemicals and correspondence to globally harmonized system (GHS) classification. J Toxicol
Sci, 34(6): 611-626.

8)

Takahashi et al. (2010) An interlaboratory study of the short time exposure (STE) test using SIRC cells forpredicting eye irritation potential. J. Cutan. Ocul. Toxicol., 29(2): 77-90.

9)JaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍㸸OECD TG491 in vitro ▷᫬㛫᭚㟢ἲ㸦The Short Time
Exposure In Vitro Test Method㸸STEἲ㸧ホ౯ሗ࿌᭩㸦2015ᖺ9᭶29᪥㸧

5

5

6

║่⃭ᛶヨ㦂௦᭰ἲࡢホ౯ሗ࿌᭩In vitro ▷᫬㛫᭚㟢ἲ
㸦Short Time Exposure In Vitro Test Method: STE ἲ㸧║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍

2015 ᖺ 9 ᭶ 29 ᪥17║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍➉ෆ ᑠⱑ 㸦P&G ࢖ࣀ࣮࣋ࢩࣙࣥྜྠ఍♫㸧ᑠᆏ ᛅྖ 㸦୍⯡㈈ᅋἲே ṧ␃㎰⸆◊✲ᡤ㸧బࠎᮌ ṇ἞ 㸦࢔ࢵࣦ࢕ྜྠ఍♫㸧᳃ᮧ ᬛ⨾ 㸦୍⯡㈈ᅋἲே 㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮㸧ྜྷᮧ ຌ 㸦ᮾி⌮⛉኱Ꮫྡ㄃ᩍᤵ㸧

2

8

␎ㄒ
AD: Applicable Domain
BCOP: Bovine Corneal Opacity and Permeability
CAS: Chemical Abstracts Services
CM: Cytosensor Microphysiometer
DMSO: Dimethyl Sulfoxide
ETDA: Ethylenediaminetetraacetic Acid
FL: Fluorescein Leakage
GHS: Globally Harmonized System of Classification and Labeling of Chemicals
ICCVAM: Interagency Coordinating Committee on the Validation of Alternative Methods
ICE: Isolated Chicken Eye
JaCVAM: Japanese Center for the Validation of Alternative Methods
kPa: kiloPascals
MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NICEATM: National Toxicology Program (NTP) Interagency Center for the Evaluation of
Alternative Toxicological Methods
OD: Optical Density
OECD: Organization for Economic Co-operation and Development
SD: Standard Deviation
SRD: Summary Review Document
SIRC: Statens Seruminstitut Rabbit Cornea
SLS: Sodium Lauryl Sulfate
STE: Short Time Exposure In Vitro Test
TG: Test Guideline
UN: United Nations

3

9

せ᪨
in vitro ▷᫬㛫᭚㟢ἲ㸦Short Time Exposure In Vitro Test Method㸸STE ἲ㸧ࡣࠊ࢘ࢧࢠ⏤᮶ࡢゅ⭷ୖ⓶⣽⬊࡟ᑐࡍࡿ໬Ꮫ≀㉁ࡢ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศ 1 ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࠾ࡼࡧ࣎ࢺ
࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࣂࣜࢹ࣮ࢩࣙࣥࡀ⾜ࢃࢀࠊ2015 ᖺ࡟ OECD ࡢࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ TG 491 ࡜ࡋ࡚᥇ᢥࡉࢀࡓࠋᮏሗ࿌᭩ࡣࠊICCVAM/NICEATM ࡢ SRD ࡸⓎ⾲ㄽᩥ࡞࡝ࢆࡶ࡜࡟ࠊ
᥇ᢥࡉࢀࡓ STE ἲࡢᴫせࢆㄝ᫂ࡋࠊJaCVAM ║่⃭ᛶ௦᭰ἲ㈨ᩱ⦅⧩ጤဨ఍ࡢពぢࢆ㏙࡭ࡓࡶࡢ࡛࠶ࡿࠋ ࢘ࢧࢠࢆ⏝࠸ࡓ Draize ║่⃭ᛶヨ㦂࡜ẚ㍑ࡋࡓ࡜ࡁࠊSTE ἲࡢࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࠾ࡼ
ࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿ୍⮴ᗘࡣ 83.2%࠾ࡼࡧ 84.6%ࠊഇ㝧ᛶ⋡ࡣ 1.2%࠾ࡼࡧ 19.3%ࠊ
ഇ㝜ᛶ⋡ࡣ 51.3%࠾ࡼࡧ 12.3%࡛࠶ࡗࡓࠋ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚㐺⏝㝖እ≀㉁ࢆ㝖࠸ࡓሙྜࡣࠊ୍⮴ᗘࠊഇ㝧ᛶ⋡ࠊഇ㝜ᛶ⋡ࡀ 90.2%ࠊ18.8%ࠊ1.9%࡛࠶ࡗࡓࠋ᪋タෆ࣭᪋タ㛫෌⌧ᛶࠊᢏ⾡⛣㌿ᛶ࡟㛵ࡋ࡚ࡣࠊᠱᛕࡉࢀࡿ⤖ᯝࡀ࡞࠿ࡗࡓࠋ
TG 491 ࡟‽ᣐࡋ࡚ᐇ᪋ࡍࢀࡤࠊSTE ἲࡣࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ UN GHS ༊ศ 1 ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲࠊ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ᮏጤဨ఍ࡣ⪃࠼ࡿࠋ

410

1. ࡲ࠼࠾ࡁ
໬Ꮫ≀㉁ࡢ║่⃭ᛶࢆホ౯ࡍࡿ᪉ἲ࡜ࡋ࡚ࡣࠊ࢘ࢧࢠࡢ║ࢆ⏝࠸ࡓ Draize ║่⃭ᛶヨ㦂ࡀᚑ᮶౑⏝ࡉࢀ࡚ࡁࡓࠋࡋ࠿ࡋࠊ㏆ᖺࡢື≀⚟♴࡟ᑐࡍࡿ㛵ᚰࡢ㧗ࡲࡾࡸḢᕞ࡟࠾ࡅࡿἲつไᨵṇ࡟ࡼࡾࠊࡑࡢ௦᭰ἲࡢ㛤Ⓨࡀ㐍ࡵࡽࢀ࣭ࣂࣜࢹ࣮ࢩࣙࣥࡀ⾜ࢃࢀ࡚ࡁࡓࠋ
⌧ᅾ OECD ࡟ࡼࡾࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚᥇ᢥࡉࢀࡓ௦᭰ἲࡣࠊ࢘ࢩ᦬ฟゅ⭷ࡢΰ⃮
࠾ࡼࡧ㏱㐣ᛶヨ㦂㸦BCOP ἲࠊTG 437㸧ࠊࢽ࣡ࢺࣜ᦬ฟ║⌫ࢆ⏝࠸ࡓ║่⃭ᛶヨ㦂ἲ㸦ICE
ἲࠊTG 438㸧ࠊ࠾ࡼࡧࣇࣝ࢜ࣞࢭ࢖ࣥ₃ฟヨ㦂ἲ㸦FL ἲࠊTG 460㸧ࡢ 3 ヨ㦂ἲ࡛࠶ࡿࠋBCOP ἲ࡜ ICE ἲࡣ㣗⏝࡞࡝ࡢ┠ⓗ࡛ฎศࡉࢀࡓື≀ࡼࡾ᦬ฟࡋࡓჾᐁ࣭⤌⧊ࢆ⏝࠸࡚໬Ꮫ≀㉁ࡢ᭚㟢࡟ࡼࡾ⏕ࡌࡿゅ⭷ࡢ≀⌮ⓗ≉ᛶࡢኚ໬ࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚㔜⠜࡞║ࡢയᐖࢆ㉳ࡇࡍ㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศ 1㸧≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚ࠊࡲࡓ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾࠸࡚║࡟ᑐࡍࡿ㔜⠜࡞ᦆയᛶࢆ
᭷ࡍࡿ≀㉁࠾ࡼࡧ║่⃭ᛶ≀㉁࡜ࡣศ㢮ࡉࢀ࡞࠸㸦ࡍ࡞ࢃࡕࠊUN GHS ༊ศእ㸧≀㉁ࢆ
᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡽࢀࡿࠋFL ἲࡣ༢ᒙᇵ㣴ࡋࡓᒀ⣽⟶ୖ⓶⣽⬊ࢆ⏝࠸࡚໬Ꮫ≀㉁ࡢ᭚㟢࡟ࡼࡾ⏕ࡌࡿ⣽⬊㛫⤖ྜࡢയᐖࢆᣦᶆ࡟║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛ࠊࢺࢵࣉࢲ
࢘ࣥ᪉ᘧ࡛ࡢࡳ⏝࠸ࡽࢀࡿࠋ
in vitro ▷᫬㛫᭚㟢ἲ㸦Short Time Exposure In Vitro Test Methodࠊ௨ୗ STE ἲ㸧ࡣࠊゅ⭷ୖ⓶ࡢᦆയࡢస⏝ᶵᗎ࡛࠶ࡿゅ⭷ୖ⓶⣽⬊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡟⏝࠸࡚ࠊ║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࡍ࡞ࢃࡕ STE ἲ࡛ࡣࠊ࢘ࢧࢠゅ⭷⏤᮶ᰴ໬⣽⬊࡛࠶ࡿ SIRC⣽⬊࡟⿕㦂≀㉁ࡢᕼ㔘ᾮࢆ 5 ศ㛫᭚㟢ࡋࡓᚋࠊMTT ࡢྲྀࡾ㎸ࡳ㔞ࢆᇶ࡟⣽⬊⏕Ꮡ⋡ࢆᐃࡋ㸦MTT 㑏ඖἲ㸧ࠊ⿕㦂≀㉁㸦ᮍᕼ㔘㸧ࡢ║่⃭ᛶࢆホ౯ࡋ࡚࠸ࡿ㸦Takahashi et al. 2008㸧ࠋ
STE ἲࡣ᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸦Sakaguchi et al. 2011㸧࡜ JaCVAM㸦Kojima et al. 2013㸧ࡢࣂࣜࢹ࣮ࢩࣙࣥࢆ⤒࡚ࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡟࠾࠸࡚ UN GHS ༊ศ 1 ≀㉁ࢆ᳨ฟࡍࡿ᪉
ἲ࠾ࡼࡧ࣎ࢺ ࣒࢔ࢵ ࣉ᪉ᘧ ࡟࠾࠸ ࡚ UN GHS ༊ศእ≀ ㉁ࢆ ᳨ฟࡍࡿ᪉ἲ ࡜ࡋ࡚
ICCVAM/NICEATM ࡟ࡼࡾ➨୕⪅ホ౯ࢆཷࡅࠊࡑࡢሗ࿌᭩㸦Summary Review Document:
SRD㸧ࡀ 2013 ᖺ 6 ᭶࡟බ⾲ࡉࢀࡓࠋࡑࡢᚋࠊ2015 ᖺ 7 ᭶࡟ OECD ࡟ࡼࡾࢺࢵࣉࢲ࣭࢘ࣥ
࣎ࢺ࣒࢔ࢵࣉ୧᪉ᘧࡢ║่⃭ᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ࡟᥇ᢥࡉࢀࡓ㸦TG 
491㸧ࠋᮏሗ࿌᭩ࡣ ICCVAM/NICEATM ࡢ SRD ࡸⓎ⾲ㄽᩥ࡞࡝ࢆࡶ࡜࡟ OECD TG 491 ࡟᥇ᢥ
ࡉࢀࡓ STE ἲࡢᴫせࢆㄝ᫂ࡋࠊᮏጤဨ఍ࡢពぢ࠾ࡼࡧホ౯ࢆ㏙࡭ࡓࡶࡢ࡛࠶ࡿࠋ
2㸬ヨ㦂ἲࡢ఩⨨࡙ࡅ
STE ἲࡣࠊUN GHS ༊ศ 1 ≀㉁࡛࠶ࡿ༢୍≀㉁࠾ࡼࡧΰྜ≀ࢆࢺࢵࣉࢲ࢘ࣥ᪉ᘧ᳨࡛
ฟࡍࡿ㝿࡟᭱ึ࡟⏝࠸ࡿヨ㦂ἲ࡛࠶ࡿࠋࡲࡓࠊSTE ἲࡣࠊUN GHS ༊ศእ≀㉁࡛࠶ࡿ༢
୍≀㉁࠾ࡼࡧΰྜ≀ࢆ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ᳨࡛ฟࡍࡿ㝿࡟᭱ึ࡟⏝࠸ࡿヨ㦂ἲ࡛ࡶ࠶ࡿࠋ
3㸬ヨ㦂ἲࡢཎ⌮║࡟␗≀ࡀධࡗࡓሙྜࠊ║ࡢ่⃭ᛶࡣ᭱⾲㠃ࡢ⣽⬊യᐖ࠿ࡽጞࡲࡿࠋSTE ἲࡣࡇࢀ࡟

511ὀ┠ࡋ࡚ࠊゅ⭷ୖ⓶⣽⬊࡟ᑐࡍࡿ⣽⬊ẘᛶࢆᣦᶆ࡜ࡋ࡚║่⃭ᛶࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋࢥࣥࣇ࢚ࣝࣥࢺ࡟༢ᒙᇵ㣴ࡋࡓ SIRC ⣽⬊࡟⿕㦂≀㉁ᕼ㔘ᾮࢆ 5 ศ㛫᭚㟢ࡋࡓᚋࠊ
MTT ࡢྲྀࡾ㎸ࡳ㔞࡛ ᐃࡋࡓ⣽⬊⏕Ꮡ⋡ࢆ࢚ࣥࢻ࣏࢖ࣥࢺ࡟⏝࠸ࡿࠋࡇࢀࡣࠊMTT ࡀ⬺Ỉ⣲㓝⣲ࡢᇶ㉁࡜࡞ࡿᛶ㉁ࢆ฼⏝ࡋࠊ⣽⬊ෆ࡟ྲྀࡾ㎸ࡲࢀࡓ MTT ࡀ࣑ࢺࢥࣥࢻࣜ࢔ෆ
⬺Ỉ⣲㓝⣲࡟ࡼࡾ㑏ඖࡉࢀࠊ⏕ᡂࡉࢀࡓ࣐࣍ࣝࢨࣥ㔞ࡀ⏕Ꮡ⣽⬊ᩘ࡟ẚ౛ࡍࡿࡇ࡜ࢆ฼
⏝ࡋ࡚࠸ࡿ㸦MTT 㑏ඖἲ㸧ࠋ║࡟⁐ᾮࡀධࡗࡓሙྜࠊࣄࢺ࡛ࡣࡑࡢ኱㒊ศࡀ 1㹼2 ศ࡛║ෆ࠿ࡽ᤼ฟࡉࢀࠊ࢘ࢧࢠ࡛
ࡣ 3㹼4 ศ࡛ 80%ࡀ᤼ฟࡉࢀࡿ࡜ሗ࿌ࡉࢀ࡚࠸ࡿࠋࡇࡢࡼ࠺࡞ᐇ㝿ࡢ᭚㟢≧ἣࢆ⪃៖ࡋ࡚ࠊ
STE ἲࡣ㏻ᖖࡢ⣽⬊ẘᛶヨ㦂࡜ẚ࡭࡚▷᫬㛫᭚㟢ࡢヨ㦂࡜ࡋ࡚タィࡉࢀ࡚࠸ࡿࠋ
4㸬ヨ㦂ᡭ㡰
 STE ἲࡢᡭ㡰ࡢヲ⣽ࡣ TG 491 ࡟᭩࠿ࢀ࡚࠸ࡿࡀࠊᴫせࡣ௨ୗࡢ㏻ࡾ࡛࠶ࡿࠋ
4-1. ⣽⬊ࡢ‽ഛ
SIRC ⣽⬊ࡣ 10%㸦v/v㸧࢘ࢩ⫾ඣ⾑Ύࠊ2 mM L-ࢢࣝࢱ࣑ࣥࠊ50㹼100 unit/mL ࣌ࢽࢩ
ࣜࣥࠊ50㹼100 μg/mL ࢫࢺࣞࣉࢺ࣐࢖ࢩࣥࢆῧຍࡋࡓ Eagle ᭱ᑠᚲ㡲ᇵᆅ࡟࡚ 37˚Cࠊ5%
CO2 Ꮡᅾୗ࡛ᇵ㣴ࡍࡿࠋࢥࣥࣇ࢚ࣝࣥࢺ࡟࡞ࡿࡲ࡛ᇵ㣴ࡋࡓ⣽⬊ࢆࢺࣜࣉࢩࣥ EDTA ⁐ᾮ࡟ࡼࡾ๤ࡀࡋࠊ⥅௦⏝ᇵ㣴ࣇࣛࢫࢥ࡟᧛✀ࡍࡿࠋヨ㦂࡟ࡣ⥅௦ᩘ 25 ᅇࡲ࡛ࡢ⣽⬊ࢆ౑⏝ࡍࡿࠋヨ㦂࡟⏝࠸ࡿ㝿ࡣࠊヨ㦂ᐇ᪋᫬࡟ࢥࣥࣇ࢚ࣝࣥࢺ㸦>80%㸧࡟࡞ࡿࡼ࠺࡟ࠊ๓ᇵ㣴ᮇ㛫
ࡀ 4 ᪥㛫࡞ࡽࡤ࢙࢘ࣝ࠶ࡓࡾ 6.0 × 103 cellsࠊ5 ᪥㛫࡞ࡽࡤ࢙࢘ࣝ࠶ࡓࡾ 3.0 × 103 cells ࡟ㄪ〇ࡋ 96 ࢙࢘ࣝᖹᯈࣉ࣮ࣞࢺ࡟ 200 μL ࡎࡘ᧛✀ࡍࡿࠋ
4-2. ヨ㦂ࢧࣥࣉࣝࡢㄪ〇⏕⌮㣗ሷỈࠊ5%㸦w/w㸧DMSO㸦CAS# 67-68-5㸧ࢆྵࡴ⏕⌮㣗ሷỈࠊ࣑ࢿࣛࣝ࢜࢖ࣝ

612

㸦CAS# 8042-47-5㸧ࡢ୰࠿ࡽ㐺ษ࡞⁐፹ࢆᅗ 1 ࡟ᚑࡗ࡚㑅ᢥࡍࡿࠋヨ㦂࡟ࡣ⿕㦂≀㉁ࡢ 5% ࠾ࡼࡧ 0.05% ᕼ㔘ᾮ㸦⁐ᾮ࠶ࡿ࠸ࡣᠱ⃮ᾮ㸧ࢆ౪ࡍࡿࠋ㝧ᛶᑐ↷࡟ࡣ SLS ࡢ 0.01% ⏕⌮㣗ሷỈ⁐ᾮࢆ⏝࠸ࡿࠋࡇࢀ࡟ຍ࠼࡚ࠊ⁐፹ᑐ↷ࠊ᧯సᑐ↷㸦ᇵᆅᑐ↷㸧࠾ࡼࡧࣈࣛࣥࢡ㸦⣽⬊࡞ࡋᇵᆅࡢࡳ㸧ࡶタࡅࡿࠋ
4-3. ᭚㟢࣭⣽⬊⏕Ꮡ⋡ࡢ⟬ฟ SIRC ⣽⬊ࢆ๓ᇵ㣴ࡋ࡚ࢥࣥࣇ࢚ࣝࣥࢺ࡜࡞ࡗࡓ 96 ࢙࢘ࣝᖹᯈࣉ࣮ࣞࢺࢆ௨ୗࡢࡼ࠺
࡟ฎ⌮ࡍࡿࠋྛ࢙࢘ࣝ࠿ࡽᇵᆅࢆ㝖ཤࡋࠊ⿕㦂≀㉁ᕼ㔘ᾮ㸦5%ࠊ0.05%㸧࠾ࡼࡧᑐ↷≀㉁ࢆࡑࢀࡒࢀ 200 μL ࡎࡘῧຍࡋ 5 ศ㛫ᐊ ࡛᭚㟢ࡍࡿࠋ᭚㟢⤊஢ᚋࠊヨ㦂ࢧࣥࣉࣝࢆ㝖ཤࡋࠊࣜࣥ㓟⦆⾪ᾮ 200 μL ࡛ 2 ᅇὙίࡍࡿࠋMTT ⁐ᾮࢆ 200 μL ῧຍࡋࠊ37˚Cࠊ5% CO2Ꮡᅾୗ࡛ 2 ᫬㛫཯ᛂࡉࡏࡿࠋ཯ᛂᚋ MTT ⁐ᾮࢆ㝖ཤࡋࠊ࣐࣍ࣝࢨࣥᢳฟᾮࢆ 200 μL ῧຍࡋࠊ1 ᫬㛫ᐊ ᬯᡤ࡛࣐࣍ࣝࢨࣥࢆᢳฟࡋࠊࡑࡢ྾ගᗘࢆࣉ࣮ࣞࢺ࣮ࣜࢲ࣮࡟ࡼࡾ 570
nm㸦OD570㸧࡛ ᐃࡍࡿࠋ ヨ㦂ࢧࣥࣉࣝẖ࡟ 3 ࢙࢘ࣝࢆ⏝࠸ࠊ3 ࢙࢘ࣝࡢᖹᆒ OD570 ࢆ⣽⬊⏕Ꮡ⋡⟬ฟ࡟⏝࠸ࡿࠋ⣽⬊⏕Ꮡ⋡ =

OD570 ヨ㦂ࢧࣥࣉࣝ㸫OD570 ࣈࣛࣥࢡOD570 ⁐፹ᑐ↷㸫OD570 ࣈࣛࣥࢡ× 100

࡞࠾ࠊヨ㦂ࢧࣥࣉࣝฎ⌮⩌ࡢ⣽⬊⏕Ꮡ⋡ࡀ࣐࢖ࢼࢫ࡟࡞ࡗࡓሙྜࡣࠊ⏕Ꮡ⋡ࢆࢮࣟ࡜ࡍ
ࡿࠋ
4-4. ║่⃭ᛶุᐃ ௨ୖࡢᡭ㡰࡛ᐇ㦂ࢆ 3 ᅇᐇ᪋ࡋࠊࡑࢀࡒࢀ࡛ᚓࡽࢀࡓ⣽⬊⏕Ꮡ⋡ࡢᖹᆒࢆ᭱⤊ุᐃ࡟⏝࠸ࡿࠋ║่⃭ᛶ GHS ศ㢮ࡢุᐃࡣ⾲ 1 ࡟♧ࡋࡓ㏻ࡾ࡛࠶ࡿࠋ
⾲ 1. ║่⃭ᛶ GHS ศ㢮ࡢุᐃ⿕㦂≀㉁ᕼ㔘⃰ᗘ

ᖹᆒ⏕Ꮡ⋡

0.05%

> 70%

5%

> 70%

0.05%

> 70%

5%

< 70%

0.05%

< 70%

5%

< 70%

⿕㦂≀㉁ࡢ GHS ศ㢮༊ศእศ㢮࡛ࡁ࡞࠸
༊ศ 1

4-5. ⤖ᯝࡢᢎㄆᇶ‽௨ୗࡢ 4 ᮲௳ࢆ‶ࡓࡋࡓሙྜࠊヨ㦂ࡢ⤖ᯝࢆᢎㄆࡍࡿࠋ
1) ᧯సᑐ↷ࡢ྾ගᗘࡀࣈࣛࣥࢡῶ⟬ᚋ 0.3 ௨ୖ࡛࠶ࡿࡇ࡜ࠋ
2) ᐇ㦂࡟⏝࠸ࡓ⁐፹ᑐ↷ࡍ࡭࡚࡟࠾࠸࡚ࠊ⣽⬊⏕Ꮡ⋡ࡀ᧯సᑐ↷ࡢ 80%௨ୖ࡛࠶ࡿࡇ
࡜ࠋ

7

13

3) 㝧ᛶᑐ↷ࡢ⣽⬊⏕Ꮡ⋡ࡀጇᙜ࡞⠊ᅖὀ㸧࡟཰ࡲࡿࡇ࡜ࠋ
4) 3 ᅇࡢᐇ㦂ࡢ⣽⬊⏕Ꮡ⋡ࡢ SD ࡀ⿕㦂≀㉁ 5%ࠊ0.05% ᕼ㔘ᾮ࡝ࡕࡽ࡛ࡶ 15%ᮍ‶࡛࠶ࡿࡇ࡜ࠋ1)㹼4) ࡢ᮲௳ࡢ࠸ࡎࢀ࠿ࡀ‶ࡓࡉࢀ࡞࠸ሙྜ࡟ࡣࢹ࣮ࢱࢆࡍ࡭࡚◚Რࡋࠊ᪂ࡓ࡟⊂❧ࡋࡓ 3 ᅇࡢᐇ㦂ࢆᐇ᪋ࡍࡿࠋ
5㸬ヨ㦂ἲࡢṇ☜ᛶ௨ୗࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂➼ࡢ⤖ᯝࡀ STE ἲࡢṇ☜ᛶࡢ᳨ウ࡟⏝࠸ࡽࢀࡓࠋ
࣭ Takahashi et al.㸦2009㸧– ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 I 44 ≀㉁࣭ Takahashi et al.㸦2010㸧– ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 II 70 ≀㉁࣭ Sakaguchi et al.㸦2011㸧– ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 I 25 ≀㉁࣭ Kojima et al.㸦2013㸧– ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 II 40 ≀㉁࣭ ⰼ⋤♫ෆࢹ࣮ࢱ – Takahashi et al.㸦2011㸧࠾ࡼࡧᮍⓎ⾲♫ෆࢹ࣮ࢱ 23 ≀㉁࣭ ⰼ⋤♫ෆࢹ࣮ࢱ㸦㏣ຍศ㸧– ⏺㠃άᛶ๣࠾ࡼࡧ⏺㠃άᛶ๣ྵ᭷ࡢฎ᪉ 52 ≀㉁࣭ Saito et al.㸦2015㸧– ΰྜ≀ 40 ≀㉁
Saito et al.ࡢࢹ࣮ࢱࢆ㝖ࡃ 169 ≀㉁ࡢࢹ࣮ࢱࢆ⏝࠸࡚ ICCVAM/NICEATM ࡟ࡼࡿṇ☜ᛶࡢ᳨ウࡀ⾜ࢃࢀࡓࠋࡑࡢ࠺ࡕࠊGHS ࡢ║่⃭ᛶศ㢮ࢆ⾜࠺ࡢ࡟㐺ษ࡞ື≀ヨ㦂ࡢࢹ࣮ࢱࡀ࡞࠸≀㉁ࡣṇ☜ᛶࡢ᳨ウᑐ㇟࠿ࡽ㝖࠿ࢀࡓࠋ1 ࡘࡢ≀㉁࡟ᑐࡋ࡚」ᩘࡢヨ㦂᪋タ࡛ࡢ
STE ἲࡢุᐃࡀ࠶ࡿሙྜࡣࠊヨ㦂᪋タᩘࡀ᭱ࡶከ࠸ุᐃࢆ᥇⏝ࡋࡓࠋࡑࡢヨ㦂᪋タᩘࡀྠᩘ࡟࡞ࡗࡓሙྜࡣࠊ่⃭ᛶࡢ㧗࠸ุᐃࡢ᪉ࢆ᥇⏝ࡋࡓࠋ࡞࠾ࠊICCVAM/NICEATM ࡢ
᳨ウ࡛ࡣ MTT ࢆ┤᥋㑏ඖࡍࡿ≀㉁㸦࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡣ༊ศእ࡜ุᐃࡉࢀࡓ≀㉁ࡢࡳ㸧ࡣ᳨ウᑐ㇟࠿ࡽእࡉࢀ࡚࠸ࡓࡀࠊ2 ḟඖ⣽⬊ᇵ㣴⣔ࢆ⏝࠸ࡿ STE ἲ࡟࠾࠸࡚ࡣὙί࡟ࡼ
ࡾ⿕㦂≀㉁ࡣ༑ศྲྀࡾ㝖࠿ࢀࡿࡢ࡛ࠊ௨ୗࡢṇ☜ᛶࡢ᳨ウ࡛ࡣ᥇⏝ࡋࡓࠋ
Saito et al. ࡢࢹ࣮ࢱࡣ 5%ᕼ㔘ࡢ⤖ᯝࡢࡳࢆྵࡳࠊ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡢΰྜ≀ࡢホ౯࡟࠾ࡅࡿṇ☜ᛶࢆ᳨ウࡋ࡚࠸ࡿࠋ
5-1. ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ㸫GHS ༊ศ 1 ࡢ≀㉁ࡢ᳨ฟ
ࢺࢵࣉࢲ࢘ࣥ᪉ᘧࡢṇ☜ᛶࡢ᳨ウ࡟ࡣ 125 ≀㉁ࡢࢹ࣮ࢱࡀ⏝࠸ࡽࢀࡓ㸦Appendix 1㸧ࠋࡑࡢ⤖ᯝࡣࠊṇ☜ᗘ 83.2%㸦104/125㸧ࠊഇ㝧ᛶ⋡ 1.2%㸦1/86㸧ࠊഇ㝜ᛶ⋡ 51.3%㸦20/39㸧࡛࠶ࡗࡓࠋഇ㝜ᛶࢆ♧ࡋࡓ 20 ≀㉁ࡢ࠺ࡕࠊ࢔ࣝࢥ࣮ࣝ㢮ࡀ 8 ≀㉁ࠊ࢝ࣝ࣎ࣥ㓟㢮ࡀ 5 ≀㉁ࠊࡲࡓሷ㢮ࡀ 3 ≀㉁࠶ࡗࡓࠋࡑࡢ࡯࠿ࠊ࢚ࢫࢸࣝ㢮ࡸ࣊ࢸࣟࢧ࢖ࢡࣜࢵࢡ໬ྜ≀࡞࡝࡛ഇ㝜ᛶࢆ♧ࡋࡓ≀㉁ࡀ࠶ࡗࡓࠋഇ㝧ᛶ࡜࡞ࡗࡓ 1 ≀㉁ࡣ㠀࢖࢜ࣥᛶ⏺㠃άᛶ๣࡛࠶ࡗ
ࡓࠋ࢔ࣝࢥ࣮ࣝ㢮ࡸ࢚ࢫࢸࣝ㢮࡞࡝ື≀ヨ㦂࡛ᚓࡽࢀࡿ║่⃭ᛶศ㢮࡜୍⮴ࡋ࡞࠸≀㉁ࡢ๭ྜࡀ㧗࠿ࡗࡓ໬Ꮫ≀㉁⩌ࢆ㝖࠸࡚ࡶࠊṇ☜ᛶࡢᩘ್࡟㢧ⴭ࡞ᨵၿࡣぢࡽࢀ࡞࠿ࡗࡓࠋὀ㸧ᐇ᪋᪋タࡢ㐣ཤࡢࢹ࣮ࢱ࠿ࡽồࡵࡽࢀࡿᖹᆒ⣽⬊⏕Ꮡ⋡s2SD ࡟཰ࡲࡿࡇ࡜ࠋୖ㝈࣭ୗ㝈್ࡣ 3 ࣨ᭶ࡈ࡜࡟᭦᪂ࡍ
࡭ࡁ࡛࠶ࡿࠋᐇ᪋ᐇ⦼ࡀ 1 ࣨ᭶࡟ 1 ᅇᮍ‶ࡢ᪋タ࡟࠾࠸࡚ࡣୖ㝈࣭ୗ㝈್ࡢ᭦᪂ࡣヨ㦂ᐇ᪋ࡈ࡜࡟⾜࠺ࡇ࡜ࠋ㐣ཤࡢࢹ࣮ࢱࡀ୙༑ศ࡞᪋タ࡟࠾࠸࡚ࡣࠊSTE ἲ㛤Ⓨ᪋タࡀタᐃࡋࡓୖ㝈࣭ୗ㝈್㸦21.1%㹼62.3%㸧ࢆ⏝࠸࡚ᵓࢃ࡞࠸ࠋ

814ࡍ࡛࡟ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢࣂࣜࢹ࣮ࢩࣙࣥࡀ⤊஢ࡋ࡚࠸ࡿ௚ࡢ௦᭰ἲ࡜ STE ἲࡢṇ☜ᛶࡢẚ㍑ࢆ⾲ 2 ࡟ࡲ࡜ࡵࡓࠋ
5-2. ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ㸫GHS ༊ศእࡢ≀㉁ࡢ᳨ฟ
࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࡢṇ☜ᛶࡢ᳨ウ࡟ࡣ 130 ≀㉁ࡢࢹ࣮ࢱࡀ⏝࠸ࡽࢀࡓ㸦Appendix 1㸧ࠋࡑࡢ⤖ᯝࡣࠊṇ☜ᗘ 84.6%㸦110/130㸧ࠊഇ㝧ᛶ⋡ 19.3%㸦11/57㸧ࠊഇ㝜ᛶ⋡ 12.3%㸦9/73㸧
࡛࠶ࡗࡓࠋ㧗࠸ഇ㝜ᛶ⋡ࢆ♧ࡋࡓ≀㉁ࡣࠊሷ㢮ࠊⅣ໬Ỉ⣲ࠊ࢔ࣝࢥ࣮ࣝ㢮࡞࡝ࡀ࠶ࡗࡓࠋ
ࡲࡓࠊᅛయ㸦13.5%㸧ࡢ᪉ࡀᾮయ㸦4.3%㸧ࡼࡾࡶഇ㝜ᛶ⋡ࡀ㧗࠿ࡗࡓࠋࡇࢀࡽࡢ໬Ꮫ≀㉁⩌ࡢࢹ࣮ࢱࢆࡑࢀࡒࢀ㝖࠸࡚ࡶࠊṇ☜ᛶࡢᩘ್࡟㢧ⴭ࡞ᨵၿࡣぢࡽࢀ࡞࠿ࡗࡓࠋ㐺⏝⠊ᅖ࡜ࡋ࡚ࠊ⵨Ẽᅽࡀ㧗࠸≀㉁㸦> 6 kPa㸧࡜⏺㠃άᛶ๣࡛ࡣ࡞࠸ᅛయࢆ㝖࠸ࡓሙྜࡣࠊṇ☜ᗘ 90.2%㸦92/102㸧ࠊഇ㝧ᛶ⋡ 18.8%㸦9/48㸧ࠊഇ㝜ᛶ⋡ 1.9%㸦1/54㸧࡛࠶ࡗࡓࠋഇ㝜ᛶࢆ♧ࡋࡓ≀㉁ࡣື≀ヨ㦂࡛ࡢ GHS ศ㢮ࡀ༊ศ 2B ࡛࠶ࡿ Toluene ࡛࠶ࡗࡓࠋࡍ࡛࡟࣎ࢺ
࣒࢔ࢵࣉ᪉ᘧ࡛ࡢࣂࣜࢹ࣮ࢩࣙࣥࡀ⤊஢ࡋ࡚࠸ࡿ௚ࡢ௦᭰ἲ࡜ STE ἲࡢṇ☜ᛶࡢẚ㍑ࢆ
⾲ 3 ࡟ࡲ࡜ࡵࡓࠋ
⾲ 2. ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡢ STE ἲ࡜ࣂࣜࢹ࣮ࢩࣙࣥࡀ⤊஢ࡋ࡚࠸ࡿ௦᭰ἲ࡜ࡢṇ☜ᛶ
ࡢẚ㍑
ヨ㦂ἲ

≀㉁ᩘ

STE
BCOP
ICE
CM
FL

125
188
144
82
151

ṇ☜ᗘ
%
No.
83.2
104/125
79.3
149/188
83.3
120/144
90.2
74/82
77.5
117/151

ഇ㝧ᛶ⋡
%
No.
1.2
1/86
23.6
29/123
7.9
9/114
2.1
1/48
6.8
7/103

ഇ㝜ᛶ⋡
%
No.
51.3
20/39
15.4
10/65
50.0
15/30
20.6
7/34
56.3
27/48

⾲ 3. ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ࡢ STE ἲ࡜ࣂࣜࢹ࣮ࢩࣙࣥࡀ⤊஢ࡋ࡚࠸ࡿ௦᭰ἲ࡜ࡢṇ☜ᛶ
ࡢẚ㍑

130
188
152
53

ṇ☜ᗘ
%
No.
84.6
110/13066.5
125/18882.2
125/152
67.9
36/53

ഇ㝧ᛶ⋡
%
No.
19.3
11/57
69.2
63/91
32.9
26/79
68.0
17/25

ഇ㝜ᛶ⋡
%
No.
12.3
9/73
0.0
0/97
1.4
1/73
0.0
0/28

102

90.2

18.8

1.9

ヨ㦂ἲ

≀㉁ᩘ

STE
BCOP
ICE
CM
STE*

92/102

9/48

1/54* ⵨Ẽᅽ > 6 kPa ࡢ≀㉁ࠊ࠾ࡼࡧࠊ⏺㠃άᛶ๣௨እࡢᅛయࢆ㝖࠸ࡓሙྜ5-3. ΰྜ≀
๓㏙ࡢ ICCVAM/NICEATM ࡟ࡼࡿṇ☜ᛶࡢ᳨ウ࡟ࡣΰྜ≀ࡀ 10 ≀㉁ྵࡲࢀ࡚࠸ࡿࠋࡑ
ࡢ࠺ࡕࠊ༊ศ 1 ࡢ 1 ≀㉁ࡣศ㢮୙ྍ࡜ุᐃࡉࢀࡓࠋ༊ศእࡢ 4 ≀㉁୰ 3 ≀㉁ࡣ༊ศእ࡜
ุᐃࡉࢀࡓࠋ

915

ICCVAM/NICEATM ࡟ࡼࡿṇ☜ᛶࡢ᳨ウ࡜ࡣู࡟ࠊSaito et al. ࡢ 40 ΰྜ≀ࢆ⏝࠸ࡓ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡟࠾ࡅࡿṇ☜ᛶࡢ᳨ウ࡟ࡼࡿ࡜ࠊṇ☜ᗘ 87.5% 㸦35/40㸧ࠊഇ㝧ᛶ⋡ 50.0%
㸦5/10㸧
ࠊഇ㝜ᛶ⋡ 0.0%㸦0/30㸧࡛࠶ࡗࡓࠋ
6㸬ヨ㦂ἲࡢಙ㢗ᛶ௨ୗࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂ࡢ⤖ᯝࡀ STE ἲࡢಙ㢗ᛶࡢ᳨ウ࡟⏝࠸ࡽࢀࡓࠋ
࣭ Takahashi et al.㸦2009㸧– ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 I
࣭ Takahashi et al.㸦2010㸧– ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 II
࣭ Sakaguchi et al.㸦2011㸧– ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 I
࣭ Kojima et al.㸦2013㸧– ࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 II
6-1. ᢏ⾡⛣㌿ᛶ
STE ἲࢆᐇ᪋ࡍࡿࡓࡵ࡟ᚲせ࡞⿦⨨࣭ჾලࡣࠊ୍⯡ⓗ࡞↓⳦໬ࡢ⣽⬊ᇵ㣴࡟⏝࠸ࡿࡶࡢ࡛ࠊධᡭࡣᐜ࡛᫆࠶ࡿࠋᡭᢏࡶᢏ⾡ⓗ࡟」㞧࡞ࡶࡢ࡛ࡣ࡞࠸ࠋ2 ࡘࡢࣉࣞࣂࣜࢹ࣮ࢩ
ࣙࣥヨ㦂࡟࠾࠸࡚ࡶᢏ⾡⛣㌿ᛶ࡟ࡘ࠸࡚ࡢၥ㢟ࡣᣦ᦬ࡉࢀ࡚࠸࡞࠸ࠋ
6-2. ᪋タෆ෌⌧ᛶSTE ἲࡣ 1 ࡘࡢヨ㦂࡛ 3 ࡘࡢ⊂❧ࡋࡓᐇ㦂ࢆ⾜࠺ࡓࡵࠊಶࠎࡢᐇ㦂࠿ࡽᚓࡽࢀࡓ⣽⬊⏕Ꮡ⋡ࡀ᪋タෆ෌⌧ᛶࡢ᳨ウ࡟⏝࠸ࡽࢀࡓࠋ༊ศእ࡜ุᐃࡉࢀࡓ≀㉁ࡢ 5%ᕼ㔘ᾮ࡛ࡢ⣽⬊⏕Ꮡ⋡ࡢኚືಀᩘࡣ 0.3㹼23.5%࡛࠶ࡗࡓࠋ༊ศእ࡜ุᐃࡉࢀ࡞࠿ࡗࡓ≀㉁࡛ࡣ⏕Ꮡ⋡ࡀᴟࡵ࡚ప࠸್࡟࡞ࡿࡓࡵࠊࡑࡢኚືಀᩘࡣ㧗࠸್ࢆ♧ࡋࡓࠋ㝧ᛶᑐ↷࡜ࡋ࡚⏝࠸ࡽࢀ
ࡿ 0.01% SLS ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡣ 41.7%㸦n = 71㸧࡛࠶ࡾࠊኚືಀᩘࡣ 24.7%࡛࠶ࡗࡓࠋ
6-3. ᪋タ㛫෌⌧ᛶ
5 ᪋タ࡛⾜ࢃࢀࡓ Sakaguchi et al.ࡢࣂࣜࢹ࣮ࢩࣙࣥヨ㦂 I ࡟࠾࠸࡚ࠊࡑࢀࡒࢀࡢ᪋タ࡛ᚓࡽࢀࡓᕼ㔘⃰ᗘࡈ࡜ࡢ 25 ≀㉁ࡢᖹᆒ⣽⬊⏕Ꮡ⋡ࡢ SD ࡣ 0.3㹼20.1%࡛࠶ࡗࡓࠋSD ࡣᇶᮏⓗ࡟ᑠࡉࡃࠊⰋዲ࡞᪋タ㛫෌⌧ᛶࡀ☜ㄆࡉࢀࡓࠋ25 ≀㉁ࡢ GHS ศ㢮ࡢุᐃ࡟ࡘ࠸
࡚ 5 ᪋タࡍ࡭ุ࡚࡛ᐃࡀ୍⮴ࡋࡓࡢࡣ 21 ≀㉁ࠊ1 ᪋タุ࡛ᐃࡀ␗࡞ࡗࡓࡢࡣ 3 ≀㉁࡛ࠊ2 ᪋タุ࡛ᐃࡀ␗࡞ࡗࡓࡢࡣ 1 ≀㉁࡛࠶ࡗࡓࠋ
 STE ἲࢆᐇ᪋ࡍࡿヨ㦂᪋タ࡬㐺ษ࡞ᢏ⾡⛣㌿ࡀ㐩ᡂ࡛ࡁࡓࡇ࡜ࢆ☜࠿ࡵࡿ࡟ࡣࠊ
Appendix 2 ࡢ⇍㐩ᗘ☜ㄆ≀㉁ࢆ⏝࠸ࡿࠋ
7㸬ヨ㦂ἲࡢ㐺⏝⠊ᅖ
STE ἲࡢ㐺⏝࡟ࡣヨ㦂ἲࡢᛶ㉁ୖࠊࡲࡓࠊṇ☜ᛶࡢほⅬ࠿ࡽ௨ୗࡢไ㝈ࡀタࡅࡽࢀࡿࠋ
1) ⏕⌮㣗ሷỈࠊ5%㸦w/w㸧DMSO ࢆྵࡴ⏕⌮㣗ሷỈࠊ࠾ࡼࡧ࣑ࢿࣛࣝ࢜࢖ࣝࡢ࠸ࡎࢀ
࡟ࡶ⁐ゎࡋ࡞࠸ࠊ࠶ࡿ࠸ࡣࡑࢀࡽ⁐፹୰࡛ᆒ୍ศᩓࡋ࡚࠸ࡿ≧ែࡀ 5 ศ௨ୖಖࡓࢀ
࡞࠸≀㉁ࡣ㐺⏝୙ྍ⬟࡛࠶ࡿࠋ2) ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧࢆ༢୍≀㉁࡟㐺⏝ࡍࡿሙྜࠊ⵨Ẽᅽࡀ 6 kPa ࢆ㉸࠼ࡿ≀㉁࠾ࡼࡧ
⏺㠃άᛶ๣࡛ࡣ࡞࠸ᅛయࡣࠊࡇࢀࡽ≀㉁⩌ࡀ㧗࠸ഇ㝜ᛶ⋡ࢆ♧ࡍࡇ࡜࠿ࡽࠊ㐺⏝࠿1016ࡽ㝖እࡉࢀࡿࠋᅛయΰྜ≀࡟㐺⏝ࡍࡿሙྜࡣࠊ⏺㠃άᛶ๣ࡢࡳ࠿ࡽᵓᡂࡉࢀࡿ≀㉁ࡣᑐ㇟࡟ࡍࡿࡀࠊࡑࢀ௨እࡢ≀㉁ࡣ㝖እࡍࡿࠋࡲࡓࠊ⵨Ẽᅽࡢ㧗࠸㸦> 6 kPa㸧≀㉁ࢆྵࡴΰྜ≀࡟㐺⏝ࡍࡿሙྜࡣ║่⃭ᛶࡀ㐣ᑠホ౯ࡉࢀࡿྍ⬟ᛶࡀ࠶ࡿࡢ࡛ࠊSTEἲࢆ⏝࠸ࡿṇᙜᛶࢆಶู࡟᳨ウࡍ࡭ࡁ࡛࠶ࡿࠋ
3) ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ࡣࠊ༢୍≀㉁ࢆ⏝࠸ࡿሙྜࡣไ㝈ࡀ࡞ࡃࠊࡲࡓࠊΰྜ≀࡟ࡘ࠸࡚ࡶࣂࣜࢹ࣮ࢩࣙࣥࢹ࣮ࢱࡣ㝈ࡽࢀ࡚ࡣ࠸ࡿࡶࡢࡢࠊ㐺⏝࠿ࡽ㝖እࡍࡿᚲせࡣ࡞࠸ࠋ
8. ᮏጤဨ఍ࡢ⤖ㄽ
STE ἲࡣࠊࡍ࡛࡟ࣂࣜࢹ࣮ࢩࣙࣥࡀ⤊஢ࡋ࡚࠸ࡿ௚ࡢ௦᭰ἲ࡜ẚ㍑ࡋࡓሙྜࠊṇ☜ᛶࠊ෌⌧ᛶࡣྠ⛬ᗘ࡛࠶ࡗࡓࠋ
▷᫬㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࠊ౑⏝⣽⬊ࡢ౪⤥※࡟ไ㝈ࡀ࡞ࡃࠊ≉Ṧ࡞ᶵᮦࡸヨ⸆ࢆᚲせ࡜ࡏࡎࠊᚲせ࡞ᡭᢏࡶ」㞧࡞ࡶࡢ࡛࡞࠸࠿ࡽࠊᢏ⾡⛣㌿ᛶࡣ㧗࠸࡜ุ᩿࡛ࡁࡿࠋ࢞࢖ࢻ
ࣛ࢖ࣥ࡟♧ࡉࢀࡓ⇍㐩ᗘ☜ㄆ≀㉁ࢆ⏝࠸࡚ࠊᐇ᪋ࡍࡿヨ㦂᪋タ࡬㐺ษ࡞ᢏ⾡⛣㌿ࡀ㐩ᡂ࡛ࡁࡓࡇ࡜ࢆ☜࠿ࡵࡿᚲせࡀ࠶ࡿࠋ౑⏝⁐፹࡟୙⁐࡞≀㉁ࠊ࠶ࡿ࠸ࡣᆒ୍࡟ศᩓࡋ࡞࠸≀㉁ࡣヨ㦂࡟㐺ࡉ࡞࠸ࠋ࣎ࢺ࣒࢔
ࢵࣉ᪉ᘧ࡛ࡣࠊ⏺㠃άᛶ๣௨እࡢᅛయࡸ⵨Ẽᅽࡢ㧗࠸㸦> 6 kPa㸧≀㉁ࢆ㝖እࡍࡿࡇ࡜࡟
࡞ࡗ࡚࠸ࡿࡀࠊࡑࡢጇᙜᛶࡢ☜ㄆࡣ༑ศ࡛࡞࠸ࠋ
TG 491 ࡟‽ᣐࡋ࡚ᐇ᪋ࡋࡓሙྜࠊࢺࢵࣉࢲ࢘ࣥ᪉ᘧ࡛ UN GHS ༊ศ 1 ≀㉁ࢆ᳨ฟࡍࡿ
᪉ἲࠊ࠾ࡼࡧ࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ࡛ UN GHS ༊ศእ≀㉁ࢆ᳨ฟࡍࡿ᪉ἲ࡜ࡋ࡚⏝࠸ࡿࡇ࡜ࡀ࡛ࡁࡿ࡜ᮏጤဨ఍ࡣ⪃࠼ࡿࠋ

1117

9. ᩥ⊩ICCVAM (2013) Short Time Exposure (STE) Test Method Summary Review Document.
Kojima et al. (2013) Second-phase validation study of the short time exposure test for assessmentof eye irritation potency of chemicals. Toxicol In Vitro 27(6): 1855-1869.
OECD (2012) Test Guideline 437. Bovine Corneal Opacity and Permeability Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring
Classification for Eye Irritation or Serious Eye Damage.
OECD (2012) Test Guideline 438. 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.
OECD (2012) Test Guideline 460. Fluorescein Leakage Test Method for Identifying OcularCorrosives and Severe Irritants.
OECD (2015) Test Guideline 491. Short Time Exposure In Vitro Test Method for Identifying 1)
Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye
Irritation or Serious Eye Damage.
Saito et al. (2015) Predictive performance of the Short Time Exposure test for identifying eye
irritation potential of chemical mixtures. Toxicol In Vitro 29(3): 617-620.
Sakaguchi et al. (2011) Validation study of the Short Time Exposure (STE) test to assess the eyeirritation potential of chemicals. Toxicol In Vitro 25(4): 796-809.
Takahashi et al. (2010) An interlaboratory study of the short time exposure (STE) test using SIRC
cells for predicting eye irritation potential. Cutan Ocul Toxicol 29(2): 77-90.
Takahashi et al. (2009) Inter-laboratory study of short time exposure (STE) test for predicting eye
irritation potential of chemicals and correspondence to globally harmonized system (GHS)
classification. J Toxicol Sci 34(6): 611-626.
Takahashi et al. (2008) Development of the short time exposure (STE) test: an in vitro eye
irritation test using SIRC cells. Toxicol In Vitro 22(3): 760-770.

1218

Appendix 1
ST(ἲࡢホ౯࡟⏝࠸ࡽࢀࡓ≀㉁㸦ࡑࡢ㸯㸧ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ

࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ

࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ
㐺⏝ไ㝈≀㉁ࢆ㝖እ

125

130

102

࢔ࣝࢥ࣮ࣝ㢮

39

40

36࢔ࣝࢹࣄࢻ㢮

2

2

0

࢔ࣝ࢝ࣜ

1

1

0

࢔࣑ࣥ㢮

9

10

8

࣍࢘⣲໬ྜ≀

4

42࢝ࣝ࣎ࣥ㓟㢮

23

24

19

ሷ⣲໬ྜ≀

1

1

0

࢚ࢫࢸࣝ㢮

17

18

17

࢚࣮ࢸࣝ㢮

16

16

16

〇๣㸦ΰྜ≀㸧

10

10

10

ࢢࣜࢥ࣮ࣝ㢮

1

11࣊ࢸࣟࢧ࢖ࢡࣜࢵࢡ໬ྜ≀

9

10

7

Ⅳ໬Ỉ⣲

23

24

18

↓ᶵ໬ྜ≀

1

1

0

ࢣࢺࣥ㢮

8

8

5

ࣛࢡࢺࣥ

1

1

1

Ἔ㢮

5

7

6

ࢽࢺࣜࣝ໬ྜ≀

1

1

1

❅⣲໬ྜ≀

1

1

1

࢜ࢽ࣒࢘໬ྜ≀

11

12

11

ࣇ࢙ࣀ࣮ࣝ㢮

110ከ⎔ᘧ໬ྜ≀

1

1

1

ሷ㢮

17

18

14

◲㯤໬ྜ≀

15

16

12

඲య
໬Ꮫ≀㉁༊ศ

ὀ㸧≀㉁࡟ࡼࡗ࡚ࡣ」ᩘࡢ໬Ꮫ≀㉁༊ศ࡟ࡲࡓࡀࡗ࡚࠸ࡿሙྜࡶ࠶ࡿࡓࡵࠊྜィࡣ≀㉁⥲ᩘ࡜ࡣ୍⮴
ࡋ࡞࠸ࠋ

13

19

Appendix 1
ST(ἲࡢホ౯࡟⏝࠸ࡽࢀࡓ≀㉁㸦ࡑࡢ㸰㸧ࢺࢵࣉࢲ࢘ࣥ᪉ᘧ

࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ

࣎ࢺ࣒࢔ࢵࣉ᪉ᘧ
㐺⏝ไ㝈≀㉁ࢆ㝖እ

125

130

102

92

93

79

⵨Ẽᅽ >6kPa

-

14

0

⵨Ẽᅽ <6kPa

-

61

61

⵨Ẽᅽ ୙᫂

-

18

18

33

37

23

⏺㠃άᛶ๣

-

23

23

㠀⏺㠃άᛶ๣

-140⵨Ẽᅽ >6kPa-

00

⵨Ẽᅽ <6kPa

-

23

10

⵨Ẽᅽ ୙᫂

-

14

13

⏺㠃άᛶ๣

45

49

49

⁐๣

34

35

26

໬Ꮫ୰㛫⏘≀

24

24

20

་⸆ရ

20

20

13

㤶ᩱ

20

20

13

ẅ⹸๣࣭ᢠ⳦๣࣭㜵⭉๣

10

10

7

ሬᩱ

11

10

7

඲య
≀㉁ࡢ≉ᛶ
ᾮయ

ᅛయ

〇ရ༊ศ

ὀ㸧〇ရ༊ศࡣ≀㉁ᩘࡢከ࠸ࡶࡢࢆᢳฟࡋࡓࠋ≀㉁࡟ࡼࡗ࡚ࡣ」ᩘࡢ〇ရ༊ศ࡟ࡲࡓࡀࡗ࡚࠸ࡿሙྜ
ࡶ࠶ࡿࠋ

14

20

Appendix 2
STE ἲࡢ⇍㐩ᗘ☜ㄆ≀㉁≀㉁ྡ
Benzalkonium chloride(10% aqueous)
Triton X-100 (100%)

STE ἲ࡛ࡢ

STE ἲ

౑⏝⁐፹

ุᐃ

༊ศ 1

⏕⌮㣗ሷỈ

༊ศ㸯

ᾮయ

༊ศ 1

⏕⌮㣗ሷỈ

༊ศ 1

ᅛయ

༊ศ 1

⏕⌮㣗ሷỈ

༊ศ 1

ᅛయ

༊ศ 1

⏕⌮㣗ሷỈ

༊ศ 1

ᾮయ

༊ศ 2A

⏕⌮㣗ሷỈ

ศ㢮୙ྍ

ᾮయ

༊ศ 2A/B㺮㺦㺵㺷㺓㺐㺷ศ㢮୙ྍᾮయ༊ศ 2A/B

⏕⌮㣗ሷỈ

ศ㢮୙ྍ

ᾮయ

༊ศእ

⏕⌮㣗ሷỈ

༊ศእ

CAS ␒ྕ

໬Ꮫศ㢮

8001-54-5

࢜ࢽ࣒࢘໬ྜ≀ᾮయ

9002-93-1

࢚࣮ࢸࣝ㢮

ᛶ≧

GHS ศ㢮

㺫㺡㺹㺙㺐㺖㺶㺍㺖໬ྜ≀㸹
Acid Red 92

18472-87-2⮯໬໬ྜ≀㸹
ሷ⣲໬ྜ≀࢔ࣝ࢝ࣜ㸹

Sodium hydroxide

1310-73-2

Butyrolactone

96-48-0

1-Octanol

111-87-5

Cyclopentanol

96-48-02-Ethoxyethyl acetate111-15-9

Dodecane112-40-3Ⅳ໬Ỉ⣲㸦㠀⎔≧㸧ᾮయ༊ศእ㺮㺦㺵㺷㺓㺐㺷

༊ศእ

Methyl isobutyl ketone108-10-1

ࢣࢺࣥ㢮

ᾮయ

༊ศእ

㺮㺦㺵㺷㺓㺐㺷

༊ศእ

n,n-Dimethylguanidine sulfate598-65-2

ᅛయ

༊ศእ

⏕⌮㣗ሷỈ

༊ศእ

↓ᶵ໬ྜ≀
ࣛࢡࢺࣥ㢮㸹
㺫㺡㺹㺙㺐㺖㺶㺍㺖໬ྜ≀࢔ࣝࢥ࣮ࣝ㢮
࢔ࣝࢥ࣮ࣝ㢮㸹
Ⅳ໬Ỉ⣲㸦⎔≧㸧࢔ࣝࢥ࣮ࣝ㢮㸹
࢚࣮ࢸࣝ㢮࢔࣑ࢪࣥ㢮㸹
◲㯤໬ྜ≀

15

21

22



OECD/OCDE

491$GRSWHG
-XO\
OECD GUIDELINE FOR THE TESTING OF CHEMICALS
Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious
Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious
Eye Damage
INTRODUCTION
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XVHGWRLGHQWLI\WHVWFKHPLFDOVQRWUHTXLULQJFODVVLILFDWLRQIRUH\HLUULWDWLRQDQGVHULRXVH\HGDPDJH 81
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RYHUN3DDQGLL 6ROLGFKHPLFDOV VXEVWDQFHVDQGPL[WXUHV RWKHUWKDQVXUIDFWDQWVDQGPL[WXUHVFRPSRVHGRQO\RIVXUIDFWDQWV6XFKFKHPLFDOVDUHH[FOXGHGIURPWKHDSSOLFDELOLW\GRPDLQRIWKH67(WHVWPHWKRG  
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24

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GDWDVHWDOVRFRQWDLQVLQKRXVHGDWDRQPL[WXUHVZKLFKZKHQFRPSDUHGWRWKH in vivo'UDL]HH\HWHVWVKRZHGDQDFFXUDF\RI  DIDOVHSRVLWLYHUDWHRI  DQGDIDOVHQHJDWLYHUDWHRI  IRU SUHGLFWLQJ PL[WXUHV WKDW GR QRW UHTXLUH FODVVLILFDWLRQ XQGHU WKH 81 *+6 FODVVLILFDWLRQV\VWHP  7KH67(WHVWPHWKRGFDQWKHUHIRUHEHDSSOLHGWRLGHQWLI\PL[WXUHVDV81*+61R&DWHJRU\LQ
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25

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26

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Table 1: List of Proficiency Substances Substance

CASRN

Chemical class1

Physicalstate

In Vivo UN
GHS Cat.2

Solvent in
STE test%HQ]DONRQLXPFKORULGH DTXHRXV 

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6DOLQH&DWHJRU\

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1,&($70
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27

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28

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29

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Table 2: Prediction model of the STE test method
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30

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31

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32

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33

491

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DEFINITIONS
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34

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35

491

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36

Short Time Exposure (STE) Test Method
Summary Review Document

Interagency Coordinating Committee on the
Validation of Alternative Methods (ICCVAM)

National Toxicology Program (NTP) Interagency Center for the
Evaluation of Alternative Toxicological Methods (NICEATM)

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

2013

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

37

Short Time Exposure Summary Review Document

This page intentionally left blankii

38

Short Time Exposure Summary Review Document

Table of Contents
List of Tables......................................................................................................................................... v
List of Abbreviations and Acronyms................................................................................................. vi
Acknowledgements............................................................................................................................. vii
Executive Summary ............................................................................................................................. 1
1.0

Introduction and Background ................................................................................................... 5

2.0

STE Test Method Database........................................................................................................ 7

3.0

STE Test Method Performance ............................................................................................... 30
3.1

GHS Classification System: STE Performance in a Top-Down Approach ................... 30
3.1.1

3.2

GHS Classification System: STE Performance in a Bottom-Up Approach................... 33
3.2.1

3.3

STE Discordant Results for GHS Classification in a Bottom-Up
Approach .......................................................................................................... 34

EPA Classification System: STE Performance in a Top-Down Approach.................... 36
3.3.1

3.4

STE Discordant Results for GHS Classification in a Top-Down
Approach .......................................................................................................... 31

STE Discordant Results for EPA Classification in a Top-Down
Approach .......................................................................................................... 37

EPA Classification System: STE Performance in a Bottom-Up Approach ................... 39
3.4.1

STE Discordant Results for EPA Classification in a Bottom-Up
Approach .......................................................................................................... 40

4.0

STE Test Method Reliability.................................................................................................... 43

5.0

Peer Review Summary.............................................................................................................. 44

6.0

References.................................................................................................................................. 46

Supplements
Supplements containing files all of the available documentation related to the short time exposure(STE) test method review have been provided on the enclosed CD-ROM. These documents are alsoavailable on the NICEATM website at http://iccvam.niehs.nih.gov/STEReview.htm. Supplement A
contains the Kao Corporation STE background review document (A1) and appendices (A2 to A10) as
submitted. Supplement B (B1 to B3) contains the data and information used by NICEATM to conduct
a technical review of the STE test method.
Supplement A
Kao Corporation Background Review Document and Appendices
Supplement A1

Kao Background Review Document: Current Status of In Vitro Test
Methods for Identifying Ocular Irritants: Short Time Exposure (STE)
Test

Supplement A2

Short Time Exposure (STE) Test Protocol

iii

39

Short Time Exposure Summary Review Document

Supplement A3

Supplement A4

Supplement A5

Chemical Classes of Substances Tested in the STE Test (Appendix B1)
In Vivo Data Source of Substances Tested in the STE Test
(Appendix B2)
Solubility of Substances Tested in the STE Test (Appendix B3)
Skin Corrosivity/Irritation of Substances Tested in the STE Test
(Appendix B4)
MTT Reduction (Appendix B5) of Substances Tested in the STE Test
In Vitro Data for Substances Tested in the STE Test Sorted by Reference(Appendix C1)
In Vitro Data for Substances Tested in the STE Test Sorted by SubstanceName (Appendix C2)
Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
Sorted by Reference (Appendix D1)
Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
Sorted by Substance Name (Appendix D2)

Supplement A6

Intralaboratory CV Analysis of STE by Study

Supplement A7

In Vitro Data for Substances Tested in the STE Test: Sorted by
Reference (Appendix F1)
In Vitro Data for Substances Tested in the STE Test: Sorted by
Substance Name with 0.05% Data (Appendix F2)

Supplement A8

Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
Sorted by Reference (Appendix G1)
Comparison of In Vivo and In Vitro Ocular Irritancy Classifications
Sorted by Substance Name with 0.05% Data (Appendix G2)

Supplement A9

EpiOcular Assay Protocol (Appendix H)

Supplement A10

In Vitro Data for Substances Tested in the EpiOcular Test

Supplement B
Comparison of In Vitro and In Vivo Ocular Irritancy Classification
Supplement B1

In Vivo Classification

Supplement B2

STE Data Sorted by Study

Supplement B3

STE Data Sorted by Substance with Consensus Classifications

iv

40

Short Time Exposure Summary Review Document

List of Tables
Table 1

Overall STE Performance in a Top-Down Approach........................................................ 2

Table 2

Top-Down Performance of Validated In Vitro Test Methods Compared to the STE
Test Method....................................................................................................................... 2

Table 3Overall STE Performance in a Bottom-Up Approach....................................................... 2

Table 4Overall STE Performance in a Bottom-Up Approach When Substances with Vapor
3UHVVXUHN3DDQG1RQVXUIDFWDQW6ROLGVDUH([FOXGHG ................................................... 3

Table 5

Bottom-Up Performance of Validated In Vitro Test Methods Compared to the STE
Test Method....................................................................................................................... 3

Table 2-1

Test Substances Used to Evaluate STE Performance in a Top-Down Approacha............. 8

Table 2-2

Test Substances Used to Evaluate STE Performance in a Bottom-Up Approacha .......... 15

Table 2-3

Test Substances Used to Evaluate STE Performance in a Bottom-Up Approach
Within the Defined Applicability Domaina,b ................................................................... 22

Table 3-1

STE Performance for GHS Classification in a Top-Down Approach ............................. 31

Table 3-2

STE False Positive and False Negative Rates by Chemical Category and Properties
of Interest for GHS Classification in a Top-Down Approach ......................................... 32

Table 3-3

STE Performance for GHS Classification in a Top-Down Approach After
Excluding Discordant Categories .................................................................................... 33

Table 3-4

STE Performance for GHS Classification in a Bottom-Up Approach ............................ 34

Table 3-5

STE False Positive and False Negative Rates by Chemical Category and Properties
of Interest for GHS Classification in a Bottom-Up Approach......................................... 35

Table 3-6

STE Performance for GHS Classification in a Bottom-Up Approach After
Excluding Discordant Categories .................................................................................... 36

Table 3-7

STE Performance for EPA Classification in a Top-Down Approach.............................. 37

Table 3-8

STE False Positive and False Negative Rates by Chemical Category and Properties
of Interest for EPA Classification in a Top-Down Approach.......................................... 38

Table 3-9

STE Performance for EPA Classification in a Top-Down Approach After
Excluding Discordant Categories .................................................................................... 39

Table 3-10 STE Performance for EPA Classification in a Bottom-Up Approach............................. 39
Table 3-11 STE False Positive and False Negative Rates by Chemical Category and Properties
of Interest for EPA Classification in a Bottom-Up Approach......................................... 40
Table 3-12 STE Performance for EPA Classification in a Bottom-Up Approach After
Excluding Discordant Categories .................................................................................... 42v

41

Short Time Exposure Summary Review Document

List of Abbreviations and Acronyms
AD

Applicability domain

BCOP

Bovine corneal opacity and permeability

BRD
CASRN

Background review document
CAS Registry Number® (American Chemical Society)CV

Coefficient of variation

EPA

U.S. Environmental Protection Agency

GHS

Globally Harmonized System of Classification and Labelling of Chemicals

ICCVAM

Interagency Coordinating Committee on the Validation of Alternative Methods

ICE

Isolated chicken eye

ILSIntegrated Laboratory Systems, Inc.

JaCVAMkPa

Japanese Center for the Validation of Alternative Methods
Kilopascals

MeSH®

Medical Subject Headings (National Library of Medicine)MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NICEATM

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

NIEHS
NLM

National Institute of Environmental Health Sciences
National Library of Medicine

NTP

National Toxicology ProgramOECD

Organisation for Economic Co-operation and Development

SIRC
STE

Statens Seruminstitut rabbit cornea
Short time exposure

TG

Test GuidelineUN

United Nations

Trademarks are the property of their respective owners.

vi

42

Short Time Exposure Summary Review Document

Acknowledgements
National Institute of Environmental Health Science
National Toxicology Program Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM)
Warren Casey, PhD, DABT
Acting Director, Project Officer
NICEATM Support Contract Staff (Integrated Laboratory Systems [ILS], Inc.)David Allen, PhD (through 2011)
Lori Rinckel, PhD
Jonathan Hamm, PhD
Elizabeth Lipscomb, PhD (through 2012)
Steven Morefield, MD
Catherine Sprankle
James Truax, MA
Linda Wilson

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43

Short Time Exposure Summary Review Document

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44

Short Time Exposure Summary Review Document

Executive Summary
The short time exposure (STE) test method is an in vitro method for identifying ocular irritants.
Developed by Takahashi et al. (2008), the STE test method assesses cytotoxicity in a rabbit corneal
epithelial cell line (SIRC cells) through a 5-minute exposure to the test substance. In March 2011,
Kao Corporation (Tochigi, Japan) submitted a background review document (BRD) titled “Current
Status of In Vitro Test Methods for Identifying Ocular Irritants: Short Time Exposure (STE) Test” to
the National Toxicology Program (NTP) Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM). NICEATM conducted a preliminary evaluation of the BRD andrequested additional information, which resulted in several revisions to the BRD. Kao Corporation
drafted a final BRD in May 2012 (Supplement A). The BRD contains all data and information thatwere available in the peer-reviewed literature and Kao Corporation in–house data to describe thecurrent validation status of the STE test method, including what was known about its accuracy andreliability.
This summary review document presents an evaluation of STE test method accuracy, sensitivity,
specificity, false positive rate, and false negative rate based on test substances with corresponding invivo data. The analysis in a top-down and a bottom-up approach was based on the Globally
Harmonized System of Classification and Labelling of Chemicals (GHS; UN 2011) and U.S.
Environmental Protection Agency (EPA 2012) classification systems for eye hazard classification andlabeling.
In a top-down approach, the STE test method is used to distinguish and label severe eyeirritants/corrosives from all other hazard categories. Any substance not identified as a severe eyeirritant/corrosive by the STE test method requires additional testing with other methods. A top-down
approach requires a low false positive rate to avoid overclassification of substances. The false
negative rate is not as critical because substances that test negative in the STE test method would betested with another method.
In contrast, a bottom-up approach is used to distinguish substances not labeled as eye irritants from allother hazard categories. Any substance that tests positive in a bottom-up approach requires additional
testing with other methods to determine the appropriate hazard classification and labeling. A bottomup approach requires a low false negative rate to avoid irritants being classified and mislabeled as
irritants when the correct eye hazard classification is GHS Not classified or EPA Category IV(minimal effects clearing in less than 24 hours). The false positive rate is not as critical because
substances that test positive in the STE test method would be tested with another method.
The Kao Corporation BRD describes their analyses of 119 tests substances in four studies (Kojima etal. [Kao BRD]; Sakaguchi et al. 2011; Takahashi et al. 2009, 2010), with additional in-house data
provided by Kao Corporation. In September 2012, Kao Corporation provided data on 52 additionalsurfactant or surfactant-containing substances, for a total of 169 substances with in vitro STE and invivo rabbit eye test data (Supplement B).
The analyses in this report used consensus calls for both STE and rabbit eye test data when results
were available from more than one laboratory or study. When equivocal results were obtained in two
or more laboratories or in different studies, the more severe hazard classification was used.
Table 1 summarizes overall performance of the STE test method in a top-down approach for all
substances in the database. The overall false positive rate in a top-down approach ranged from
1.2% (1/84) for GHS classification to 2.3% (2/87) for EPA classification. Exclusion of alcohols
reduced the rate to 0% (data not shown). The performance of the STE test method is compared toother validated in vitro test methods in Table 2.

1

45

Short Time Exposure Summary Review Document

Table 1
Regulatory
SystemOverall STE Performance in a Top-Down Approach
AccuracyN

Sensitivity

Specificity

False
Positive
Rate

False
Negative Rate

%

No.

%

No.

%

No.

%

No.

%

No.

GHS

120

85

102/120

53

19/36

99

83/84

1.2

1/84

47

17/36

EPA

120

87

104/120

58

19/33

98

85/87

2.3

2/87

42

14/33

Abbreviations: EPA = U.S. Environmental Protection Agency; GHS = Globally Harmonized System of Classification andLabelling of Chemicals; N = number of substances; STE = short time exposure.

Table 2

Top-Down Performance of Validated In Vitro Test Methods Compared to the
STE Test Method

GHS

N

BCOP

Accuracy

False Positive Rate

False Negative Rate

%

No.

%

No.

%

No.

188

79

149/18824

29/123

15

10/65

ICE

144

83

120/144

8.0

9/114

50

15/30

CM

82

90

74/82

2.0

1/48

21

7/34

STE

120

85

102/120

1.2

1/84

47

17/36

Abbreviations: BCOP = bovine corneal opacity and permeability; CM = Cytosensor microphysiometer; GHS = Globally
Harmonized System of Classification and Labelling of Chemicals; ICE = isolated chicken eye; N = number of substances.

The STE overall performance in a bottom-up approach is shown in Table 3. The overall false
negative rate in a bottom-up approach ranged from 12.3% (9/73) for GHS classification to 24.7%(24/97) for EPA classification.
Table 3Overall STE Performance in a Bottom-Up Approach

Regulatory
SystemN

GHS
EPA

Accuracy

Sensitivity

Specificity

False Positive
Rate

False Negative
Rate

%

No.

%

No.

%

No.

%

No.

%

No.

129

85

109/129

88

64/73

80

45/56

20

11/56

12

9/73

129

80

103/129

75

73/97

94

30/32

6.3

2/32

25

24/97

Abbreviations: EPA = U.S. Environmental Protection Agency; GHS = Globally Harmonized System of Classification andLabelling of Chemicals; N = number of substances; STE = short time exposure.

The applicability domain was evaluated to reduce the false positive rate and increase performance forboth GHS and EPA classifications in a bottom-up approach. Improvements in the applicability
domain were determined by analyzing assay performance by chemical class and physical properties.
As a result, two applicability domains were evaluated based on excluding certain chemical andproduct classes, or physical characteristics. Applicability domain one excludes liquids with vapor
SUHVVXUHV kilopascals (kPa) solid alcohols, hydrocarbons, and salts while applicability domain two
excludes liquids with vapor pressures  kilopascals (kPa) and nonsurfactant solids (Table 4). The
performance of the STE test method is compared to other validated in vitro test methods in Table 5.

2

46

Short Time Exposure Summary Review Document

Table 4Overall STE Performance in a Bottom-Up Approach After Excluding
Discordant Categories

Regulatory
System

Accuracy

N%

False Positive Rate

False Negative Rate

%

%

No.

No.

No.

Applicability Domain 1 ([FOXVLRQRIOLTXLGVZLWKYDSRUSUHVVXUHV kilopascals (kPa) solid alcohols, hydrocarbons, and salts
GHS

94

90

85/94

18

8/45

2

1/49

EPA

94

83

78/94

7.7

2/26

21

14/68

Applicability Domain 2 ([FOXVLRQRIOLTXLGVZLWKYDSRUSUHVVXUHV kilopascals (kPa) and nonsurfactant solids
GHS

101

90

91/101

19

9/47

1.9

1/54

EPA

101

85

86/101

7.1

2/28

18

13/73

Abbreviations: EPA = U.S. Environmental Protection Agency; GHS = Globally Harmonized System of Classification andLabelling of Chemicals; No. = number; STE = short time exposure.

Table 5

Bottom-Up Performance of Validated In Vitro Test Methods Compared to the
STE Test Method

GHS

N

BCOP

Accuracy

False Positive Rate

False Negative Rate

%

No.

%

No.

%

No.

188

66

125/188

69

63/91

0

0/97

CM

53

68

36/53

68

17/25

0

0/28

STE – AD1

94

91

85/94

18

8/45

2

1/49

STE – AD2101

90

91/101

19

9/47

1.9

1/54

Abbreviations: AD = applicability domain; BCOP = bovine corneal opacity and permeability; CM = Cytosensor
microphysiometer; N = number of substances; STE = short time exposure.

This evaluation of the STE performance shows that this method is able to distinguish substances as
severe irritants or corrosives (i.e., GHS Category 1 or EPA Category I) from all other hazard
categories (GHS Category 2A, 2B, Not Classified or EPA Category II, III, IV) in a top-down
approach, with false positive rates ranging from 1.2% (1/84) to 2.3% (2/87) for the GHS and EPA
classification systems, respectively. Exclusion of discordant chemical classes (e.g., alcohols, ethers,
hydrocarbons, or nonionic surfactants) reduced the false positive rate to 0%. In a bottom-up approachto distinguish substances that were either not classified or minimal irritants (i.e., GHS Not Classified
or EPA Category IV) from all other hazard categories (i.e., GHS Category 1, 2A, 2B or EPA
Category I, II, III), the STE false negative rates ranged from 12.3% (9/73) to 24.7% (24/97). The
range of false negative rates in a bottom-up approach was decreased to 2% (1/49) and 21% (14/68)
for the GHS and EPA classification systems, respectively, when liquids with vapor pressures >6kPa
solid alcohols, hydrocarbons, and salts were excluded. The range of false negative rates in a bottomup approach was decreased to 1.9% (1/54) and 18% (13/73) for the GHS and EPA classification
systems, respectively, when liquids with vapor pressures >6kPa and nonsurfactant solids were
excluded.
This SRD along with the original Kao BRD and other supporting documentation were forward by
NTP to four external scientific reviewers. The reviewers were provided a list of questions that
included a request to comment on the adequacy of the database used for evaluating STE, theadequacy of the performance evaluation, and to provide comments for regulators using the test3

47

Short Time Exposure Summary Review Document

method. In response, the reviewers indicated that the database of compounds was generally sufficientand the review thorough. A summary of reviewer comments is provided in Section 5.0.

448

Short Time Exposure Summary Review Document

1.0

Introduction and Background

The Draize rabbit eye test has been the primary method used to determine the ocular irritation
potential of chemicals (Draize et al. 1944). However, public interest in animal welfare has increased
the pressure to develop non-animal alternatives. The development of alternative methods is also
accelerating due to regulations banning animal ocular irritation tests for cosmetics in the European
Union (Directive 2003/15/EC; European Union 2003). As a result, numerous alternative ocular
irritation methods that use cell lines and tissues are being developed around the world (Balls et al.
1999; Eskes et al. 2005; Ohno et al. 1999). The test guidelines for the bovine corneal opacity andpermeability (BCOP) test method and isolated chicken eye (ICE) test method were accepted by theOrganisation for Economic Co-operation and Development (OECD) for predicting severe ocular
irritation (OECD 2012a, 2012b). However, no other test guidelines have been accepted for in vitro
ocular irritation tests.
The short time exposure (STE) test method is an alternative ocular irritation method developed by
Kao Corporation (Takahashi et al. 2008). The STE test method uses a cultured cell line (SIRC cells)
derived from rabbit cornea and uses shorter exposure times than many other cytotoxicity-based
methods. Generally, cytotoxicity tests using cultured cells have the advantage of being simple, quick
procedures with a low evaluation cost. The facility requirements necessary to conduct the STE test
include a standard laboratory setup for cell culture. The cornea is one of the main targets during
accidental eye exposures, and damage to the cornea can result in visual impairment. A final advantageof the STE test method is that it can be used to evaluate poorly water-soluble chemicals like toluene,
octanol, and hexanol by using mineral oil as the vehicle (Takahashi et al. 2008).
The STE test method involves exposing SIRC cells to 5% and 0.05% concentrations of test substancefor 5 minutes. Following exposure to 5% test substance concentration, substances that reduce cell
viability below 70% are classified as irritants. Using this classification scheme, Kao Corporationassessed the performance of the STE test method in a bottom-up approach to distinguish substances
not labeled as irritants from all other categories. Kao Corporation also proposed a second approach toestablish an ocular irritation potency ranking that differentiates severely irritating substances frommild and moderate irritants. This approach uses a point system based on the test concentration andrelative viability resulting from an exposure to 5% or 0.05% of test material (Takahashi et al. 2008).
This second approach was used to review the STE test method in a top-down approach to distinguishcorrosives/severe irritants from all other categories.
In March 2011, the Japanese Center for the Validation of Alternative Methods (JaCVAM), as part of
the International Cooperation on Alternative Test Methods agreement, requested that the National
Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods(NICEATM) and the Interagency Coordinating Committee on the Validation of Alternative Methods(ICCVAM) conduct a technical review of the STE test method. In support of the review, Kao
Corporation submitted their STE test method BRD and subsequently provided NICEATM with arevised May 2012 BRD (Supplement A). The BRD contains STE and rabbit eye test data for
119 substances from four in vitro–in vivo comparative studies, with additional in-house data on
23 substances provided by Kao Corporation. After the preliminary analysis, additional data wererequested and provided for 52 surfactants or surfactant-containing formulations that increased the
STE database to 169 substances.
To assess the ability of the STE test method to predict the regulatory hazard classification identified
in the rabbit eye test, the STE rank results were converted to Globally Harmonized System ofClassification and Labelling of Chemicals (GHS) and U.S. Environmental Protection Agency (EPA)
classifications (UN 2011, EPA 2012). An STE rank of 1 (nonirritant), 2 (mild or moderate irritant), or
3 (severe irritant) was converted to GHS Not Classified, Category 2A/2B, or Category 1, respectively,

549

Short Time Exposure Summary Review Document

or to the U.S. EPA Label Review Manual classification of eye irritation as Category IV, Category
III/II, or Category I, respectively.
STE test method performance was also evaluated in a top-down approach (i.e., distinguishing GHS
Category 1 or EPA Category I substances from those in all other categories) or in a bottom-up
approach (i.e., distinguishing GHS Not Classified or EPA Category IV substances from all othercategories) for substances with corresponding in vitro–in vivo data. For a top-down approach,120 substances had corresponding in vitro and in vivo classification data using the GHS or EPA
classification systems, respectively, that were suitable for accuracy analysis. For a bottom-up
approach, 129 substances had suitable in vitro–in vivo data for GHS and EPA classifications.
A variety of chemical categories were tested in the STE test method, and the chemical categories withthe greatest amount of test data are alcohols, carboxylic acids, esters, ethers/polyethers, heterocyclic
compounds, ketones/lactones, onium compounds, and salts. Physical properties of these substances
have also been evaluated (pH, solids, liquids, and surfactants [nonionic, anionic, cationic]). Thissummary review document describes evaluations of the STE test method performance in a top-down
or bottom-up approach.

650

Short Time Exposure Summary Review Document

2.0

STE Test Method Database

The May 2012 BRD submitted by Kao Corporation (Supplement A) includes information on the
STE test method, the test method protocol, and data and performance analyses based on:
x
x
x
x
x

Takahashi et al. (2009) – Prevalidation study 1 on 44 substances
Takahashi et al. (2010) – Prevalidation study 2 on 70 substances
Sakaguchi et al. (2011) – Phase I validation study on 25 substances
Kojima et al. (Kao BRD) – Phase II validation study on 40 substances, then combined with Phase
I substances for a total of 63 substances
Kao in-house data on 22 of 23 substances in the original BRD

Additional information on the test substances are found in Supplement B. Supplement B1 contains
the in vivo data used to develop consensus in vivo classifications for substances evaluated in the STE
test method. Supplement B2 shows the test substances along with CAS Registry Number®
(American Chemical Society), concentration tested, STE test data (mean viability value, standard
deviation, number of replicates), category classification, and the reference. Supplement B3 provides
the same information but indicates the consensus STE classification.
The STE database includes test substances in the Kao Corporation BRD, with additional data on
52 surfactants and surfactant-containing formulations provided by Kao Corporation. However, thedatabase used to assess performance consists of consensus classifications when a single substance
was tested in multiple laboratories or in different studies. In vivo data are typically generated by
testing neat chemicals. Twenty-three substances that were tested in the STE test method at aconcentration less than 100% and that did not produce a severe irritant effect were excluded fromthese analyses because a mild/moderate irritant or nonirritant classification of a diluted chemical may
be classified as a severe irritant when tested neat in vitro.
Chemicals that directly reduce MTT in the absence of cells have been shown to artificially inflate
viability measures and underpredict cytotoxicity (Huang 2004; Sims and Plattner 2009). Kao
Corporation assessed chemicals for their ability to directly reduce MTT by incubating the test
substances with MTT and visually inspecting for color development. Test substances that wereidentified as direct MTT reducers were removed from top-down analysis and those classified as STE
nonirritants were removed from bottom-up analysis, as these could be false negative.
Finally, in vivo data were analyzed to calculate the appropriate ocular irritation hazard classification.
These data include cornea, iris, and conjunctiva scores for each animal at 24, 48, and 72 hours
following test substance administration and/or assessment of lesions at 7, 14, and 21 days. Some test
substances had insufficient in vivo data to assign a hazard classification. Thus, these substances were
not used to evaluate STE accuracy and reliability.
The STE database contains 169 test substances representing a variety of chemical classes andphysicochemical properties. Table 2-1 provides information on the test substances evaluated in a
top-down approach to identify severe eye irritants or corrosives. Table 2-2 provides information
evaluated in a bottom-up approach to identify GHS Not Classified or EPA Category IV substances.
These substances had corresponding in vivo data, were assigned a GHS (UN 2011) or EPA (EPA
2012) eye hazard classification, and met other assay criteria as discussed in Section 3.2. Table 2-3
shows the substances used to assess the STE test method in a bottom-up approach applicability
domain one excluding liquids with vapor pressure >6 kilopascals alcohols, hydrocarbons, and salts
and applicability domain two excluding liquids with vapor pressure >6 kilopascals and nonsurfactant
solids.

7

51

52

111-87-5

99-62-7

592-42-7

1647-16-1

622-08-2

111-15-9

104-76-721245-02-3

105-30-6

116-53-0

107-83-5

1-Octanol

1,3-Di-isopropylbenzene

1,5-Hexadiene

1,9-Decadiene

2-Benzyloxyethanol

2-Ethoxyethyl acetate(Cellosolve acetate)

2-Ethyl-1-hexanol

2-Ethylhexyl pdimethylamino benzoate

2-Methyl-1-pentanol

2-Methylbutyric acid

2-Methylpentane

3970-62-5

135-98-8

1-Methylpropyl benzene

2,2-Dimethyl-3-pentanol

13197-76-7

1-Dodecanaminium, N-(2hydroxy-3-sulfopropyl)N,N-dimethyl-, inner salt

85255-76-1

6940-78-9

1-Bromo-4-chlorobutane

2-Naphthalenesulfonicacid,6-hydroxy,monosodium salt,
polymer withformaldehyde andhydroxymethylbenzenesul
fonic aid monosodium salt

CASRN

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidPhysical Formas Tested

0.413

NA

27.8

0.149

0.191

4.72E-06

0.025

0.397

2.9E-4

0.320

28.6

0.041

0.013

0.176

1.16E-21

0.164

Vapor
Pressure(kPa 25°C)b

8

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Not Classified

Category 1

Not Classified

Category 1

Category 2B

Not Classified

Category 2A

Not Classified

Category 2A

Not Classified

Not Classified

Not Classified

Category 2A

Not Classified

Category 1

Not Classified

GHS
Consensusd

Test Substances Used to Evaluate STE Performance in a Top-Down Approacha

Test Substance

Table 2-1

Short Time Exposure Summary Review Document

Category III

Category II

Category IV

Category I

Category III

Category IV

Category II

Category III

Category II

Category IV

Category III

Category IV

Category II

Category IV

Category I

Category IV

EPA
Consensusd

1

2

1

2

2

1

2

1

2

1

1

1

2

1

2

1

Overall
Consensus
STE Ranke

53

96-48-025638-17-9Butylnaphthalenesulfonicacid sodium salt

Butyrolactone

111-76-2

NA

Body shampoo A

Butyl cellosolve

100-51-6

Benzyl alcohol

123-86-4

119345-049

Benzene, 1,1'-oxybis-,
tetrapropylene derivatives,
sulfonated, sodium salts

Butyl acetate

63449-41-2

Benzalkonium chloride(10%)

71-36-38001-54-5

Benzalkonium chloride

Butanol

6484-52-2

67-64-1

AcetoneAmmonium nitrate562-49-2

3,3-Dimethylpentane

9003-04-7

589-34-4

3-Methylhexane

Acrylic acidhomopolymer sodium salt

623-39-2

3-Methoxy-1,2propanediol

18472-87-2

110-03-2

2,5-Dimethyl-2,5hexanediol

Acid red 92

625-69-4

CASRN

2,4-Pentanediol

Test Substance

LiquidSolid

LiquidLiquidLiquidLiquidLiquid

Solid

Solid

Liquid

Solid

Solid

Solid

LiquidLiquidLiquidLiquidSolid

LiquidPhysical Formas Tested

0.0394NA

0.0633

1.59

1.04

NA

7.14E-03

NA

NA

NA

4.48E-16

4.56E-04

5.71E-24

33.2

10.1

8.29

1.92E-03

5.78E-04

7.3E-03

Vapor
Pressure(kPa 25°C)b

9

100%

100%

100%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Category 2ACategory 1

Category 1

Not Classified

Category 1

Category 2ACategory 1

Category 1

Category 1

Category 1

Category 2B

Not Classified

Category 1

Category 2A

Not Classified

Not Classified

Not Classified

Category 1

Not Classified

GHS
Consensusd

Short Time Exposure Summary Review Document

Category II

Category I

Category II

Category III

Category I

Category II

Category I

Category I

Category I

Category I

Category III

Category IV

Category I

Category II

Category IV

Category IV

Category IV

Category I

Category IV

EPA
Consensusd

2

2

2

1

2

2

2

3

3

3

1

1

3

2

1

1

1

1

1

Overall
Consensus
STE Ranke

54

79-92-5

140-72-7

6004-24-6

57-09-0

108-93-0

108-94-1

96-41-3

629-19-6

577-11-7

29911-27-1

108-83-8

67-68-5

112-40-3

538-71-6

64-17-5

609-14-3

141-78-6

3938-95-2

118-60-5

56-81-5

106-91-2

Cetylpyridinium bromide(10%)

Cetylpyridinium chloride

Cetyltrimethylammonium
bromide (10%)

Cyclohexanol

Cyclohexanone

Cyclopentanol

Di-n-propyl disulphideDi(2-Ethylhexyl) sodium
sulfosuccinate

Di(propylene glycol)
propyl ether

Diisobutyl ketone

Dimethyl sulfoxide

Dodecane

Domiphen bromide

Ethanol

Ethyl 2methylacetoacetate

Ethyl acetate

Ethyl trimethyl acetate

Ethylhexyl salicylate

Glycerol

Glycidyl methacrylate

CASRN

Camphene

Test Substance

LiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

LiquidLiquidLiquidLiquidSolid

LiquidLiquidLiquidLiquid

Solid

Solid

Solid

Solid

Physical Formas Tested

0.08291.06E-05

9.51E-07

2.24

13.1

0.0915812

NA0.03150.08290.287

2.38E-04

1.63E-15

0.0664

0.307

0.539

0.087

NA

NA

3.47E-07

0.237

Vapor
Pressure(kPa 25°C)b

10

100%

100%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

10%

100%

100%

100%

100%

10%

10%

10%

100%

In Vivo Conc(%)c

Not Classified

Not Classified

Not Classified

Not Classified

Not Classified

Category 2B

Category 2ACategory 1

Not Classified

Not Classified

Not Classified

Category 2B

Category 1

Not Classified

Category 2B

Not Classified

Category 1

Category 1

Category 1

Category 1

Category 2B

GHS
Consensusd

Short Time Exposure Summary Review Document

Category III

Category IV

Category IV

Category III

Category III

Category III

Category I

Category I

Category III

Category III

Category IV

Category III

Category I

Category IV

Category II

Category III

Category I

Category I

Category I

Category I

Category III

EPA
Consensusd

2

1

1

1

2

2

1

3

1

1

1

2

3

1

2

2

2

3

3

3

1

Overall
Consensus
STE Ranke

55

67-63-0

75-26-3

110-27-0

50-21-5Isopropyl alcohol

Isopropyl bromide

Isopropyl myristate

Lactic acid108-45-2

3121-61-7

79-20-9

110-43-0

105-34-0

96-37-7

78-93-3

108-10-1

598-98-1

112-72-1

111-27-3

111-25-1

Methoxyethyl acrylate

Methyl acetate

Methyl amyl ketone

Methyl cyanoacetate

Methyl cyclopentane

Methyl ethyl ketone (2Butanone)

Methyl isobutyl ketone

Methyl trimethyl acetate

Myristyl alcoholn-Hexanoln-Hexyl bromideNAm-Phenylene diamine

Lotion A

1643-20-5

78-83-1

Isobutyl alcohol

Lauryldimethylamine oxide78-84-2

29590-42-9Iso-octyl acrylate

Isobutanal

288-32-4

CASRN

Imidazole

Test Substance

LiquidLiquidSolid

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

LiquidSolid

LiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

Physical Formas Tested

0.541

0.117

269E-05

4.76

2.90

13.1

17.8

0.047

0.655

7.03

0.598

251E-04NA

1.68E-15

3.81E-03

1.08E-04

28.5

6.61

1.78

21.9

0.0204

5.78E-04

Vapor
Pressure(kPa 25°C)b

11

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Not Classified

Category 2ACategory 2A

Not Classified

Not Classified

Category 2A

Not classified

Category 2A

Not Classified

Category 2ACategory 1

Category 1

Not Classified

Category 1

Category 1

Not Classified

Not Classified

Category 2ACategory 1

Category 2B

Not Classified

Category 1

GHS
Consensusd

Short Time Exposure Summary Review Document

Category IV

Category II

Category III

Category IV

Category III

Category III

Category III

Category II

Category III

Category II

&DWHJRU\,,,Category I

Category IV

Category I

Category I

Category IV

Category IV

Category III

Category I

Category III

Category IV

Category I

EPAConsensusd

1

2

1

1

1

2

1

2

1

1

2

2

1

3

2

1

1

1

2

2

1

2

Overall
Consensus
STE Ranke

56
Solid

LiquidLiquidLiquidSolid

Naphthalenesulfonic acid,butyl-, polymer withformaldehyde and 2188070-49-7
naphthalenesulfonic acid,sodium salt

25322-68-3

9004-81-3

61788-85-0

68439-51-0

104376-75-2

104376-75-2

9002-92-0

Polyethyleneglycol
monolaurate (10 E.O.)

Polyoxyethylenehydrogenated castor oil(60E.O.)

Polyoxyethylene(10) polyoxypropylene(1.5) laurylmyristyl ether

Polyoxyethylene(13) (mono-, di-, tri-)styrenated phenyl ether

Polyoxyethylene(14) tribe
116998-28-8
nzylated phenyl ether

54392-28-8

Polyethylene glycol 400

Polyoxyethylene(160) sor
bitan triisostearate

Polyoxyethylene(19) (mono-, di-, tri-)styrenated phenyl ether

Polyoxyethylene(23) laury
l ether
Solid

LiquidLiquidSolid

LiquidSolid598-65-2

n,n-Dimethylguanidine
sulfateLiquidPhysical Formas Tested

111-83-1

CASRN

n-Octyl bromide

Test Substance

2.03E-13

NA

NA

NA

NA

NA

NA

0

NA

NA

4.04

0.0691

Vapor
Pressure(kPa 25°C)b

12

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Category 2A

Not Classified

Not Classified

Not Classified

Not Classified

Category 1

Not Classified

Not Classified

Not Classified

Category 2A

Not Classified

Not Classified

GHS
Consensusd

Short Time Exposure Summary Review Document

Category III

Category II

Category IV

Category IV

Category III

Category I

Category IV

Category IV

Category IV

Category II

Category III

Category IV

EPA
Consensusd

2

2

1

1

3

3

1

2

1

2

1

1

Overall
Consensus
STE Ranke

57

NA
NA
NA
NA
NA
NA

Rinse C

Rinse D

Shampoo A

Shampoo B

Shampoo C

Shampoo D

3926-62-3

NA

Rinse B

Sodiummonochloroacetate

NA

Rinse A

151-21-3

110-86-1

Pyridine

Sodium lauryl sulfate(15%)

57-55-6

Propylene glycol

151-21-3

1569-01-3

Propasol solvent P

Sodium lauryl sulfate58-33-3

Promethazinehydrochloride

1310-73-2

143-18-0

Potassium oleate

Sodium hydroxide

10124-65-9

Potassium laurate

532-02-5

61788-85-0

Polyoxyethylene(40) hydr
ogenated castor oil

Sodium 2naphthalenesulfonate

CASRN

Test Substance

Solid

Solid

Solid

Solid

Solid

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid

Solid

Solid

Solid

LiquidPhysical Formas Tested

4.23E-09

NA

2.40E-13

6.53E-21

NA

NA

NA

NA

NA

NA

NA

NA

NA

2.58

0.01480.180

04.93E-10

0

NA

Vapor
Pressure(kPa 25°C)b

13

100%

15%

100%

10%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

10%

100%

In Vivo Conc(%)c

Category 2B

Category 1

&DWHJRU\$Category 1

Not Classified

Category 2ACategory 2ACategory 1

Category 2A

Not Classified

Not Classified

Category 2B

Not Classified

Category 1

Not Classified

Category 2B

Category 1

Not Classified

Category 1

Not Classified

GHS
Consensusd

Short Time Exposure Summary Review Document

Category III

Category I

Category III

Category I

Category III

Category II

Category II

Category I

Category II

Category III

Category IV

Category III

Category III

Category I

Category IV

Category II

Category I

Category III

Category I

Category IV

EPA
Consensusd

2

3

3

3

2

2

2

2

2

1

1

2

2

2

1

2

3

2

3

1

Overall
Consensus
STE Ranke

58

9004-82-4

54-21-7

1338-39-2

112-03-8

100-42-5

108-88-3

102-71-6

27028-82-6

9002-93-1

9002-93-1

9005-64-5

9005-65-6

Sodiumpolyoxyethylene(3) lauryl
ether sulfate

Sodium salicylate

Sorbitan monolaurate

Stearyltrimethylammoniu
m chloride

StyreneTolueneTriethanolamine

Triethanolamine
polyoxyethylene(3.0)
lauryl ether sulfate

Triton X-100

Triton X-100 (10%)

Tween 20

Tween 80®

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

LiquidSolid

LiquidPhysical Formas Tested

00

9.32E-04

02.50E-10

4.51E-07

3.16

0.673

NA

1.25E-15

4.84E-12

2.27E-13

Vapor
Pressure(kPa 25°C)b

100%

100%

10%

100%

100%

100%

100%

100%

10%

100%

100%

100%

In Vivo Conc(%)c

Not Classified

Not Classified

Category 1

Category 1

Category 1

Not Classified

&DWHJRU\%Not Classified

Category 1

Not Classified

Category 1

Category 1

GHS
Consensusd

Category IV

Category III

Category I

Category I

Category I

Category III

Category III

Category III

Category I

Category IV

Category I

Category I

EPAConsensusd

1

2

2

3

3

1

1

1

3

2

1

3

Overall
Consensus
STE Ranke

14

Abbreviations: CASRN = CAS Registry Number (American Chemical Society); EPA = U.S. Environmental Protection Agency; GHS = United Nations Globally Harmonized
System of Classification and Labelling of Chemicals; JaCVAM = Japanese Center for the Validation of Alternative Methods; kPa = kilopascals; NA = not available;
STE = short time exposure.
a
A top-down approach is used to distinguish severe eye irritants or corrosives (i.e., GHS Category 1, EPA Category I, or STE Rank 3) from all other hazard or no hazard
categories (i.e., GHS Category 2A, 2B, Not Classified; EPA Category II, III, IV; or STE Rank 1 or 2).
b
Vapor pressure is expressed in kilopascals at 25°C. Vapor pressures were found using the Hazardous Substances Data Bank (HSDB® [U.S. National Library of Medicine]),available at http://toxnet.nlm.nih.gov (accessed 2/25/2013) or from ChemSpider (available at www.chemspider.com [accessed 2/25/2013]). If actual values were not available,
predicted values were obtained from the U.S. EPA EPI (Estimation Programs Interface) Suite™ for Microsoft® Windows, v. 4.11) or ACD/Labs’ ACD/PhysChem Suiteavailable at http://www.acdlabs.com/products/pc_admet/physchem/physchemsuite/ (accessed 2/25/2013). Data from the EPI Suite and ACD/PhysChem Suite programs werealso available in ChemSpider.
c
The concentration as tested in the rabbit eye test, based on NICEATM data. For substances tested at 100%, the starting material was tested neat/undiluted.
d
The consensus classification of two or more studies. When there was no consensus using either the GHS (UN 2011) or EPA (EPA 2012) eye hazard classification system (e.g.,
one GHS Category 2A and one GHS Category 2B), the more hazardous classification (i.e., GHS Category 2A) was used as the consensus classification.

CASRN

Test Substance

Short Time Exposure Summary Review Document

59

99-62-7
592-42-7
1647-16-1
622-08-2
111-15-9
104-76-7

1,3-Di-isopropylbenzene

1,5-Hexadiene

1,9-Decadiene

2-Benzyloxyethanol

2-Ethoxyethyl acetate(Cellosolve acetate)

2-Ethyl-1-hexanol

107-83-5

2-Methylpentane

85255-76-1

116-53-0

2-Methylbutyric acid

2-Naphthalenesulfonicacid,6-hydroxy-,monosodium
salt, polymer withformaldehyde andhydroxymethylbenzenesulfon
ic aid monosodium salt

105-30-6

2-Methyl-1-pentanol

21245-02-3

111-87-5

1-Octanol

2-Ethylhexyl pdimethylamino benzoate

135-98-813197-76-7

1-Methylpropyl benzene

6940-78-9

1-Dodecanaminium, N-(2hydroxy-3-sulfopropyl)-N,Ndimethyl-, inner salt

CASRN

1-Bromo-4-chlorobutane

Test Substance

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidPhysicalForm asTested

NA

27.8

0.149

0.191

15

4.72E-06

0.245

0.397

294E-04

0.320

28.6

0.041

0.0132

0.176

1.16E-21

0.164

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Category 1

Not Classified

Category 1

Category 2B

Not Classified

Category 2A

Not Classified

Category 2A

Not Classified

Not Classified

Not Classified

Category 2A

Not Classified

Category 1

Not Classified

GHS
Consensusd

Test Substances Used to Evaluate STE Performance in a Bottom-Up Approacha

Category II

Category IV

Category I

Category III

Category IV

Category II

Category III

Category II

Category IV

Category III

Category IV

Category II

Category IV

Category I

Category IV

EPA
Consensusd

2

1

2

2

1

2

1

2

1

1

1

2

1

2

1

Overall
Consensus
STE Ranke

STE rank scores from Kao Corporation were equated to the GHS or EPA classification of eye hazard (i.e., UN 2011 and EPA 2012) such that an STE rank of 3 was considered asevere eye irritant or corrosive (i.e., GHS Category 1 or EPA Category I); an STE rank of 2 was considered a moderate to mild eye irritant (i.e., GHS Category 2A or 2B or EPA
Category II or III); and an STE rank of 1 was considered to be equivalent to GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours).

Table 2-2e

Short Time Exposure Summary Review Document

60
111-76-2

Butyl cellosolve

Calcium thioglycolate

Butyrolactone
5793-98-6

96-48-025638-17-9

123-86-4

Butyl acetate

Butylnaphthalenesulfonicacid sodium salt

71-36-3

ButanolNA

Body shampoo A

119345-04-9

Benzene, 1,1'-oxybis-,
tetrapropylene derivatives,
sulfonated, sodium salts
100-51-6

63449-41-2

Benzalkonium chloride(10%)

Benzyl alcohol

8001-54-5

67-64-1

AcetoneBenzalkonium chloride

64-19-7

Acetic acid6484-52-2

562-49-2

3,3-Dimethylpentane

Ammonium nitrate589-34-4

3-Methylhexane

9003-04-7

623-39-2

3-Methoxy-1,2-propanediol

Acrylic acid homopolymer
sodium salt

110-03-2

2,5-Dimethyl-2,5-hexanediol

18472-87-2

625-69-4

2,4-Pentanediol

Acid red 92

3970-62-5

CASRN

2,2-Dimethyl-3-pentanol

Test Substance

Solid

LiquidSolid

LiquidLiquidLiquidLiquidLiquid

Solid

Solid

Liquid

Solid

Solid

Solid

LiquidLiquidLiquidLiquidLiquidSolid

LiquidLiquidPhysicalForm asTested16

4.20E-03

0.0394NA

0.0633

1.595

1.04

NA

7.14E-03

NA

NA

NA

4.48E-16

4.56E-04

5.71E-24

33.2

2.29

10.1

8.29

1.92E-03

578E-04

7.3E-03

0.413

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

100%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Category 1

Category 2ACategory 1

Category 1

Not Classified

Category 1

Category 2ACategory 1

Category 1

Category 1

Category 1

Category 2B

Not Classified

Category 1

Category 2ACategory 1

Not Classified

Not Classified

Not Classified

Category 1

Not Classified

Not Classified

GHS
Consensusd

Short Time Exposure Summary Review Document

Category I

Category II

Category I

Category II

Category III

Category I

Category II

Category I

Category I

Category I

Category I

Category III

Category IV

Category I

Category II

Category I

Category IV

Category IV

Category IV

Category I

Category IV

Category III

EPA
Consensusd

2

2

2

2

1

2

2

2

3

3

3

1

1

3

2

2

1

1

1

1

1

1

Overall
Consensus
STE Ranke

61140-72-7
6004-24-6
57-09-0
57-09-0
108-93-0
108-94-1
96-41-3
629-19-6
577-11-7

Cetylpyridinium bromide

Cetylpyridinium chloride

Cetyltrimethylammonium
bromideCetyltrimethylammonium
bromide (10%)

Cyclohexanol

Cyclohexanone

Cyclopentanol

Di-n-propyl disulphideDi(2-Ethylhexyl) sodium
sulfosuccinate

141-78-6

538-71-6

Domiphen bromide

Ethyl acetate112-40-3

Dodecane
64-17-5

107-64-2

Distearyldimethylammonium
chloride609-14-3

67-68-5

Dimethyl sulfoxide

Ethyl 2-methylacetoacetate108-83-8

Diisobutyl ketone

Ethanol

100-37-8

Diethylethanolamine

29911-27-1

112-02-7

Cetyl trimethyl ammonium
chlorideDi(propylene glycol) propyl
ether

79-92-5

CASRN

Camphene

Test Substance

LiquidLiquidLiquidSolid

LiquidSolid

LiquidLiquidLiquidLiquidSolid

LiquidLiquidLiquidLiquid

Solid

Solid

Solid

Solid

LiquidSolid

PhysicalForm asTested13.1

0.09158.12

NA0.031517

2.55E-15

0.08290.287

0.0863

2.38E-04

1.63E-15

0.0664307

539

0.0866NA

NA

NA

3.47E-07

NA

0.237

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

10%

100%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

10%

10%

10%

10%

5%

100%

In Vivo Conc(%)c

Not Classified

Category 2B

Category 2ACategory 1

Not Classified

Category 1

Not Classified

Not Classified

Category 1

Category 2B

Category 1

Not Classified

Category 2B

Not Classified

Category 1

Category 1

Category 1

Category 1

Category 1

Category 1

Category 2B

GHS
Consensusd

Short Time Exposure Summary Review Document

Category III

Category III

Category I

Category I

Category III

Category I

Category III

Category IV

Category I

Category III

Category I

Category IV

Category II

Category III

Category I

Category I

Category I

Category I

Category I

Category I

Category III

EPA
Consensusd

2

2

1

3

1

2

1

1

2

2

3

1

2

2

2

3

3

3

3

2

1

Overall
Consensus
STE Ranke

6250-21-5
143-07-7
1643-20-5

Lactic acid

Lauric acid

Lauryldimethylamine oxide79-20-9
110-43-0
105-34-0
96-37-7
78-93-3
108-10-1
598-98-1

Methyl amyl ketone

Methyl cyanoacetate

Methyl cyclopentane

Methyl ethyl ketone (2Butanone)

Methyl isobutyl ketone

Methyl trimethyl acetate

3121-61-7

Methoxyethyl acrylate

Methyl acetate

108-45-2

m-Phenylene diamineNA110-27-0

Isopropyl myristate

Lotion A

75-26-3Isopropyl bromide

29590-42-9Iso-octyl acrylate

67-63-0

288-32-4

Imidazole

Isopropyl alcohol

106-91-2

Glycidyl methacrylate

78-83-1

56-81-5GlycerolIsobutyl alcohol

118-60-5Ethylhexyl salicylate

78-84-23938-95-2Ethyl trimethyl acetate

Isobutanal

CASRN

Test Substance

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

Liquid

Solid

Solid

LiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

LiquidLiquidLiquidLiquidPhysicalForm asTested

4.76

2.90

13.1

17.8

0.0469

0.655

7.03

0.598

18

2.51E-04

NA

1.68E-15

2.13E-09

3.81E-03.108E-04

28.5

6.61

1.78

21.9

0.0204

5.78E-04

0.08291.06E-05

9.51E-07

2.240

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Not Classified

Not Classified

Category 2A

Not Classified

Category 2A

Not Classified

Category 2ACategory 1

Category 1

Not Classified

Category 1

&DWHJRU\$Category 1

Not Classified

Not Classified

Category 2ACategory 1

Category 2B

Not Classified

Category 1

Not Classified

Not Classified

Not Classified

Not Classified

GHS
Consensusd

Short Time Exposure Summary Review Document

Category IV

Category III

Category III

Category III

Category II

Category III

Category II

&DWHJRU\,,,Category I

Category IV

Category I

&DWHJRU\,,Category I

Category IV

Category IV

Category III

Category I

Category III

Category IV

Category I

Category III

Category IV

Category IV

Category III

EPA
Consensusd

1

1

2

1

2

1

1

2

2

1

3

2

2

1

1

1

2

2

1

2

2

1

1

1

Overall
Consensus
STE Ranke

63

9004-81-3
61788-85-0

68439-51-0

104376-75-2
116998-28-8
54392-28-8

Polyoxyethylenehydrogenated castor oil(60E.O.)

Polyoxyethylene(10) polyoxy
propylene(1.5) laurylmyristyl ether

Polyoxyethylene(13) (mono-,
di-, tri)styrenated phenyl ether

Polyoxyethylene(14) tribenzy
lated phenyl ether

Polyoxyethylene(160) sorbita
n triisostearate598-65-2

n,n-Dimethylguanidine
sulfatePolyethyleneglycol
monolaurate (10 E.O.)

111-83-1

n-Octyl bromide

25322-68-3

111-25-1

n-Hexyl bromide

Polyethylene glycol 400

111-27-3

n-Hexanol

188070-49-7

123-72-8

n-Butanal

Naphthalenesulfonic acid,butyl-, polymer withformaldehyde and 2naphthalenesulfonic acid,sodium salt

112-72-1

CASRN

Myristyl alcohol

Test Substance

Solid

LiquidLiquidLiquidSolid

LiquidLiquid

Solid

Solid

LiquidLiquidLiquidLiquidSolid

PhysicalForm asTested

NA

NA

NA

NA

NA

0

NA

NA

4.04

0.0691

0.541

0.117

14.4

19

2.69E-05

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

In Vivo Conc(%)c

Not Classified

Not Classified

Not Classified

Category 1

Not Classified

Not Classified

Not Classified

Category 2A

Not Classified

Not Classified

Not Classified

Category 2ACategory 2B

Category 2A

GHS
Consensusd

Short Time Exposure Summary Review Document

Category IV

Category IV

Category III

Category I

Category IV

Category IV

Category IV

Category II

Category III

Category IV

Category IV

Category II

Category III

Category III

EPA
Consensusd

1

1

3

3

1

2

1

2

1

1

1

2

2

1

Overall
Consensus
STE Ranke

64
143-18-0
58-33-3

Potassium oleate

Promethazine hydrochloride

NA
NA
NA
NA
NA
NA
NA

Rinse B

Rinse C

Rinse D

Shampoo A

Shampoo B

Shampoo C

68910-56-5

Quaternary ammonium
compounds, di-C12-15alkyldimethyl, chlorides

Rinse A

68424-85-1

Quaternary ammonium
compounds, benzyl-C12-16alkyldimethyl, chlorides

110-86-1

10124-65-9

Potassium laurate

Pyridine

61788-85-0

Polyoxyethylene(40) hydroge
nated castor oil

57-55-69002-92-0

Polyoxyethylene(23) lauryl et
her

Propylene glycol

61790-82-7

Polyoxyethylene(20) hydroge
nated tallow amine

1569-01-3

104376-75-2

Polyoxyethylene(19) (mono-,
di-, tri-)styrenated phenyl ether

Propasol solvent P

CASRN

Test Substance

LiquidLiquidLiquidLiquidLiquidLiquidLiquid

Solid

Solid

LiquidLiquidLiquid

Solid

Solid

Solid

Liquid

Solid

Solid

LiquidPhysicalForm asTested

NA

NA

NA

NA

NA

NA

NA

NA

NA

2.58

0.0148

0.180

0

20

4.93E-100NA

2.03E-13

NA

NA

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

100%

100%

100%

100%

10%

1%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

In Vivo Conc(%)c

Category 2ACategory 1

Category 2A

Not Classified

Not Classified

Category 2B

Not Classified

Category 1

Category 1

Category 1

Not Classified

Category 2B

Category 1

Not Classified

Category 1

Not Classified

Category 2A

&DWHJRU\$Not Classified

GHS
Consensusd

Short Time Exposure Summary Review Document

Category II

Category I

Category II

Category III

Category IV

Category III

Category III

Category I

Category I

Category I

Category IV

Category II

Category I

Category III

Category I

Category IV

Category III

&DWHJRU\,,Category II

EPAConsensusd

2

2

2

1

1

2

2

2

2

2

1

2

3

2

3

1

2

3

2

Overall
Consensus
STE Ranke

65151-21-3
151-21-3
3926-62-3
9004-82-4

Sodium lauryl sulfate

Sodium lauryl sulfate (15%)

Sodium monochloroacetate

Sodium polyoxyethylene(3)lauryl ether sulfate

100-42-5

Styrene9002-93-1
9005-64-5
9005-65-6
1330-20-7

Triton X-100

Tween 20

Tween 80

XyleneLiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

LiquidSolid

LiquidSolid

Liquid

Solid

Solid

Solid

Solid

Solid

LiquidPhysicalForm asTested

0.883

0

0

0

2.50E-10

4.51E-07

3.160

NA

0.673

NA

1.25E-15

4.84E-12

2.27E-13

4.23E-09

NA

2.40E-13

6.53E-22

NA

NA

Vapor
Pressure(kPa 25°C)b

100%

100%

100%

100%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

15%

100%

10%

100%

100%

In Vivo Conc(%)c

Not Classified

Not Classified

Not Classified

Category 1

Category 1

Not Classified

&DWHJRU\%&DWHJRU\$Not Classified

Category 1

Not Classified

Category 1

Category 1

Category 2B

Category 1

&DWHJRU\$Category 1

Not Classified

Category 2A

GHS
Consensusd

Category II

Category IV

Category III

Category I

Category I

Category III

Category III

&DWHJRU\,,Category III

Category I

Category IV

Category I

Category I

Category III

Category I

Category III

Category I

Category III

Category II

EPAConsensusd

1

1

2

3

3

1

1

2

1

3

2

1

3

2

3

3

3

2

2

Overall
Consensus
STE Ranke

21

Abbreviations: CASRN = CAS Registry Number® (American Chemical Society); EPA = U.S. Environmental Protection Agency; GHS = United Nations Globally Harmonized
System of Classification and Labelling of Chemicals; JaCVAM = Japanese Center for the Validation of Alternative Methods; kPa = kilopascals; NA = not available;
STE = short time exposure.

27028-82-6

102-71-6

Triethanolamine

Triethanolamine
polyoxyethylene(3.0) lauryl
ether sulfate

108-88-3

TolueneNA

112-03-8

Stearyltrimethylammonium
chloride

Sucrose fatty acid ester

1338-39-2

Sorbitan monolaurate

54-21-71310-73-2

Sodium hydroxide

Sodium salicylate532-02-5

NA

CASRN

Sodium 2naphthalenesulfonate

Shampoo D

Test Substance

Short Time Exposure Summary Review Document

66
135-98-8
111-87-5
99-62-7
1647-16-1
622-08-2
111-15-9
104-76-7

1-Octanol

1,3-Di-isopropylbenzene

1,9-Decadiene

2-Benzyloxyethanol

2-Ethoxyethyl acetate(Cellosolve acetate)

2-Ethyl-1-hexanol

13197-76-7

1-Dodecanaminium, N-(2hydroxy-3-sulfopropyl)-N,Ndimethyl-, inner salt

1-Methylpropyl benzene

6940-78-9

CASRN

1-Bromo-4-chlorobutane

Substance

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

App
Domainb

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidPhysicalForm asTested222.460E-02

3.970E-01

2.940E-04

3.200E-01

4.100E-02

1.320E-02

1.760E-01

1.160E-21

1.640E-01

Vapor
Pressure(kPa25ºC)c

100%

100%

100%

100%

100%

100%

100%

neat

100%

NICEATMIn Vivo
Concd(%)

Category 2A

Not classified

Category 2A

Not classified

Not classified

Category 2A

Not classified

Category 1

Not classified

GHSNICEATM
Consensuse

Category II

Category III

Category II

Category IV

Category IV

Category II

Category IV

Category I

Category IV

EPANICEATM
Consensuse

2

1

2

1

1

2

1

2

1

JaCVAM
Overall
Consensus
STE Rankf

Test Substances Used to Evaluate STE Performance in a Bottom-Up Approach Within the Defined Applicability Domaina,bA bottom-up approach is used to distinguish GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours) and STE Rank 1 from all other hazard
categories (i.e., GHS Category 1, 2A, 2B; EPA Category I, II, III; or STE Rank 2 and 3).
Vapor pressure is expressed in kilopascals at 25°C. Vapor pressures were found using the Hazardous Substances Data Bank (HSDB® [U.S. National Library of Medicine]),available at http://toxnet.nlm.nih.gov (accessed 2/25/2013) or from ChemSpider (available at www.chemspider.com [accessed 2/25/2013]). If actual values were not available,
predicted values were obtained from the U.S. EPA EPI (Estimation Programs Interface) Suite™ for Microsoft® Windows, v. 4.11) or ACD/Labs’ ACD/PhysChem Suiteavailable at http://www.acdlabs.com/products/pc_admet/physchem/physchemsuite/ (accessed 2/25/2013). Data from the EPI Suite and ACD/PhysChem Suite programs werealso available in ChemSpider.
The concentration as tested in the rabbit eye test, based on NICEATM data. For substances tested at 100%, the starting material was tested neat/undiluted.
The consensus classification of two or more studies. When there was no consensus using either the GHS (UN 2011) or EPA (EPA 2012) eye hazard classification system (e.g.,
one GHS Category 2A and one GHS Category 2B), the more hazardous classification (i.e., GHS Category 2A) was used as the consensus classification.
STE rank scores from Kao Corporation were equated to the GHS or EPA classification of eye hazard (i.e., UN 2011 and EPA 2012) such that an STE rank of 3 was considered asevere eye irritant or corrosive (i.e., GHS Category 1 or EPA Category I); an STE rank of 2 was considered a moderate to mild eye irritant (i.e., GHS Category 2A or 2B or EPA
Category II or III); and an STE rank of 1 was considered to be equivalent to GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours).

Table 2-3

e

d

c

b

a

Short Time Exposure Summary Review Document

67

71-36-3
123-86-4

Butanol

Butyl acetate

NA119345-04-9

Benzene, 1,1'-oxybis-,
tetrapropylene derivs.,
sulfonated, sodium salts

Body shampoo A

63449-41-2

Benzalkonium chloride (10%)

100-51-6

8001-54-5

Benzalkonium chloride

Benzyl alcohol

9003-04-7

623-39-2

3-Methoxy-1,2-propanediol

Acrylic acid homopolymer
sodium salt

625-69-4

2,4-Pentanediol

18472-87-2

3970-62-5

2,2-Dimethyl-3-pentanol

Acid red 92

85255-76-1

116-53-0

2-Methylbutyric acid

2-Naphthalenesulfonic acid,6hydroxy-,monosodium salt,
polymer with formaldehyde andhydroxymethylbenzenesulfonic
aid monosodium salt

105-30-621245-02-3

CASRN

2-Methyl-1-pentanol

2-Ethylhexyl p-dimethylamino
benzoate

Substance

1,2

1,2

1,2

1,2

2

2

1,2

2

1

1,2

1,2

1,2

1,2

1,2

1,2

1,2

App
Domainb

LiquidLiquidLiquidLiquid

Solid

Solid

Liquid

Solid

Solid

LiquidLiquidLiquidLiquidLiquidLiquidLiquidPhysicalForm asTested23

1.587E+00

1.040E+00

NA

7.140E-03

NA

NA

NA

4.560E-04

5.710E-24

1.920E-03

7.300E-03

4.130E-01

NA

1.490E-01

1.910E-01

4.720E-06

Vapor
Pressure(kPa25ºC)c

100%

100%

100

100%

neat

10

100%

neat

100%

100%

100%

100%

neat

100%

100%

100%

NICEATMIn Vivo
Concd(%)

Short Time Exposure Summary Review Document

Not classified

Category 1

Category 2ACategory 1

Category 1

Category 1

Category 1

Not classified

Category 1

Not classified

Not classified

Not classified

Category 1

Category 1

Category 2B

Not classified

GHSNICEATM
Consensuse

Category III

Category I

Category II

Category I

Category I

Category I

Category I

Category IV

Category I

Category IV

Category IV

Category III

Category II

Category I

Category III

Category IV

EPANICEATM
Consensuse

1

2

2

2

3

3

3

1

3

1

1

1

2

2

2

1

JaCVAM
Overall
Consensus
STE Rankf

6896-41-3
629-19-6
577-11-7

Cyclopentanol

Di-n-propyl disulphideDi(2-Ethylhexyl) sodium
sulfosuccinate

100-37-8
108-83-8
67-68-5
107-64-2
112-40-3

Diethylethanolamine

Diisobutyl ketone

Dimethyl sulfoxide

Distearyldimethylammonium
chloride

Dodecane

29911-27-1

108-94-1

Cyclohexanone

Di(propylene glycol) propyl
ether

108-93-0

57-09-0

Cetyltrimethylammonium
bromide (10%)

Cyclohexanol

6004-24-6

Cetylpyridinium chloride

5793-98-6

140-72-7

Cetylpyridinium bromide (10%)

Calcium thioglycolate

96-48-025638-17-9

111-76-2

CASRN

Butyrolactone

Butylnaphthalenesulfonic acidsodium salt

Butyl cellosolve

Substance

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1

2

2

2

1,2

2

1,2

App
Domainb

LiquidSolid

LiquidLiquidLiquidLiquidSolid

LiquidLiquidLiquidLiquid

Solid

Solid

Solid

Solid

LiquidSolid

LiquidPhysicalForm asTested24

3.150E-02

2.550E-15

8.290E-02

2.870E-01

8.630E-02

2.380E-04

1.630E-15

6.640E-02

3.070E-01

5.390E-01

8.660E-02

4.200E-03

NANA

3.470E-07

3.940E-02

NA

6.330E-02

Vapor
Pressure(kPa25ºC)c

100%

100%

100%

100%

100%

100%

10%

100%

100%

100%

100%

100%

10%

10%

10%

100%

neat

100%

NICEATMIn Vivo
Concd(%)

Short Time Exposure Summary Review Document

Not classified

Category 1

Not classified

Not classified

Category 1

Category 2B

Category 1

Not classified

Category 2B

Not classified

Category 1

Category 1

Category 1

Category 1

Category 1

Category 2ACategory 1

Category 1

GHSNICEATM
Consensuse

Category III

Category I

Category III

Category IV

Category I

Category III

Category I

Category IV

Category II

Category III

Category I

Category I

Category I

Category I

Category I

Category II

Category I

Category II

EPANICEATM
Consensuse

1

2

1

1

2

2

3

1

2

2

2

2

3

3

3

2

2

2

JaCVAM
Overall
Consensus
STE Rankf

691643-20-5

Lauryldimethylamine oxide

108-45-2
3121-61-7
110-43-0
105-34-0
108-10-1
598-98-1

m-Phenylene diamine

Methoxyethyl acrylate

Methyl amyl ketone

Methyl cyanoacetate

Methyl isobutyl ketone

Methyl trimethyl acetateNA

143-07-7

Lauric acid

Lotion A

50-21-5Lactic acid288-32-4

Imidazole

110-27-0

106-91-2

Glycidyl methacrylate

Isopropyl myristate56-81-5Glycerol78-83-1

118-60-5Ethylhexyl salicylate

Isobutyl alcohol3938-95-2Ethyl trimethyl acetate

29590-42-9

609-14-3Ethyl 2-methylacetoacetate

Iso-octyl acrylate

538-71-6

CASRN

Domiphen bromide

Substance

1,2

1,2

1,2

1,2

1,2

1

1,2

2

1,2

1,2

1,2

1,2

1,2

1

1,2

1,2

1,2

1,2

1,2

1,2

App
Domainb

LiquidLiquidLiquidLiquidLiquidSolid

Liquid

Solid

Solid

LiquidLiquidLiquidLiquidSolid

LiquidLiquidLiquidLiquidLiquidSolid

PhysicalForm asTested25

4.760E+00

2.900E+00

4.690E-02

6.550E-01

5.980E-01

2.510E-04

NA

1.680E-15

2.130E-09

3.810E-03

1.080E-04

1.780E+00

2.040E-02

5.780E-04

8.290E-02

1.060E-05

9.510E-07

2.240E+00

9.150E-02

NA

Vapor
Pressure(kPa25ºC)c

100%

100%

100%

100%

100%

100%

100

neat

neat

100%

100%

100%

100%

100%

100%

100%

100%

100%

100%

10%

NICEATMIn Vivo
Concd(%)

Short Time Exposure Summary Review Document

Not classified

Not classified

Category 2A

Not classified

Category 1

Category 1

Not classified

Category IV

Category III

Category II

Category III

&DWHJRU\,,,Category I

Category IV

Category I

&DWHJRU\,,&DWHJRU\2A
Category 1

Category I

Category IV

Category I

Category IV

Category I

Category III

Category IV

Category IV

Category III

Category III

Category I

EPANICEATM
Consensuse

Category 1

Not classified

Category 1

Not classified

Category 1

Not classified

Not classified

Not classified

Not classified

Category 2B

Category 1

GHSNICEATM
Consensuse

1

1

2

1

2

2

1

3

2

2

1

2

1

2

2

1

1

1

2

3

JaCVAM
Overall
Consensus
STE Rankf

70111-83-1

n-Octyl bromide

25322-68-3
9004-81-3
61788-85-0
68439-51-0
104376-75-2
116998-28-8
54392-28-8
104376-75-2
61790-82-7
9002-92-0

Polyethyleneglycol monolaurate(10 E.O.)

Polyoxyethylene hydrogenatedcastor Oil (60E.O.)

Polyoxyethylene(10) polyoxypro
pylene(1.5) lauryl-myristyl ether

Polyoxyethylene(13) (mono-, di, tri-)styrenated phenyl ether

Polyoxyethylene(14) tribenzylate
d phenyl ether

Polyoxyethylene(160) sorbitan tr
iisostearate

Polyoxyethylene(19) (mono-, di, tri-)styrenated phenyl ether

Polyoxyethylene(20) hydrogenat
ed tallow amine

Polyoxyethylene(23) lauryl ether

598-65-2

Polyethylene glycol 400

n,n-Dimethylguanidine sulfate

188070-49-7

111-25-1

n-Hexyl bromide

Naphthalenesulfonic acid, butyl-,
polymer with formaldehyde and2-naphthalenesulfonic acid,sodium salt

111-27-3

CASRN

n-Hexanol

Substance

2

1,2

1,2

1,2

1,2

1,2

1,2

2

1,2

1,2

1

2

1,2

1,2

1,2

App
Domainb

Solid

Solid

LiquidSolid

LiquidLiquidLiquidSolid

LiquidLiquid

Solid

Solid

LiquidLiquidLiquidPhysicalForm asTested26

2.030E-13

NA

NA

NA

NA

NA

NA

NA

0.000E+00

NA

4.040E+00

NA

6.910E-02

5.410E-01

1.170E-01

Vapor
Pressure(kPa25ºC)c

neat

neat

neat

neat

neat

neat

neat

100%

100%

100%

100%

neat

100%

100%

100%

NICEATMIn Vivo
Concd(%)

Short Time Exposure Summary Review Document

Category 2A

&DWHJRU\
2A

Not classified

Not classified

Not classified

Not classified

Category 1

Not classified

Not classified

Not classified

Not classified

Category 2A

Not classified

Not classified

Category 2A

GHSNICEATM
Consensuse

Category III

&DWHJRU\,,Category II

Category IV

Category IV

Category III

Category I

Category IV

Category IV

Category IV

Category III

Category II

Category IV

Category IV

Category II

EPANICEATM
Consensuse

2

3

2

1

1

3

3

1

2

1

1

2

1

1

2

JaCVAM
Overall
Consensus
STE Rankf

71

NA

Shampoo D

9004-82-4

NA

Shampoo C

Sodium polyoxyethylene(3)lauryl ether sulfate

NA

Shampoo B

151-21-3

NA

Shampoo A

Sodium lauryl sulfate (15%)

NA

Rinse D

151-21-3

NA

Rinse C

Sodium lauryl sulfate

NA

Rinse B

1310-73-2

NA

Rinse A

Sodium hydroxide

110-86-1

Pyridine

58-33-3

Promethazine hydrochloride

57-55-6

143-18-0

Potassium oleate

Propylene glycol

10124-65-9

Potassium laurate

1569-01-3

61788-85-0

Polyoxyethylene(40) hydrogenat
ed castor oil

Propasol solvent P

CASRN

Substance

1,2

2

2

1

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1

2

1,2

1,2

App
Domainb

Liquid

Solid

Solid

Solid

LiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquidLiquid

Solid

Solid

Solid

LiquidPhysicalForm asTested27

2.270E-13

NA

2.400E-13

6.530E-22

NA

NA

NA

NA

NA

NA

NA

NA

2.580E+00

1.480E-02

1.800E-01

0.000E+00

4.930E-10

0.000E+00

NA

Vapor
Pressure(kPa25ºC)c

neat

15

100%

10%

100

100

100

100

100

100

100

100

100%

100%

100%

100%

neat

10%

neat

NICEATMIn Vivo
Concd(%)

Short Time Exposure Summary Review Document

Category 1

Category I

Category I

Category III

&DWHJRU\2A
Category 1

Category I

Category II

Category II

Category I

Category II

Category III

Category IV

Category III

Category III

Category I

Category IV

Category II

Category I

Category III

Category I

Category IV

EPANICEATM
Consensuse

Category 1

Category 2ACategory 2ACategory 1

Category 2A

Not classified

Not classified

Category 2B

Not classified

Category 1

Not classified

Category 2B

Category 1

Not classified

Category 1

Not classified

GHSNICEATM
Consensuse

3

3

3

3

2

2

2

2

1

1

2

2

2

1

2

3

2

3

1

JaCVAM
Overall
Consensus
STE Rankf

72
112-03-8
100-42-5

Stearyltrimethylammonium
chloride

Styrene9002-93-1
9005-64-5
9005-65-6
1330-20-7

Triton X-100

Tween 20

Tween 80

Xylene

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

1,2

®

LiquidLiquidLiquidLiquidLiquidLiquidLiquidSolid

LiquidSolid

LiquidPhysicalForm asTested8.826E-01

0.000E+00

0.000E+00

0.000E+00

2.500E-10

4.510E-07

3.160E+00

NA

6.730E-01

NA

1.250E-15

Vapor
Pressure(kPa25ºC)c

100%

100%

100%

100%

neat

100%

100%

100%

100%

10%

neat

NICEATMIn Vivo
Concd(%)

Not classified

Not classified

Not classified

Category 1

Category 1

Not classified

Category II

Category IV

Category III

Category I

Category I

Category III

Category III

&DWHJRU\,,&DWHJRU\
2A
&DWHJRU\%Category III

Category I

Category IV

EPANICEATM
Consensuse

Not classified

Category 1

Not classified

GHSNICEATM
Consensuse

1

1

2

3

3

1

1

2

1

3

2

JaCVAM
Overall
Consensus
STE Rankf

28

Abbreviations: App = applicability; CASRN = CAS Registry Number (American Chemical Society); EPA = U.S. Environmental Protection Agency; GHS = United Nations
Globally Harmonized System of Classification and Labelling of Chemicals; JaCVAM = Japanese Center for the Validation of Alternative Methods; kPa = kilopascals; NA = notavailable; NICEATM = National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods; STE = short time exposure.
a
A bottom-up approach is used to distinguish GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours) and STE Rank 1 from all other hazard
categories (i.e., GHS Category 1, 2A, 2B; EPA Category I, II, III; or STE Rank 2 and 3).
b
There are two defined applicability domains (AD) for the STE test method. AD 1 (n=94 VXEVWDQFHV LQFOXGHVDOOVXEVWDQFHVZLWKYDSRUSUHVVXUHVNLORSDVFDOVEXWH[FOXGHV
solids that are alcohols, hydrocarbons, or salts. AD 2 (n = 101 VXEVWDQFHV LQFOXGHVDOOOLTXLGVZLWKYDSRUSUHVVXUHVNLORSDVFDOVDQGVROLGVXUIDFWDQWVRUVXUIDFWDQW-containing
formulations (i.e., nonsurfactant solids and substances with vapor pressures >6 kilopascals are excluded).
cVapor pressure is expressed in kilopascals at 25°C. Vapor pressures were found using the Hazardous Substances Data Bank (HSDB® [U.S. National Library of Medicine]),available at http://toxnet.nlm.nih.gov (accessed 2/25/2013) or from ChemSpider (available at www.chemspider.com [accessed 2/25/2013]). If actual values were not available,
predicted values were obtained from the U.S. EPA EPI (Estimation Programs Interface) Suite™ for Microsoft® Windows, v. 4.11) or ACD/Labs’ ACD/PhysChem Suiteavailable at http://www.acdlabs.com/products/pc_admet/physchem/physchemsuite/ (accessed 2/25/2013). Data from the EPI Suite and ACD/PhysChem Suite programs werealso available in ChemSpider.

27028-82-6

102-71-6

Triethanolamine

Triethanolamine
polyoxyethylene(3.0) lauryl
ether sulfate

108-88-3

TolueneNA1338-39-2

Sorbitan monolaurate

Sucrose fatty acid ester

CASRN

Substance

App
Domainb

Short Time Exposure Summary Review Document

73

f

e

d

29

The concentration as tested in the rabbit eye test, based on NICEATM data.
The consensus classification of two or more studies. When there was no consensus using either the GHS (UN 2011) or EPA (EPA 2012) eye hazard classification system (e.g.,
one GHS Category 2A and one GHS Category 2B), the more hazardous classification (i.e., GHS Category 2A) was used as the consensus classification.
STE rank scores were equated to the GHS or EPA classification of eye hazard (i.e., UN 2011 and EPA 2012) such that an STE rank of 3 was considered a severe eye irritant or
corrosive (i.e., GHS Category 1 or EPA Category I); an STE rank of 2 was considered a moderate to mild eye irritant (i.e., GHS Category 2A or 2B or EPA Category II or III);and an STE rank of 1 was considered to be equivalent to GHS Not Classified or EPA Category IV (minimal effects clearing in less than 24 hours).

Short Time Exposure Summary Review Document

Short Time Exposure Summary Review Document

3.0

STE Test Method Performance

Test method performance is typically evaluated by calculating the following (ICCVAM 2003):
x
x
x
x
x
x
x

Accuracy (concordance): the proportion of correct outcomes (positive and negative) of a test
methodSensitivity: the proportion of all positive substances that are classified correctly as positive
Specificity: the proportion of all negative substances that are classified correctly as negative
Positive predictivity: the proportion of correct positive responses among substances testing
positive
Negative predictivity: the proportion of correct negative responses among substances testingnegative
False positive rate: the proportion of all negative substances that are falsely identified as positive
False negative rate: the proportion of all positive substances that are falsely identified as negative

The STE test method performance was evaluated for each study, and the data set is provided in
Supplement B. An overall STE ocular irritation classification was assigned for each test substance inthe database based on the majority of ocular irritation classification calls. When a test substance had
an even number of different irritation classifications (e.g., two tests classified a substance as amoderate irritant and two tests classified a substance as a severe irritant), the more severe hazard
classification was used for its overall classification (e.g., severe irritant). Using the consensus ocular
irritation classification for each substance, the STE test method was evaluated in a top-down approachto distinguish ocular corrosives and severe irritants (i.e., GHS Category 1 or EPA Category I) from allother categories (i.e., GHS Category 2A, 2B, Not Classified or EPA Category II, III, IV). The STE
test method was also evaluated in a bottom-up approach to identify GHS Not Classified substances or
EPA Category IV (minimally irritant) substances from all other irritant categories (i.e., GHS
Category 1, 2A, or 2B or EPA Category I, II, or III).
The overall accuracy of the STE test method in a top-down approach ranged from 70% to 96%, andthe accuracy in a bottom-up approach ranged from 80% to 85% depending on the classification. The
predictive capacity of the STE test method was assessed by identifying the chemical classes orphysical properties that increased the false positive rate in a top-down approach and those that
increased the false negative rate in a bottom-up approach. Excluding discordant chemical classes orphysical properties optimized the applicability domain for a top-down or bottom-up approach.

3.1

GHS Classification System: STE Performance in a Top-Down Approach

The performance of the STE test method was evaluated for GHS ocular hazard classification in a topdown approach. STE accuracy, sensitivity, specificity, false positive rate, and false negative rate were
determined based on available in vivo reference data for the test substances. Test substances that wereidentified as direct MTT reducers were removed from the analyses. These include two substancesfrom Kojima et al. (Kao BRD), two substances from Takahashi et al. (2010), and one substance fromthe Kao in-house studies. These analyses were performed for each of the five studies as well as for
120 unique substances from these five studies that remained after duplicates were removed andconsensus classifications were assigned (Table 3-1). The GHS classification for each test substance islisted in Supplement B.

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Short Time Exposure Summary Review Document

Table 3-1

Data Source

STE Performance for GHS Classification in a Top-Down ApproachN

Accuracy

Sensitivity

Specificity

False
Positive
Ratea

False Negative
Rateb

%

No.c

%

No.c

%

No.c

%

No.c

%

No.c

Kojima et al.
(Kao BRD)

30

70

21/30

10

1/10

100

20/20

0

0/20

90

9/10

Sakaguchi et al.
2011

23

96

22/23

80

4/5

100

18/18

0

0/18

20

1/5

Takahashi et al.
2009

37

84

31/37

65

11/17

100

20/20

0

0/20

35

6/17

Takahashi et al.
2010

47

83

39/47

58

11/19

100

28/28

0

0/28

42

8/19

Kao In-House

22

96

21/22

0

0/1

100

21/21

0

0/21

100

1/1

Kao New
Surfactants

39

69

27/39

45

9/20

95

18/19

5.3

1/19

55

11/20

UniqueSubstancesd

120

85

102/120

53

19/36

99

83/84

1.2

1/84

47

17/36

Abbreviations: BRD = background review document; GHS = Globally Harmonized System of Classification and Labellingof Chemicals (UN 2011); N = number of substances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.
d
Substances from all of the above studies remaining after duplicates were removed and consensus classifications wereassigned.

3.1.1

STE Discordant Results for GHS Classification in a Top-Down Approach

The STE results that were discordant with in vivo results were analyzed further. These analyses were
performed on specific categories of chemicals, as well as on certain physicochemical properties
potentially relevant to ocular toxicity testing (e.g., surfactants, pH, physical form).
Several trends were noted in STE performance among these subgroups of substances (Table 3-2).
Only one of 84 substances was overpredicted (i.e., false positive) and slightly affected theoverprediction of its constituent chemical classes (3.6% to 8.3% overprediction). The chemical
categories of substances that were most consistently underpredicted (i.e., false negatives) by the STE
test method were alcohols and carboxylic acids. Of the 17 underpredicted substances, 7 werealcohols, 4 were carboxylic acids, and 3 were salts. Additional chemical categories represented
among the underpredicted substances were esters (2) and heterocyclic compounds (2).
With regard to the physical form of the substances underpredicted by the STE test method, 12 wereliquids and 5 were solids. Considering the proportion of the total available database, solids (16%;5/31) and liquids (13%; 12/89) were underpredicted at a similar rate by the STE test method.

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Short Time Exposure Summary Review Document

Table 3-2

STE False Positive and False Negative Rates by Chemical Category andProperties of Interest for GHS Classification in a Top-Down Approach

CategoryN

False Positive Ratea

False Negative Rateb

%

No.

c

%

No.c

120

1.2

1/84

47

17/36

Alcohol

39

3.6

1/28

64

7/11

Amine/Amidine

8

0

0/2

17

1/6

Carboxylic acid

21

0

0/14

57

4/7

Ester

17

0

0/14

67

2/3

Ether/Polyether

16

8.3

1/12

0

0/4

Heterocyclic compound

9

0

0/3

33

2/6

Hydrocarbon

23

5.0

1/20

33

1/3

Ketone

8

0

0/8

-

0/0

Onium compound

10

0

0/1

11

1/9

Salt

17

0

0/6

27

3/11

Liquids

89

1.4

1/72

71

12/17

Solids

31

0

0/12

26

5/19

Surfactants – Total

44

4.2

1/24

20

4/20

-nonionic

14

8.3

1/12

0

0/2

-anionic

11

0

0/3

25

2/8

-cationic

7

-

0/0

0

0/7

2

-

0/0

50

1/2

27

0

0/10

41

7/17

-acidic (pH < 7.0)

19

0

0/8

36

4/11

-basic (pH > 7.0)

7

0

0/1

50

3/6

-equals 7

1

0

0/1

-

0/0

90

0

0/66

58

14/24

13

0

0/13

-

0/0

77

0

0/53

58

14/24

OverallChemical CategorydProperties of Interest-ampholyic
pH – Totale

Vapor Pressure – Total
>6kPa
6kPa

Abbreviations: GHS = Globally Harmonized System of Classification and Labelling of Chemicals (UN 2011);
kPa = kilopascals; N = number of substances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.
d
One or more chemical categories were assigned to each test substance based on the chemical categories outlined in thetree structure provided for that chemical in the National Library of Medicine’s Medical Subject Headings (MeSH®) for
inorganic or organic chemicals when available (http://www.nlm.nih.gov/mesh) and on the presence of common organic
functional groups (i.e., ketones) if that functional group was not available in the MeSH tree structure.
e
Total number of substances with pH data available.

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Short Time Exposure Summary Review Document

Table 3-3 shows the STE test method performance in a top-down approach when problematic
categories are excluded that gave the most discordant results in the GHS classification system. In
general, exclusion of alcohols, ethers/polyethers, hydrocarbons, or nonionic surfactants individually
reduced false positive rates to 0% and marginally reduced or slightly increased false negative rates.
The performance of validated in vitro methods for GHS classification is included for comparison.
Table 3-3

STE Performance for GHS Classification in a Top-Down Approach After
Excluding Discordant Categories

Method EvaluatedAccuracy

False Positive Ratea

False Negative Rateb

%

No.c

%

No.c

%

No.c

STE Overall

85

102/120

1.2

1/84

47

17/36

STE w/o Alcohols

87

71/82

0

0/56

42

11/26

STE w/o Ethers/Polyethers

84

87/104

0

0/72

53

17/32

STE w/o Hydrocarbons

84

81/97

0

0/64

49

16/33

STE w/o Nonionicsurfactants

84

89/106

0

0/73

50

17/34

BCOP

79

149/18824

29/123

15

10/65

ICE

83

120/144

8

9/114

50

15/30

CM

90

74/82

2

1/48

21

7/34

Abbreviations: BCOP = bovine corneal opacity and permeability; CM = Cytosensor microphysiometer; GHS = Globally
Harmonized System of Classification and Labelling of Chemicals (UN 2011); ICE = isolated chicken eye; N = number ofsubstances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.

3.2

GHS Classification System: STE Performance in a Bottom-Up Approach

The performance of the STE test method was evaluated for GHS ocular hazard classification in abottom-up approach. STE accuracy, sensitivity, specificity, false positive rate, and false negative rate
were determined based on available in vivo reference data for the test substances. Test substances thatwere identified as direct MTT reducers and classified as STE nonirritants were removed from thebottom-up analysis, as these could be false negative. These include two substances from Kojima et al.
(Kao BRD), two substances from Takahashi et al. (2010), and one substance from the Kao in-housestudies. These analyses were performed for each of the five studies as well as for 129 uniquesubstances from these five studies that remained after duplicates were removed and consensus
classifications were assigned (Table 3-4). The GHS classification for each test substance is listed in
Supplement B.

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Short Time Exposure Summary Review Document

Table 3-4

Data Source

STE Performance for GHS Classification in a Bottom-Up ApproachN

Accuracy

Sensitivity

Specificity

False
Positive
Ratea

False
Negative
Rateb

%

No.c

%

No.c

%

No.c

%

No.c

%

No.c

Kojima et al.
(Kao BRD)

31

71

22/31

73

19/26

60

3/5

40

2/5

27

7/26

Sakaguchi et al.
2011

24

88

21/24

77

10/13

100

11/11

0

0/11

23

3/13

Takahashi et al.
2009

39

87

34/39

85

23/27

92

11/12

8.3

1/12

15

4/27

Takahashi et al.
2010

52

83

43/52

85

28/33

79

15/19

21

4/19

15

5/33

Kao In-House

22

96

21/22

100

1/1

95

20/21

4.8

1/21

0

0/1

Kao New
Surfactants

34

85

29/34

100

22/22

58

7/12

42

5/12

0

0/22

UniqueSubstancesd

129

85

109/129

88

64/73

80

45/56

20

11/56

12

9/73

Abbreviations: BRD = background review document; GHS = Globally Harmonized System of Classification and Labellingof Chemicals (UN 2011); N = number of substances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.
d
Substances from all of the above studies remaining after duplicates were removed and consensus classifications wereassigned.

3.2.1

STE Discordant Results for GHS Classification in a Bottom-Up Approach

The STE results that were discordant with in vivo results were analyzed further. These analyses were
performed on specific categories of chemicals, as well as on certain physicochemical properties
potentially relevant to ocular toxicity testing (e.g., surfactants, pH, physical form).
Several trends were noted in STE performance among these subgroups of substances (Table 3-5).
The overall false positive rate was 20%. The chemical categories of substances that the STE test
method most consistently underpredicted for GHS classification (i.e., false negatives) were salts(13%; 2/15), hydrocarbons (33%; 2/6), and alcohols (16%; 4/25).
With regard to the physical form of the substances underpredicted by the STE test method, four wereliquids and five were solids. Considering the proportion of the total available database, solids (14%;5/37) appear more likely than liquids (4.3%; 4/92) to be underpredicted by the STE test method.

34

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Short Time Exposure Summary Review Document

Table 3-5

STE False Positive and False Negative Rates by Chemical Category andProperties of Interest for GHS Classification in a Bottom-Up Approach

CategoryN

False Positive Ratea

False Negative Rateb

%

No.

c

%

No.c

129

20

11/56

12

9/73

Alcohol

41

31

5/16

16

4/25

Amine/Amidine

17

0

0/2

0

0/15

Carboxylic acid

33

28

3/11

9.1

2/22

Ester

18

46

5/11

14

1/7

Ether/Polyether

16

38

3/8

0

0/8

Heterocyclic compound

10

50

1/2

0

0/8

Hydrocarbon

24

17

3/18

33

2/6

Ketone

8

20

1/5

0

0/3

Onium compound

12

-

0/0

8.3

1/12

Salt

18

67

2/3

13

2/15

Liquids

92

18

9/50

9.5

4/42

Solids

37

33

2/6

16

5/31

Surfactants – Total

49

41

7/17

0

0/32

-nonionic

16

46

5/11

0

0/5

-anionic

12

50

1/2

0

0/10

-cationic

8

-

0/0

0

0/8

33

25

2/8

13

3/24

-acidic (pH < 7.0)

23

17

1/6

18

3/17

-basic (pH > 7.0)

8

0

0/1

0

0/7

-equals 7

1

0

0/1

-

0/0

97

16

7/44

17

9/53

14

14

1/7

43

3/7

83

16

6/37

13

6/46

OverallChemical CategorydProperties of Interest

pH – Totale

Vapor Pressure – Total
>6kPa
6kPa

Abbreviations: GHS = Globally Harmonized System of Classification and Labelling of Chemicals (UN 2011);
kPa = kilopascals; N = number of substances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = The proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.
d
One or more chemical categories were assigned to each test substance based on the chemical categories outlined in thetree structure provided for that chemical in the National Library of Medicine’s Medical Subject Headings (MeSH®) for
inorganic or organic chemicals when available (http://www.nlm.nih.gov/mesh) and on the presence of common organic
functional groups (i.e., ketones) if that functional group was not available in the MeSH tree structure.
e
Total number of substances with pH data available.

Table 3-6 shows the STE test method performance in a bottom-up approach when problematic
categories are excluded that gave the most discordant results in the GHS classification system. In

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Short Time Exposure Summary Review Document

general, exclusion of alcohols, hydrocarbons, salts, or solids individually resulted in small changes inassay performance. However, two applicability domains were evaluated based on excluding certain
chemical and product classes, or physical characteristics. When substances with high vapor pressures,
solid alcohols, hydrocarbons, and salts were excluded, the false negative rate was reduced to 2.0%
(1/49). When substances with high vapor pressures and nonsurfactant solids were excluded, the false
negative rate was reduced to 1.9% (1/54). The single false negative substance using the restricted
applicability domains was toluene. In the NICEATM database, an in vivo study from the European
Centre for Ecotoxicology and Toxicology of Chemicals classifies toluene as GHS Not Classified(ECETOC 1998), whereas a study submitted to the EPA under the Toxic Substances Control Act(TSCA) classifies it as GHS Category 2B (eye irritation data made available by the U.S.
Environmental Protection Agency). These data suggest toluene is a mild ocular irritant and mitigatesconcern about the false negative classification. The performance of validated in vitro methods for
GHS classification is included in Table 3-6 for comparison.
Table 3-6

STE Performance for GHS Classification in a Bottom-Up Approach After
Excluding Discordant Categories

Method EvaluatedFalse Positive Ratea

Accuracy

False Negative Rateb

%

No.c

%

No.c

%

No.c

STE Overall

85

109/129

20

11/56

12

9/73

STE w/o Alcohols

89

78/89

15

6/40

10

5/49

STE w/o Hydrocarbons

86

90/105

21

8/38

10

7/67

STE w/o Salts

86

95/111

17

9/53

12

7/58

STE w/o Solids

86

79/92

18

9/50

9.5

4/42

STE w/o Vapor Pressure >6kPa,
solid alcohols, hydrocarbons,and salts

90

85/94

18

8/45

2.0

1/49

STE w/o Vapor Pressure >6kPa
and Nonsurfactant Solids

90

91/101

19

9/47

1.9

1/54

BCOP

66

125/188

69

63/91

0

0/97

CM

68

36/53

68

17/25

0

0/28

Abbreviations: BCOP = bovine corneal opacity and permeability; CM = Cytosensor microphysiometer; GHS = Globally
Harmonized System of Classification and Labelling of Chemicals (UN 2011); kPa = kilopascals; N = number ofsubstances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.

3.3

EPA Classification System: STE Performance in a Top-Down Approach

The performance of the STE test method was evaluated for EPA ocular hazard classification in a
top-down approach. STE accuracy, sensitivity, specificity, false positive rate, and false negative rate
were determined based on available in vivo reference data for the test substances. Test substances thatwere identified as direct MTT reducers were removed from the analyses. These include two
substances from Kojima et al. (Kao BRD), two substances from Takahashi et al. (2010), one
substance from the Kao in-house studies, and five substances from the combined overall data set.
These analyses were performed for each of the five studies as well as for the overall data set of
120 test substances from all these studies (Table 3-7). The EPA classification for each test substanceis listed in Supplement B.

36

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Short Time Exposure Summary Review Document

Table 3-7

STE Performance for EPA Classification in a Top-Down Approach

Data
SourceN

Kojima etal. (Kao
BRD)

Accuracy

Sensitivity

Specificity

False Positive
Ratea

False Negative
Rateb

%

No.c

%

No.c

%

No.c

%

No.c

%

No.c

30

70

21/30

11

1/9

95

20/21

4.8

1/21

89

8/9

Sakaguchi
et al. 2011

24

92

22/24

67

4/6

100

18/18

0

0/18

33

2/6

Takahashi
et al. 2009

38

82

31/38

61

11/18

100

20/20

0

0/20

39

7/18

Takahashi
et al. 2010

49

82

40/49

58

11/19

97

29/30

3.3

1/30

42

8/19

Kao InHouse

21

100

21/21

-

0/0

100

21/21

0

0/21

-

0/0

Kao New
Surfactants

39

72

28/39

47

9/19

95

19/20

5.0

1/20

53

10/19

Overall120

87104/120

58

19/33

98

85/87

2

2/87

42

14/33

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); N = number of substances; STE = short time
exposure.
aFalse positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.

3.3.1

STE Discordant Results for EPA Classification in a Top-Down Approach

The STE results that were discordant with in vivo results were analyzed further. These analyses were
performed on specific categories of chemicals, as well as on certain physicochemical properties
potentially relevant to ocular toxicity testing (e.g., surfactants, pH, physical form).
Several trends were noted in STE performance among these subgroups of substances (Table 3-8).
Two of 87 substances were overpredicted (i.e., false positives) and affected its representative
chemical classes, with false positive rates ranging from 4.8% to 12.5%. The chemical categories ofsubstances that were most consistently underpredicted for EPA classification (i.e., false negatives)
were alcohols (64%; 7/11) and carboxylic acids (50%; 3/6). Of the 14 underpredicted substances,seven were alcohols, three were carboxylic acids, two were heterocyclic compounds, and two were
salts.
With regard to the physical form of the substances overpredicted by the STE test method, 1.4% (1/74)
were liquids and 7.7% (1/13) were solids. With regard to the physical form of the substances
underpredicted by the STE test method, 10 were liquids and 5 were solids. Considering the proportionof the total available database, solids (16%; 5/32) appear more likely than liquids (11%; 10/89) to beunderpredicted by the STE test method.

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Table 3-8

STE False Positive and False Negative Rates by Chemical Category andProperties of Interest for EPA Classification in a Top-Down Approach

CategoryN

False Positive Ratea

False Negative Rateb

%

No.

c

%

No.c

120

2.3

2/87

42

14/33

Alcohol

40

6.9

2/29

64

7/11

Amine/Amidine

8

0

0/2

17

1/6

Carboxylic acid

20

0

0/14

50

3/6

Ester

16

0

0/15

0

0/1

Ether/Polyether

16

8.3

1/12

0

0/4

Heterocyclic compound

9

0

0/3

33

2/6

Hydrocarbon

24

4.8

1/21

33

1/3

Ketone

8

0

0/8

-

0/0

Onium compound

10

0

0/1

11

1/9

Salt

18

12.5

1/8

20

2/10

Liquids

89

1.4

1/74

67

10/15

Solids

32

7.7

1/13

26

5/19

Surfactants – Total

45

7.7

2/26

16

3/19

-nonionic

14

8.3

1/12

0

0/2

-anionic

12

20

1/5

14

1/7

-cationic

7

-

0/0

0

0/7

-ampholytic

2

-

0/0

50

1/2

e

28

10

1/10

44

8/18

-acidic (pH < 7.0)

20

13

1/8

42

5/12

-basic (pH > 7.0)

7

0

0/1

50

3/6

-equals 7

1

0

0/1

-

0/0

Vapor Pressure – Total

91

1.5

1/68

57

13/23

>6kPa

13

0

0/12

100

1/1

6kPa

78

1.8

1/56

55

12/22

OverallChemical CategorydProperties of Interest

pH – Total

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); kPa = kilopascals; N = number of substances;
STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.
d
One or more chemical categories were assigned to each test substance based on the chemical categories outlined in thetree structure provided for that chemical in the National Library of Medicine’s Medical Subject Headings (MeSH®) for
inorganic or organic chemicals when available (http://www.nlm.nih.gov/mesh) and on the presence of common organic
functional groups (i.e., ketones) if that functional group was not available in the MeSH tree structure.
e
Total number of substances with pH data available.

Table 3-9 shows the STE test method in a top-down approach when problematic categories are
excluded that gave the most discordant results for the EPA classification system. Exclusion of38

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Short Time Exposure Summary Review Document

alcohols reduced the false positive rate from 2.3% (2/87) to 0% (0/58) with only a slight reduction in
the false negative rate (42%, 14/33 to 38%, 9/24). In general, removal of other individual chemicalclasses with false positive rates produced higher false negative rates.
Table 3-9

STE Performance for EPA Classification in a Top-Down Approach After
Excluding Discordant Categories
False Positive Ratea

Accuracy

Method EvaluatedFalse Negative Rateb

%

No.c

%

No.c

%

No.c

STE Overall

87

104/120

2.3

2/87

42

14/33

STE w/o Alcohols

89

73/82

0

0/58

38

9/24

STE w/o Ethers/Polyethers

85

89/105

1.3

1/75

50

15/30

STE w/o Hydrocarbons

85

82/97

1.5

1/66

45

14/31

STE w/o Salts

86

89/103

1.3

1/79

54

13/24

STE w/o Solids

88

78/89

1.4

1/74

67

10/15

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.

3.4

EPA Classification System: STE Performance in a Bottom-Up Approach

The performance of the STE test method was evaluated for EPA ocular hazard classification in abottom-up approach to identify EPA Category IV substances (i.e., minimal effects clearing in lessthan 24 hours). STE accuracy, sensitivity, specificity, false positive rate, and false negative rate were
determined based on available in vivo reference data for the test substances. Test substances that wereidentified as direct MTT reducers and classified as STE nonirritants were removed from bottom-up
analysis, as these could be false negative. These include two substances from Kojima et al. (KaoBRD) and two substances from Takahashi et al. (2010). These analyses were performed for each of
the five studies as well as for the overall data set of 129 test substances from all these studies(Table 3-10). The EPA classification for each test substance is listed in Supplement B.
Table 3-10

Data Source

STE Performance for EPA Classification in a Bottom-Up ApproachN

Accuracy

Sensitivity

Specificity

False
Positive
Ratea

False Negative
Rateb

%

No.c

%

No.c

%

No.c

%

No.c

%

No.c

Kojima et al.
(Kao BRD)

31

77

24/31

75

21/28

100

3/3

0

0/3

25

7/28

Sakaguchi et al.
2011

24

67

16/24

56

10/18

100

6/6

0

0/6

44

8/18

Takahashi et al.
2009

39

80

31/39

75

24/32

100

7/7

0

0/7

25

8/32

Takahashi et al.
2010

52

77

40/52

74

31/42

90

9/10

10

1/10

26

11/42

Kao In-House

22

68

15/22

22

2/9

100

13/13

0

0/13

78

7/9

39

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Short Time Exposure Summary Review Document

Kao New
Surfactants

34

94

32/34

96

26/27

86

6/7

14

1/7

3.7

1/27

Overall

12980103/129

75

73/97

94

30/32

6.3

2/32

25

24/97

Abbreviations: BRD = background review document; EPA = U.S. Environmental Protection Agency (EPA 2012);
N = number of substances; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.

3.4.1

STE Discordant Results for EPA Classification in a Bottom-Up Approach

The STE results that were discordant with in vivo results were analyzed further. These analyses were
performed on specific categories of chemicals, as well as on certain physicochemical properties
potentially relevant to ocular toxicity testing (e.g., surfactants, pH, physical form).
Several trends were noted in STE performance among these subgroups of substances (Table 3-11).
Two substances were overpredicted. The overall false positive rate was 6.3% (2/32), with alcohols(20% 2/10), esters (33%; 2/6), and heterocyclic compounds (50%; 1/2) overpredicted. The chemical
categories of substances that the STE test method most consistently underpredicted for EPA
classification (i.e., false negatives) were hydrocarbons, ketones, and esters. Of the 24 underpredicted
substances, seven were alcohols, five were carboxylic acids, and six were hydrocarbons. Additional
chemical categories represented among the underpredicted substances were amines/amidines (2),esters (3), ethers/polyethers (1), ketones (3), onium compounds (1), and salts (2).
With regard to the physical form of the substances in a bottom-up approach, two liquids were
overpredicted (6.9%; 2/29), which was 2.2% (2/92) of the entire database. With regard to the physicalform of the substances underpredicted by the STE test method, 18 were liquids and six were solids.
Considering the proportion of the total available database, liquids (20%; 18/92) appear more likely
than solids (16%; 6/37) to be underpredicted by the STE test method.
Table 3-11

STE False Positive and False Negative Rates by Chemical Category andProperties of Interest for EPA Classification in a Bottom-Up Approach

CategoryN

False Positive Ratea
%

No.c

%

No.c

129

6.3

2/32

25

24/97

Alcohol

41

20

2/10

23

7/31

Amine/Amidine

11

-

0/0

18

2/11

Carboxylic acid

33

0

0/5

18

5/28

Ester

18

33

2/6

25

3/12

Ether/Polyether

16

0

0/4

8.3

1/12

Heterocyclic compound

10

50

1/2

0

0/8

Hydrocarbon

24

0

0/11

46

6/13

Ketone

8

-

0/0

38

3/8

Onium compound

12

-

0/0

8.3

1/12

Salt

18

0

0/1

12

2/17

OverallChemical Category

d

40

84

False Negative Rateb

Short Time Exposure Summary Review Document

Properties of Interest
Liquids

92

6.9

2/29

29

18/63

Solids

37

0

0/3

18

6/34

Surfactants – Total

49

18

2/11

2.6

1/38

-nonionic

16

25

2/8

0

0/8

-anionic

12

0

0/1

0

0/11

8

-

0/0

0

0/8

32

17

1/6

15

4/26

-acidic (pH < 7.0)

23

17

1/6

18

3/17

-basic (pH > 7.0)

8

-

0/0

12

1/8

-equals 7

1

-

0/0

100

1/1

Vapor Pressure – Total

97

8.0

2/25

32

23/72

>6kPa

14

0

0/4

50-

5/10

<6kPa

83

9.5

2/21

29

18/62

-cationic
pH – Totale

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); kPa = kilopascals; N = number of substances;
STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.
d
One or more chemical categories were assigned to each test substance based on the chemical categories outlined in thetree structure provided for that chemical in the National Library of Medicine’s Medical Subject Headings (MeSH®) for
inorganic or organic chemicals when available (http://www.nlm.nih.gov/mesh) and on the presence of common organic
functional groups (i.e., ketones) if that functional group was not available in the MeSH tree structure.
e
Total number of substances with pH data available.

Table 3-12 shows the STE test method performance in a bottom-up approach when problematic
categories are excluded that gave the most discordant results in the EPA classification system. In
general, exclusion of alcohols, hydrocarbons, salts, or solids individually resulted in small changes inassay performance. However, two applicability domains were evaluated based on excluding certain
chemical and product classes, or physical characteristics. When substances with high vapor pressures,
solid alcohols, hydrocarbons, and salts were excluded, the false negative rate was slightly reduced to21% (14/68). When substances with high vapor pressures, and nonsurfactant solids were excluded,
the false negative rate was slightly reduced to 18% (13/73).

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Short Time Exposure Summary Review Document

Table 3-12

STE Performance for EPA Classification in a Bottom-Up Approach After
Excluding Discordant Categories

Method EvaluatedFalse Positive Ratea

Accuracy
c

%

No.

c

False Negative Rateb
%

No.c

%

No.

STE Overall

80

103/129

6.3

2/32

25

24/97

STE w/o Alcohols

89

72/89

0

0/22

25

17/67

STE w/o Hydrocarbons

81

85/105

9.5

2/21

21

18/84

STE w/o Solids

78

72/92

6.9

2/29

29

18/63

STE w/o Salts

78

87/111

6.5

2/31

28

22/80

STE w/o Vapor Pressure >6kPa,
solid alcohols, hydrocarbons, and
salts

83

78/94

7.7

2/26

21

14/68

STE w/o Vapor Pressure >6kPa
and Nonsurfactant Solids

85

86/101

7.1

2/28

18

13/73

Abbreviations: EPA = U.S. Environmental Protection Agency (EPA 2012); kPa = kilopascals; STE = short time exposure.
a
False positive rate = the proportion of all negative substances that are falsely identified as positive in vitro.
b
False negative rate = the proportion of all positive substances that are falsely identified as negative in vitro.
c
Data used to calculate the percentage.

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Short Time Exposure Summary Review Document

4.0

STE Test Method Reliability

Test method reliability (intralaboratory repeatability and intra- and interlaboratory reproducibility) isan essential element of an evaluation of assay performance (ICCVAM 2003). Repeatability refers to
the closeness of agreement between test results obtained within a single laboratory when theprocedure is performed on the same substance under identical conditions within a given time period(ICCVAM 1997, 2003). Intralaboratory reproducibility refers to the extent to which qualified
personnel within the same laboratory can replicate results using a specific test protocol at different
times. Interlaboratory reproducibility refers to the extent to which different laboratories can replicate
results using the same protocol and test chemicals and indicates the extent to which a test method canbe transferred successfully among laboratories. A reliability assessment includes (1) reviewing therationale for selecting the substances used to evaluate test method reliability, (2) discussing the extent
to which the substances tested represent the range of possible test outcomes and the properties of thevarious substances for which the test method is proposed for use, and (3) performing a quantitative
and/or qualitative analysis of repeatability and intra- and interlaboratory reproducibility.
Background information, data, and the performance (i.e., accuracy and reliability) analyses of the
STE test method conducted by Kao Corporation are provided in the BRD (Supplement A). STE test
data were available for replicates within individual experiments repeated three times for each test
substance in two to five different laboratories. Coefficient of variation (CV) analyses were performedon within-experiment and between-laboratory STE data, using the cell viability value obtained foreach test substance within each of the two to five testing laboratories.
The %CV values for intralaboratory reliability for substances classified as nonirritants ranged from
0.3% to 23.5% in the four studies evaluated. Substances classified in vitro as irritants tended to havegreater %CV values, as expected, because the cell viability for these chemicals was often quite low.
Further, the mean viability for the positive control, 0.01% sodium lauryl sulfate, was 41.7% (N = 71)
with %CV of 24.7%.
In terms of interlaboratory agreement, the laboratories recorded 100% agreement for 83% to 100% of
the substances for GHS classification and 87% to 100% of the substances for EPA classification.

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5.0

Peer Review Summary

To ensure the completeness of the NICEATM STE performance review, NTP provided the STE
Summary Review Document along with the original Kao BRD and other supporting documentation
to four external scientific reviewers who were asked to:xComment on whether the protocol is complete and adequatexComment on the adequacy of the database used for evaluating STEx

Provide any additional published STE studies or datax

Comment on the adequacy of the test method reliabilityxComment on the adequacy of the performance evaluationx

Provide any additional comments on the protocol or analysisx

Provide comments for regulatory agencies considering using data from this test method

The reviewers commented that the evaluation of the STE test method performance was thoroughly
conducted. The reviewers remarked that the evaluation not only examined performance of the test
method based upon the entire set of chemicals but also had a secondary assessment of STE
performance with select chemical classes removed from the applicability domain. Given thethoroughness of the review, the reviewers stated that no other analysis is necessary. As with theperformance analysis, the reviewers commented that the reliability analysis was thorough with noneed for additional analysis.
The reviewers commented that the STE database was adequate for its intended purpose and addedthat they were not aware of additional STE data that could be used in this evaluation. The reviewers
did however suggest that the STE database would require further development if the test method were
to be used in the evaluation of pesticides.
The reviewers made a number of comments directed towards regulatory agencies considering using
data obtained from the STE method:
When compared to other in vitro or in vivo assays currently available for eye irritation assessment,
STE has a number of advantages, including time and cost required to do the assay, the use of a cellline rather than ex vivo tissue, its ability to assess poorly water-soluble substances, a low false
positive rate, and protocol simplicity
The analysis highlights that the performance of a test method is dependent upon the classification
system to which it is being compared. Specifically, STE “false negatives”, after all poorly compatible
substances are excluded, are EPA Cat III: mild irritants.
Acceptance and use of data from the STE method is suggested by the developers as part of an in vitro/ ex vivo battery of tests designed to offer an alternative to the in vivo OECD Test Guideline 405
Acute Eye Irritation/Corrosion assay in rabbits. As such, results from the STE when considered alone
would most often be seen as “screening data” and best interpreted as part of a systematic evaluationof hazard.
There are likely to be circumstances in which the predictive nature of the STE assay is unknown (i.e.,
new chemical domains, mixtures, etc.). Submission of useful STE data relies on careful and44

88Short Time Exposure Summary Review Document

consistent conduct of the assay and verification of the performance of the STE method when extended
beyond the currently available chemical domain or space.
At present, there is no in vitro alternative test to definitively and accurately distinguish non-irritantand irritant chemicals. In a bottom-up approach aimed at identifying non-irritants, it is important to
reduce the false negative substances as much as possible. The false negative rate of BCOP and ICE
are 0%, but their false positive rates are high, 68 to 69%. While the false negative rate in STE is not
0%, when either applicability domain is adopted, the rate is 1.9 to 2%. Test systems that obtain highaccuracy, low false positive rate, and low false negative rate are desirable and based upon its
performance, STE is suitable for use in a bottom-up approach.
As a result of the reviewers comments, minor edits were made to the NICEATM SRD including
updating OECD TG references and clarifying the evaluation of direct MTT reducers. The reviewers
also had a number of comments and suggestions for the STE protocol that were compiled andprovided to Kao Corporation.

STE Peer Reviewers
Michael Joseph Olson
Director, Occupational Toxicology, Hazard Assessment and Communication, Corporate Environment
Health and Safety, GlaxoSmithKline
Research Triangle Park, NC
Seung Hyeok Seok
Assistant Professor, Department of Microbiology and Immunology, Institute for Experimental
Animals, College of Medicine, Seoul National University
Seoul, Korea
Kristie Sullivan
Director, Regulatory Testing Issues, Physicians Committee for Responsible Medicine
Washington, D.C.
Kohji Yamakage
Chief of Division of alternative toxicology testing, Hatano Research Institute, Food Drug Safety
CenterKanagawa, Japan

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6.0

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Draize J, Woodard G, Calvery H. 1944. Methods for the study of irritation and toxicology of
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EPA. 2012. Label Review Manual [Internet]. Available: http://www.epa.gov/oppfead1/labeling/lrm/;
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