新規試験法評価書

SHE細胞形質転換試験法（SHE CTA）

平成30年 3 月

国立医薬品食品衛生研究所

᪂つヨ㦂ἲホ౯᭩             ᖹᡂ 30 ᖺ 3 ᭶ 30 ᪥                                 No. 2017-05

SHE⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧࡟㛵ࡍࡿホ౯ ᖹᡂ 30 ᖺ 2 ᭶ 21 ᪥䛻ᕝᓮࠊᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ࡟࡚㛤ദࡉࢀࡓ᪂つヨ㦂ἲホ౯఍㆟㸦㏻⛠㸸JaCVAM ホ౯఍㆟㸧࡟࠾࠸࡚௨ୗࡢホ౯ࡀ࡞ࡉࢀࡓࠋ  ホ౯ෆᐜ㸸 SHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ
⿕㦂≀㉁ࢆࡀࢇཎᛶ≀㉁࡜ุᐃࡍࡿࡇ࡜ࡣ⛉Ꮫⓗ࡟ࡶࠊࡲࡓ⾜ᨻⓗ࡟ࡶᅔ㞴࡛࠶ࡾࠊࡋࡓࡀࡗ࡚
ᮏヨ㦂ࢆࡀࢇཎᛶヨ㦂࡟⨨ࡁ᥮࠼ࡿࡇ࡜ࡣ⌧≧࡛ࡣ୙㐺ษ࡛࠶ࡿࠋᮏヨ㦂ࡢ⾜ᨻୖࡢ฼⏝࡟࠶ࡓࡗ࡚ࡣࠊᵓ㐀άᛶ┦㛵࡞࡝࡜⤌ࡳྜࢃࡏ࡚⿵ຓⓗ࡞ヨ㦂ࠊ౛࠼ࡤ WoE ࡟ᇶ࡙ࡃホ౯ࡢ㝿ࡢ୍ࡘࡢ㡯┠ࠊ࠶ࡿ࠸ࡣࡀࢇཎᛶヨ㦂ᐇ᪋ࡢඃඛ㡰఩௜ࡅࡢࡓࡵࡢࢫࢡ࣮ࣜࢽࣥࢢヨ㦂࡜ࡋ࡚఩⨨௜ࡅࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋࡇࡢホ౯᭩ࡣࠊOrganisation for Economic Co-operation and Development (OECD) GuidanceDocument on the In Vitro Syrian Hamster Embryo (SHE) Cell Transformation Assay, Series on
Testing & Assessment No 214➼ࢆࡶ࡜࡟ࠊᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕSHE⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧ホ౯ሗ࿌᭩ࠖࢆ⏝࠸࡚ࠊJaCVAMホ౯఍㆟ࡀホ౯࠾ࡼࡧ᳨
ウࡋࡓ⤖ᯝࠊࡑࡢ఩⨨௜ࡅࡀ☜ㄆࡉࢀࡓࡇ࡜࠿ࡽసᡂࡉࢀࡓࠋ
኱㔝Ὀ㞝                      すᕝ⛅ె኱㔝Ὀ㞝JaCVAM ホ౯఍㆟ ㆟㛗               JaCVAM 㐠Ⴀጤဨ఍ ጤဨ㛗

i

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 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㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧㔝 ཱྀ ┿ ᕼ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧**᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵ච␿࢔ࣞࣝࢠ࣮Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥*㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 29 ᖺ 3 ᭶ 31 ᪥**㸸ᖹᡂ 29 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥

                 
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JaCVAM 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኱ ཎ  ᣅ㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ㸧ᑠᕝஂ⨾Ꮚ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⑓⌮㒊㸧ຍ ⸨  ⠜㸦ᅜ❧ឤᰁ⑕◊✲ᡤ㸧ㅋ ⏣ Ὀ ᡂ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ⸆⌮㒊㸧ᑠụ⣫୍㑻㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧⠛ ⏣ ࿴ ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧㧗 ᮌ ⠜ ஓ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊 ື≀⟶⌮ᐊ㸧᮰ 㔝 ṇ ᫂㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᖹ ᯘ ᐜ Ꮚ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ẘᛶ㒊㸧ᗈ ℩ ᫂ ᙪ㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶண ホ౯㒊㸧ᘅ ⏣ ග ᜨ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ῲ ᒸ  Ꮫ㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ ໬Ꮫ≀㉁Ᏻ඲ᑐ⟇ᐊ㸧ᮏ 㛫 ṇ ඘ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ ኚ␗㑇ఏ㒊㸧Ώ 㑔 ఙ ୍㸦ཌ⏕ປാ┬ ་⸆࣭⏕ά⾨⏕ᒁ ་⸆ရᑂᰝ⟶⌮ㄢ㸧ᑠ ᓥ  ⫕ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮ Ᏻ඲ᛶணホ౯㒊➨஧ᐊ㸧㸸஦ົᒁ

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JaCVAM Statement on
the Syrian Hamster Embryo Cell Transformation Assay

At a meeting held on 21 February 2018 at the National Institute of Health Sciences (NIHS) in
Kawasaki, Japan, the Japanese Center for the Validation of Alternative Methods (JaCVAM)
Regulatory Acceptance Board unanimously endorsed the following statement:
Proposal: We do not consider the present Syrian Hamster Embryo Cell Transformation Assay (SHE
CTA) to be suitable as a replacement for established carcinogenicity tests, nor is it
suitable for predicting the carcinogenicity of a test chemical, either in a scientific or aregulatory context, even when positive test results are obtained. The SHE CTA can only
be considered for regulatory use as a supplementary test in combination with structureactivity relationships, such as one item to be considered in a weight-of-evidence
approach to evaluation or part of screening to determine prioritization for the
implementation of carcinogenicity tests.
This statement was prepared following a review of the Guidance Document on the In Vitro Syrian
Hamster Embryo (SHE) Cell Transformation Assay, Series on Testing & Assessment No. 214
together with other materials prepared by the Cell Transformation Testing JaCVAM Editorial
Committee to acknowledge that the results of a review and study by the JaCVAM Regulatory
Acceptance Board have failed to confirm the usefulness of this assay.

Yasuo Ohno

Akiyoshi Nishikawa

Chairperson

Chairperson

JaCVAM Regulatory Acceptance BoardJaCVAM Steering Committee

30 March 2018v

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 (Kihara Memorial Yokohama Foundation for the Advancement of Life
Sciences) : Chairperson
Mr. Naofumi Iizuka (Pharmaceuticals and Medical Devices Agency)*
Mr. Yoshiaki Ikarashi (National Institute of Health Sciences: NIHS)
Mr. Noriyasu Imai (Japanese Society for Alternatives to Animal Experiments)
Mr. Tomoaki Inoue (Japanese Society of Immunotoxicology)
Mr. Yuji Ishii (Biological Safety Research Center: BSRC, NIHS)
Ms. Yumiko Iwase (Japan Pharmaceutical Manufacturers Association)
Mr. Takeshi Morita (Japanese Environmental Mutagen Society)
Mr. Shunji Nakai (Japan Chemical Industry Association)
Ms. Ruriko Nakamura (National Institute of Technology and Evaluation)
Mr. Akiyoshi Nishikawa (BSRC, NIHS)
Ms. Maki Noguchi (Pharmaceuticals and Medical Devices Agency)**
Mr. Satoshi Numazawa (Japanese Society of Toxicology)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Ms. Mariko Sugiyama (Japan Cosmetic Industry Association)
Mr. Hiroo Yokozeki (Japanese Society for Cutaneous Immunology and Allergy)
Term: From 1st April 2016 to 31st March 2018*: From 1st April 2016 to 31st March 2017**: From 1st April 2017 to 31st March 2018vi

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. Manabu Fuchioka (Ministry of Health, Labour and Welfare)
Ms. Yoko Hirabayashi (Division of Toxicology, BSRC, NIHS)
Mr. Akihiko Hirose (Division of Risk Assessment, BSRC, NIHS)
Ms. Mitsue Hirota (Pharmaceutical & Medical Devices Agency)
Mr. Masamitsu Honma (Division of Genetics and Mutagenesis, BSRC, NIHS)
Mr. Yasunari Kanda (Division of Pharmacology, BSRC, NIHS)
Mr. Atsushi Kato (National Institute of Infectious Diseases)
Mr. Kouichirou Koike (Ministry of Health, Labour and Welfare)
Ms. Kumiko Ogawa (Division of Pathology, BSRC, NIHS)
Mr. Taku Oohara (Ministry of Health, Labour and Welfare)
Mr. Kazutoshi Shinoda (Pharmaceuticals and Medical Devices Agency)
Mr. Atsuya Takagi (Animal Management Section of the Division of Toxicology, BSRC,
NIHS)
Mr. Masaaki Tsukano (Ministry of Health, Labour and Welfare)
Mr. Shinichi Watanabe (Ministry of Health, Labour and Welfare)Mr. Hajime Kojima (Division of Risk Assessment, BSRC, NIHS): Secretary

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SHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧┠  ḟ
ホ౯఍㆟ሗ࿌᭩ ------------------------------------------------------------------------------------ 1
ホ౯ሗ࿌᭩ ------------------------------------------------------------------------------------------ 7
GUIDANCE DOCUMENT ON THE IN VITRO SYRIAN HAMSTER EMBRYO (SHE)
CELL TRANSFORMATION ASSAY ------------------------------------------------------------33ix

xホ౯఍㆟ሗ࿌᭩

SHE⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧JaCVAM ホ౯఍㆟

ᖹᡂ 30 ᖺ㸦2018 ᖺ㸧2 ᭶ 21 ᪥

1

1

JaCVAM ホ౯఍㆟኱ 㔝 Ὀ 㞝 㸦බ┈㈈ᅋἲே ᮌཎグᛕᶓ὾⏕࿨⛉Ꮫ᣺⯆㈈ᅋ㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 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㸧஭ ୖ ᬛ ᙲ 㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒ ஭ ᩍ Ᏻ 㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ 㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛ ⏣ ࿴ ಇ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ 㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰ ஭ ಇ ྖ 㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ 㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧す ᕝ ⛅ ె 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟ ⃝  ⪽ 㸦᪥ᮏẘᛶᏛ఍㸧㔝 ཱྀ ┿ ᕼ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧**᳃ ⏣  ೺ 㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ 㛵 ༤ 㞝 㸦᪥ᮏ⓶⭵ච␿࢔ࣞࣝࢠ࣮Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥*㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 29 ᖺ 3 ᭶ 31 ᪥**㸸ᖹᡂ 29 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥

           22⣽⬊ᙧ㉁㌿᥮ヨ㦂㸦Cell Transformation Assay㸸CTA㸧ࡣࠊᇵ㣴⣽⬊࡟໬Ꮫ≀㉁ࢆ᭚㟢ࡍࡿࡇ࡜࡛㉳ࡁࡿࠕ⣽⬊ࡢᙧែࠖࡸࠕ⾲⌧ᙧ㉁ࠖࡢኚ໬ࢆᣦᶆ࡜ࡋ࡚⭘⒆ㄏⓎᛶࡢ᭷↓ࢆㄪ࡭ࡿヨ㦂ἲࡢ୍ࡘ࡛࠶ࡿࠋ
ࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⫾ඣ㸦Syrian Hamster Embryo㸸SHE㸧⣽⬊ࢆ⏝࠸ࡿ CTA ࡛ࡣࠊึ௦ᇵ㣴⣽⬊ࢆ෾⤖࣭ゎ෾ࡋࡓṇᖖ஧ಸయ⣽⬊ࢆ⏝࠸࡚ࠊ┠ⓗ࡜ࡍࡿ໬Ꮫ≀㉁ࢆ᭚㟢ࡋࡓᚋࠊᙧែᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢ
ฟ⌧⋡࡟ࡼࡗ࡚ࠊ໬Ꮫ≀㉁ࡢᙧ㉁㌿᥮⬟ࡢ᭷↓ࢆホ౯ࡍࡿࠋSHE CTA ࡟ࡘ࠸࡚ࡣࠊOECD ࡟ࡼࡾ Detailed Review Paper 31㸦DRP31㸧࡛ 245 ࡢ໬Ꮫ≀㉁ࡀホ౯ࡉࢀ࡚࠸ࡿ 1)ࠋEuropean Union Reference Laboratory for Alternatives to Animal Testing㸦EURL ECVAM㸧࡟ࡼࡿࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀ 2-3)ࠊEURL ECVAM Scientific Advisory Committee㸦ESAC㸧࡟ࡼࡿ➨
3 ⪅ホ౯ࡀ⾜ࢃࢀ 4)ࠊ࢞࢖ࢻࣛ࢖ࣥ໬ࡀ᥎ዡࡉࢀࡓ 5)ࠋࡑࡢᚋࠊSHE CTA ᑓ㛛ᐙ఍㆟࡟ࡼࡿ෌ホ౯ࡀ⾜ࢃࢀ 6-7)ࠊ2015 ᖺ 5 ᭶࡟ OECD ࢞࢖ࢲࣥࢫ࡜ࡋ࡚බ⾲ࡉࢀ࡚࠸ࡿ 8)ࠋJaCVAM ホ౯఍㆟ࡣࠊJaCVAM ᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕSHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧ホ౯ሗ࿌᭩ࠖ9)ࢆ
⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 SHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 ࡆࡗṑ㢮ࢆ⏝࠸ࡿࡀࢇཎᛶヨ㦂
ヨ㦂ἲࡢᴫ␎㸸 ᮏヨ㦂ἲ࡛ࡣࠊ⫾⏕ 13 ᪥┠ࡢࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮࠿ࡽㄪ〇ࡋࡓึ௦ᇵ㣴⣽⬊࠿ࡽస〇ࡋࡓ 2 ௦┠ࡢ⣽⬊㸦ṇᖖ஧ಸయ䠅ࢆ⏝࠸ࡿࠋ୍㒊ࡣ X ⥺↷ᑕ࡟ࡼࡾቑṪ⬟ࢆ୙ά໬ࡉࡏࡓࣇ࢕࣮
ࢲ࣮⣽⬊㸦ᨭᣢ⣽⬊㸧࡜ࡋ࡚ࠊ௚ࡣᙧ㉁㌿᥮⬟ࢆㄪ࡭ࡿᶆⓗ⣽⬊࡜ࡋ࡚౑⏝ࡍࡿࠋࣇ࢕࣮ࢲ࣮⣽⬊ୖ࡟ᶆⓗ⣽⬊ࢆ᧛✀ࡋࡓᚋࠊ⿕㦂≀㉁ࢆ 7 ᪥㛫᭚㟢ࡋࡓᚋࠊ⣽⬊ࢆὙί࣭ᅛᐃࡋ࡚ᰁⰍࡋࠊᐇయ㢧ᚤ㙾ୗ࡛ࢥࣟࢽ࣮ࡢᙧែࢆㄪ࡭ࡿࠋ⣽⬊ẘᛶࡣࠊࢥࣟࢽ࣮ᙧᡂຠ⋡࡜ࢥࣟࢽ࣮ࡢ኱ࡁࡉࡸ⣽⬊ᐦᗘࡢῶᑡ࠿ࡽホ౯ࡍࡿࠋᙧ㉁㌿᥮⬟ࡣࠊ⿕㦂≀㉁᭚㟢⩌࡜⁐፹ᑐ↷⩌࡟ࡘ࠸࡚ࠊࡑࢀࡒࢀࢥࣟࢽ࣮ࡢ⥲ᩘ࡟ᑐࡍࡿᙧែᙧ㉁㌿᥮ࢥࣟࢽ࣮ᩘࡢ๭ྜࢆ⟬ฟࡋ࡚ẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾホ౯ࡍࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶᮏヨ㦂ἲࡣࠊEURL ECVAM ࡟ࡼࡿ 6 ✀㢮ࡢ໬Ꮫ≀㉁ࢆ⏝࠸ࡓࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 2-3)࡜ࡑࢀ࡟⥆ࡃ ESAC ࡟ࡼࡿ➨୕⪅ホ౯࡟ࡼࡾࠊ့ங㢮ࢆ⏝࠸ࡓࡀࢇཎᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚ホ౯ࡉࢀ࡚࠸ࡿ 4)ࠋ
OECD ࢞࢖ࢲࣥࢫ 34 ࡟ᇶ࡙࠸ࡓࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡣᐇ᪋ࡉࢀ࡚࠸࡞࠸ࡀࠊ2015 ᖺ 5 ᭶࡟බ⾲ࡉࢀࡓ
OECD ࢞࢖ࢲࣥࢫ 8)࡛ࡣࠊࡀࢇཎᛶࢆホ౯ࡍࡿヨ㦂ᡓ␎ࡢ୍⎔࡜ࡋ࡚ࠊ࠶ࡿ࠸ࡣࡀࢇཎᛶ࡟࠿࠿ࡿドᣐࡢ㔜ࡳ࡙ࡅ㸦Weight of Evidence㸸WoE㸧࡟ᮏヨ㦂ࡢ⤖ᯝࢆ฼⏝ࡍࡿࡇ࡜ࡣ᭷⏝࡛࠶ࡿ࡜ࡉࢀ࡚࠸ࡿࠋJaCVAM ᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡛ࡣࠊ⌧ᅾࡲ࡛බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗ 1-8)ࢆᇶ࡟ᮏヨ㦂ἲࡢࡀࢇཎ
ᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ホ౯ࡋࡓࠋSHE ⣽⬊ࡢᙧែᙧ㉁㌿᥮ࡣࠊከẁ㝵Ⓨࡀࢇ㐣⛬ࡢ᪩ᮇ࡟㉳ࡁࡿ⌧㇟࡟┦ᙜࡋ࡚࠾ࡾࠊᙧ㉁㌿᥮⣽⬊ࡣࠊ⥅௦ᇵ㣴ᚋࠊྠ⣔⤫ࡢື≀ࡸࢾ࣮ࢻ࣐࢘ࢫ࡟ᑐ
ࡋ࡚⣙ 50㹼100㸣ࡢ๭ྜ࡛㐀⭘⒆ᛶࢆ♧ࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿ 10-11)ࠋDRP31 ࡟ࡣࠊ」ᩘࡢ pH ࡢᇵ㣴ᾮ33㸦౛࠼ࡤࠊpH 6.7ࠊpH 7.0 ࠶ࡿ࠸ࡣࡑࢀ௨ୖ㸧࡛ヨ㦂ࡉࢀࡓᩘከࡃࡢ໬Ꮫ≀㉁ࡢ SHE CTA ⤖ᯝࡀࡲ࡜ࡵ
ࡽࢀࠊ㑇ఏẘᛶࡀࢇཎ≀㉁࠾ࡼࡧ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᭚㟢࡟ࡼࡾࠊSHE ⣽⬊ࡣᙧែᙧ㉁㌿᥮ࡍࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠋEURL ECVAM ࡣࠊᶆ‽ࣉࣟࢺࢥࣝࠊᢏ⾡⛣㌿ᛶࠊ࠾ࡼࡧ᪋タෆ෌⌧ᛶ୪ࡧ࡟᪋タ㛫෌⌧ᛶࢆホ౯ࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࢆㄆࡵ࡚࠸ࡿࠋࡲࡓࠊEURL ECVAM ࡢᶆ‽ⓗࣉࣟࢺࢥࣝ࡜ DRP31
࡟♧ࡉࢀࡓࣉࣟࢺࢥࣝ࡜ࡢ㛫࡟ࡣᇶᮏⓗ࡞ᕪ␗ࡣ࡞ࡃࠊᅇ㢳ⓗホ౯࡟࠾࠸࡚ࠊ࠸ࡎࢀࡢࣉࣟࢺࢥࣝ࡟ࡼ
ࡾᚓࡽࢀࡓࢹ࣮ࢱࡶࠊཷࡅධࢀྍ⬟࡛࠶ࡿ࡜ࡉࢀࡓࠋ
⣽⬊ࡢᙧ㉁㌿᥮ࡢṇ☜࡞ศᏊ⏕≀Ꮫⓗ࣓࢝ࢽࢬ࣒ࡣ༑ศ࡟ࡣゎ᫂ࡉࢀ࡚࠾ࡽࡎࠊSHE ⣽⬊ࡢᙧ㉁㌿᥮࣓࢝ࢽࢬ࣒࡟ࡶ୙᫂࡞Ⅼࡀከ࠸ࡀࠊࡑࡢ࣓࢝ࢽࢬ࣒ࢆゎᯒࡍࡿㄽᩥࡣᩘከࡃ࠶ࡿࠋ⣽⬊࿘ᮇไᚚࢩࢫࢸ
࣒ࠊࢤࣀ࣒Ᏻᐃᛶࠊ࠾ࡼࡧศ໬࣭ቑṪ㐣⛬࡛ࡢ㑇ఏᏊⓎ⌧ࡢኚ໬ࡸ␗ᖖࡀᙧ㉁㌿᥮ࡢ୺ࡓࡿせᅉ࡜ࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽࡢ㐣⛬࡟ᙳ㡪ࢆཬࡰࡍ㑇ఏⓗኚ␗ࡣ┤᥋ⓗ࡞㑇ఏẘᛶస⏝࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ
ࡉࡽ࡟ࠊDNA ࡢ㧗࣓ࢳࣝ໬࠶ࡿ࠸ࡣప࣓ࢳࣝ໬ࠊࣄࢫࢺࣥಟ㣭࠾ࡼࡧࢾࢡࣞ࢜ࢯ࣮࣒ࣜࣔࢹࣜࣥࢢ࡟
ࡼࡿ㑇ఏᏊⓎ⌧ࡢኚ໬ࡸ㑇ఏⓗ୙Ᏻᐃᛶࡀࠊ࢚ࣆࢪ࢙ࢿࢸࢵࢡ࡞ኚ໬ࢆㄏ㉳ࡋࠊᝏᛶᙧ㉁㌿᥮࡬࡜㐍⾜ࡉࡏࡿᘬࡁ㔠࡟࡞ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡶࡲࡓࠊከẁ㝵Ⓨࡀࢇ㐣⛬࡟㛵୚ࡋࠊṇᖖ⣽⬊ࢆᙧ㉁㌿᥮ࡉࡏࡿࡶࡢ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡍ࡞ࢃࡕࠊࢠࣕࢵࣉࢪࣕࣥࢡࢩࣙࣥ࡟ࡼࡿ⣽⬊㛫ࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥࢆ㜼ᐖࡍࡿ㓟໬ⓗࢫࢺࣞࢫࠊ⣽⬊ศ⿣ᅇᩘࡢቑຍࠊ࢔࣏ࢺ࣮ࢩࢫࡢῶᑡࠊࢳࣗࣈࣜࣥྜ
ᡂ㜼ᐖࠊࢸ࣓࣮ࣟࣛࢮࡢάᛶ໬࡟ࡼࡿ⪁໬ࡢ㜼ᐖࠊࢩࢢࢼࣝఏ㐩㐣⛬ࡢ㜼ᐖࠊࡲࡓࠊ࣍ࣝࣔࣥ௓ᅾ㐣⛬ࡸ࣌ࣝ࢜࢟ࢩࢰ࣮࣒ࡢቑṪ࡟㛵୚ࡍࡿཷᐜయ࡬ࡢ⤖ྜ࡞࡝࡛࠶ࡿࠋࡇࡢࡼ࠺࡞⛉Ꮫⓗ▱ぢࡢከࡃࡀࠊࡀ
ࢇ໬㐣⛬ุ࡛᫂ࡋ࡚࠸ࡿ஦ᐇ࡜ࡑࡢ୍㒊ࡀᩚྜࡍࡿࡇ࡜࡞࡝࠿ࡽࠊSHE CTA ࡢ⛉Ꮫⓗ࡞᳨ドࡣࠊ᏶඲࡛
ࡣ࡞࠸ࡶࡢࡢࠊ⌧ᅾࡢศᏊ⏕≀ᏛⓗỈ‽࡟ᇶ࡙࠸࡚࠶ࡿ⛬ᗘ࡞ࡉࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ

㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣࠊᇵ㣴ṇᖖ⣽⬊ࡀᙧែᙧ㉁㌿᥮ࡍࡿࡇ࡜ࢆᣦᶆ࡟ࡋ࡚ࠊ໬Ꮫ≀㉁ࡢ⭘⒆ㄏⓎᛶࡢ᭷↓ࢆணࡍࡿヨ㦂ἲࡢ୍ࡘ࡛࠶ࡾࠊ௚ࡢࡀࢇཎᛶண

ヨ㦂⤖ᯝ࡜࡜ࡶ࡟ࠊWoE ࡢ୍ࡘ࡜ࡋ࡚ SHE CTA ࡢ⤖ᯝࢆ฼⏝ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋᮏヨ㦂ἲࡣࠊ⌧⾜ࡢࡀࢇཎᛶヨ㦂࡟᏶඲࡟⨨ࡁ᥮ࢃࡿヨ㦂ἲ࡜ࡋ࡚ࡢ఩⨨࡙ࡅ࡛ࡣ࡞ࡃࠊ⿵ຓⓗ࡞ヨ㦂㸦౛࠼ࡤࠊࡀࢇཎᛶヨ㦂ࡢᐇ᪋㡰఩ࢆỴࡵࡿࡓࡵࡢࢫࢡ࣮ࣜࢽࣥࢢ࡞࡝㸧࡜ࡋ࡚఩⨨࡙ࡅࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿࠋᮏヨ㦂ἲ࡛ࡣࠊື≀⫾ඣ࠿ࡽึ௦ᇵ㣴⣽⬊ࢆㄪ〇ࡍࡿᚲせࡀ࠶ࡿࡀࠊ
⏝࠸ࡿື≀ᩘࡣࠊࡀࢇཎᛶヨ㦂࡟ẚ࡭࡚ࡣࡿ࠿࡟ᑡ࡞ࡃࠊ3Rs ࡢほⅬ࠿ࡽ᭷⏝࡛࠶ࡿࠋࡲࡓࠊᮏヨ㦂ἲࡣࠊึ௦ᇵ㣴⣽⬊࠿ࡽస〇ࡋࡓ 2 ௦┠ࡢ⣽⬊㸦ṇᖖ஧ಸయ䠅ࢆ⏝࠸ࡿࡇ࡜࠿ࡽࠊ୙Ṛ໬ᇵ㣴⣽⬊ࢆ⏝࠸ࡿ CTA ࡜ẚ㍑ࡋ࡚ࠊࡑࡢ཯ᛂࡣࡼࡾṇᖖ⣽⬊࡟㏆࠸≧ែࢆ཯ᫎࡋࡓ⣔࡜⪃࠼ࡽࢀࡿࠋ
DRP31 ࡛ࡢ 245 ࡢ໬Ꮫ≀㉁ࢆホ౯ࡋࡓ⤖ᯝ࡟࠾࠸࡚ࠊSHE CTA ࡢ⤖ᯝ࡜ࡀࢇཎᛶヨ㦂⤖ᯝ࡜ࡢ┦㛵ࡀㄆࡵࡽࢀࡿࠋSHE CTA ࡢṇ☜ᛶ࡟ࡘ࠸࡚ࡣࠊື≀ࢆ⏝࠸ࡓࡀࢇཎᛶヨ㦂࡜ࡢṇ☜ᗘࠊឤᗘࠊ≉␗ᗘࠊഇ㝜ᛶ⋡࠾ࡼࡧഇ㝧ᛶ⋡ࡀࠊࡀࢇཎ≀㉁ࢆ᳨ฟࡍࡿ㑇ఏẘᛶヨ㦂ࢹ࣮ࢱ࡜ᴫࡡྠ➼ཪࡣࡑࢀ௨ୖࡢ⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿࠋࡍ࡞ࢃࡕࠊ␗࡞ࡿᇵ㣴ᾮ pH ࡟࠾ࡅࡿ SHE CTA ࡢṇ☜ᛶࡢ෌ホ౯࡛ࡣࠊpH 6.7 ἲ㸦93໬Ꮫ≀㉁㸧ࡣࠊṇ☜ᗘ 73㸣ࠊឤᗘ 72㸣ࠊ≉␗ᗘ 74㸣࡛ࠊpH 7.0 ἲ㸦42 ໬Ꮫ≀㉁㸧ࡣࠊṇ☜ᗘ 83㸣ࠊឤ

44

ᗘ 80㸣ࠊ≉␗ᗘ 92㸣࡛࠶ࡗࡓࠋ࠸ࡎࢀࡢ pH ࡛ࡶࠊᴫࡡྠ➼ࡢ⤖ᯝࡀᚓࡽࢀ࡚࠾ࡾࠊඹ࡟ࡑࡢ᭷⏝ᛶࡣ㧗࠸࡜⪃࠼ࡽࢀࡿࠋ
୍᪉ࠊᮏヨ㦂࡟ࡘ࠸࡚ࡣከ✀㢮ࡢ໬Ꮫ≀㉁ࢆ⏝࠸ࡓࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀ࡚࠾ࡽࡎࠊヨ㦂᪉ἲࡀ༑ศ࡟᳨ドࡉࢀࡓ࡜ࡣゝ࠸㞴࠸ࠋࡲࡓࠊᮏヨ㦂ࡢ⤖ᯝࡀ㝧ᛶ࡛࠶ࡗ࡚ࡶࠊࡑࡢ໬Ꮫ≀㉁ࡢື≀࡟࠾ࡅࡿࡀࢇཎᛶࡢᙉࡉࠊ⮚ჾ≉␗ᛶࠊ✀≉␗ᛶ࠾ࡼࡧస⏝ᶵᗎ㸦࢖ࢽࢩ࢚࣮ࢩࣙࣥ/ࣉ࣮ࣟࣔࢩࣙࣥస⏝㸧࡟㛵ࡍࡿ᝟ሗࡣᚓࡽࢀ࡞࠸ࠋࡉࡽ࡟ࠊᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢุู࡟ࡣᐇ㦂⪅ࡢ⩦⇍㸦༑ศ࡞ᩍ⫱カ⦎࠾ࡼࡧ⤒㦂㸧ࡀᚲせ࡛࠶ࡿࠋヨ㦂ࢆ㐺ษ࡟ᐇ᪋ࡍࡿ࡟ࡣࠊ௨ୗࡢ஦㡯ࢆ⪃៖ࡍࡿᚲせࡶ࠶ࡿ㸸1) ㄪ〇ࡋࡓSHE ⣽⬊࠾ࡼࡧ౑⏝ࡍࡿ࢘ࢩ⫾ඣ⾑Ύࡢ㐺ษᛶࠊ2) ࣇ࢕࣮ࢲ࣮⣽⬊ࡢ㐺ษᛶ࡜ࡑࡢㄪ〇࡟࠾ࡅࡿ X ⥺↷ᑕ⿦⨨ࡢ౑⏝ࠊ3) ヨ㦂ᡂ❧ᇶ‽࠾ࡼࡧ⤖ᯝุᐃᇶ‽ࡢタᐃࠋ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣࠊᐇ᪋࡟㝿ࡋ✀ࠎࡢ᮲௳ࡀ࠶ࡿࡶࡢࡢࠊ⣽⬊ᇵ㣴࡞ࡽࡧ࡟ᙧ㉁㌿᥮ࢥࣟࢽุู࣮࡟⩦⇍ࡋࡓ᪋タ࡛࠶ࢀࡤᐇ᪋࡛ࡁࠊᇵ㣴ᮇ㛫ࡶ㸯㐌㛫⛬ᗘ࡜▷ࡃࠊࡲࡓヨ㦂㈝⏝ࡶẚ㍑ⓗᏳ౯࡛࠶ࡿࠋ୍༉ࡢዷ
ፎື≀⏤᮶ࡢ⫾ඣ⣽⬊࠿ࡽ 50 ᅇࡢホ౯ࡀྍ⬟࡞ᇵ㣴⣽⬊ࡀᚓࡽࢀࡿ࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠾ࡾࠊ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲ࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊࡑࡢ⿕㦂≀㉁ࢆࡀࢇཎᛶ≀㉁࡜ุᐃࡍࡿࡇ࡜ࡣ⛉Ꮫⓗ࡟ࡶࠊࡲࡓ⾜ᨻⓗ࡟ࡶᅔ㞴࡛࠶ࡾࠊࡋࡓࡀࡗ࡚ᮏヨ㦂ࢆࡀࢇཎᛶヨ㦂࡟⨨ࡁ᥮࠼ࡿࡇ࡜ࡣ⌧≧࡛ࡣ୙㐺ษ࡛࠶ࡿࠋᮏヨ㦂ࡢ⾜ᨻୖࡢ฼⏝࡟࠶ࡓࡗ࡚ࡣࠊᵓ㐀άᛶ┦㛵࡞࡝࡜⤌ࡳྜࢃࡏ࡚⿵ຓⓗ࡞ヨ㦂ࠊ౛࠼ࡤ WoE ࡟ᇶ࡙ࡃホ౯ࡢ㝿ࡢ୍ࡘࡢ㡯┠ࠊ࠶ࡿ࠸ࡣࡀࢇཎᛶヨ㦂ᐇ᪋ࡢඃඛ㡰఩௜ࡅࡢࡓࡵࡢࢫࢡ࣮ࣜ
ࢽࣥࢢヨ㦂࡜ࡋ࡚఩⨨௜ࡅࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ

ཧ⪃ᩥ⊩
1)

OECD (2007). Detailed review paper on cell transformation assays for detection of chemical carcinogens,OECD Environment, Health and Safety Publications, Series on Testing and Assessment, No. 31.

2)

ECVAM - The Validation Management Team (2010). Syrian Hamster Embryonic (SHE) cell pH 6.7 Cell
Transformation Assay - Prevalidation study Report.

3)

ECVAM - The Validation Management Team (2010). Syrian Hamster Embryonic (SHE) cell pH 7.0 Cell
Transformation Assay - Prevalidation study Report.

4)

ECVAM (2011). ESAC Working Group Peer Review Consensus Report on an ECVAM-coordinated55

prevalidation study concerning three protocols of the Cell Transformation Assay (CTA) for in vitro
carcinogenicity testing.
5)

EURL ECVAM (2012). Recommendation on three Cell Transformation Assays using Syrian Hamster
Embryo Cells (SHE) and the BALB/c 3T3 Mouse Fibroblast Cell Line for In Vitro Carcinogenicity Testing.

6)

OECD (2014). Results of An Analysis of The Performance of The Syrian Hamster Embryo Cell
Transformation Assay at PH 6.7 and PH 7.0, 26th Meeting of the Working Group of National Coordinators of
the Test Guidelines Program (WNT), ENV/JM/TG/RD (2014) 16.

7)

OECD (2014). Report on the Evaluation of Genotoxic or Non-genotoxic Activity of the Organic Chemicals
Tested in the SHE Cell Transformation Assay, 26th Meeting of the Working Group of National Coordinatorsof the Test Guidelines Program (WNT), ENV/JM/TG/RD (2014) 17.

8)OECD (2015). Guidance Document on the In Vitro Syrian Hamster Embryo (SHE) Cell Transformation
Assay, Series on Testing & Assessment, No 214, ENV/JM/MONO (2015)18.

9)JaCVAM ᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍㸸SHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧ホ౯ሗ࿌᭩㸦2017ᖺ 3 ᭶ 31 ᪥㸧㸬10) Pienta RJ et al. (1977). Morphological transformation of early passage Golden Syrian Hamster embryo cells
derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying diverse
carcinogens. Int. J. Cancer, 19, 642-655.
11) Watanabe M et al., (1991). Expression dynamics of transforming phenotypes in X-irradiated Syrian golden
hamster embryo cells. Mutat. Res., 249, 71-80.

66

ホ౯ሗ࿌᭩
SHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂ἲ㸦SHE CTA㸧ᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍

ᖹᡂ 29 ᖺ㸦2017 ᖺ㸧3 ᭶ 31 ᪥

1

7

ᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍

ጤဨ㛗

ὸ㔝ဴ⚽

㸦ඖ᪥ᮾ㟁ᕤᰴᘧ఍♫㸪⌧኱㜰ಙឡዪᏛ㝔▷ᮇ኱Ꮫ㸧

ጤ ဨ

⟄஭೺ᶵ

㸦᪥ᮏṑ⛉኱Ꮫ㸧ᒣᙳᗣḟ㸦୍⯡㈈ᅋἲே㣗ရ⸆ရᏳ඲ࢭࣥࢱ࣮ ⛙㔝◊✲ᡤ㸧໭ᮏᖾᏊ㸦ఫ཭໬Ꮫᰴᘧ఍♫㸧➟ᯇಇኵ㸦ⰼ⋤ᰴᘧ఍♫㸧ᒣᮏ⨾ె㸦࢔ࢫࢸࣛࢫ〇⸆ᰴᘧ఍♫㸧

2

8

┠ ḟ

1.

せ᪨

2.

ヨ㦂᪉ἲࡢ⛉Ꮫⓗጇᙜᛶ࡜つไヨ㦂ἲ࡜ࡋ࡚ࡢጇᙜᛶ

3.

ヨ㦂᪉ἲࡢࣉࣟࢺࢥࣝࡢጇᙜᛶ

4.

ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟⏝࠸ࡽࢀࡓ≀㉁ࡢศ㢮࡜ጇᙜᛶ5.

ヨ㦂ࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵ࡟⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ࡢ in vivo ཧ↷ࢹ࣮ࢱ࡞ࡽࡧ࡟ཧ↷ࢹ࣮ࢱసᡂࡢ
⤒⦋

6.

ヨ㦂ࢹ࣮ࢱ࡜⤖ᯝࡢ฼⏝ᛶ

7.

ヨ㦂᪉ἲࡢṇ☜ᛶ

8.

ヨ㦂᪉ἲࡢಙ㢗ᛶ

9.

ヨ㦂᪉ἲࡢࢹ࣮ࢱࡢ㉁10.

ヨ㦂᪉ἲ࡟㛵ࡍࡿ௚ࡢ⛉Ꮫⓗ࡞ሗ࿌11.

3Rs ࡬ࡢ㛵୚㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧12.

ヨ㦂᪉ἲࡢ᭷⏝ᛶ࡜㝈⏺

13.

⤖ㄽ

14.

ཧ⪃ᩥ⊩

3

9

Abbreviations
3Rs:

Reduction, Replacement, and Refinement

CTA:

Cell Transformation Assay

CGM:

Complete Growth Medium

CIM:

Cell Isolation Medium

DB-ALM: DataBase service on Alternative Methods to Animal Experimentation
DRF:

Dose Range Finding assay

DRP:

Detailed Review Paper

DMEM:

Dulbecco’s modified Eagle’s medium

ESAC:

ECVAM Scientific Advisory Committee

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

Foetal Bovine Serum

GD:

Guidance Document

IARC:

International Agency for Research on Cancer

MoA:

Mode of Action

MTF:

Morphological Transformation Frequency

NTP:

National Toxicology ProgramOECD:

Organization for Economic Co-operation and Development

SHE:

Syrian Hamster Embryo

UVBCs:

Substances of Unknown or Variable Composition, Complex Reaction Products or Biological Materials

WoE:

Weight of Evidence

WNT:

Working Group of National Coordinators of the Test Guidelines Program

4

10

1.

せ᪨
⣽⬊ࡢᙧ㉁㌿᥮ヨ㦂㸦Cell Transformation Assay : CTA㸧ࡣࠊin vitro ࡟࠾࠸࡚ࠊ໬Ꮫ≀㉁ࢆ᭚㟢ࡍࡿࡇ࡜࡛㉳ࡁࡿࠕ⣽⬊ࡢᙧែࠖࡸࠕ⾲⌧ᙧ㉁ࠖࡢᝏᛶ໬ࢆᣦᶆ࡜ࡋ࡚⭘⒆ㄏⓎᛶࡢ᭷↓ࢆㄪ࡭ࡿヨ㦂ἲࡢ୍ࡘ࡛࠶ࡿࠋ
JaCVAM SHE ⣽⬊ᙧ㉁㌿᥮ヨ㦂㈨ᩱ⦅⧩ጤဨ఍ࡣࠊOECD ࢞࢖ࢲࣥࢫ 1)࡜ࡋ࡚ 2015 ᖺ 5 ᭶࡟බ⾲ࡉࢀࡓࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⫾௘㸦Syrian Hamster Embryo: SHE㸧⣽⬊ࢆ⏝࠸ࡿ CTA ࡀ໬Ꮫ≀㉁ࡢࡀࢇཎᛶࡢ
ண࡟᭷⏝࠿ྰ࠿࡟ࡘ࠸᳨࡚ウࡋࠊࡑࡢ⤖ᯝࢆࡲ࡜ࡵࡓࠋSHE ⣽⬊ࢆ⏝࠸ࡿ CTA ࡢ≉ᚩ࡜ࡋ࡚ࡣࠊ⏝࠸ࡿ⣽⬊ࡀึ௦ᇵ㣴⣽⬊࡛࠶ࡿࠊᇶᮏⓗ࡞௦ㅰ⬟ࢆ⥔ᣢࡋ࡚࠸ࡿࠊᇵ㣴ᮇ㛫ࡣ㸯㐌㛫⛬ᗘࡢ▷ᮇ࡛࠶ࡿࠊヨ㦂㈝⏝ࡣẚ㍑ⓗᏳ౯࡛࠶ࡿࠊࡋ࠿ࡋࠊᙧ㉁㌿᥮ࡋࡓࢥࣟࢽ࣮㸦⣽⬊㞟ⴠ㸧ࢆぢᴟࡵࡿ࡟ࡣ༑ศ࡞ᩍ⫱࡜カ
⦎ࡀᚲせ࡛࠶ࡿࡇ࡜࡞࡝ࡀᣲࡆࡽࢀࡿࠋ
SHE CTA ࡣࠊヨ㦂᪉ἲࡸࡀࢇཎᛶ࡜ࡢ┦㛵࡟㛵ࡍࡿㄽᩥࡀከࡃฟ∧ࡉࢀࠊࡑࡢ᭷⏝ᛶ࡟ࡘ࠸࡚ࡢ㆟ㄽࡶከࡃ࡞ࡉࢀ࡚࠸ࡿࠋEuropean Union Reference Laboratory for Alternatives to Animal Testing㸦EURL ECVAM㸧
࡟ࡼࡗ࡚ࠊࡑࡢヨ㦂᪉ἲ࡟㛵ࡍࡿࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࡓࠋࡋ࠿ࡋࠊ⿕㦂≀㉁ࡢᩘࡀᑡ࡞
ࡃࠊࡋ࠿ࡶヨ㦂㛤ጞ᫬࡟ࡣ㠀㑇ఏẘᛶࡀࢇཎ≀㉁࡜⪃࠼ࡽࢀ࡚࠸ࡓ≀㉁ࡀ୍✀㢮ᏑᅾࡋࡓࡶࡢࡢࠊOECD࡟ࡼࡿ SHE CTA ᑓ㛛ᐙ఍㆟࡟ࡼࡿ෌ホ౯⤖ᯝ 2)࡛ࡣࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡣྵࡲࢀ࡚࠸࡞࠸࡜ࡢ⤖ㄽࢆᚓࡓࠋ᭱⤊ⓗ࡟ࡣ⿕㦂≀㉁ࡢᩘࢆቑࡸࡋ࡚ࡢṇᘧ࡞ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡣᐇ᪋ࡉࢀ࡞࠿ࡗࡓࡓࡵࠊヨ㦂᪉ἲࡀࣂࣜࢹ࣮ࢺࡉࢀࡓ࡜ࡣゝ࠸㞴࠸ࠋ ࡋ࠿ࡋ࡞ࡀࡽࠊࡀࢇཎ≀㉁࠾ࡼࡧ㠀ࡀࢇཎ≀㉁ࢆ⏝࠸ࡓ◊✲࡟ࡼࡿ࡜ࠊSHE CTA ࡢヨ㦂᪉ἲࡢṇ☜ᛶ࡟ࡘ࠸࡚ࡣࠊື≀ࢆ⏝࠸ࡓࡀࢇཎᛶヨ㦂࡜ࡢ୍⮴⋡ࠊឤᗘࠊ≉␗ᗘࠊഇ㝜ᛶ⋡࠾ࡼࡧഇ㝧ᛶ⋡ࡀࠊᴫࡡࠊ
ࡀࢇཎ≀㉁ࢆ᳨ฟࡍࡿ㑇ఏẘᛶヨ㦂ࢹ࣮ࢱ࡜ྠ➼ཪࡣࡑࢀ௨ୖࡢ⤖ᯝࡀᚓࡽࢀ࠾ࡾࠊࡑࡢ᭷⏝ᛶࡣ㧗࠸࡜⪃࠼ࡽࢀࡿࠋ
࡞࠾ࠊᮏ㉁ⓗ࡟ࡣࠊ⣽⬊ࡀᙧ㉁㌿᥮ࡍࡿ࣓࢝ࢽࢬ࣒ࡣᮍࡔᮍゎ᫂࡞ⅬࡀከࡃࠊCTA ࢆࡶࡗ࡚ື≀ࢆ⏝࠸ࡿࡀࢇཎᛶヨ㦂࡟⨨ࡁ᥮࠼ࡿࡇ࡜ࡣ⌧ẁ㝵࡛ࡣ୙㐺ษ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ
ࡇࡢࡼ࠺࡟ SHE CTA ࡀᣢࡘᅛ᭷ࡢၥ㢟ⅬࡣᏑᅾࡍࡿࡀࠊ ໬Ꮫ≀㉁࡟ࡼࡿⓎࡀࢇᶵᵓࡢ㔜せ࡞ࢫࢸࢵࣉ࡛࠶ࡿ⣽⬊ࡢᙧ㉁㌿᥮⬟ࢆホ౯࡛ࡁࡿ᪉ἲ࡛࠶ࡾࠊᐇ㝿ࠊࡀࢇཎ≀㉁ࡢ᭚㟢࡟ࡼࡾᙧ㉁㌿᥮ࢥࣟࢽ࣮
ࡢᩘࡀቑ࠼ࡿ࡜࠸࠺஦ᐇࢆ⪃៖ࡍࢀࡤࠊࡀࢇཎ≀㉁ࢆ᳨ฟࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ≉࡟ International
Agency for Research on Cancer (IARC) ࢢ࣮ࣝࣉ 1 ࠾ࡼࡧ 2A ࡟ศ㢮ࡉࢀࡿ໬Ꮫ≀㉁࡟࠾࠸࡚ࠊSHE CTA ࡜ࣄࢺࡢࡀࢇཎᛶ࡜࡟┦㛵ࡀ࠶ࡾࠊᵓ㐀┦㛵࡞࡝࡜⤌ࡳྜࢃࡏࡿࡇ࡜࡟ࡼࡾࠊࡀࢇཎᛶࢆ␲ࢃࡏࡿ໬Ꮫ≀㉁࡞࡝ࡢࢫࢡ࣮ࣜࢽࣥࢢ⣔࡜ࡋ࡚ᛂ⏝ࡍࡿࡇ࡜࡟ࡼࡾࠊື≀ᩘࡢ኱ᖜ࡞๐ῶࡀྍ⬟࡜࡞ࡾࠊ3Rs ࡢ⢭⚄࡟ࡶ㐺࠺ヨ㦂᪉ἲ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋࡇࡢࡼ࠺࡞ほⅬ࠿ࡽࠊSHE CTA ࡣࡉࡽ࡟ࢹ࣮ࢱࢆ✚ࡳ㔜ࡡࡿࡇ࡜࡟ࡼࡾࠊ໬Ꮫ≀㉁ࡢࡀࢇཎᛶண

ヨ㦂ࡢ᭷ᮃ࡞୍ࡘࡢヨ㦂ἲ࡟࡞ࡿ࡜⪃࠼ࡽࢀࡿࠋ

5

11

2.

ヨ㦂᪉ἲࡢ⛉Ꮫⓗጇᙜᛶ࡜つไヨ㦂ἲ࡜ࡋ࡚ࡢጇᙜᛶ

1䠅 ⛉Ꮫⓗጇᙜᛶ୍⯡ⓗ࡟ in vitro ࡟࠾ࡅࡿ CTA ࡜ࡣࠊ໬Ꮫ≀㉁ࡢ᭚㟢࡟ࡼࡾᇵ㣴⣽⬊ࡀ୙ྍ㏫ⓗ࡟ᙧែⓗኚ໬ࢆ㉳ࡇࡍ࠿࡝࠺࠿ࢆㄪ࡭ࡿヨ㦂࡛࠶ࡿࠋSHE CTA ࡣࠊࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⫾௘࡟⏤᮶ࡍࡿึ௦⣽⬊ࢆ෾⤖࣭ゎ෾ࡋࡓ 2 ௦┠ࡢ⣽⬊㸦ṇᖖ஧ಸయ⣽⬊㸧ࢆ⏝࠸࡚ࠊ┠ⓗ࡜ࡍࡿ໬Ꮫ≀㉁ࢆ in vitro ࡛᭚㟢ࡋࡓᚋࠊᙧែᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢฟ⌧⋡࡟ࡼࡗ࡚ࠊ໬Ꮫ≀㉁ࡢᙧ㉁㌿᥮⬟ࡢ᭷↓ࢆホ౯ࡍࡿヨ㦂ἲ࡛࠶ࡿࠋSHE ⣽⬊ࡢᙧែᙧ㉁㌿᥮ࡣࠊከẁ㝵ⓗ⭘⒆ᙧᡂ㐣⛬ࡢ᪩ᮇ࡟㉳ࡁࡿ⌧㇟࡟┦ᙜࡋ࡚࠾ࡾࠊᙧ㉁㌿᥮⣽⬊ࡣࠊ⥅௦ᇵ㣴ᚋࠊྠ⣔⤫ࡢື≀ࡸࢾ࣮ࢻ࣐
࢘ࢫ࡟ᑐࡋ࡚⣙ 50㸣ࠥ100㸣ࡢ๭ྜ࡛㐀⭘⒆ᛶ 3,4)ࢆ♧ࡍࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ
OECD Detailed Review Paper31 5) 㸦௨ୗ DRP31㸧࡟ࡣࠊ」ᩘࡢ pH ࡢᇵ㣴ᾮ࡛ヨ㦂ࡉࢀࡓᩘከࡃࡢ໬Ꮫ≀㉁ࡢ SHE CTA ⤖ᯝࡀࡲ࡜ࡵࡽࢀࠊ㑇ఏẘᛶࡀࢇཎ≀㉁࠾ࡼࡧ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᭚㟢࡟ࡼࡾ㸪
SHE ⣽⬊ࡣᙧែᙧ㉁㌿᥮ࡍࡿࡇ࡜ࡀ♧ࡉࢀ࡚࠸ࡿࠋEURL ECVAM ࡣࠊᶆ‽ࣉࣟࢺࢥࣝࠊᢏ⾡⛣㌿ᛶࠊ࠾ࡼࡧ᪋タෆ෌⌧ᛶ࡞ࡽࡧ࡟᪋タ㛫෌⌧ᛶࢆホ౯ࡋࡓ⤖ᯝࠊࡑࡢ᭷⏝ᛶࢆㄆࡵ࡚࠸ࡿࠋࡲࡓࠊEURL ECVAMࡢᶆ‽ⓗࣉࣟࢺࢥࣝ࡜ DRP31 ࡟♧ࡉࢀࡓࣉࣟࢺࢥࣝ࡜ࡢ㛫࡟ࡣᇶᮏⓗ࡞ᕪ␗ࡣ࡞ࡃࠊࡇࢀࡽࡢᚓࡽࢀࡓ
ࢹ࣮ࢱࡣᅇ㢳ⓗホ౯࡟࠾࠸࡚ࠊཷࡅධࢀྍ⬟࡛࠶ࡿ࡜ࡉࢀࡓࠋ
⣽⬊ࡢᙧ㉁㌿᥮ࡢṇ☜࡞ศᏊ⏕≀Ꮫⓗ࣓࢝ࢽࢬ࣒ࡣ༑ศ࡟ࡣゎ᫂ࡉࢀ࡚࠸࡞࠸ࡢࡀ⌧≧࡛࠶ࡿࠋSHE⣽⬊ࡀᙧ㉁㌿᥮ࡍࡿ࣓࢝ࢽࢬ࣒࡟ࡣ୙᫂࡞Ⅼࡀከ࠸ࡀࠊࡑࡢ࣓࢝ࢽࢬ࣒ࢆゎᯒࡍࡿㄽᩥࡣᩘከࡃ࠶ࡾࠊ
ᙧ㉁㌿᥮ࡣ༢୍ࡢ࣓࢝ࢽࢬ࣒࡛ㄏᘬࡉࢀ࡞࠸ࡇ࡜ࡶ᫂ࡽ࠿࡟࡞ࡗ࡚ࡁࡓࠋࡍ࡞ࢃࡕࠊ⣽⬊࿘ᮇไᚚࢩࢫ
ࢸ࣒ࠊࢤࣀ࣒Ᏻᐃᛶࠊ࠾ࡼࡧศ໬࣭ቑṪ㐣⛬࡛ࡢ㑇ఏᏊⓎ⌧ࡢኚ໬ࡸ␗ᖖࡀᙧ㉁㌿᥮ࡢ୺ࡓࡿせᅉ࡜ࡋ࡚⪃࠼ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽࡢ㐣⛬࡟ᙳ㡪ࢆཬࡰࡍ㑇ఏⓗኚ␗ࡣ┤᥋ⓗ࡞㑇ఏẘᛶస⏝࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ
ࡉࡽ࡟ࠊDNA ࡢ㧗࣓ࢳࣝ໬࠶ࡿ࠸ࡣప࣓ࢳࣝ໬ࠊࣄࢫࢺࣥಟ㣭࠾ࡼࡧࢾࢡࣞ࢜ࢯ࣮࣒ࣜࣔࢹࣜࣥࢢ࡟ࡼ
ࡿ㑇ఏᏊⓎ⌧ࡢኚ໬ࡸ㑇ఏⓗ୙Ᏻᐃᛶࡀࠊ࢚ࣆࢪ࢙ࢿࢸࢵࢡ࡞ኚ໬ࢆㄏ㉳ࡋࠊᝏᛶᙧ㉁㌿᥮࡬࡜㐍⾜ࡉࡏࡿᘬࡁ㔠࡟࡞ࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋ
ࡑࡢ௚ࡢ▱ぢ࡜ࡋ࡚ࠊ䞉୙Ṛ໬ࡸᝏᛶ໬ࡋࡓᙧ㉁㌿᥮⣽⬊࡟࠾ࡅࡿᚋᮇẁ㝵࡛ࡣࠊࡀࢇ㑇ఏᏊ ras ࡸ myc ࡟ᗈỗⓗࡶࡋࡃࡣ㒊఩≉␗ⓗ࡟ప࣓ࢳࣝ໬ࡀࡳࡽࢀ࡚࠸ࡿࡇ࡜ࠋ
䞉⣽⬊࿘ᮇࢳ࢙ࢵࢡ࣏࢖ࣥࢺ㑇ఏᏊࡢ࣓ࢳࣝ໬࡟ࡼࡿ㑇ఏᏊࡢⓎ⌧ᢚไࡸ p53 㑇ఏᏊࡢ✺↛ኚ␗࡜ྠᵝࠊ୙Ṛ໬ࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⓶⭵⣽⬊࡟࠾࠸࡚ࠊࡀࢇᢚไ㑇ఏᏊ ink4a ࡸ ink4b ࡢᑐ❧㑇ఏᏊ࡟࣊ࢸࣟ
ࡲࡓࡣ࣍ࣔࡢḞኻࡀほᐹࡉࢀ࡚࠸ࡿࡇ࡜ࠋ
䞉ᙧ㉁㌿᥮ࡋࡓ SHE ⣽⬊ᰴ࡛ࡣ⣽⬊࿘ᮇࡢ G2 ࢳ࢙ࢵࢡ࣏࢖ࣥࢺ࡟㞀ᐖࡀ⏕ࡌ࡚࠸ࡿࡇ࡜ࠋ
䞉௚ࡢ⣽⬊ࢆᙧ㉁㌿᥮ࡉࡏࡿࡼ࠺࡞άᛶ໬ࣉࣟࢺ࢜ࣥࢥࢪ࣮ࣥ cph ࡀᝏᛶᙧ㉁㌿᥮⣽⬊࠿ࡽศ㞳ࡉࢀ࡚࠸ࡿࡇ࡜ࠋ࡞࡝ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋ
㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡶࡲࡓࠊከẁ㝵Ⓨࡀࢇ㐣⛬࡟㛵୚ࡋࠊṇᖖ⣽⬊ࢆᙧ㉁㌿᥮ࡉࡏࡿࡶࡢ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋࡍ࡞ࢃࡕࠊࢠࣕࢵࣉࢪࣕࣥࢡࢩࣙࣥ࡟ࡼࡿ⣽⬊㛫ࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥࢆ㜼ᐖࡍࡿ㓟໬ⓗࢫࢺ
612ࣞࢫࠊ⣽⬊ศ⿣ᅇᩘࡢቑຍࠊ࢔࣏ࢺ࣮ࢩࢫࡢῶᑡࠊࢳࣗࣈࣜࣥྜᡂ㜼ᐖࠊࢸ࣓࣮ࣟࣛࢮࡢάᛶ໬࡟ࡼࡿ⪁໬ࡢ㜼ᐖࠊࢩࢢࢼࣝఏ㐩㐣⛬ࡢ㜼ᐖࠊࡲࡓࠊ࣍ࣝࣔࣥ௓ᅾ㐣⛬ࡸ࣌ࣝ࢜࢟ࢩࢰ࣮࣒ࡢቑṪ࡟㛵୚ࡍࡿཷᐜ
య࡬ࡢ⤖ྜ࡞࡝࡛࠶ࡿࠋ
ࡇࡢࡼ࠺࡞⛉Ꮫⓗ▱ぢࡢከࡃࡀࠊࡀࢇ໬㐣⛬ุ࡛᫂ࡋ࡚࠸ࡿ஦ᐇ࡜ࡑࡢ୍㒊ࡀᩚྜࡍࡿࡇ࡜࡞࡝࠿ࡽࠊ
SHE CTA ࡢ⛉Ꮫⓗ࡞᳨ドࡣࠊ᏶඲࡛ࡣ࡞࠸ࡀࠊ⌧ᅾࡢศᏊ⏕≀ᏛⓗỈ‽࡟ᇶ࡙࠸࡚࠶ࡿ⛬ᗘ࡞ࡉࢀ࡚࠸
ࡿヨ㦂࡜⪃࠼ࡽࢀࡿࠋ

2䠅 つไୖࡢጇᙜᛶ
SHE CTA ࡣ༢୍ࡢ໬Ꮫ≀㉁࡟ࡘ࠸࡚ࡢࡳࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀ࡚࠾ࡾࠊ࠸ࡃࡘ࠿ࡢ໬Ꮫ≀㉁࠿ࡽᵓᡂࡉࢀ࡚࠸ࡿ≀㉁ࠊUVCBsࠊᡈ࠸ࡣΰྜ≀࡟ࡘ࠸࡚ࡣࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀ࡚࠸࡞
࠸ࡇ࡜ࢆ␃ពࡍ࡭ࡁ࡛࠶ࡿࠋࡇ࠺ࡋࡓ≧ἣࢆ⪃៖ࡋࡓୖ࡛ࠊOECD ࡢ࢞࢖ࢲࣥࢫࢻ࣓࢟ࣗࣥࢺ 1)ࡀ᫂☜࡟ࡋ࡚࠸ࡿࡇ࡜ࡣࠊࡀࢇཎᛶࢆホ౯ࡍࡿヨ㦂ᡓ␎ࡢ୍⎔࡜ࡋ࡚ࠊᡈ࠸ࡣࡀࢇཎᛶ࡟࠿࠿ࡿ WoE㸦ドᣐࡢ㔜ࡳ௜ࡅ㸧࡟ SHE CTA ⤖ᯝࢆ฼⏝ࡍࡿࡇ࡜ࡣ᭷⏝࡛࠶ࡿ࡜ࡋ࡚࠸ࡿࠋࡲࡓࠊSHE CTA ࡣ㑇ఏẘᛶࡀࢇཎ≀㉁࡟ᑐࡋ࡚㧗ឤཷᛶࢆࡶࡘࡀࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁࡟ᑐࡋ࡚ࡣᮍࡔ㆟ㄽࡢవᆅࡀ࠶ࡾࠊࡼࡾከࡃࡢ᝟
ሗࡀồࡵࡽࢀ࡚࠸ࡿࡢࡶ஦ᐇ࡛࠶ࡿࠋᮏヨ㦂ἲࡀ OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ᢎㄆࡉࢀ࡞࠿ࡗࡓ⌮⏤࡜ࡋ࡚ᣦ᦬ࡉࢀ࡚࠸ࡿࡇ࡜ࡣࠊᙧ㉁㌿᥮⣽⬊ࡢホ౯࡟㛵ࡋ࡚ᮏ㉁ⓗ࡞ၥ㢟ࡀࡑࡇ࡟ࡣᏑᅾࡍࡿࡇ࡜ࠊ
ࡍ࡞ࢃࡕࠊᙧ㉁㌿᥮࡟⮳ࡿศᏊ⏕≀Ꮫⓗస⏝ᶵᵓࡢ⌮ゎࡀ㝈ᐃⓗ࡛࠶ࡿࡇ࡜࡛࠶ࡿࠋࡲࡓ SHE CTA ࡢṇ☜ᛶศᯒ࡛⏝࠸ࡓ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡀᑡ࡞࠿ࡗࡓࡇ࡜ 1,2)ࠊSHE CTA ࡢ⤖ᯝ࠿ࡽࡣࠊin vivo ࡟࠾ࡅࡿࡀࢇཎᛶࡢᙉࡉࠊ✀≉␗ᛶࡲࡓࡣ⤌⧊≉␗ᛶ࡞࡝ࡢ᝟ሗࡣᚓࡽࢀ࡞࠸ࡇ࡜࡞࡝ࡀᣦ᦬ࡉࢀ࡚࠸ࡿࠋ
ᮏヨ㦂ἲࡣ⌧⾜ࡢࡀࢇཎᛶヨ㦂࡟᏶඲࡟⨨ࡁ᥮ࢃࡿヨ㦂ἲ࡜ࡋ࡚ࡢ఩⨨࡙ࡅ࡛ࡣ࡞ࡃࠊ໬Ꮫ≀㉁➼ࡢࡀࢇཎᛶ࡟㛵ࡋ࡚ࡢ⾜ᨻⓗ࡞ุ᩿ࡀᚲせ࡟࡞ࡗࡓሙྜࠊ⿵ຓⓗ࡞ヨ㦂㸦౛࠼ࡤ Weight of Evidence㸸 WoE㸧࡜ࡋ࡚఩⨨࡙ࡅࡿࡇ࡜ࡀࡑࡢ฼⏝౯್ࢆ㧗ࡵࡿࡶࡢ࡜⪃࠼ࡽࢀࡿࠋ

3.

ヨ㦂᪉ἲࡢࣉࣟࢺࢥࣝࡢጇᙜᛶ

1㸧 ヨ㦂ἲࡢᴫ␎
SHE ⣽⬊ࡣ⫾⏕ 13 ᪥┠ࡢࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⫾௘ࡢึ௦ᇵ㣴࠿ࡽᚓࡽࢀࡓṇᖖ஧ಸయ⣽⬊࡛ࠊ⸆≀௦ㅰ⬟ࡸ p53 άᛶࢆ᭷ࡍࡿࠋSHE ⣽⬊ࡣࠊ✀ࠎࡢࢱ࢖ࣉࡢ⣽⬊ᆺ࠿ࡽᵓᡂࡉࢀࡿࡓࡵࠊSHE CTA ࡣᰴ⣽⬊࠿ࡽ࡞ࡿ௚ࡢ CTA ἲ࡜ẚ㍑ࡋ࡚ࠊࡼࡾ⏕య࡟㏆࠸ṇᖖ⣽⬊࡟㉳ࡇࡿኚ໬ࢆᤊ࠼ࡿࡇ࡜ࡀ࡛ࡁࡿࠋከ✀ከᵝ࡞ࡀࢇཎ≀㉁ࢆ SHE ⣽⬊࡟᭚㟢ࡍࡿ࡜ࡲࡎᙧែᙧ㉁㌿᥮ࡀㄏᑟࡉࢀࠊᙧ㉁㌿᥮⣽⬊ࡣࠊ⥅௦ᇵ㣴ᚋࠊ኱㒊ศࡀ㐀⭘⒆ᛶ࣐࣮࣮࡛࢝࠶ࡿ㊊ሙ㠀౫ᏑᛶቑṪ⬟ࢆ⋓ᚓࡋࠊឤཷᛶࢆᣢࡘື≀࡟ᝏᛶ⭘⒆ࢆᙧᡂࡍ
ࡿࡇ࡜ࡀ▱ࡽࢀ࡚࠸ࡿࠋ
SHE CTA ࡣ⿕㦂≀㉁ࡢ᭚㟢࡟ࡼࡗ࡚ࠊከẁ㝵ⓗ࡟㐍⾜ࡍࡿ⭘⒆ᙧᡂ㐣⛬ࡢึᮇ࡟㉳ࡇࡿᙧ㉁㌿᥮ࢆ᳨ฟࡍࡿ in vitro ヨ㦂ἲ࡛࠶ࡿࠋᮏヨ㦂ἲ࡟ࡼࡗ࡚ࠊᚑ᮶ࡢ in vitro 㑇ఏẘᛶヨ㦂࠿ࡽࡣධᡭ࡛ࡁ࡞࠸⿕㦂≀㉁࡟₯ᅾࡍࡿ⣽⬊ᙧ㉁㌿᥮⬟ࢆ᳨ฟࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ
SHE CTA ࡛ᚓࡽࢀࡓ⤖ᯝࢆໟᣓⓗ࡞ヨ㦂ᡓ␎ࡢ୍㒊ࡸ WoE ࡟ά⏝ࡍࢀࡤࠊ⿕㦂≀㉁ࡢࡶࡘ₯ᅾⓗᙧ
713

㉁㌿᥮⬟ࡢホ౯࡟౑࠼ࡿࡤ࠿ࡾ࡛࡞ࡃࠊSHE CTA ࡣࠊᚑ᮶ࡢື≀ࢆ⏝࠸ࡓࡀࢇཎᛶヨ㦂ࡢ୍㒊⨨ࡁ᥮࠼
㸦Replacement㸧࡞ࡽࡧ࡟ື≀ᩘࡢ๐ῶ㸦Reduction㸧࡟㈉⊩ࡍࡿࡇ࡜࡟࡞ࡾࠊࡑࡢព⩏ࡣ኱ࡁ࠸ࠋ

2㸧 ヨ㦂ἲࡢཎ⌮(1) SHE ⣽⬊ࡣ⫾⏕ 13 ᪥┠ࡢࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⫾௘⣽⬊ࡢึ௦ᇵ㣴࠿ࡽᚓࡿࠋ㓝⣲࡛⫾௘⤌⧊ࢆᾘ໬ᚋࠊ⣽⬊ࢆ཰㞟ࡋᾮయ❅⣲୰࡛ಖᏑࡍࡿࠋ෾⤖⣽⬊ࡢ୍㒊ࡣࣇ࢕࣮ࢲ࣮⣽⬊㸦ᨭᣢ⣽⬊㸧࡟ࠊ௚ࡣᶆⓗ⣽⬊࡟౑⏝ࡍࡿࠋX ⥺↷ᑕ࡛ቑṪ⬟ࢆ୙ά໬ࡉࡏࡓ⣽⬊ࢆࣇ࢕࣮ࢲ࣮⣽⬊࡟⏝࠸ࡿࠋࣇ࢕
࣮ࢲ࣮⣽⬊ࡣᶆⓗ⣽⬊࡬ࡢᰤ㣴⿵⤥࡜⸆≀௦ㅰάᛶࢆࢧ࣏࣮ࢺࡍࡿ┠ⓗ࡛᧛✀ࡉࢀࠊᶆⓗ⣽⬊ࡣᙧ
ែᙧ㉁㌿᥮⬟ࢆㄪ࡭ࡿࡓࡵ࡟᧛✀ࡉࢀࡿࠋ(2) ᶆⓗ⣽⬊ࡣᙧᡂࡉࢀࡿࢥࣟࢽ࣮ࡢⓎ⫱ࢆ⪃៖ࡋ࡚ࠊCTA ࡢヨ㦂ᡂ❧ᇶ‽ࢆ‶ࡓࡍ࡟ᚲせ࡞ࢡ࣮ࣟࢽࣥࢢຠ⋡ࢆ㐩ᡂ࡛ࡁࡿࢡ࣮ࣟࣥᐦᗘ࡛ࣇ࢕࣮ࢲ࣮⣽⬊ᒙୖ࡬᧛✀ࡍࡿࠋ᧛✀ᚋࠊ⿕㦂≀㉁ࢆ 7 ᪥㛫᭚㟢ࡍࡿࠋࡑࡢᚋࠊ⣽⬊ࢆὙί࣭ᅛᐃࡋᰁⰍࡍࡿࠋࢹ࢕ࢵࢩࣗࡣࢥ࣮ࢻ໬ࡋࠊᐇయ㢧ᚤ㙾᳨ᰝ࡛ࢥࣟࢽ࣮ࡢᙧែⓗ⾲⌧ᆺࢆグ㘓ࡍࡿࠋ(3) ⣽⬊ẘᛶࡣࢥࣟࢽ࣮ᙧᡂຠ⋡࡜ࢥࣟࢽ࣮ࡢ኱ࡁࡉࡸ⣽⬊ᐦᗘࡢῶᑡ࠿ࡽホ౯ࡍࡿࠋグ㘓ࡋࡓࢥࣟࢽ
࣮ࡢ⥲ᩘ࡟ᑐࡍࡿᙧែᙧ㉁㌿᥮ࢥࣟࢽ࣮ᩘࡢ๭ྜࢆྛヨ㦂⃰ᗘ࡛⟬ฟࡍࡿࠋ⿕㦂≀㉁᭚㟢⩌ࡢ⥲ࢥࣟࢽ࣮ᩘ࡟ᑐࡍࡿᙧែᙧ㉁㌿᥮ࢥࣟࢽ࣮ᩘࡢ๭ྜࢆ⁐፹ᑐ↷⩌ࡢࡑࡢ๭ྜ࡜ẚ㍑ࡍࡿࠋ࡞࠾ࠊࢥࣟ
ࢽ࣮ࢧ࢖ࢬ࡜ᐦᗘ➼ࡢ≧ែࡣᚲせ࡟ᛂࡌ࡚グ㘓࡟ṧࡋࠊホ౯ࡢཧ⪃࡜ࡍࡿࡀࠊᙧ㉁㌿᥮⋡࡟ࡣᐤ୚ࡋ࡞࠸ࠋ

3㸧 ࣉࣟࢺࢥࣝࡢጇᙜᛶ
௨ୗࡢ㡯┠ࡢၥ㢟Ⅼࡀ᫂☜࡟ࡉࢀ࡚࠾ࡾࠊጇᙜᛶࡣᴫࡡ☜ಖࡉࢀ࡚࠸ࡿࠋ
(1)

ᐇ㦂⣔㸸
୍⭡ࡢࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮⫾௘࠿ࡽᚓࡽࢀࡓึ௦ᇵ㣴⣽⬊ࢆ౑⏝ࡍࡿࠋࡇࡢࡇ࡜ࡣ᭱ప࡛ࡶ 1 ༉ࡢዷፎࣁ࣒ࢫࢱ࣮ࡢᏳᴦṚࢆព࿡ࡍࡿࡀࠊ⣽⬊ࡢ㐺ษ࡞෾⤖ಖᏑ࡟ࡼࡗ࡚ࠊ1 ༉ࡢዷፎࣁ࣒ࢫࢱ࣮
࠿ࡽᑡ࡞ࡃ࡜ࡶ 50 ᅇศࡢ CTA ࢆᐇ᪋ࡍࡿࡢ࡟ᚲせ࡛༑ศ࡞⣽⬊ࢆᚓࡿࡇ࡜ࡀ࡛ࡁࡿࠋ

(2) SHE ⣽⬊࡜࢘ࢩ⫾௘⾑Ύ㸦FBS㸧ࡢࣟࢵࢺヨ㦂㸸
SHE ⣽⬊ࠊFBS ࡜ࡶ౑⏝࡟ඛ❧ࡗ࡚ࠊ᪂ࡋ࠸ࣟࢵࢺẖ࡟ CTA ࡟㐺ษ࠿࡝࠺࠿ࢆࢳ࢙ࢵࢡࡍࡿࠋ᭦
࡟ࠊ㐺ษ࡛࠶ࡿ࡜ุ᫂ࡋࡓ⣽⬊࡜ FBS ࢆ⤌ࡳྜࢃࡏ࡚ CTA ࢆᐇ᪋ࡋࠊᐃࡵࡽࢀࡓヨ㦂ᡂ❧ᇶ‽ࢆ‶ࡓࡍ࠿࡝࠺࠿ࡢヨ㦂ࢆ⾜࠺ࠋ(3) ⿕㦂≀㉁ࡢࣇ࢕࣮ࢲ࣮⣽⬊㞀ᐖᛶ☜ㄆ㸸(a) CTA ࡬ࡢ౑⏝࡟ඛ❧ࡗ࡚ࠊࣇ࢕࣮ࢲ࣮⣽⬊ࡔࡅࢆࢹ࢕ࢵࢩࣗ࡟᧛✀࣭ᇵ㣴ࡋࠊࢥࣟࢽ࣮ᙧᡂ⬟ࡢ࡞࠸ࡇ࡜ࢆ☜ㄆࡍࡿࠋ(b) ⿕㦂≀㉁࡛㑅ᢥⓗ࡟㞀ᐖࡉࢀࡓሙྜࢆ㝖࠸࡚ࠊ⿕㦂≀㉁᭚㟢⩌ࡢࢹ࢕ࢵࢩࣗୖࡢࣇ࢕࣮ࢲ࣮⣽⬊ࢆྍど࡛ࡁ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ

8

14

(4) CTA ᢏ⾡ࡢ⇍㐩ᗘ☜ㄆヨ㦂㸸
CTA ᐇ᪋᪋タࡣᐃࡵࡽࢀࡓస⏝ᶵᗎࡢ␗࡞ࡿ㝧ᛶ࠾ࡼࡧ㝜ᛶᑐ↷≀㉁ࢆ⏝࠸࡚ CTA ࢆᐇ᪋ࡋࠊ
CTA ᢏ⾡ࡢ⇍㐩ᗘࢆ☜ಖࡍࡿࠋ(5) ౑⏝ࡍࡿᇵᆅࡢ pH㸦pH6.7 ࠾ࡼࡧ pH7.0)㸸
CTA ࡢᐇ᪋࡟ඛ❧ࡕࠊ㐺⏝ࡍࡿ pH ᮲௳࡛๓㏙ࡋࡓ CTA ᢏ⾡ࡢ⇍㐩ᗘ☜ㄆヨ㦂ࢆᐇ᪋ࡋࠊ⇍㐩ᗘࢆド᫂ࡍࡿࠋ
(6) ୧ pH 㛫ࡢ SHE CTA ࣉࣟࢺࢥࣝࡢᕪ␗㸸ᇵᆅ୰ࡢ FBS ⃰ᗘ࡟ᕪࡣ࠶ࡿࡀࠊࡑࢀ௨እ࡛ࡢᕪࡣㄆࡵࡽࢀ࡞࠸ࠋ
(7) ୧ pH 㛫࡟࠾ࡅࡿ SHE CTA ࣃࣇ࢛࣮࣐ࣥࢫࡢᕪࡢ᭷↓㸸࠸ࡎࢀࡢ pH ࡛ࡶ CTA ࡢ⤖ᯝࡣࠊឤᗘࠊ≉␗ᗘ࡜ࡶྠ⛬ᗘ࡛࠶ࡾࠊ኱ࡁ࡞ᕪࡣㄆࡵࡽࢀ࡞࠸ࠋ
(8) ヨ㦂ᡂ❧ᇶ‽㸸
CTA ࡢ⤖ᯝࡣࣉࣟࢺࢥ࡛ࣝつᐃࡋ࡚࠸ࡿヨ㦂ᡂ❧ᇶ‽࡟ᚑ࠸ࠊุᐃࡍࡿࠋ
(9) CTA ホ౯ࡢᐈほᛶࡢ☜ಖ㸸
CTA ࡟࠶ࡓࡗ࡚ࡣࠊ⿕㦂≀㉁ࡸᚓࡽࢀࡓࢹ࣮ࢱ࡟ࡘ࠸࡚ࢥ࣮ࢻ໬ࡋ࡚ホ౯ࡍࡿࠋ(10) ࢥࣟࢽ࣮ࡢ୺ほⓗࢫࢥ࢔ࣜࣥࢢ࡟ࡼࡿࢹ࣮ࢱࡢኚື㸸ࡇࡢᑐ⟇࡜ࡋ࡚ࠊ௨ୗࡢࡇ࡜ࢆᐇ᪋ࡍࡿࠋ
(a) ᪋タෆእࡢ CTA ⤒㦂⪅࡟ࡼࡿࢫࢥ࢔࣮ࣛࡢࢺ࣮ࣞࢽࣥࢢ(b) ✀ࠎࡢṇᖖ࠾ࡼࡧᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢ෗┿ぢᮏࡢ฼⏝㸦ࢫࢥ࢔ࣜࣥࢢ࡟࠶ࡓࡗ࡚ࡣࠊᖖ᫬෗┿ぢᮏࢆཧ⪃࡟ࡍࡿ㸧(c) ࢭ࢝ࣥࢻ࢜ࣆࢽ࢜ࣥ㸦௚ࡢࢫࢥ࢔࣮ࣛࡲࡓࡣࢫࢥ࢔⤒㦂⪅ࡢពぢࢆཧ⪃࡟ࡍࡿ㸧ࡸ௚ࡢࢫࢥ࢔
࣮ࣛ࡟ࡼࡿ஧㔜ࢫࢥ࢔ࣜࣥࢢ(d) ࢹ࢕ࢵࢩࣗࡢࢥ࣮ࢻ໬
(11) ⤖ᯝุᐃᇶ‽CTA ࡢ⤖ᯝࢆࣉࣟࢺࢥ࡛ࣝᐃࡵࡓ⤖ᯝุᐃᇶ‽࡟ᚑ࠸ࠊุᐃࡍࡿࠋ

4.

ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟⏝࠸ࡽࢀࡓ≀㉁ࡢศ㢮࡜ጇᙜᛶࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 6,7)࡟౪ࡉࢀࡓ 6 ≀㉁ࡢ࠺ࡕࠊ4 ≀㉁ࡀࡀࢇཎ≀㉁࡛ࠊࡑࡢ࠺ࡕࡢ 3 ≀㉁ࡣ㑇ఏẘᛶ≀㉁ࠊ1 ≀㉁ࡣ㠀㑇ఏẘᛶ≀㉁࡛࠶ࡿ࡜ヨ㦂㛤ጞ᫬࡟ࡣホ౯ࡉࢀ࡚࠸ࡓࡶࡢࡢࠊSHE CTA ᑓ㛛ᐙ఍㆟࡟ࡼࡿ෌᳨ウ⤖ᯝ࠿ࡽࠊ4 ≀㉁ࡣ඲࡚㑇ఏẘᛶ≀㉁࡛࠶ࡗࡓ࡜ุ᩿ࡉࢀࡓ 2)ࠋṧࡿ 2 ≀㉁ࡣ㠀㑇ఏẘᛶ㠀ࡀࢇཎ≀㉁࡛࠶ࡿࠋ඲య࡛ 6 ≀㉁࡜໬ྜ≀ᩘࡣᑡ࡞ࡃࠊ኱㒊ศࡀከ⎔ⰾ㤶᪘Ⅳ໬Ỉ⣲࡜ⰾ㤶᪘࢔࣑࡛ࣥࠊ⿕㦂≀㉁ࡢస⏝࣓࢝ࢽࢬ࣒࡟೫ࡾࡀࡳࡽࢀࡓࠋࡲࡓࠊ㝧ᛶ≀㉁ࡣ᫂ࡽ࠿࡞㝧ᛶ཯ᛂࢆ♧ࡍ໬ྜ≀ࡀከࡃ㸦4/6㸧ࠊࡀࢇཎᛶࡢ࠶࠸ࡲ࠸࡞཯ᛂࢆ♧ࡍ໬ྜ≀ࡣྵࡲࢀ࡞࠿ࡗࡓࡶࡢࡢࠊ㝜ᛶ≀㉁ࡶ 33㸣㸦2/6㸧
ࢆ༨ࡵ࡚࠾ࡾࠊ㑅ࡤࢀࡓ໬ྜ≀ࡣጇᙜ࡛࠶ࡿ࡜⪃࠼ࡽࢀࡓࠋ

915

㸺Genotoxic carcinogens㸼䞉Benzo(a)pyrene䞉2.4-Diaminotoluene䞉3-Methylcholanthrene䞉o-Toluidine㸨㸨ヨ㦂㛤ጞᙜ᫬ࡣ equivocal ࡲࡓࡣ inconclusive genotoxic carcinogen ࢆ non-genotoxic carcinogen ࡛࠶ࡿ࡜ゎ㔘ࡋ࡚࠸ࡓࠋ
㸺Non-genotoxic non-carcinogen㸼䞉Anthracene䞉Phthalic anhydride5.

ヨ㦂ࡢṇ☜ᛶࢆホ౯ࡍࡿࡓࡵ࡟⏝࠸ࡽࢀࡓ໬Ꮫ≀㉁ࡢ in vivo ཧ↷ࢹ࣮ࢱ࡞ࡽࡧ࡟
ཧ↷ࢹ࣮ࢱసᡂࡢ⤒⦋㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࢆ᳨ฟࡍࡿ in vitro ヨ㦂ἲࡢ㛤Ⓨࡢ㔜せᛶࡀᣦ᦬ࡉࢀࡓࡇ࡜ࢆཷࡅࠊ1997 ᖺ࠿ࡽࠕNon-Genotoxic Carcinogens Detection: the Performance of In Vitro Cell Transformation Assaysࠖ࡜㢟ࡍࡿ
Detailed Review Paper ࡢసᡂࡀ㛤ጞࡉࢀࡓࠋࡑࢀࡣࠊSHE ⣽⬊ࠊBALB/c 3T3 ⣽⬊࠾ࡼࡧ C3H10T1/2 ⣽⬊ࢆ⏝࠸ࡿ CTA ࡟↔Ⅼࡀᙜ࡚ࡽࢀࡓࠋ2001 ᖺ࡟᭱ึࡢ᱌ࡀ The Working Group of National Coordinators of the
Test Guidelines Program㸦WNT㸧࡟ᅇぴࡉࢀࠊྛᅜࡢᑓ㛛ᐙ࠿ࡽࡢᗈ⠊ᅖ࡟ࢃࡓࡿࢥ࣓ࣥࢺࢆࡶ࡜࡟ᨵᐃ
∧ࡀసᡂࡉࢀࡓࡀࠊ᭱⤊໬࡟ࡣ⮳ࡽ࡞࠿ࡗࡓࠋ2002 ᖺ࡟ࡣࠊ➨ 2 ᱌ࡀᅇぴࡉࢀࠊ෌ࡧከᩘࡢࢥ࣓ࣥࢺࡀ
ฟࡉࢀࡓࡇ࡜࠿ࡽࠊࣇࣛࣥࢫ㸦SHE ⣽⬊㸧ࠊ᪥ᮏ㸦BALB/c3T3 ⣽⬊㸧ࠊ࢝ࢼࢲ㸦C3H10T1/2 ⣽⬊㸧ࡢ 3 ᅜࡀࠊࡑࢀࡒࢀࡢ⣽⬊ࢆ⏝࠸ࡓྛヨ㦂ἲ࡟ࡘ࠸࡚ᨵᐃసᴗࢆ⥅⥆ࡋ࡚㐍ࡵࡓ⤖ᯝࠊ2006 ᖺ࡟➨ 3 ᱌ࡀᥦ♧ࡉࢀࡓࠋ➨ 3 ᱌࡟ᑐࡍࡿࢥ࣓ࣥࢺ࡬ࡢ㆟ㄽ㸦࣡ࢩࣥࢺࣥ఍㆟㸧ࢆ⤒࡚➨ 4 ᱌ࡀసᡂࡉࢀࡓࠋࡇࡢ➨ 4 ᱌
ࠕDETAILED REVIEW PAPER ON CELL TRANSFORMATION ASSAYS FOR DETECTION OF CHEMICALCARCINOGENSࠖࡀ࠸ࢃࡺࡿ DRP31 ࡜ࡋ࡚ 2007 ᖺ 4 ᭶࡟㛤ദࡉࢀࡓ WNT19 ࡛ᢎㄆࡉࢀ᭱⤊໬ࡉࢀࡓࠋ
⾲㢟ࡀኚ᭦ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽศ࠿ࡿࡼ࠺࡟ࠊࡇࡢ DRP31 ࡣࠊࡀࢇཎ≀㉁ࡢ᳨ฟ࡟࠾ࡅࡿ CTA ࡢព⩏ࢆ୺┠ⓗ࡜ࡋࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ in vitro ᳨ฟ⣔࡜ࡋ࡚ࡢศᯒࡣḟࡢㄢ㢟࡜ࡉࢀࡓࠋ
CTA ࡟㛵ࡋ࡚ࡣࠊୖ㏙ࡢ DRP31 సᡂ⤒⦋࡟࠶ࡿࡼ࠺࡟ SHE ⣽⬊௨እ࡟ BALB/c 3T3 ⣽⬊࠶ࡿ࠸ࡣ
C3H10T1/2 ࡞࡝ࡢ⣽⬊ࡀ⏝࠸ࡽࢀ࡚࠸ࡿࠋࡇࢀࡽࡢ⣽⬊࡛ࡢᙧ㉁㌿᥮⬟࡜ in vivo ࡀࢇཎᛶ࡜ࡢ┦㛵࡟ࡘ࠸࡚ࡣࠊDRP31 ࡀཧ↷ࡋᚓࡿࢹ࣮ࢱ㞟࡛࠶ࡿࠋ
DRP31 ࡛ࡣࠊࡆࡗṑ㢮࡟࠾ࡅࡿࡀࢇཎᛶヨ㦂ࢹ࣮ࢱࠊ㑇ఏẘᛶࢹ࣮ࢱ࡜ 3 ✀㢮ࡢ CTA ࢹ࣮ࢱࡀࡲ࡜ࡵ
ࡽࢀ࡚࠸ࡿࠋࡀࢇཎᛶヨ㦂ࢹ࣮ࢱ࡟㛵ࡋ࡚ࡣࠊIARC ࠾ࡼࡧ NTP ࡢࢹ࣮ࢱࡀ᥇⏝ࡉࢀ࡚࠸ࡿࠋ
DRP31 ࡛ࡣࠊSHE CTA ࡢ⤖ᯝ࡜ࡆࡗṑ㢮࡛ࡢࡀࢇཎᛶヨ㦂⤖ᯝࡢ୧᪉ࡀグ㍕ࡉࢀࡓ໬Ꮫ≀㉁ࡢᩘࡣ
245 ࡛࠶ࡿࠋࡑࡢෆヂࡣࠊࡀࢇཎ≀㉁ࡀ 175 ≀㉁ࠊ㠀ࡀࢇཎ≀㉁ࡀ 70 ≀㉁࡛࠶ࡿࠋෆヂࡣࠊ᭷ᶵ໬ྜ≀
ࡀ 190 ≀㉁㸦ࡀࢇཎ≀㉁㸸127 ≀㉁ࠊ㠀ࡀࢇཎ≀㉁㸸63 ≀㉁㸧ࠊ↓ᶵ໬ྜ≀ࡀ 55 ≀㉁㸦ࡀࢇཎ≀㉁㸸48≀㉁ࠊ㠀ࡀࢇཎ≀㉁㸸7 ≀㉁㸧࡛࠶ࡗࡓ㸦Appendix 1㸧ࠋ1016

SHE CTA ࢞࢖ࢻࣛ࢖ࣥࡢసᡂ㐣⛬࡟࠾࠸࡚ࠊࢹ࣮ࢱࡢ෌ホ౯ࡀᐇ᪋ࡉࢀࡓࠋChemical Carcinogenesis
Research Information System, NTP, IARC Monographs (1-106), PubMed,࠾ࡼࡧ CLP/GHS ࡢࢹ࣮ࢱ࣮࣋ࢫࢆ⏝࠸࡚ࠊSHE CTA ࡢࢹ࣮ࢱࡢࡳ࡞ࡽࡎࠊ㑇ఏẘᛶ࡟㛵ࡍࡿࢹ࣮ࢱ࠾ࡼࡧࡀࢇཎᛶࡢࢹ࣮ࢱ࡟ࡘ࠸࡚ࡶ෌ホ౯ࡉࢀࡓࠋࡑࡢ⤖ᯝࠊᑐ㇟࡜࡞ࡗࡓ 108 ≀㉁㸦ࡀࢇཎ≀㉁ 66 ≀㉁ࠊ㠀ࡀࢇཎ≀㉁ 42 ≀㉁㸧࡟ࡘ࠸࡚ࠊ෌ホ౯ࡉࢀࡓࢹ࣮ࢱࡀ♧ࡉࢀࡓ 2)ࠋ

6.

ヨ㦂ࢹ࣮ࢱ࡜⤖ᯝࡢ฼⏝ᛶࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡟⏝࠸ࡓ 6 ≀㉁㸦㝧ᛶ 4 ≀㉁ࠊ㝜ᛶ 2 ≀㉁㸧ࡢࢹ࣮ࢱࡣࠊヨ㦂᪉ἲࡢಙ㢗ᛶࡢホ౯࡟⏝࠸ࡿࡇ࡜ࡀฟ᮶ࡿࠋ᪋タ㛫෌⌧ᛶࡢホ౯࡟ࡣ 6 ≀㉁඲࡚ࡢࢹ࣮ࢱࡀ⏝࠸ࡽࢀࡓࠋ୍᪉ࠊ᪋タෆ෌⌧ᛶࡢホ౯࡟ࡣ㝧ᛶᑐ↷≀㉁࡜ࡋ࡚౑ࢃࢀࡓ Benzo[a]pyrene ࡢࡳࡢ⤖ᯝࡀ⏝࠸ࡽࢀࠊ1 ᪋タ࡛ࡣࢥ࣮ࢻ໬ࡋ࡚⧞ࡾ㏉ࡋ 3 ᅇᐇ᪋㸦pH7.0 ἲ㸧ࡋࠊⰋዲ࡞⤖ᯝࡀᚓࡽࢀࡓࡀࠊ௚ࡢ 3 ᪋タ࡟ࡘ࠸࡚ࡣࠊẖᅇࢥ࣮ࢻ໬ࡏࡎ࡟㝧ᛶᑐ↷࡜ࡋ࡚ᐇ᪋ࡋࠊᚓࡽࢀࡓ್ࢆẚ㍑ࡋࡓ⤖ᯝ㸦pH6.7 ἲ㸧ࡋ࠿♧ࡉࢀ࡚࠸࡞࠸ࠋࡇࡢࡼ
࠺࡟ࠊᙉ࠸㝧ᛶ཯ᛂࢆ♧ࡍ 1 ໬ྜ≀ࡢࡳ࡛᪋タෆ෌⌧ᛶヨ㦂ࡀᐇ᪋ࡉࢀ࡚࠸ࡿࡇ࡜ࠊࡲࡓࠊྠ୍ࣉࣟࢺ
ࢥ࡛ࣝᐇ᪋ࡉࢀࡓヨ㦂࡛ࡣ࡞࠸ࡇ࡜࠿ࡽࠊ᪋タෆ෌⌧ᛶࢆホ౯ࡍࡿ࡟ࡣ༑ศ࡜ࡣゝ࠸㞴࠸ࠋ
DRP31 ࡛ホ౯ࡋࡓ≀㉁ࡢࢹ࣮ࢱࡣࠊヨ㦂᪉ἲࡢṇ☜ᛶࡢホ౯࡟⏝࠸ࡿࡇ࡜ࡀฟ᮶ࡿࠋ265 ≀㉁ࡢ࠺ࡕࠊ
157 ≀㉁ࡣࢻࣛࣇࢺࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ㸦OECD, Feb. 2013㸧࡛ᐃࡵࡽࢀࡓ pH ࡸ⿕㦂≀㉁ฎ⌮᫬㛫࡛ᐇ᪋ࡉࢀ࡚࠸࡞࠸ࡓࡵࠊṇ☜ᛶࡢホ౯࠿ࡽ㝖እࡉࢀࡓࠋṧࡿ 108 ≀㉁ࡀࢻࣛࣇࢺࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥࡢࢡࣛ
࢖ࢸࣜ࢔࡟ᚑࡗ࡚෌ホ౯ࡉࢀࠊࡑࡢ⤖ᯝࡀヨ㦂᪉ἲࡢṇ☜ᛶࡢホ౯ 8)࡟౑ࢃࢀࡓࠋ

7.

ヨ㦂᪉ἲࡢṇ☜ᛶ
DRP31 ࡟ࡣࠊ㑇ఏẘᛶࢹ࣮ࢱࠊࡆࡗṑ㢮ື≀ࡢࡀࢇཎᛶヨ㦂ࢹ࣮ࢱ࡜ 3 ✀㢮ࡢ in vitro CTA ࢹ࣮ࢱࡀࡲ

࡜ࡵࡽࢀࠊࡀࢇཎᛶホ౯࡟࠾ࡅࡿ CTA ࢹ࣮ࢱࡢṇ☜ᛶ࡟ࡘ࠸࡚ሗ࿌ࡉࢀ࡚࠸ࡿࠋSHE CTA ࡢ⤖ᯝ࡟ࡘ࠸࡚ࡣࠊᇵ㣴ᾮࡢ pH ࡀ␗࡞ࡿヨ㦂ࢹ࣮ࢱࡀ⵳✚ࡉࢀ࡚࠸ࡿࡇ࡜࠿ࡽࠊpH6.7 ࠾ࡼࡧ pH7.0 ௨ୖࡢ 2 ࡘ࡟ศࡅ࡚ゎᯒࡉࢀ࡚࠸ࡿ㸦⾲ 1ࠊ⾲ 2㸧ࠋࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA ࠾ࡼࡧྛ✀㑇ఏẘᛶヨ㦂ࢹ࣮ࢱ࡟ࡘ࠸࡚ࡣࠊDRP31 ࡢ Annex ࡟ሗ࿌ࡉࢀ࡚࠾ࡾࠊࡑࢀࢆ⦅㞟ࡋࡓ⾲ 3 ࡟♧ࡋࡓ㏻ࡾࠊࡀࢇཎᛶ࡜ SHE CTA ࡢ୍⮴⋡ࡣྛ✀ࡢ㑇ఏẘᛶヨ㦂ࡢࡑࢀ࡜ྠ➼௨ୖ࡛࠶ࡾࠊഇ㝧ᛶ⋡࠾ࡼࡧഇ㝜ᛶ⋡ࡣప࠸࡜ゝ࠺⤖ᯝ࡜࡞ࡗ࡚࠸ࡿࠋࡲࡓࠊ⾲ 4 ࡟♧ࡍ㏻ࡾࠊྛ✀㑇ఏẘᛶヨ㦂࡜ CTA ࡟ඹ㏻ࡍࡿ≀㉁ࡢヨ㦂⤖ᯝ࡟ࡘ࠸࡚ࡀࢇཎᛶ࡜ࡢ୍⮴⋡ࢆࡳ࡚ࡶࠊSHE CTAࡣⰋዲ࡞⤖ᯝࢆ♧ࡋ࡚࠸ࡿࠋ

1117

⾲ 1 ࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA㸦pH6.7 ἲ㸧ࡢṇ☜ᛶ
In vivo ࡀࢇཎᛶࡀࢇཎ≀㉁㠀ࡀࢇཎ≀㉁SHE CTA

㝧ᛶ

36

5

㝜ᛶ

18

29

୍⮴⋡㸦Concordance㸧㸸(36+29)/(36+5+18+29)×100㸻65/88×100㸻74%ឤᗘ㸦Sensitivity㸧㸸

36/(36+18) ×100㸻36/54×100㸻67%≉␗ᗘ㸦Specificity㸧:

29/(5+29) ×100㸻29/34×100㸻85%ഇ㝜ᛶ⋡㸦False Negative㸧㸸

18/(36+18) ×100㸻18/54×100㸻33%ഇ㝧ᛶ⋡㸦False Positive㸧㸸

5/(5+29) ×100㸻5/34×100㸻15%

⾲ 2 ࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA㸦pH7.0 ἲ㸧ࡢṇ☜ᛶ
In vivo ࡀࢇཎᛶࡀࢇཎ≀㉁㠀ࡀࢇཎ≀㉁SHE CTA

㝧ᛶ

131

17

㝜ᛶ

11

33

୍⮴⋡㸦Concordance㸧㸸(131+33)/(131+17+11+33)×100㸻164/192×100㸻85%ឤᗘ㸦Sensitivity㸧㸸

131/(131+11) ×100㸻131/142×100㸻92%≉␗ᗘ㸦Specificity㸧:

33/(17+33) ×100㸻33/50×100㸻66%ഇ㝜ᛶ⋡㸦False Negative㸧㸸

11/(131+11) ×100㸻11/142×100㸻8%ഇ㝧ᛶ⋡㸦False Positive㸧㸸

17/(17+33) ×100㸻17/50×100㸻34%

⾲ 3 ࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA ࡜ྛ✀㑇ఏẘᛶヨ㦂ࡢẚ㍑
SHE CTA

Ames

ML

pH6.7 ἲpH7.0 ἲ୍⮴⋡㸦%㸧

74

85

52

72

ឤᗘ㸦%㸧

67

92

39.5

≉␗ᗘ㸦%㸧

8566

ഇ㝜ᛶ⋡㸦%㸧

33ഇ㝧ᛶ⋡㸦%㸧

HPRT

CA

MN

In vitro

In vivo

In vivo

78

62

60

58

86

80

65

57

58

79

26

70

59

68

55

8

60.5

14

20

35

43

42

15

34

21

74

30

41

32

45

ホ౯≀㉁⥲ᩘ

88

192

315

215

135

238

110

198

㠀ࡀࢇཎ≀㉁⋡㸦%㸧

39

26

29

30

22

30

29

25

Ames㸸᚟ᖐ✺↛ኚ␗ヨ㦂ࠊML㸸࣐࢘ࢫࣜࣥࣇ࢛࣮࣐ TK ヨ㦂ࠊ
HPRT㸸ᇵ㣴⣽⬊ࢆ⏝࠸ࡿ㑇ఏᏊ✺↛ኚ␗ヨ㦂ࠊCA㸸ᰁⰍయ␗ᖖヨ㦂ࠊMN㸸ᑠ᰾ヨ㦂1218

⾲ 4 ඹ㏻≀㉁ࡢࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA ࡜ྛ✀㑇ఏẘᛶヨ㦂ࡢẚ㍑
SHE

Ames

SHE

ML

SHE

HPRT

ホ౯≀㉁⥲ᩘ

242

166

111

㠀ࡀࢇཎ≀㉁⋡㸦%㸧

29

28

21

୍⮴⋡㸦%㸧

86

48

86

75

92

78

ឤᗘ㸦%㸧

90.5

36

88

86

96

79

≉␗ᗘ㸦%㸧

75

78

80

35

74

71

ഇ㝜ᛶ⋡㸦%㸧

10

64

12

14

4

21

ഇ㝧ᛶ⋡㸦%㸧

25

22

20

65

26

29

SHE

CA

SHE

CA

SHE

MN

In vitro

In vivo

In vivoホ౯≀㉁⥲ᩘ

180

78

154

㠀ࡀࢇཎ≀㉁ᩘ

29

29

26

୍⮴⋡㸦%㸧

86

62

86

66

83

56

ឤᗘ㸦%㸧

89

63

91

62.5

87

57

≉␗ᗘ㸦%㸧

77

59

75

71

71

54

ഇ㝜ᛶ⋡㸦%㸧

11

37

9

37.5

13

43

ഇ㝧ᛶ⋡㸦%㸧

23

41

25

29

29

46

SHE㸸SHE CTAࠊAmes㸸᚟ᖐ✺↛ኚ␗ヨ㦂ࠊML㸸࣐࢘ࢫࣜࣥࣇ࢛࣮࣐ TK ヨ㦂ࠊ
HPRT㸸ᇵ㣴⣽⬊ࢆ⏝࠸ࡿ㑇ఏᏊ✺↛ኚ␗ヨ㦂ࠊCA㸸ᰁⰍయ␗ᖖヨ㦂ࠊMN㸸ᑠ᰾ヨ㦂ࡇࡢࡼ࠺࡟ࠊOECD ࡣᩥ⊩ㄪᰝ࡟ࡼࡗ࡚ CTA ࡟࠾ࡅࡿࡀࢇཎ≀㉁ࡢண

ᛶࢆ᳨ドࡋࠊDRP31 ࡜ࡋ࡚ࡲ࡜ࡵࡓࠋࡑࡢ⤖ㄽ࡜ࡋ࡚ SHE CTA ࡢ࢞࢖ࢻࣛ࢖ࣥ໬ࡀ᥎ዡࡉࢀࡓࠋࡑࡢ⤖ᯝࠊุᐃἲࢆྵࡴヨ㦂ἲࡢᶆ‽໬࡞ࡽࡧ࡟࢞࢖ࢻࣛ࢖ࣥ໬࡟ྥࡅࠊOECD ࢞࢖ࢲࣥࢫ㸦OECD GD34ࠊ2005㸧9)࡟ᚑ࠸ࠊEURL ECVAM
୺ᑟ࡛ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࡓࠋࡑࢀࡽࡢ⤖ᯝࢆホ౯ࡋࡓ EURL ECVAM Scientific Advisory
Committee㸦ESAC㸧ࡢሗ࿌᭩ 10)࡜ࡑࡢሗ࿌᭩ࢆཷࡅ࡚సᡂࡉࢀࡓ EURL ECVAM RECOMMENDATION11)ࡣ࡜ࡶ࡟ࠊSHE CTA ࡢ OECD ࢞࢖ࢻࣛ࢖ࣥ࡜ࡍࡿࡇ࡜ࢆ᥎ዡࡋࡓࠋࡇࡢࡼ࠺࡞⤒⦋ࢆ⤒࡚ࠊSHE CTA ࡢ࢞࢖ࢻࣛ࢖ࣥ᱌ࡀ 2013 ᖺ࡟බ⾲ࡉࢀࡓࠋࡋ࠿ࡋ࡞ࡀࡽࠊᥦ᱌ࡉࢀࡓヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ᱌࡟ᑐࡋ࡚ࠊ㛵ಀྛᅜ࠿ࡽࡢࢥ࣓ࣥࢺ࡜ࡋ࡚ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ᱌ࡢุᐃᇶ‽࡜ DRP31 ࡢุᐃ⤖ᯝ࡜ࡢᩚྜᛶࡢ෌☜ㄆࡀồࡵࡽࢀࡓࠋ
ࡇࢀࢆཷࡅ࡚ࠊpH6.7 ἲ࡜ pH7.0 ἲ㸦pH7.0 ࢆ㉸࠼ࡿ᮲௳࡛ᐇ᪋ࡉࢀࡓࢹ࣮ࢱࡣ㝖እ㸧࡛ᐇ᪋ࡉࢀ࡚࠸
ࡿࢹ࣮ࢱࡢ෌ホ౯ࡀ⾜ࢃࢀࡓ 8)ࠋࡍ࡞ࢃࡕࠊpH6.7 ἲ࡛ࡣ 93 ≀㉁ࠊpH7.0 ἲ࡛ࡣ 42 ≀㉁࡟ࡘ࠸࡚෌ホ౯ࡀ⾜ࢃࢀࡓ㸦⾲ 5ࠊ⾲ 6㸧ࠋ

1319

⾲ 5 ࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA㸦pH6.7 ἲ㸧ࡢṇ☜ᛶ㸦෌ホ౯⤖ᯝ㸧In vivo ࡀࢇཎᛶSHE CTAࡀࢇཎ≀㉁㠀ࡀࢇཎ≀㉁

㝧ᛶ

42㸦6㸧

8㸦1㸧

㝜ᛶ

15㸦1㸧

26㸦0㸧

୙☜ᐃ㸦Equivocal㸧

1㸦0㸧

1㸦0㸧

୍⮴⋡㸦Concordance㸧㸸(42+26)/(42+8+15+26+1+1)×100㸻68/93×100㸻73%ឤᗘ㸦Sensitivity㸧㸸

42/(42+15+1) ×100㸻42/58×100㸻72%≉␗ᗘ㸦Specificity㸧:

26/(8+26+1) ×100㸻26/35×100㸻74%ഇ㝜ᛶ⋡㸦False Negative㸧㸸

15/(42+15+1) ×100㸻15/58×100㸻26%ഇ㝧ᛶ⋡㸦False Positive㸧㸸

8/(8+26+1) ×100㸻8/35×100㸻23%ᣓᘼෆࡢᩘ್ࡣ↓ᶵ≀㉁ࡢホ౯ᩘ⾲ 6 ࡀࢇཎᛶホ౯࡟࠾ࡅࡿ SHE CTA㸦pH7.0 ἲ㸧ࡢṇ☜ᛶ㸦෌ホ౯㸧
In vivo ࡀࢇཎᛶSHE CTAࡀࢇཎ≀㉁㠀ࡀࢇཎ≀㉁㝧ᛶ24㸦11㸧

1㸦0㸧

㝜ᛶ

6㸦2㸧

11㸦0㸧

୍⮴⋡㸦Concordance㸧㸸(24+11)/(24+1+6+11)×100㸻35/42×100㸻83%ឤᗘ㸦Sensitivity㸧㸸

24/(24+6) ×100㸻24/30×100㸻80%≉␗ᗘ㸦Specificity㸧:

11/(11+1) ×100㸻11/12×100㸻92%ഇ㝜ᛶ⋡㸦False Negative㸧㸸

6/(24+6) ×100㸻6/30×100㸻20%ഇ㝧ᛶ⋡㸦False Positive㸧㸸

1/(11+1) ×100㸻1/12×100㸻8%ᣓᘼෆࡢᩘ್ࡣ↓ᶵ≀㉁ࡢホ౯ᩘ
ࡲࡓࠊ2 ࡘࡢ᪉ἲ࡟ඹ㏻ࡢ≀㉁㸦ࡀࢇཎ≀㉁㸸14 ≀㉁ࠊ㠀ࡀࢇཎ≀㉁㸸4 ≀㉁㸧࡟ࡘ࠸࡚ࡢ⤖ᯝࡀẚ㍑
ࡉࢀࠊ14 ࡢࡀࢇཎ≀㉁ࡢෆ 1 ≀㉁㸦diethylstilbestrol㸧ࡢࡳ 2 ࡘࡢヨ㦂㛫࡛⤖ᯝࡀ୍⮴ࡋ࡚࠸࡞࠿ࡗࡓࡀࠊ
13 ≀㉁ࡣࡍ࡭࡚⤖ᯝࡀ୍⮴㸦12 ≀㉁㸸㝧ᛶࠊtitanium dioxide ࡢ 1 ≀㉁㸸㝜ᛶ㸧ࡋ࡚࠸ࡓࠋࡲࡓࠊ㠀ࡀࢇཎ≀㉁ࡢෆ 2 ≀㉁ࡣ 2 ヨ㦂࡛࡜ࡶ࡟㝜ᛶࠊṧࡾࡢ 2 ≀㉁ࡢ࠺ࡕࠊCaprolactam ࡣ pH6.7 ἲ࡛ࡣ㝜ᛶ࡛࠶ࡗ
ࡓࡀࠊpH7.0 ἲ࡛ࡣ㝧ᛶ࡛࠶ࡗࡓࠋࡲࡓࠊPhthalic anhydride ࡣ pH6.7 ἲ࡛ࡣヨ㦂᪋タ㛫࡛⤖ᯝ࡟ᕪࡀ࠶ࡾࠊ
pH7.0 ἲ࡛ࡣ㝜ᛶࡢ⤖ᯝ࡛࠶ࡗࡓࠋࡇࡢࡼ࠺࡟ࠊ2 ࡘࡢ᪉ἲ࡟ࡣ pH ࡢ㐪࠸ࡣ࠶ࡿࡀࠊ࡯ࡰྠ➼ࡢ⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ
ࡀࢇཎ≀㉁ࢆ᭷ᶵ໬ྜ≀࡜↓ᶵ໬ྜ≀࡟ศࡅ࡚ࡑࡢ᳨ฟ⋡࡟ࡘ࠸࡚ࡶ᳨ウࡋ࡚࠸ࡿࠋpH6.7 ἲ࡟ࡘ࠸࡚
ࡣ↓ᶵ໬ྜ≀ࡀ 1 ≀㉁࡛࠶ࡾࠊ᭷⏝࡞᝟ሗ࡜ࡣ⪃࠼ࡽࢀ࡞࠸ࠋ୍᪉ࠊpH7.0 ἲ࡟ࡘ࠸࡚ࡣ᭷ᶵ໬ྜ≀ࡀ 17≀㉁ࠊ↓ᶵ໬ྜ≀ࡀ 13 ≀㉁࠶ࡾࠊ᳨ฟ⋡ࡣࡑࢀࡒࢀ 76.5% ࠾ࡼࡧ 84.6% ࡛࠶ࡗࡓࠋ᭦࡟ࠊSHE CTA ࡟࠾ࡅࡿ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᳨ฟ⋡࡟ࡘ࠸࡚ࡶホ౯ࡋ࡚࠸ࡿ 12)㸦⾲ 7㸧ࠋ

14

20

⾲ 7

SHE CTA ࡟࠾ࡅࡿ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᳨ฟ⋡㠀㑇ఏẘᛶࡀࢇཎ≀㉁᳨ฟ⋡㸦%㸧

SHE CTA

㝧ᛶ

14㸦1㸧

58

(pH6.7 ἲ)

㝜ᛶ

10㸦0㸧

42

ィ

24㸦1㸧

SHE CTA

㝧ᛶ

8㸦4㸧

67

(pH7.0 ἲ)

㝜ᛶ

4㸦1㸧

33

ィ

12㸦5㸧

ᣓᘼෆࡢᩘ್ࡣ↓ᶵ≀㉁ࡢホ౯ᩘࡇࡢ⤖ᯝࡣࠊ㑇ఏẘᛶ≀㉁ࡢᐃ⩏ࢆࠕ࠸ࡎࢀ࠿ࡢ㑇ఏẘᛶヨ㦂࡛㝧ᛶ㸦ࡓࡔࡋࠊ࣐࢘ࢫࣜࣥࣇ࢛࣮࣐
TK ヨ㦂ࡢࡳ࡛㝧ᛶࡢሙྜࢆ㝖ࡃ㸧ࡢ≀㉁ࠖ࡜ࡋࠊ㠀㑇ఏẘᛶ≀㉁ࢆ௨ୗࡢ࠸ࡎࢀ࠿࡟ヱᙜࡍࡿ≀㉁࡜ᐃ
⩏ࡋ࡚ࡀࢇཎ≀㉁ࢆศ㢮ࡋࡓሙྜࡢ⤖ᯝ࡛࠶ࡿ 12)ࠋ䞉Ames ヨ㦂㸦᚟ᖐ✺↛ኚ␗ヨ㦂㸧ࡀ㝜ᛶ࡛ in vivo 㑇ఏẘᛶヨ㦂ࢹ࣮ࢱࡀ↓࠸ࠋ䞉Ames ヨ㦂㸦᚟ᖐ✺↛ኚ␗ヨ㦂㸧ࢹ࣮ࢱࡀᚓࡽࢀ࡞࠸ࡀࠊ௚ࡢ㑇ఏẘᛶヨ㦂࡛㝜ᛶ࡛࠶ࡿࠋ
䞉ᚓࡽࢀ࡚࠸ࡿࡍ࡭࡚ࡢ㑇ఏẘᛶヨ㦂࡛㝜ᛶ࡛࠶ࡿࠋ
ࡋ࠿ࡋ࡞ࡀࡽࠊ㑇ఏẘᛶࡢホ౯ࡣ Ames ヨ㦂ࡢࡳ࡛ホ౯ࡉࢀ࡚ࡣ࠸࡞࠸ࡇ࡜࠿ࡽࠊ㑇ఏẘᛶ≀㉁࠾ࡼࡧ
㠀㑇ఏẘᛶ≀㉁ࡢᐃ⩏ࢆ௨ୗࡢࡼ࠺࡟ኚ᭦ࡋ 2,12)ࠊ㑇ఏẘᛶ࡜ࡀࢇཎᛶࡢᩥ⊩ࢆ෌ㄪᰝࡋࠊ᭷ᶵ໬ྜ≀ࢆ෌ศ㢮ࡋࡓࠋ
<㑇ఏẘᛶ≀㉁>
䞉In vitro ヨ㦂ࢹ࣮ࢱࡀ㝧ᛶ࡛࠶ࡾࠊࡑࢀࡀ in vivo 㑇ఏẘᛶヨ㦂࡛☜ㄆࡉࢀ࡚࠸ࡿࠋ
䞉In vivo ヨ㦂ࢹ࣮ࢱࡀ฼⏝࡛ࡁ࡞࠸ሙྜ࡟ࡣࠊin vitro ヨ㦂⤖ᯝࡀ౑ࢃࢀࡿࠋ

<㠀㑇ఏẘᛶ≀㉁>䞉Ames ヨ㦂㸦᚟ᖐ✺↛ኚ␗ヨ㦂㸧࡛㝜ᛶ࡛࠶ࡾࠊᇵ㣴⣽⬊ࢆ⏝࠸ࡿヨ㦂㸦ᰁⰍయ␗ᖖヨ㦂/ᑠ᰾ヨ㦂/㑇ఏᏊ✺↛ኚ␗ヨ㦂/࣐࢘ࢫࣜࣥࣇ࢛࣮࣐ TK ヨ㦂࡟ࡘ࠸࡚ࡣ ICH ࡟ࡼࡿ⌧⾜ࡢᇶ‽ุ࡛᩿㸧࡛㝜ᛶࠊ࠶ࡿ࠸ࡣᶆ‽ⓗ in vivo ヨ㦂࡛㝜ᛶ㸦ྠࡌ࢚ࣥࢻ࣏࢖ࣥࢺࡢሙྜࠊin vivo ヨ㦂⤖ᯝࢆ᥇⏝ࡍࡿ㸧࡛࠶ࡿࠋࡑࡢ⤖ᯝࠊ෌ホ౯ࡉࢀࡓ᭷ᶵ໬ྜ≀ࡣ pH6.7 ἲ࡛ࡣ 85 ≀㉁ࠊpH7.0 ἲ࡛ࡣ 32 ≀㉁࡛࠶ࡾࠊࡑࢀࡽࡣ㑇ఏẘᛶࡀࢇཎ≀㉁ࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁࠾ࡼࡧ㠀ࡀࢇཎ≀㉁࡟ศ㢮ࡉࢀࠊࡑࢀࡽࡢ SHE CTA ⤖ᯝࡣ⾲ 8 ࡟♧ࡋࡓࡼ࠺࡟ࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᳨ฟ⋡ࡣࠊpH6.7 ἲ࡛ࡣ 56.5%ࠊpH7.0 ἲ࡛ࡣ 83.3%࡛࠶ࡗࡓ 2)ࠋ

15

21

⾲ 8

SHE CTA ࡟࠾ࡅࡿ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᳨ฟ⋡㑇ఏẘᛶࡀࢇཎ≀㉁㠀㑇ఏẘᛶࡀࢇཎ≀㉁㠀ࡀࢇཎ≀㉁

≀㉁ᩘ

᳨ฟ⋡
㸦%㸧

≀㉁ᩘ

᳨ฟ⋡
㸦%㸧

≀㉁ᩘ

᳨ฟ⋡
㸦%㸧

SHE CTA

㝧ᛶ

20

71.4

13

56.5

3

8.8

㸦pH6.7 ἲ㸧

㝜ᛶ

3

10.7

10

43.7

26

76.5

Equivocal

5

17.9

0

0.0

5

14.7

ィ

28

SHE CTA

㝧ᛶ

9

64.3

5

83.3

1

8.3

㸦pH7.0 ἲ㸧

㝜ᛶ

4

28.6

1

16.7

11

91.7

Equivocal

1

7.1

0

0.0

0

0.0

ィ

14

23

6

34

12

DRP31 ࡛ࡣࠊᩥ⊩ㄪᰝࡋࡓ໬Ꮫ≀㉁࡟ࡘ࠸࡚ࠊIARC ࡀሗ࿌ࡋ࡚࠸ࡿࣄࢺ࡟ᑐࡍࡿࡀࢇཎᛶࣜࢫࢡศ㢮ࢆグ㍕ࡋ࡚࠸ࡿࡀࠊࡑࡢศ㢮࡜ CTA ⤖ᯝࡢ┦㛵ᛶ࡟ࡘ࠸࡚ࡢヲ⣽ࡣグ㍕ࡉࢀ࡚࠸࡞࠸ࠋ
ࡑࡇ࡛ࠊᙜ㈨ᩱ⦅⧩ጤဨ఍࡛ࡣࠊDRP31 ࡢ Table 11㹼13 ࡟グ㍕ࡉࢀ࡚࠸ࡿ≀㉁ࡢ࠺ࡕࠊࣄࢺ࡟ᑐࡍࡿ
ࡀࢇཎᛶࣜࢫࢡศ㢮ࡢグ㍕ࡀ࠶ࡾࠊࡋ࠿ࡶ SHE CTA ࡢ⤖ᯝࡀ᫂ࡽ࠿࡞ 169 ≀㉁࡟ࡘ࠸࡚ࠊSHE CTA ⤖ᯝ࡜ࣄࢺࡀࢇཎᛶ࡜ࡢ┦㛵ࢆㄪᰝࡋࡓࠋ࡞࠾ࠊIARC ࡢศ㢮࡟ࡘ࠸࡚ࡣࠊBenigni ࡽ 13) ࡢሗ࿌ࡶཧ⪃࡟ࡋࡓࠋࡑࡢ⤖ᯝࠊࢢ࣮ࣝࣉ 1㸦ࣄࢺ࡟ᑐࡋ࡚ࡀࢇཎᛶࡀ࠶ࡿ㸧࠾ࡼࡧࢢ࣮ࣝࣉ 2A㸦ࣄࢺ࡟ᑐࡋ࡚࠾ࡑࡽࡃ
ࡀࢇཎᛶࡀ࠶ࡿ㸧ࢆࣄࢺࡀࢇཎ≀㉁࡜ࡋࡓሙྜࠊSHE CTA㸦pH6.7 ἲࡲࡓࡣ pH7.0 ἲࡢ࠸ࡎࢀ࠿୍᪉࡛ࡶ
㝧ᛶࡢሙྜࢆ SHE CTA 㝧ᛶ࡜ุᐃ㸧࡟࠾ࡅࡿ୍⮴⋡ࡣ 55.0%ࠊឤᗘࡣ 93.7%ࠊ≉␗ᗘࡣ 32.1%࡜ࣄࢺࡀࢇཎ≀㉁ࡢ᳨ฟឤᗘࡢ㧗࠸⤖ᯝ࡛࠶ࡗࡓ㸦Appendix II㸧ࠋࡉࡽ࡟ࠊIARC ศ㢮ẖࠊ SHE CTA ࠾ࡼࡧࡀࢇཎᛶヨ㦂⤖ᯝࡢヨ㦂ἲẖ࡟ࡑࢀࡽࡢ┦㛵ࢆ⾲ 9-1 ࡟♧ࡋ
ࡓࠋ

16

22

⾲ 9-1 ࣄࢺ࡟࠾ࡅࡿࡀࢇཎᛶࣜࢫࢡศ㢮㸦IARC ศ㢮㸧࡜ SHE CTA ࠾ࡼࡧࡀࢇཎᛶヨ㦂⤖ᯝ࡜ࡢ┦㛵ᛶ
࢔ࢵࢭ࢖⣔

ุᐃ

ࣄࢺ࡟ᑐࡍࡿࡀࢇཎᛶ
ࢢ࣮ࣝࣉ 1ࢢ࣮ࣝࣉ 2A

ࢢ࣮ࣝࣉ 2B

ࢢ࣮ࣝࣉ 3

SHE CTA㝧ᛶ

6 (66.7%)

6 (100.0%)

12 (75.0%)

8 (36.4%)(pH 6.7 ἲ)㝜ᛶ

3 (33.3%)

0 (0.0%)4 (25.0%)

14 (63.8%)

ィ

9

6

16

22

SHE CTA㝧ᛶ

43 (95.6%)

13 (92.9%)

40 (87.0%)

24 (51.1%)(pH 7.0 ἲ)㝜ᛶ

2 (4.4%)1 (7.1%)6 (13.0%)23 (48.9%)

ィ

45

14

46

47

ᐇ㦂ື≀

㝧ᛶ

48 (100.0)

16 (94.1%)

50 (94.3%)

22 (39.3%)

࡟࠾ࡅࡿ

㝜ᛶ

0 (0.0)1 (5.9%)3 (5.7%)34 (60.7%)

ࡀࢇཎᛶヨ㦂

ィ

48

17

53

56

ࡇࢀࡽ≀㉁࡟ࡣࠊࡆࡗṑ㢮ࡢࡀࢇཎᛶヨ㦂࡛㝧ᛶࡢ 136 ≀㉁㸦᭷ᶵ໬ྜ≀㸸94 ≀㉁ࠊ↓ᶵ໬ྜ≀㸸42≀㉁㸧࡜㝜ᛶࡢ 38 ≀㉁㸦᭷ᶵ໬ྜ≀㸸34 ≀㉁ࠊ↓ᶵ໬ྜ≀㸸4 ≀㉁㸧ࡀྵࡲࢀ࡚࠸ࡿࡀࠊࡉࡽ࡟᭷ᶵ໬
ྜ≀࡜↓ᶵ໬ྜ≀࡟ศ㢮ࡋ࡚⾲ 9-2 ࡟♧ࡋࡓࠋ

1723

⾲ 9-2 ࣄࢺ࡟࠾ࡅࡿࡀࢇཎᛶࣜࢫࢡศ㢮㸦IARC ศ㢮㸧࡜ SHE CTA ࠾ࡼࡧࡀࢇཎᛶヨ㦂⤖ᯝ࡜ࡢ┦㛵ᛶ㸦᭷ᶵ໬ྜ≀㸧࢔ࢵࢭ࢖⣔ุᐃࣄࢺ࡟ᑐࡍࡿࡀࢇཎᛶ
ࢢ࣮ࣝࣉ 1ࢢ࣮ࣝࣉ 2A

ࢢ࣮ࣝࣉ 2B

ࢢ࣮ࣝࣉ 3

SHE CTA㝧ᛶ

3 (50.0%)

6 (100.0%)

10 (76.9%)

7 (33.3%)(pH 6.7 ἲ)㝜ᛶ

3 (50.0%)0 (0.0%)3 (23.1%)

14 (66.7%)

ィ

6

6

13

21

SHE CTA㝧ᛶ

11 (84.6%)

12 (92.3%)

34 (87.2%)

21 (50.0%)(pH 7.0 ἲ)㝜ᛶ

2 (15.4%)

1 (7.7%)5 (12.8%)

21 (50.0%)

ィ

13

13

39

42

ᐇ㦂ື≀

㝧ᛶ

15 (100.0)

15 (93.8%)

43 (93.5%)

21 (41.2%)

࡟࠾ࡅࡿ

㝜ᛶ

0 (0.0)1 (6.2%)3 (6.5%)30 (58.8%)

ࡀࢇཎᛶヨ㦂

ィ

15

16

46

51

㸦↓ᶵ໬ྜ≀㸧࢔ࢵࢭ࢖⣔ุᐃࣄࢺ࡟ᑐࡍࡿࡀࢇཎᛶ
ࢢ࣮ࣝࣉ 1ࢢ࣮ࣝࣉ 2A

ࢢ࣮ࣝࣉ 2B

ࢢ࣮ࣝࣉ 3

SHE CTA㝧ᛶ

3 (100.0%)

0 (0.0%)2 (66.7%)

1 (100.0%)(pH 6.7 ἲ)㝜ᛶ

0 (0.0%)0 (0.0%)1 (33.3%)

0 (0.0%)

ィ

3

0

3

1

SHE CTA㝧ᛶ

32(100.0%)

1 (100.0%)

6 (85.7%)

3 (60.0%)(pH 7.0 ἲ)㝜ᛶ

0 (0.0%)0 (0.0%)1 (14.3%)

2 (40.0%)

ィ

32

1

7

5

ᐇ㦂ື≀

㝧ᛶ

33 (100.0)

1 (100.0%)

7 (100.0%)

1 (20.0%)

࡟࠾ࡅࡿ

㝜ᛶ

0 (0.0)0 (0.0%)0 (0.0%)4 (80.0%)ࡀࢇཎᛶヨ㦂

ィ

33

1

7

5

ࢢ࣮ࣝࣉ 1 ࡟࠾ࡅࡿ≀㉁࡛ࡢ SHE CTA ࡢ᭷ᶵ໬ྜ≀ࡢ㝧ᛶ⋡ࡣࠊpH 6.7 ἲ࡛ࡣ 50.0%ࠊpH 7.0 ἲ࡛ࡣ
84.6%࡛࠶ࡾࠊᐇ㦂ື≀࡟࠾ࡅࡿࡀࢇཎᛶヨ㦂ࡢ⤖ᯝ㸦100.0%㸧࡜ẚ㍑ࡍࡿ࡜౛ᩘࡀᑡ࡞࠸ࡶࡢࡢప࠸್
࡛࠶ࡗࡓࠋ୍᪉ࠊ↓ᶵ໬ྜ≀࡟ࡘ࠸࡚ࡣࠊSHE CTA ࠾ࡼࡧࡀࢇཎᛶヨ㦂࡜ࡶ࡟ 100.0%ࡢ㝧ᛶ⋡࡛࠶ࡗࡓࠋ
ࢢ࣮ࣝࣉ 2A ࡟࠾ࡅࡿ᭷ᶵ໬ྜ≀ࡢ㝧ᛶ⋡ࡣࠊpH 6.7 ἲ࡛ࡣ 100.0%ࠊpH 7.0 ἲ࡛ࡣ 92.3%࡛࠶ࡾࠊࡀࢇ
ཎᛶヨ㦂ࡢ⤖ᯝ㸦93.8%㸧࡜ྠ➼࡛࠶ࡗࡓࠋ↓ᶵ໬ྜ≀࡟ࡘ࠸࡚ࡣ 1 ≀㉁ࡢࡳ࡛࠶ࡿࡀࠊ࠸ࡎࢀࡢ pH ࡛ࡶ㝧ᛶ࡛࠶ࡗࡓࠋ
ࢢ࣮ࣝࣉ 2B㸦ࣄࢺ࡟ᑐࡋ࡚ࡀࢇཎᛶࡢྍ⬟ᛶࡀ࠶ࡿ㸧࡟࠾ࡅࡿ᭷ᶵ໬ྜ≀ࡢ SHE CTA ࡢ㝧ᛶ⋡㸦76.9
18

24

࠾ࡼࡧ 87.2%㸧ࡣࠊࡀࢇཎᛶヨ㦂ࡢ⤖ᯝ㸦93.5%㸧ࡼࡾࢃࡎ࠿࡟పୗࡋࡓࠋ↓ᶵ໬ྜ≀ࡶྠᵝ࡟ࠊSHE CTAࡢ㝧ᛶ⋡㸦66.7 ࠾ࡼࡧ 85.7%㸧ࡣࡀࢇཎᛶヨ㦂ࡢ⤖ᯝ㸦100.0%㸧ࡼࡾపୗࡋࡓࠋ
ࢢ࣮ࣝࣉ 3㸦ࣄࢺ࡟ᑐࡍࡿⓎ⒴ᛶࡀศ㢮࡛ࡁ࡞࠸㸧࡟࠾ࡅࡿ᭷ᶵ໬ྜ≀ࡢ SHE CTA ࡢ㝧ᛶ⋡㸦ഇ㝧ᛶ⋡࡜ྠࡌ㸧ࡣࠊpH 6.7 ἲ࡛ࡣ 33.3%ࠊpH 7.0 ἲ࡛ࡣ 50.0%࡛࠶ࡾࠊࡀࢇཎᛶヨ㦂ࡢ⤖ᯝ㸦41.2%㸧࡜࡯ࡰྠ➼࡛࠶ࡗࡓࠋࡑࡢ㝜ᛶ⋡㸦≉␗ᗘ࡜ྠࡌ㸧ࡶࠊpH 6.7 ἲ࡛ࡣ 66.7%ࠊpH 7.0 ἲ࡛ࡣ 50.0%࡛ࠊࡀࢇཎᛶヨ㦂㸦58.6%㸧࡜࡯ࡰྠ➼࡛࠶ࡗࡓࠋ

8.

ヨ㦂᪉ἲࡢಙ㢗ᛶ1) ᪋タ㛫ኚື(1) ECVAM ࡢࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲SHE CTA ࡢ᪋タ㛫ࡢ෌⌧ᛶࡣࠊpH6.7 ࠾ࡼࡧ pH7.0 ἲࡢ࡝ࡕࡽࡶࠊ⾲ 10 ࡟♧ࡍࡼ࠺࡟ 4 ࡘࡢࡀࢇཎ≀㉁࡜ 2 ࡘࡢ㠀ࡀࢇཎ≀㉁ࠊィ 6 ≀㉁ࢆ⏝࠸᳨࡚ドࡉࢀࡓ㸦Benzo[a]pyrene ࡣ㝧ᛶᑐ↷≀㉁࡜ࡋ࡚ࡢホ౯㸧6,7)ࠋ

⾲ 10 ECVAM ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲᳨࡛ウࡉࢀࡓ⿕㦂≀㉁໬Ꮫ≀㉁CAS ␒ྕ

ࡆࡗṑ㢮ࡀࢇཎᛶ㸦ฟ඾㸧

Benzo[a]pyrene

50-32-8

࠶ࡾ㸦IARC, 2009㸧

Anthracene120-12-7

࡞ࡋ㸦IARC, 2009㸧

2,4-Diaminotoluene

95-80-7

࠶ࡾ㸦IARC, 2009㸧

3-Methylcholanthrene

56-49-5

࠶ࡾ㸦Gold and Zeiger, 1997㸧o-Toluidine HCl

636-21-5

࠶ࡾ㸦NTP㸧

Phthalic anhydride

85-44-9࡞ࡋ㸦NTP㸧

pH7.0 ἲ࡟ࡘ࠸࡚ࡣࠊ4 ᪋タ࡟࡚ヨ㦂ࢆ⾜࠸ࠊホ౯ࡋࡓ⤖ᯝࠊPhthalic anhydride ࢆ㝖ࡃ඲࡚ࡢ⿕㦂≀㉁࡟
ࡘ࠸ุ࡚ᐃ⤖ᯝࡀ୍⮴ࡋࠊື≀ࡀࢇཎᛶࡢ⤖ᯝ࡜ࡶྜ⮴ࡋࡓࠋPhthalic anhydride ࡟ࡘ࠸࡚ࡣࠊ1 ᪋タ࡟࡚
1 ⃰ᗘࡢࡳᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢ᭷ព࡞ቑຍࡀ࠶ࡾࠊഴྥ᳨ᐃ࡛ࡶ᭷ព࡞ഴྥࢆ♧ࡋࡓࡇ࡜࠿ࡽ㝧ᛶ࡜ࡢุᐃ࡟࡞ࡗࡓࡀࠊ௚ࡢ᪋タ࡛ࡣື≀ࡀࢇཎᛶࡢホ౯⤖ᯝ࡜ྠࡌࡃࠊ㝜ᛶ࡜ุᐃࡉࢀࡓࠋ
pH6.7 ἲ࡟ࡘ࠸࡚ࡣࠊ3 ᪋タ࡟࡚ヨ㦂ࢆ⾜࠸ࠊホ౯ࡋࡓ⤖ᯝࠊ඲࡚ࡢ⿕㦂≀㉁ࡢุᐃ⤖ᯝࡀ୍⮴ࡋࠊPhthalicanhydride ࢆ㝖࠸࡚ࡣື≀ࡀࢇཎᛶヨ㦂⤖ᯝ࡜ࡶྜ⮴ࡋࡓࠋPhthalic anhydride ࡢ SHE CTA ࡢホ౯⤖ᯝࡣྛ᪋タ࡜ࡶ㝧ᛶ࡜ุᐃࡋࡓࡀࠊື≀ࡀࢇཎᛶࢆ♧ࡉ࡞࠸࡜ࡍࡿ NTP ࡢሗ࿌࡜▩┪ࡋࠊࡲࡓୖグࡢ pH7.0 ἲࡢ SHE CTA ࡟࠾ࡅࡿホ౯⤖ᯝ࡜ࡶ␗࡞ࡿࠋࡇࡢ⌮⏤࡜ࡋ࡚ Phthalic anhydride ࡢ␗࡞ࡿ pH ࡛ࡢᏳᐃᛶࡢ
㐪࠸ࡀᣦ᦬ࡉࢀ࡚࠸ࡿࠋ
ࡘࡲࡾ Phthalic anhydride ࡣỈ⎔ቃୗ࡛㏿ࡸ࠿࡟ຍỈศゎࡋࠊ㠀㑇ఏẘᛶࡢ Phthalicacid ࡟ኚ໬ࡍࡿࡀࠊSHE CTA ࡛ࡣ౑⏝ࡍࡿ⦆⾪ᾮࡀ␗࡞ࡾࠊࡼࡾప࠸ pH ࡛ࡣ Phthalic anhydride ࡢ༙ῶᮇࡀ㛗ࡃ࡞ࡿࡇ࡜࠿ࡽࠊ␗࡞ࡗࡓヨ㦂⤖ᯝࡀᚓࡽࢀࡓྍ⬟ᛶࡀ♧ࡉࢀ࡚࠸ࡿࠋ ୖグࡢ᪋タ㛫ࡢヨ㦂⤖ᯝࢆ⥲ྜⓗ࡟ホ౯ࡋࡓ⤖ᯝࠊྛ᪋タ࡛ࡢ SHE CTA ࡢホ౯⤖ᯝࡣᇶᮏⓗ࡟୍⮴ࡍ1925ࡿࡶࡢ࡛࠶ࡾࠊpH6.7 ἲ࠾ࡼࡧ pH7.0 ἲ࡜ࡶࡑࡢ᪋タ㛫෌⌧ᛶࡣ㧗࠸࡜ุ᩿ࡉࢀࡓࠋ
ุᐃ⤖ᯝ࡟㛵ࡋ࡚ࠊࢥࣟࢽ࣮ࡢ࢝࢘ࣥࢺࡀ୺ほⓗ࡛࠶ࡿࡇ࡜࡟ࡼࡿุᐃࡢࡤࡽࡘࡁࡀᠱᛕࡉࢀ࡚࠸ࡓ
ࡀࠊ෗┿ᶆᮏࡢᥦ౪ࡀุᐃࡢࡤࡽࡘࡁࡢᢚไ࡟኱ࡁࡃᐤ୚ࡋ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡓࠋ
(2) ࡑࡢ௚ࡢᩥ⊩ሗ࿌࡜ࡢ୍⮴ᛶ
pH7.0 ἲ࡟ࡘ࠸࡚ࡣࠊ㢮ఝࣉࣟࢺࢥࣝ࡟ࡼࡿ෌⌧ᛶࢆホ౯ࡋࡓᩥ⊩ሗ࿌ 14)࡞ࡽࡧ࡟ 2 ᶵ㛵௨ୖ࡛ホ౯ࡉࢀࡓ≀㉁ࡢ 87.7㸣㸦57/65㸧ࡀ୍⮴ࡍࡿ࡜ࡋࡓ DRP31 ࢆཧ↷ࡋࡓࠋࡲࡓ pH6.7 ἲ࡟ࡘ࠸࡚ࡣࠊ㢮ఝࣉࣟࢺࢥࣝ࡟ࡼࡿ෌⌧ᛶࢆホ౯ࡋࡓᩥ⊩ሗ࿌ 15,16)࠾ࡼࡧୖグ DRP31 ࢆཧ↷ࡋࡓࠋࡇࢀࡽࡢሗ࿌࠿ࡽࠊⰋዲ࡞᪋タ㛫෌⌧ᛶࢆ♧ࡍ࡜⪃࠼ࡽࢀࡓࠋ

2)

᪋タෆኚື
᪋タෆ෌⌧ᛶ࡟ࡘ࠸࡚ࡣࠊpH6.7 ἲ࠾ࡼࡧ pH7.0 ἲࡢ୧᪉࡟ࡘ࠸࡚ࠊࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀホ౯ࡉࢀ࡚࠸ࡿࡀࠊ㝧ᛶᑐ↷࡜ࡋ࡚౑⏝ࡋࡓ 1 ໬ྜ≀㸦Benzo[a]pyrene㸧ࡢ⤖ᯝࡢࡳࡢホ౯࡜࡞ࡗ࡚࠸ࡿ 6,7)ࠋ
pH6.7 ἲ࡛ࡣࠊࢥ࣮ࢻ໬ࡏࡎ࡟ᐇ᪋ࡋࡓ⤖ᯝ࡜ࢥ࣮ࢻ໬ࡋ࡚ᐇ᪋ࡋࡓ 3 ᪋タࡢ⤖ᯝࢆẚ㍑ࡋ࡚ホ౯ࡋ࡚࠸ࡿࠋࡑࡢ⤖ᯝࠊ1 ᪋タ࡛ࢥࣥࢺ࣮ࣟࣝࡢᙧ㉁㌿᥮⋡ࡀ௚ࡢ 2 ᪋タ࡜␗࡞ࡿ್ࡀᚓࡽࢀ࡚࠸ࡿࡀࠊ㝧ᛶᑐ↷≀㉁࡛࠶ࡿ Benzo[a]pyrene ࡢ⤖ᯝࡣ࡯ࡰྠᵝࡢ್ࡀᚓࡽࢀ࡚࠸ࡿࠋ୍᪉ࠊpH7.0 ἲ࡛ࡣࢥ࣮ࢻ໬ࡋࡓ
Benzo[a]pyrene ࢆ⏝࠸࡚ 1 ᪋タࡢࡳࡀ 3 ᅇࡢ⧞ࡾ㏉ࡋᐇ㦂ࢆ⾜࠸ࠊⰋዲ࡞⤖ᯝࡀᚓࡽࢀ࡚࠸ࡿࠋ
ESAC ࡢሗ࿌ 10)࡟࠶ࡿࡼ࠺࡟ࠊࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛ࡣ᪋タෆ෌⌧ᛶࢆㄪ࡭ࡿࡓࡵࡢ㐺ษ࡞ᐇ㦂ࢹࢨ࢖ࣥࡀ⤌ࡲࢀ࡚࠾ࡽࡎࠊ⌧ᅾࡢホ౯ᇶ‽࡛⪃࠼ࡓሙྜࠊ᪋タෆ෌⌧ᛶ࡟ࡘ࠸࡚ࡣ༑ศ࡞ホ౯ࡀ⾜ࢃࢀ࡚࠸ࡿ࡜ࡣ⤖ㄽ࡛ࡁ࡞࠸ࠋ

9.

ヨ㦂᪉ἲࡢࢹ࣮ࢱࡢ㉁䞉ࣉࣞࣂࣜࢹ࣮ࢩࣙࣥሗ࿌᭩࡟ࡼࡿ࡜ࠊࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢ᪋タෆ෌⌧ᛶࡢ☜ㄆࢆ」ᩘࡢ໬ྜ
≀࡛ᐇ᪋ࡋ࡞࠿ࡗࡓ࡞࡝᪉ἲ࡟୙༑ศ࡞Ⅼࡀ࠶ࡗࡓࡶࡢࡢࠊヨ㦂ᐇ᪋࡟ᚲせ࡞ࢫ࢟ࣝࡢカ⦎ࢆཷࡅࡓ᪋タ࡛ࡢ᪋タෆ/᪋タ㛫෌⌧ᛶࡣ☜ಖࡉࢀ࡚࠾ࡾࠊࢹ࣮ࢱࡢ㉁ࡣᢸಖࡉࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ䞉ホ౯ୖ᭱ࡶ㔜せ࡞ࢹ࣮ࢱࡣࠊᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢィ್࡛࠶ࡿࠋ⏬ീ࢔ࢺࣛࢫ࡟ࡼࡾᶆ‽໬ࡉࢀ࡚࠸ࡿࡶࡢࡢࠊᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡣ┠どุู࡛ࡍࡿࡓࡵࠊุูἲࡢᐈほᛶ࡟ஈࡋ࠸ࠋࡇࡢࡓࡵࠊࢹ࣮
ࢱࡢ㉁ࡢᢸಖ࡟ࡣࠊࢥ࣮ࢻ໬ࡋ࡚ྲྀᚓࡉࢀࡓࢹ࣮ࢱ࡛࠶ࡿࡇ࡜ࡀ㔜せ࡜࡞ࡿࠋࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ
◊✲ࡢ໬ྜ≀ࡣࢥ࣮ࢻ໬ࡉࢀࠊᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢィࡣࢥ࣮ࢻ໬ࡋ࡚⾜ࢃࢀࡓࠋᚑࡗ࡚ࠊྲྀᚓࡉ

ࢀࡓࢹ࣮ࢱࡢ㉁ࡣᢸಖࡉࢀ࡚࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ
䞉ヨ㦂ࡢጇᙜᛶࢆᢸಖࡍࡿࡓࡵࡢࡶ࠺୍ࡘࡢ㔜せ࡞ DRP31 ࡛᥇⏝ࡉࢀࡓࢹ࣮ࢱࡣࢥ࣮ࢻ໬ࡋ࡚ྲྀᚓ
ࡉࢀࡓࡶࡢ࡛ࡣ࡞࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡢุᐃᇶ‽ࢆ௨࡚ DRP31 ࢹ࣮ࢱࢆ෌ホ౯ࡋࡓ⤖ᯝࠊឤᗘࡸ≉␗ᗘ࡟኱ᕪࡀ࡞࠿ࡗࡓࡇ࡜࠿ࡽࠊࡇࢀࡽ࡟ࡘ࠸࡚ࡶࢹ࣮ࢱࡢ㉁ࡣᢸಖࡉࢀ࡚
࠸ࡿ࡜⪃࠼ࡽࢀࡿࠋ

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26

10.

ヨ㦂᪉ἲ࡟㛵ࡍࡿ௚ࡢ⛉Ꮫⓗ࡞ሗ࿌

SHE CTA ࡜ྠᵝ࡟⣽⬊ࡢ CTA ࡟ࡣ௨ୗࡢ᪉ἲࡀ㛤Ⓨࡉࢀ࡚࠸ࡿࠋ
1) ⣽⬊ᰴ BALB/c3T3 ࢆ⏝࠸ࡿ CTA2) ⣽⬊ᰴ Bhas42 ࢆ⏝࠸ࡿ CTAͤ3) ⣽⬊ᰴ C3H10T1/2 ࢆ⏝࠸ࡿ CTA
4) JB6 ⣽⬊ࢆ⏝࠸ࡿ CTA
2) Bhas42 ࢆ⏝࠸ࡿ CTA ࡀ 2016 ᖺ 1 ᭶ OECD ࢞࢖ࢲࣥࢫ 17)࡜࡞ࡗࡓࠋͤୖグࡢ 1) ࠥ3) ࡟ࡘ࠸࡚ࡣࠊSHE CTA ࡜ྠᵝ࡟ᙧ㉁㌿᥮ࡋࡓ⣽⬊࡟ࡘ࠸࡚ࡢホ౯࡛࠶ࡿࡀࠊᙧᡂࡉࢀ
ࡓࢥࣟࢽ࣮୰ࡢᙧ㉁㌿᥮ࡋࡓࢥࣟࢽ࣮ᩘࢆᩘ࠼ࡿࡢ࡛ࡣ࡞ࡃࠊ༢ᒙࡢṇᖖ⣽⬊୰࡛ቑṪࡋࡓᙧ㉁㌿᥮⣽⬊ࡀᙧᡂࡍࡿࣇ࢛࣮࢝ࢫ㸦ᙧ㉁㌿᥮⣽⬊ࡢ㞟ྜయ㸧ࡢᩘࢆィࡍࡿࡶࡢ࡛࠶ࡿࠋ4㸧ࡢ JB6 ⣽⬊࡟ࡘ࠸࡚ࡣࠊ㌾ᐮኳᇵᆅ࡛ࡢቑṪ⬟㸦㊊ሙ౫Ꮡᛶࡢ႙ኻ㸧ࢆᣦᶆ࡜ࡍࡿヨ㦂࡛࠶ࡿࠋ

11.

3Rs ࡬ࡢ㛵୚㸦ື≀⚟♴㠃࠿ࡽࡢጇᙜᛶ㸧SHE CTA ࡛ࡣࢩࣜ࢔࣭ࣥࣁ࣒ࢫࢱ࣮ࡢ⫾௘࠿ࡽᚓࡓึ௦ᇵ㣴⣽⬊ࢆ⏝࠸ࡿࡓࡵࠊື≀ࢆ≛≅࡟ࡍࡿᚲせࡀ࠶ࡿࠋࡑࡢࡓࡵࠊ௚ࡢᇵ㣴⣽⬊㸦Bhas42㸪BALB/c 3T3㸧ࢆ⏝࠸ࡿ CTA ࡜ẚ㍑ࡋ࡚ࠊື≀⚟♴㠃࡛ࡣ୙฼࡛࠶ࡿࡇ࡜ࡣྰࡵ࡞࠸ࠋࡋ࠿ࡋ࡞ࡀࡽࠊ୍༉ࡢዷፎື≀⏤᮶ࡢ⫾௘⣽⬊࡛⣙ 50 ᅇࡢ CTA ࡀᐇ᪋ྍ⬟࡛࠶ࡿࡇ࡜ࠊ࠾ࡼࡧᮏヨ㦂ࢆྵࡴ CTA ࡣࡀࢇ໬㐣⛬࡟࠾ࡅࡿ⏕≀Ꮫⓗࣉࣟࢭࢫࢆホ౯ࡍࡿࡓࡵࠊ㐺ษ࡟ᐇ᪋ࡍࡿࡇ࡜࡟ࡼࡗ࡚ࠊከᩘࡢື≀ࢆ౑⏝ࡍࡿࡀࢇཎᛶヨ㦂࠾ࡼࡧ㛵㐃ヨ㦂ࢆᅇ㑊ࠊ࠶ࡿ࠸ࡣ┬␎ࡍࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿࡓࡵࠊື≀⚟♴࡬ࡢᐤ୚ࡣ኱ࡁ࠸࡜⪃࠼ࡽࢀࡿࠋ

12. ヨ㦂᪉ἲࡢ᭷⏝ᛶ࡜㝈⏺
1)᭷⏝ᛶ(1) CTA䞉ࡀࢇ໬ࡢ㐣⛬ࢆㄝ᫂ࡍࡿከẁ㝵ㄝ࡟ࡼࢀࡤࠊኚ␗ཎ≀㉁࡟ࡼࡾ࢖ࢽࢩ࢚࣮ࢩࣙࣥస⏝ࢆཷࡅࡓ⣽⬊ࡣࠊ᭦࡟ࡑࡢᚋࡉࡲࡊࡲ࡞せᅉ࡟ࡼࡗ࡚ᝏᛶᙧ㉁ࢆ⋓ᚓࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿࠋCTA ࡣࠊᇵ㣴ṇᖖ⣽⬊ᡈ࠸ࡣᰴ⣽⬊ࡀᙧែᙧ㉁㌿᥮ࡍࡿࡇ࡜ࢆᣦᶆ࡟ࡋ࡚ࠊ໬Ꮫ≀㉁ࡢᙧ㉁㌿᥮⬟ࡢ᭷↓ࢆணࡍࡿ

in vitro ヨ㦂ἲࡢ୍ࡘ࡜ࡋ࡚ࡢ≉ᚩࡀ࠶ࡿࠋ
䞉௚ࡢࡀࢇཎᛶண

ヨ㦂⤖ᯝ࡜࡜ࡶ࡟ࠊWoE ࡢ୍ࡘ࡜ࡋ࡚ CTA ࡢ⤖ᯝࢆ฼⏝ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋᮏ

ヨ㦂ἲࡣ⌧⾜ࡢࡀࢇཎᛶヨ㦂࡟᏶඲࡟⨨ࡁ᥮ࢃࡿヨ㦂ἲ࡜ࡋ࡚ࡢ఩⨨࡙ࡅ࡛ࡣ࡞ࡃࠊ⿵ຓⓗ࡞ヨ㦂㸦౛࠼ࡤࠊࡀࢇཎᛶヨ㦂ࡢᐇ᪋㡰఩ࢆỴࡵࡿࡓࡵࡢࢫࢡ࣮ࣜࢽࣥࢢࠊࡀࢇཎᛶヨ㦂ࡀせồࡉࢀ࡞࠸࢝ࢸࢦ࣮ࣜࡢ་⸆ရࡢࡀࢇཎᛶ㛵㐃᝟ሗࡢྲྀᚓ࡞࡝㸧࡜ࡋ࡚఩⨨࡙ࡅࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿࠋ䞉3Rs ࡢほⅬ࠿ࡽࡶ᭷⏝࡛࠶ࡿࠋ

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(2) SHE CTACTA ࡢ᭷⏝ᛶ࡟ຍ࠼࡚௨ୗࡢ᭷⏝ᛶࢆㄆࡵࡿࠋ
䞉DRP31 ࡢ 245 ໬Ꮫ≀㉁࡛ホ౯ࡉࢀࡓ⤖ᯝ࡟࠾࠸࡚ࠊSHE CTA ࡢ⤖ᯝ࡜ࡀࢇཎᛶヨ㦂⤖ᯝ࡜ࡢ┦㛵ࡀㄆࡵࡽࢀࡿࠋ
䞉SHE CTA ࡢ⣽⬊ㄪ〇ࡣ෾⤖ಖᏑࡋࡓึ௦஧ಸయ⣽⬊ࢆ⏝࠸ࡿࡀࠊᚲせ࡜ࡍࡿື≀ᩘࡣࡀࢇཎᛶヨ㦂࡟⏝࠸ࡿື≀ᩘ࡟ẚ࡭ࡣࡿ࠿࡟ᑡ࡞࠸ࠋ
䞉3Rs ࡢほⅬ࠿ࡽ᭷⏝࡛࠶ࡿࡇ࡜࡟ຍ࠼࡚ࠊ୙Ṛ໬ᇵ㣴⣽⬊ࢆ⏝࠸ࡿ CTA ࡜ẚ㍑ࡋ࡚ࠊึ௦ᇵ㣴⣽⬊ࢆ⏝࠸ࡿࡇ࡜࠿ࡽࠊࡼࡾṇᖖ⣽⬊࡟㏆࠸≧ែࢆ⥔ᣢࡋࡓᐇ㦂⣔࡛࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋ

2)

ၥ㢟Ⅼ㸦㝈⏺ࠊ୙฼Ⅼࠊ㐺⏝㝈⏺㸧(1) CTA䞉CTA ࡛⤖ᯝࡀ㝧ᛶ࡛࠶ࡗ࡚ࡶࠊ໬Ꮫ≀㉁ࡢᙧ㉁㌿᥮⬟࠿ࡽࠊࡑࡢ໬Ꮫ≀㉁ࡢ in vivo ࡟࠾ࡅࡿࡀࢇཎ
ᛶࡢᙉࡉࠊ⮚ჾ≉␗ᛶࠊ࠾ࡼࡧ✀≉␗ᛶ࡟㛵ࡍࡿ᝟ሗࡣᚓࡽࢀ࡞࠸ࠋ
䞉CTA ࡢ࢚ࣥࢻ࣏࢖ࣥࢺ࡛࠶ࡿᙧ㉁㌿᥮ࡢⓎ⏕ᶵᗎࡀ᫂ࡽ࠿࡟࡞ࡗ࡚࠸࡞࠸ࡓࡵࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢస⏝ᶵᗎ࡟㛵ࡍࡿ᝟ሗࡣᚓࡽࢀ࡞࠸ࠋ
䞉୍⯡ⓗ࡞ in vitro ヨ㦂࡜ྠᵝ࡟ࠊ⤖ᯝࡢጇᙜᛶࡣᶆ‽໬ࡉࢀࡓࣉࣟࢺࢥࣝ࡟ᚑࡗ୍࡚㐃ࡢヨ㦂᧯సࡀṇ☜࡟⾜ࢃࢀࡿࡇ࡜࡟ࡼࡗ࡚ಖドࡉࢀࡿࠋCTA ࡢ⤖ᯝࡢṇ☜ᛶࢆಖドࡍࡿࡓࡵࠊヨ㦂᧯సࡣࡶࡕࢁ
ࢇࠊᙧ㉁㌿᥮ࢥࣟࢽ࣮/ࣇ࢛࣮࢝ࢫࡢุูࢫ࢟ࣝࡢ⩦⇍ࡀ㔜せ࡛࠶ࡿࠋᶆ‽≀㉁࡛ㄏᑟࡉࢀࡓᙧ㉁㌿᥮ࢥࣟࢽ࣮/ࣇ࢛࣮࢝ࢫࡢ⏬ീ࢔ࢺࣛࢫ➼ࢆ฼⏝ࡋࡓࢫ࢟ࣝ☜ㄆࡣ㔜せ࡛࠶ࡿࠋ䞉ᙧ㉁㌿᥮ࢥࣟࢽ࣮/ࣇ࢛࣮࢝ࢫࡢ⮬ືุูἲࡢ㛤Ⓨࡀ㐍ࢇ࡛࠸࡞࠸ࡓࡵࠊ⌧᫬Ⅼ࡛ࡢከ᳨య㧗㏿ࢫࢡ࣮ࣜࢽࣥࢢࡢሗ࿌ᩘࡣᑡ࡞࠸ࠋᗈ⠊࡞໬Ꮫ≀㉁ࡢᙧ㉁㌿᥮⬟ࢆ CTA ୍࡛ḟࢫࢡ࣮ࣜࢽࣥࢢࡍࡿࡓࡵ
࡟ࡣࠊヨ㦂⣔ࡢ᭦࡞ࡿᨵⰋࠊᡈ࠸ࡣࠊᙧ㉁㌿᥮ࢥࣟࢽ࣮/ࣇ࢛࣮࢝ࢫࡢ㠉᪂ⓗุู᪉ἲࡢ㛤Ⓨࡀᚲせ࡛࠶ࡿࠋ(2) SHE CTACTA ࡟࠾ࡅࡿၥ㢟Ⅼ࡟ຍ࠼࡚௨ୗࡢၥ㢟Ⅼࢆㄆࡵࡿࠋ
䞉SHE CTA ࡟ࡣࠊ࢖ࢽࢩ࢚࣮ࢩࣙࣥ࡜ࣉ࣮ࣟࣔࢩࣙࣥస⏝࡜ࢆ༊ูࡍࡿヨ㦂ἲࡀグ㍕ࡉࢀ࡚࠸࡞࠸ࡓ
ࡵࠊ࢖ࢽࢩ࢚࣮ࢩࣙࣥ/ࣉ࣮ࣟࣔࢩࣙࣥస⏝࡜࠸ࡗࡓほⅬ࡛ࡢ⿕㦂≀㉁ࡢస⏝ᶵᗎࢆ᫂ࡽ࠿࡟ࡍࡿࡇ
࡜ࡣ࡛ࡁ࡞࠸ࠋ
䞉SHE CTA ࡛ࡣࠊࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀ⾜ࢃࢀࡓ 6 ≀㉁㸦ణࡋࠊ㑅ᢥࡉࢀࡓ≀㉁࡟ࡣࠊస⏝࣓࢝ࢽࢬ࣒࡟೫ࡾࡀ࠶ࡿ࡜ࡉࢀ࡚࠸ࡿ㸧࡟ࡘ࠸࡚ࡣࠊࡀࢇཎᛶヨ㦂⤖ᯝ࡜ࡢ┦㛵㸦ឤᗘࠊ≉␗ᗘࠊഇ㝧ᛶ࣭ഇ㝜ᛶࡢ㆑ูࡢ⤖ᯝ㸧ࡣ☜ㄆࡉࢀ࡚࠸ࡿࡀࠊ㑅ᢥࡉࢀࡓ≀㉁࡟ࡣࠊస⏝ᶵస࡟೫ࡾࡀ࠶ࡾ㸪໬Ꮫ≀㉁ࡢᩘࡸ✀㢮ࡶ༑ศ࡛ࡣ࡞࠸ࠋホ౯ࡉࢀࡓ໬Ꮫ≀㉁࡟ࡣ㑇ఏẘᛶࢆ᭷ࡍࡿࡀࢇཎ≀㉁ࡀከࡃྵ
ࡲࢀࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁㸦͆┿ࡢ㸦bona fide㸧㠀㑇ఏẘᛶࡀࢇཎ≀㉁͇ࡢᐃ⩏࡟ࡣ㆟ㄽࡢవᆅࡀ
࠶ࡿ㸧ࡀᑡ࡞࠸ࡓࡵࠊ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢ᳨ฟ⣔࡜࠸࠺ほⅬ࡛ࡢ᭷⏝ᛶࡢ᳨ドࡣ୙༑ศ࡛࠶ࡿࠋ

22

28

3)

௒ᚋࡢㄢ㢟
⣽⬊ࡀᙧ㉁㌿᥮ࡍࡿస⏝ᶵᵓࡢゎ᫂ࡀ㔜せ࡛࠶ࡿࠋࡋ࠿ࡋࠊࡇࡢㄢ㢟ࡣࣁ࣮ࢻࣝࡀ㧗࠸ࠋ䞉ࣄࢺ⣽⬊࡛ࡶ㉳ࡇࡾ࠺ࡿ࠿ࢆ෌⌧ࡍࡿࡓࡵ࡟ࡣࠊࣄࢺ⣽⬊ࢆ⏝࠸ࡓ CTA ࡀ㛤Ⓨࡉࢀࡿࡇ࡜ࡀᮃࡲࡋ
࠸ࠋ㸦In vivo ࡟࠾ࡅࡿࠊື≀࠿ࡽࣄࢺ࡬ࡢእᤄ࡜ྠࡌၥ㢟ࢆྵࢇ࡛࠸ࡿࠋ㸧䞉ᙧែᙧ㉁㌿᥮ࢥࣟࢽ࣮ࡢุᐃࡢᐈほᛶࡢ☜ಖࡀᐇົୖᚲせ࡛࠶ࡿࠋ䞉┿ࡢ㸦bona fide㸧㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢᩘࢆቑࡸࡋ࡚ CTA ࢆከࡃᐇ᪋ࡋࠊከࡃࡢࢹ࣮ࢱࢆ཰㞟ࡍࡿࠋࣉࣞࣂࣜࢹ࣮ࢩࣙࣥ◊✲࡛⏝࠸ࡓ⿕㦂≀㉁࡟ࡣ┿ࡢ㠀㑇ఏẘᛶࡀࢇཎ≀㉁ࡢᩘࡀ㑇ఏẘᛶࡀࢇཎ≀㉁࡜ẚ㍑ࡋ࡚ᴟࡵ࡚ᑡ࡞࠿ࡗࡓࡇ࡜࡞࡝ࡀࠊOECD ࡢヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ᢎㄆ㸦ྠព㸧ࡉࢀ࡞࠿ࡗࡓ⌮⏤ࡢ୍ࡘ࡜ゝࢃࢀ࡚࠸ࡿࠋࡇࢀࡽࡢ⛉Ꮫⓗ࣭ᢏ⾡ⓗ࡞ၥ㢟Ⅼࡀඞ᭹ࡉࢀࠊࡲࡓṇᘧ࡞ࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀࢀࡤࠊCTA ࡀࢸࢫࢺ࢞࢖ࢻࣛ࢖ࣥ໬ࡉࢀࡿྍ⬟ᛶࡣ࠶ࡿࠋ
䞉SHE CTA ࡛ࡣࠊᇶᮏⓗ࡞⸆≀௦ㅰᶵ⬟ࢆಖᣢࡋ࡚࠸ࡿ⣽⬊ࢆ⏝࠸ࡿࡇ࡜࠿ࡽࠊ௦ㅰάᛶ໬ࢆᚲせ࡜
ࡍࡿᵝࠎ࡞໬Ꮫ≀㉁ࡢᙧ㉁㌿᥮⬟ࡀ᳨ฟྍ⬟࡛࠶ࡿࠋࡲࡓࠊእ㒊௦ㅰάᛶ໬⣔ࢆ㐺⏝ࡍࡿࡇ࡜࡟ࡼ
ࡾࠊᙧ㉁㌿᥮⬟ࡢቑᙉࡸ᪂ࡓ࡞ᙧ㉁㌿᥮⬟ࢆᣢࡘ໬Ꮫ≀㉁ࢆ᳨ฟ࡛ࡁࡿ࡜ࡋࡓሗ࿌ 18-21) ࡀᑡᩘ࡛ࡣ
࠶ࡿࡀබ⾲ࡉࢀ࡚࠸ࡿࠋࡍ࡞ࢃࡕࠊ㏻ᖖࡢᙧ㉁㌿᥮ヨ㦂࡛㝜ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓ≀㉁࡟㛵ࡋ࡚ࡶࠊእ
㒊௦ㅰάᛶ໬⣔ࢆ㐺⏝ࡍࡿࡇ࡜࡟ࡼࡾࠊ᭦࡟ࡇࡢヨ㦂⣔ࡢṇ☜ᛶࡀ㧗ࡲࡿྍ⬟ᛶࡀ࠶ࡿࠋ

13.

⤖ㄽ

1) CTA ࡣ in vitro ࡟࠾࠸࡚ࠊ໬Ꮫ≀㉁ࢆ᭚㟢ࡍࡿࡇ࡜࡛㉳ࡁࡿࠕ⣽⬊ࡢᙧែࠖࡸࠕ⾲⌧ᙧ㉁ࠖࡢᝏᛶ໬ࢆ
ᣦᶆ࡜ࡋ࡚⭘⒆ㄏⓎᛶࡢ᭷↓ࢆㄪ࡭ࡿヨ㦂ἲࡢ୍ࡘ࡛࠶ࡿࠋ
2) ᙧ㉁㌿᥮ࡋࡓ⣽⬊ࡀᝏᛶ໬ࡍࡿࡇ࡜࡛ࠊࢾ࣮ࢻ࣐࢘ࢫࡸྠ⣔⤫ࡢື≀࡬ࡢ㐀⭘⒆ᛶࡀㄆࡵࡽࢀࡿࡓࡵࠊ
ࡀࢇཎᛶ࡜ࡣఱࡽ࠿ࡢ㛵ಀࡀ࠶ࡿࡇ࡜ࡀ᥎ᐹࡉࢀࡿࠋ
3) ໬Ꮫ≀㉁ࢆ⏝࠸ࡓ CTA ࡢ⤖ᯝ࡜ࠊࡆࡗṑ㢮ࡀࢇཎᛶヨ㦂ࡢ⤖ᯝ࡟ࡣ┦㛵ࡀ࠶ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸
ࡿࠋ4) SHE CTA ࡢ⤖ᯝ࡜ࡆࡗṑ㢮࡛ࡢࡀࢇཎᛶヨ㦂࡜ࡢ୍⮴⋡ࠊឤᗘࠊ≉␗ᗘ࡞࡝ࡢᩘ್ࠊ࠾ࡼࡧ IARC ࡢࢢ࣮ࣝࣉูࡢࡀࢇཎ≀㉁࠿ࡽุ᩿ࡍࡿ࡜ࠊSHE CTA ࡣࡀࢇཎᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚ࡢ఩⨨࡙ࡅ࡛ࡣ࡞ࡃࠊࡀࢇཎ≀㉁᳨ฟࡢࢫࢡ࣮ࣜࢽࣥࢢヨ㦂ἲࡢ୍ࡘ࡜ࡋ࡚఩⨨࡙ࡅࡿࡇ࡜ࡀ࡛ࡁࡿࠋ

23

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14.

ཧ⪃ᩥ⊩

1)

Guidance Document on the In Vitro Syrian Hamster Embryo (SHE) Cell Transformation Assay, Series on
Testing & Assessment No 214, ENV/JM/MONO (2015)18,OECD (2015), Paris

2)

Report on the Evaluation of Genotoxic or Non-genotoxic Activity of the Organic Chemicals Tested in the SHE
Cell Transformation Assay, 26th Meeting of the Working Group of National Coordinators of the Test Guidelines
Program (WNT), ENV/JM/TG/RD (2014) 17

3)

Pienta, R. J., J. A. Poiley, and W. B. Lebherz III. Morphological transformation of early passage Golden Syrian
embryo cells derived from cryopreserved primary cultures as a reliable in vitro bioassay for identifying divers
carcinogens. Int. J. Cancer: 19, 642-655 (1977)

4)

Watanabe, Masami and Keiji Suzuki, Expression dynamics of transforming phenotypes in X-irradiated Syrian
golden hamster embryo cells. Mutat. Res., 249:71-80 (1991)

5)

Detailed review paper on cell transformation assays for detection of chemical carcinogens, OECD Environment,
Health and Safety Publications, Series on Testing and Assessment, No. 31 OECD (2007)

6)

Report of the Validation Management Team on the ECVAM prevalidation study concerning the SHE pH6.7
Cell Transformation Assay, EC-ECVAM (2010a)

7)

Report of the Validation Management Team on the ECVAM prevalidation study concerning the SHE pH7.0
CTA, EC-ECVAM (2010b)

8)

Results of An Analysis of The Performance of The Syrian Hamster Embryo Cell Transformation Assay at PH
6.7 and PH 7.0, 26th Meeting of the Working Group of National Coordinators of the Test Guidelines Program(WNT), ENV/JM/TG/RD (2014) 16

9)Guidance document on the validation and international acceptance of new or updated test methods for hazard
assessment, OECD Environment, Health and Safety Publications, Series on Testing and Assessment, No. 34
OECD (2005)

10) ESAC Working Group Peer Review Consensus Report on an ECVAM-coordinated prevalidation study
concerning three protocols of the Cell Transformation Assay (CTA) for in vitro carcinogenicity testing (2011)11) EURL ECVAM Recommendation on three Cell Transformation Assays using Syrian Hamster Embryo Cells(SHE) and the BALB/c 3T3 Mouse Fibroblast Cell Line for In Vitro Carcinogenicity Testing (2012)12) Draft Summary Record of a Meeting on Cell Transformation Assay, 14-16th January 2014, Paris, France,
ENV/JM/TG/M(2014)1/REV2

13) Benigni R, Bossa C, Battistelli CL, Tcheremenskaia O, IARC Classes 1 and 2 carcinogens are successfully
identified by analternative strategy that detects DNA-reactivity and celltransformation ability of chemicals.
Mutat. Res., 758 56– 61 (2013)14) Isfort RJ, Kerckaert GA, LeBoeuf RA, Comparison of the standard and reduced pH Syrian Hamster Embryo(SHE) cell in vitro transformation assays in predicting the carcinogenic potential of chemicals. Mutat. Res.,
356: 11-63 (1996)2430

15) LeBoeuf RA, Kerckaert KA, Poiley JA and Raineri R, An interlaboratory comparison of enhanced
morphological transformation of Syrian hamster embryo cells cultured under conditions of reduced bicarbonate
concentration and pH. Mutat. Res., 222: 205-18 (1989)16) Engelhardt G, Schwind KR and Mußler B, The testing of chemicals in Syrian hamster embryo (SHE) celltransformation assay for assessment of carcinogenic potential. Toxicol. in vitro, 18: 213-8 (2004)17) Guidance Document on the In Vitro Bhas42 cells Transformation Assay. Series on Testing & Assessment No
231, ENV/JM/MONO (2016) 1, OECD (2016)18) Tsutsui T, Suzuki N, Maizumi H and Barrett J C, Characterization of an unscheduled DNA synthesis assay with
Syrian hamster embryo cells, Mutat. Res., 129:111-7 (1984)

19) Tsutsui T, Suzuki N, Maizumi H, McLachlan, J A and Barrett J C, Alteration in diethylstilbestrol-induced
mutagenicity and cell transformation by exogenous metabolic activation, Carcinogenesis, 7:1415-8 (1986)20) Tsutsui T, Watanabe E and Barrett J C, Ability of peroxisome proliferators to induce cell transformation,
chromosome aberrations and peroxisome proliferation in cultured Syrian hamster embryo cells, Carcinogenesis,
14: 611-8 (1993)21) Hayashi N, Hasegawa K, Komine A, Tanaka Y, McLachlan J A, Barrett J C and Tsutsui T, Estrogen-induced
cell transformation and DNA adduct formation in cultured Syrian hamster embryo cells, Mol Carcinogenesis,
16:149-56 (1996)

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Unclassified

ENV/JM/MONO(2015)18

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

22-May-2015___________________________________________________________________________________________English - Or. English
ENVIRONMENT DIRECTORATEENV/JM/MONO(2015)18
Unclassified

JOINT MEETING OF THE CHEMICALS COMMITTEE AND
THE WORKING PARTY ON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYGUIDANCE DOCUMENT ON THE IN VITRO SYRIAN HAMSTER EMBRYO (SHE) CELL
TRANSFORMATION ASSAY
Series on Testing & Assessement
No. 214English - Or. English

JT03376959
Complete document available on OLIS in its original format
This document and any map included herein are without prejudice to the status of or sovereignty over any territory, to the delimitation ofinternational frontiers and boundaries and to the name of any territory, city or area.

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ENV/JM/MONO(2015)18

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ENV/JM/MONO(2015)18
OECD Environment, Health and Safety Publications
Series on Testing and Assessment
No. 214

GUIDANCE DOCUMENT ON THE IN VITRO SYRIAN HAMSTER EMBRYO (SHE) CELL
TRANSFORMATION ASSAY

Environment Directorate
ORGANISATION FOR ECONOMIC CO-OPERATION AND DEVELOPMENT
Paris 2015

335

ENV/JM/MONO(2015)18
About the OECDThe Organisation for Economic Co-operation and Development (OECD) is an intergovernmentalorganisation in which representatives of 34 industrialised countries in North and South America, Europe
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ENV/JM/MONO(2015)18
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ENV/JM/MONO(2015)18
FOREWORD
This document presents guidance for conducting the Syrian Hamster Embryo Cells Transformation
Assay (SHE CTA).
This document was preceded by the development of the Detailed Review Paper (DRP) 31 on “Cell
Transformation Assays for Detection of Chemical Carcinogens” (OECD, 2007), pre-validation study led
by ECVAM, ESAC peer review, and then from 2011 by work aimed at the development of a Test
Guideline. Despite support from some countries, concerns were expressed by others regarding the SHE
CTA and the approval of the draft TG at the April 2013 WNT meeting was considered premature. Efforts
were undertaken to try to address remaining scientific, technical issues, but nevertheless the draft Test
Guideline did not reach the stage of regulatory acceptance.
In November 2014, the Joint Meeting discussed options for moving forward in the area of nongenotoxic carcinogenicity under the Test Guidelines Programme. The Joint Meeting advised 1) to proceedwith the development of guidance documents on the SHE CTA and Bhas-42 CTA, mainly to describe thetest procedures, and 2) to develop a guidance document at the OECD level outlining a conceptual
framework for the identification of non-genotoxic carcinogens for priority setting.
The draft document has been through two WNT commenting rounds in July and in December 2014.
Since all countries who commented either indicated approval or indicated that their comments did not
impede approval of the document and were only of editorial nature, this document was approved by written
procedure.
This document is published under the responsibility of the Joint Meeting of the Chemicals Committee
and the Working Party on Chemicals, Pesticides and Biotechnology.

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ENV/JM/MONO(2015)18
GUIDANCE DOCUMENT ON THE
IN VITRO SYRIAN HAMSTER EMBRYO (SHE) CELL TRANSFORMATION ASSAYPURPOSE1.
The purpose of this Guidance document is to allow the regulatory community to use thedescribed method as part of a weight of evidence approach in the testing of substances for carcinogenic
potential. There are a number of issues which have impeded consensus on the approval of the Test
Guideline; these issues mainly include the subjective nature of evaluating transformed phenotypicmorphology, the limited understanding of causal molecular mechanisms leading to the transformed SHEcolonies, the relatively small number of bona fide non-genotoxic carcinogens, as compared to genotoxic
carcinogens, that have been tested in the SHE cell transformation assay, and the way the assay might beused in a regulatory framework.
Background2.
In vitro cell transformation refers to the induction of phenotypic alterations in cultured cells. Celltransformation is an event in the multi-step process of tumour induction (1) (2). Transformed cells arephenotypically different from normal cells and have the ability to induce tumours in susceptible animals(3) (4) (5). It has been shown that SHE cells can be morphologically transformed by treatment withgenotoxic and non genotoxic carcinogens (6) (7) (8). Exposure results in an increase of morphologicallytransformed (MT) colonies, which are characterised by disorganised growth patterns and mimicking an
early stage in the carcinogenic process.
3.
The performance of the Syrian Hamster Embryo (SHE) cell transformation assay conducted at avariety of pHs to detect transforming activity has been established on a large set of substances and has
been reviewed in a database summarized in the OECD Detailed Review Paper (DRP) 31 (6) (9). In
addition, the European Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) study(10) (11) addressed the availability of standardized protocols, their transferability, within- and betweenlaboratories reproducibility. The EURL ECVAM work also included the analysis of the degree ofsimilarity between protocols. It concluded that there are no elements suggesting that the EURL ECVAM
experiments differ notably from the experiments reported in the OECD DRP 31, thus making these data
acceptable for use in a retrospective evaluation.
4.
When SHE CTA results are used as part of a testing strategy (not as results from a stand-alone
assay) and/or in a weight of evidence approach, they may contribute to the assessment of carcinogenic
potential of test chemicals (12). While the available data (see paragraph 14) would suggest that the SHE
CTA has greater sensitivity for carcinogens acting via genotoxic mechanisms, for non-genotoxic
carcinogens the added value of the SHE CTA is still debated and more information is required.
5.
This Guidance Document (GD) provides an in vitro procedure of the SHE cell transformation
assay, as specified in Maire et al. (13) or in the EURL ECVAM DB-ALM protocol on SHE CTA (14),conducted at pH 6.7 and 7.0. The assay can be performed at either pH 6.7 or 7.0 (see paragraphs 14)
provided proficiency has been demonstrated at the chosen pH (see paragraph 54-55). The morphology ofthe normal colonies differs slightly at physiological pH compared to acidic pH, however, the conduct of
the assay at either pH has been shown to give similar results. Other than the difference in the pH, theexperimental protocol for both versions of the assay is the same.

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ENV/JM/MONO(2015)18
Current knowledge and understanding about mechanisms involved in cell transformation/
6.
The exact molecular mechanisms involved in cell transformations are only partially understood(15) (16) 17). Although there are uncertainties regarding the causal mechanisms leading to thetransformed SHE colonies, the following paragraphs review current knowledge and understanding basedon the literature.
7.
Evidence indicates that cell transformation results from alterations and changes in the expressionof genes involved in cell cycle control, genomic stability, proliferation and differentiation. Genetic changes
affecting these processes may result from direct genotoxic mechanisms. Also, disturbances of gene
expression and genomic stability through hyper- or hypomethylation of DNA, histone modifications andnucleosomal remodelling are epigenetic mechanisms considered as fundamental in triggering a
carcinogenic process (18). Consistent with these diverse mechanisms, some SHE cell transformants havebeen shown to harbour biallelic, inactivated p53 tumour suppressor genes (19). Carcinogens such as DES
can suppress DNA methylation in short-term treatments (20). The initial transformants induced bypolycyclic aromatic hydrocarbons frequently display DNA methylation-associated suppression of gene
expression known to be associated with embryonic differentiation and engineered re-expression suppresses
the transformed phenotype (21). SHE cell transformation by diethanolamine is driven by altered choline
metabolism, an important methyl donor in one-carbon metabolism leading to DNA methylation (22).
Introduction of an activated oncogene (v-Ha-ras), by transfection, will morphologically transform normal
SHE cells (23). Increased frequency of akinetochoric chromosome disjunction occurs during the growth of
the initial transformants (24) which could contribute to the aneuploid characteristic of immortalized clones
arising from such populations (reviewed in Ahmadzai et al., 2012 (25)).
8.
Among later stage immortal and malignantly transformed descendents, global DNA
hypomethylation and site specific hypomethylation in ras and myc oncogenes have been observed (20)(26). Also, methylation-associated suppression of cell cycle checkpoint gene expression, or mono- orbiallelic losses of these genes (ink4a, ink4b), as well as mutations in p53 have been found in immortal
Syrian hamster dermal cells (27). In morphologically transformed SHE cell lines, cell cycle checkpoint
control (G2) is often compromised (28). An activated proto-oncogene (cph) capable of transforming othercells has been isolated from malignantly transformed SHE cells (29).
9.
Non-genotoxic carcinogens have been postulated to act via a number of mechanisms such as
inhibition of gap junction intercellular communication oxidative stress, increased mitogenesis, decreased
apoptosis, interference with tubulin polymerization, inhibition of senescence through activation of
telomerase, interference with signal transduction pathways, and binding to receptors involved in hormonemediated processes, and in peroxisome proliferation. Instances of several of these mechanisms have been
demonstrated in SHE cell transformation. Oxidative stress was shown to be causally involved inmorphological transformation (30) (31). Imbalance of cell proliferation via an inhibition of apoptosis has
been related to cell transforming effects of some hepatic peroxisome proliferators and other transforming
agents in SHE cells (32) (33). Growth factor treatments of SHE cells, presumably acting through signaltransduction pathways, can also drive transformation (34) and gap junctional cell-to-cell communication isfrequently impaired by non-genotoxic carcinogens (35).

INITIAL CONSIDERATIONS AND LIMITATIONS
10.
The SHE cells are normal diploid, metabolically and p53-competent primary cells, which retain
the ability to biotransform a wide range of xenobiotics as evidenced by studies with substances requiring
metabolic activation (6) (9) (36) (37) (38). From a 3Rs perspective, the use of primary cells means that a

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ENV/JM/MONO(2015)18
small number of pregnant hamsters are euthanized; one hamster provides sufficient cells to perform at least
50 to 100 CTAs providing the cells are adequately preserved for future use. The metabolic capability of thecells should be considered and discussed in the light of interpretation of test results. This is particularly
important when the test chemical requires metabolic activation. Exposure to test chemicals withtransforming capacity results in an increased number of morphologically transformed (MT) colonies,
which are characterised by disorganised growth patterns.
11.
Transformation of primary, diploid SHE cells appears to follow a staged process. The
transformation assay in the SHE cells is based upon identifiable colonies of morphologically transformed
cells with irregular growth patterns. The transformants are thought to be stem cells with blockages in theirdifferentiation pathways (39). Upon further passages in vitro, transformed colonies clonally isolated fromtreated cultures frequently generate cells with an infinite cellular lifespan or an ability to form tumours insyngenic hosts. Untransformed clones become senescent (40) (41) (42). High frequencies of progression
to immortality and anchorage independence were also observed in bulk cultures of SHE cells (4).
12.
Although conducted blindly, identification of morphologically transformed colonies by
microscopic scoring is subjective, as for any cytohistochemical endpoint. This subjectivity may, to someextent, be overcome with appropriate training, and the use of photo catalogues (43) (44). However, toimprove the reliability of the scoring a second opinion or duplicate independent scoring is highly
recommended, especially for ambiguous colonies/or borderline pictures.
13.
The assay would be improved by the development of objective measures for scoringtransformation, when these are validated. Some examples include biospectroscopy, which is being
explored to provide an objective determination of transformed colonies (45). In addition, molecular tools
such as gene expression changes promise to provide useful molecular markers for morphologicaltransformation, like those associated with cytoskeleton effects in the SHE cells (38).
14.
To date comparative sensitivity and specificity of the pH 6.7 and 7.0 versions of the assay are
limited to a small chemical database (see annex to DRP 31(7)). An analysis of the sensitivity andspecificity of the assay for genotoxic and non-genotoxic carcinogens that were fully tested in in vitro andin vivo genotoxicity assays was performed in 2014 and is available in an annex to the DRP.
15.
When planning the experiment, careful choice of the optimum pH needs to be taken into account.
Parameters might include ionisable nature of the compounds as affecting the differences in reactivity or
bioavailability. The historical experience of the laboratory with the scoring at either pH should also be
considered. This needs to be taken into account until a wider chemical database has been generated.
16.
At this time, the assay conducted as described in this Guidance Document does not provide
information on in vivo potency, or species-specificity or tissue-specificity of the cell transformations.
17.
It should be noted that this method has been validated for mono-constituent substances only andnot multi-constituent substances, UVCBs (substances of unknown or variable composition, complex
reaction products or biological materials) or mixtures. Before use of the assay for the testing of a mixture
intended for a regulatory purpose, it should be considered whether, and if so why, it may provide adequate
results for that purpose. Such considerations are not needed, when there is a regulatory requirement for
testing of the mixture.
PRINCIPLE OF THE TEST METHOD18.
SHE cells are obtained from primary cultures of Syrian hamster embryos at 13 days of gestation.
After enzymatic tissue digestion, cells are collected, grown for 24 to 48 hours and then cryopreserved, andstored in liquid nitrogen. One part of cryopreserved SHE cells is used as feeder cells, the other part as
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ENV/JM/MONO(2015)18
target cells. The feeder cells are x-ray irradiated to inactivate their capability to replicate, and seeded as
nutrient base and support of metabolic activity. The target cells are used to assess morphologicaltransformation of colonies.
19.
SHE cells (target cells) are seeded at clonal density onto a feeder layer of x-ray-irradiated cells in
culture conditions allowing for the development of colonies, and achieving the necessary cloning
efficiency for fulfilling the acceptability criteria for the CTA (paragraph 60). After plating the cells, they
are exposed to the test chemical for 7 days. Thereafter, cells are washed, fixed and stained. Dishes arecoded and colonies are scored for their morphological phenotype by stereomicroscopy.
20.
Cytotoxicity is evaluated by inhibition of cloning efficiency and reduction in size/density of thecolonies. The number of morphologically transformed (MT) colonies relative to the total number ofscorable colonies is calculated for each concentration tested. The frequency of morphologicallytransformed colonies relative to total number of colonies in the test chemical-treated groups is compared to
the frequency of MT colonies in the solvent control group.
DESCRIPTION OF THE METHOD
Preparations
Culture media, reagents and solutions
21.

The culture medium, reagent and solutions used for cell preparation are described in Annex 1.

Culture conditions and counting of viable cells
22.
Cell cultures are incubated in a humidified incubator at 37°C and 10 r 0.5 % CO2. All
centrifugation steps are carried out at 180-250 g for 10 minutes at 4°C. Viable cells are counted using thetrypan blue dye exclusion test using 0.4% to 0.5% (w/v) trypan blue in buffered saline.
Preparation and cryopreservation of SHE cell stocks
23.
SHE cells are isolated from 13-days gestation embryos of pregnant healthy female(s) humanely
euthanized. Embryos are washed, transferred into sterile culture dishes containing wash solution, and thedifferentiated organs (head, viscera, and limbs) are discarded from each embryo. Cells can be preparedfrom single embryos, embryos pooled from a single dam or embryos pooled from different dams sacrificed
at the same time.
24.
The remainder of the embryo is minced and dissociated by enzymatic digestion in dissociation
solution under gentle stirring for 10 min at room temperature or at 37°C. The first wash is discarded, andthe dissociation is repeated 2-4 times. Cell suspensions are collected, centrifuged (at 4°C) and resuspended in cell isolation medium (CIM). Viable cells are counted and seeded (2 x106 /100 mm diameter
culture dish, or 0.133 x 106 cells/cm2 area of 150 or 225 cm2 culture flasks) in CIM and incubated (37°C
and 10 ± 0.5 % CO2) until 60-80% cell growth confluency is achieved (usually within 24 to 48 hours).
Then, cells are rinsed with buffered saline, detached with the appropriate detachment solution, andcollected by centrifugation.
25.
The cell pellet is suspended in CIM, viable cells are counted and pelleted by centrifugation. Cells
are re-suspended in cryopreservation medium, dispensed into storage vials (1.0 or 2.0 x 106 cells/vial),
step frozen (i.e., successively for 30 minutes at 4°C, 4 hours at -20°C and one night at -80°C), and kept
frozen under liquid nitrogen until use.

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ENV/JM/MONO(2015)18
Checking of the SHE cells/FBS suitability
26.
Before use, each new cell batch should be checked for spontaneous transformation rate, plating
efficiency (colony forming ability) and morphological transformation using a positive control chemical.
Likewise, any new batch of foetal bovine serum (FBS) should be checked for suitability. The combination“cell batch/FBS batch” should fulfil the acceptability criteria described in paragraph 60.
Preparation of feeder (irradiated) SHE cells
27.
Cryopreserved SHE cells in frozen vials are thawed at 37°C, pelleted by centrifugation and resuspended in fresh cell growth medium (CGM). This also eliminates most of the dimethylsulfoxide(DMSO) used in the cryopreservation medium. Ten or 40 mL aliquots of cells are transferred torespectively 100 mm culture dishes or T225 culture flasks (2.0 x 106 cells/dish or 8.0 x 106 cells in T225
culture flask) and cultured in a humidified incubator at 37°C and 10 ± 0.5 % CO 2 for 2-4 days to achieve
50-90% confluence.
28.
On the day of x-ray irradiation, cells are rinsed, detached and immediately re-suspended in CGM.
Cells are exposed to irradiation (5000 rads or 50 grays) so that they remain viable, but no longer capable ofreplication. . Before and after irradiation, cells should be maintained on ice, and preferably also during
irradiation.
29.
These freshly irradiated cells can be directly used for the experiments soon after irradiation. In
case of cryopreservation of the irradiated cells, cells are centrifuged and the supernatant is removed. The
pellet is re-suspended in an appropriate volume of cold (hold on wet ice) cryopreservation medium. The
viable cells are counted and dispensed into storage vials (5 x 106 cells/vial) on wet ice, and step frozen (asdescribed in paragraph 25) prior to being stored frozen under liquid nitrogen. The cryopreservation step isa good way of keeping irradiated cells for a longer period if an x-ray machine is not readily available.
Preliminary cytotoxicity and dose range finding (DRF) assay
30.
The maximum dose of the test chemical should be determined taking into account the solubility
and any relevant cytotoxicity information available for the test chemical. In the DRF, a range of at least 10
concentrations to achieve a wide toxicity range should be tested in parallel to the solvent control. At least
five, preferably ten dishes should be seeded per concentration tested. The number of target cells seeded isthe same in all treatment groups. The conditions of testing (test medium, incubation conditions and time)
are the ones described for the main experiment for cell transformation (see paragraphs 41-44).
Test conditions
Solvent use
31.
The solvent should be chosen to optimize the solubility of the test chemical without adversely
impacting the assay conduct, e.g. cell growth, integrity of the test material, reaction with culture vessels. It
is recommended that, wherever possible, the use of an aqueous solvent should be considered first. Wellestablished solvents are for example water, cell culture medium, and dimethyl sulfoxide. Generally the
final concentration of organic solvents in the tissue culture medium should not exceed 0.2% (v/v). Thismay be achieved by diluting concentrated solutions (500 x) of the test chemical in CGM to prepare
ultimate dosing solutions at a concentration 2 x, so as to obtain the final concentration (1 x) in the test
medium after addition of an equal volume of the test medium (see Table 1 and paragraph 33 as examples).
If other than well-established solvents are used, their use should be supported by data indicating theircompatibility with the test chemical, the test system, and their lack of transforming potency. In such cases,
untreated controls should also be included.
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ENV/JM/MONO(2015)18
Selection of test concentrations
32.
The maximum concentrations to be tested in cell transformation assay depend on test chemicalsolubility and cytotoxicity. For test chemicals of defined composition the highest dose level should be 0.01
M, 2 mg/mL or 2 μL/mL, whichever is the lowest. For test chemical of non-defined composition, e.g.
complex mixtures (plant extracts, tars, environmental extracts etc.), the top concentration should be at least
5 mg/mL. Poorly soluble chemicals should be tested up to the first concentration producing a visible
opacity in the final test medium observable by the unaided eye.
33.
In addition to the controls, at least 5 test chemical concentrations should be used in the main
experiment. These are deducted from the range finding study and should include:

A high test concentration inducing no more than 50% cytotoxicity expressed by decrease inrelative plating efficiency and/or reduction in relative colonies density/size (by visualappearance). If the test chemical does not show a cytotoxic effect, the highest dose is selected asindicated in paragraph 32 above for soluble test chemicals, or as the visible solubility limit in the
final test medium for insoluble test chemicals;
At least one concentration which has no apparent effect on plating efficiency;
3 or 4 intermediate concentrations.

Table 1: Recommended concentrations of the test chemical in the intermediate solutions and in the final
test medium

Solvent*

Intermediate solution(4 mL test medium)

Final test medium(8 mL= 4mLintermediate
solution+4mL GCM)

Concentration of the test
chemical
500x2x1xConcentrationofthesolvent
100%
0.4%
0.2%
*For water insoluble test chemicals, concentrated solutions (500x) may be prepared in Dimethyl
sulfoxide (DMSO). For water soluble test chemicals, an aqueous solvent is recommended.

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ENV/JM/MONO(2015)18
Day 0
1
2
9
---------------------------------------------- //--------------------------------nnnpFeederTargetTreatment
Fixingcells
cells
with test chemical
staining(2 mL)(2 mL)(4 mL)
Figure 1: Timeline of the SHE CTA assay (the volumes are per each 60 mm culture dish)Preparation of test cultures
Feeder layer
34.
On day 0 (feeder cells day), the irradiated SHE cells are seeded. The cell concentration isadjusted to 20,000 – 30,000 cells/mL in CGM, and 2 mL of the cell suspension are added into each 60 mm
culture dish (4 to 6 x 104 feeder cells/dish). In case of cryopreservation of irradiated cells, cryopreserved
cells are thawed at 37°C, and pelleted by centrifugation. The cell pellet is re-suspended in fresh CGM andthe viable cells are counted. Freshly irradiated cells can also be used for seeding of the feeder layer.
35.
The culture dishes are incubated in a humified incubator at 37°C and 10 r 0.5% CO2 for 24 hours
before adding the target cells. For each test, at least 5 dishes filled with feeder cells only will be usedconcurrently as controls for the inability of the feeder cells to replicate and to form colonies. No colony
should form in these dishes.
Target cells
36.
Cryopreserved SHE cells are thawed at 37°C and seeded for growth in culture flasks. After an
incubation period (usually of 24 hours although shorter duration e.g. 5 hours can be used), the target cells
are detached, counted and the cell concentration is adjusted with CGM to a concentration whereapproximately 25 - 45 colonies/dish can be obtained at the end of the test (see paragraph 60). Two mL of
the target cell suspension will be added to each culture dish containing feeder cells. Dishes will be
incubated in a humidified incubator at 37°C and 10 r 0.5% CO2 for 24 hours prior to treatment with thetest and control chemicals.
37.
At cytotoxic dose levels, as determined in the Dose Range Finding (DRF) experiment, the target
cell number should be adjusted in order to yield the recommended number of 25 to 45 colonies per dish at
the end of the test to fulfil acceptance criteria (see paragraph 60). The adjustment of target cell number is
explained in paragraph 37.
Treatment of cultures
38.
For practicality, one way of preparing dosing solution is to start with a concentration representing
twice (2x) the final concentration (Table 1). Each dosing solution (4 mL) will be transferred to individual
culture dishes (60 mm) already containing the CGM (4 mL) with feeder and target cells (final volume
8 mL) (Table 1). The cultures will be incubated in a humidified incubator at 37°C and 10 r 0.5% CO2 for 7
days without disturbance.

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ENV/JM/MONO(2015)18
PROCEDURE
Preparation of test solutions
39.
The test chemical solutions are prepared on the day of treatment. Solid test chemicals should be
dissolved in appropriate solvents and diluted, if appropriate, prior to treatment of the cells. Liquid test
chemicals may be added directly to the final test medium and/or diluted prior to treatment. Gaseous or
volatile chemicals should be tested by appropriate methods, determined on a case by case basis. Freshpreparations of the test chemical should be used unless stability data demonstrate the acceptability ofstorage. A series of solutions at different concentrations of the test chemical should be prepared under UVfiltered lights or protected from light.
Description of the cell transformation assay
40.
A sufficient number of target cells (around 150 cells/dish, but the number of target cells isdependent on the cell batch/FBS batch) to produce an average of 25 - 45 colonies at the end of the test will
be dispensed in 2 mL of complete medium per 60 mm culture dish, each of which was seeded
approximately 24 hours earlier with 4-6 x 104 feeder cells in 2 mL of CGM. For cytotoxic concentrations,the number of target cells seeded should be increased so as to maintain the target range of 25-45 colonies
per dish (paragraph 60). For instance, an approximate 30 % reduction in the number of colonies may
require to adjust the number of target cells to 1.5x the number of cells seeded in the cytotoxicity assay; anapproximate 50 % reduction, would lead to seed twice (2x) the number of target cells seeded forcytotoxicity.
41.
Twenty four hours after the seeding of the target cells, test and control chemical treatment will be
initiated by addition to the test media (4 mL) of the appropriate dosing solution (4 mL) so as to obtain the
final concentration of 0.2% (Table 1).
42.
The treated cell cultures should be incubated for a period of 7 days in a humidified incubator(37°C, 10 r 0.5 % CO2) following treatment initiation to allow colony development. The culture dishes
should be labelled as appropriate for identification. The relative cytotoxicity of each treatment groupshould be measured by the reduction in plating efficiency and/or colony density and size of the treated
SHE cells compared to the solvent control.
43.
After the incubation period of 7 days, the medium should be discarded from the dishes by
aspiration, and the cells attached to the dishes should be rinsed with buffered saline. After removal of thebuffered saline, cells should be covered with fixing solution (ethanol or methanol) and kept for at least 10
minutes at room temperature. The fixative is removed and the dishes are stained for approximately 20
minutes with 3-5 mL Giemsa solution. The stain is poured off and the dishes are rinsed with tap water
before the stained colonies are air-dried.
Morphological cell transformation
44.
All dishes, including those of positive and negative controls, should be coded before microscopic
analysis. The stained colonies are evaluated and scored under stereomicroscope for plating efficiency (PE)and morphological transformation (MT). The scorer should be unaware of the coding. Morphologicallytransformed colonies are characterized by a criss-cross pattern of growth and piling up of cells. Individual
cells within the colony are more basophilic relative to their normal counterparts, and have a decreased
cytoplasm-to-nucleus ratio. Pictures of normal and morphologically-transformed colonies obtained at pH
6.7 and 7.0, which can be found in the photo catalogue of Bohnenberger et al. (43) and Maire et al. (44),respectively, should be used routinely.

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ENV/JM/MONO(2015)18
45.
Sparse colonies are not scored for MT (i.e. if a colony contains less than 50 cells, it is notcounted); however, they are included in the total number of colonies for plating efficiency determination..
Colonies at the edge of the dishes should be scored for MT if clearly morphologically transformed.
Generally, for each treatment group t 1000 colonies should be evaluated for morphological celltransformation (MT).
46.
For each treatment group, normal (non-transformed) colonies and transformed colonies will be
enumerated to evaluate the plating efficiency (PE), the relative plating efficiency (RPE) and themorphological transformation frequency (MTF) criteria detailed in paragraphs 56-58.
Controls
Solvent control
47.
In case the test chemical is not water soluble, an appropriate solvent control should be used. If
DMSO or other organic solvents are selected, they should be used at a final concentration that does notexceed 0.2% (see paragraph 31). The final concentration of any solvent should be the same in all solvent
control and treated dishes.
Positive control
48.
Because of the high amount of data on Benzo[a]pyrene (B[a]P), it should be used as positive
control at the recommended concentrations of 1.0 to 5.0 μg/mL in dimethyl sulfoxide (DMSO) todemonstrate the sensitivity of the assay . However, if justified, other chemicals can also be considered as
positive controls. Each laboratory should establish the performance of the positive control under their own
laboratory conditions (see paragraph 49).
Feeder cells control
49.
For each test, at least 5 dishes with feeder cells only should be used concurrently to confirm theinability of these cells to replicate and to form colonies. For a valid test, no colony should be formed inthese dishes at the end of the test period.
Solubility, pH, and osmolality
50.
The solubility/precipitation of the test chemical in the solvent and in the test culture (medium)should be visually assessed and documented in the test report.
51.
The pH of the test chemical dosing solutions should be measured at the time of preparation of thetreatment medium and after at least four hours of undisturbed incubation in an incubator, in humidified
atmosphere at 37°C and 10 r 0.5 % CO2. In case of deviation from the expected pH at any time point, thepH of the medium should be adjusted to the selected pH. Any deviation should be reported and consideredin the interpretation of the results.
52.
The osmolality of the treatment medium should be measured prior to or at the time of performing
the preliminary cytotoxicity determination or the main experiment. Osmolality of the treatment medium
should be compared to the control medium, and any change used in the interpretation of results.
Proficiency of the laboratory
53.
In order to demonstrate proficiency, a laboratory should perform tests with the four positive
chemicals acting via different mechanisms, and two negative chemicals, included in Table 2. Proficiency
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ENV/JM/MONO(2015)18
should be demonstrated at the pH the assay will be conducted, i.e. pH 6.7 and/or pH 7.0. The choice of thenegative chemicals will depend of the version of the assay the laboratory demonstrates proficiency with.
During the course of these tests, the laboratory should establish:-

A historical negative (untreated, solvent) control range and distribution
A historical positive control range and distribution.

54.
Re-evaluation of laboratory proficiency is recommended if major changes to the experimental
conditions are proposed for the assay (e.g. use of automated instead of manual scoring techniques). Before
starting to use this method, it is recommended that personnel are trained in a laboratory experienced in theperformance of this assay.

Table 2: Substances for Assessing Laboratory Proficiency
Category
Substance
CASRN
1. Carcinogens1 (responsive in the SHE CTA (pH 6.7 and pH 7.0))
Benzo[a]pyrene
50-32-8
2,4-diaminotoluene
95-80-7o-toluidine
636-21-5
Non-genotoxic
Di (2-Ethylhexyl)phthalate (DEHP)
117-81-7
carcinogen
2. Non carcinogens2 (non-responsive in the SHE CTA ((pH 6.7 and pH 7.0))
Anthracene120-12-7
d-manitol (only for pH 6.7)
1 naphtylamine (only for pH 6.7)
69-65-8
Benzoin (only for pH 7.0)
119-53-9
1

Rodent carcinogen: sufficient evidence in animals.
Rodent non-carcinogen: inadequate or no evidence in animals.

2

DATA AND REPORTING
Morphological transformation
55.
The morphological transformation frequency (MTF) should be calculated for each treatmentgroup, using the data of one trial, as follows:

MTF =total number of transformed colonies
total number of scorable colonies

x 100

Cytotoxicity
56.
The average number of colonies per dish, the plating efficiency (PE) and the relative plating
efficiency (RPE) should be determined for each treatment group.
57.
The plating efficiency (% PE) and the relative plating efficiency (% RPE) should be calculated asfollows:

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ENV/JM/MONO(2015)18

PE =total number of colonies per dishtotal number of target cells seeded per dishRPE =PE of treatment group
PE of the solvent control group

x 100

x 100

58.
In addition to the RPE, the colony size and density (number of cells per colony) should berecorded as parameters of cytotoxicity. The size and density is observed and recorded as three categories:- Normal (+)- Slightly reduced (++ ; 20 – 39 % reduction)- Greatly reduced (+++; 40 – 60 % reduction)
Acceptability Criteria and historical controls
59.

The following criteria have to be fulfilled for the validity of the assay:x At least 1000 colonies per experimental group should be available for morphologicaltransformation scoring. Occasionally, in case of a significant increase in morphologicaltransformation rate, less than 1000 colonies are acceptable. However the average number ofcolonies per dish should normally not be less than 25.
x An average of 25-45 colonies per dish should be available (46). Occasionally, in case of a negative
result, dishes with less than 25 colonies per dish are acceptable. Likewise, in case of a positive
result, dishes with more than 45 colonies are acceptable.
x Cloning efficiency of the negative/solvent control is t 20%.
x No colony formation should be observed in the feeder cell dishes. Feeder cells should be visible inthe chemical treatment groups except if they are affected selectively by the test chemical. If thefeeder cells are affected by the test chemical, then this observation should be recorded, reported,and considered in the interpretation of the results.
x Transformation frequency in the negative controls (untreated and solvent) is within the
distribution of historical control data of the laboratory (e.g. 95% confidence interval). Based on
historical data from experienced laboratories and data from the EURL ECVAM validation study,
the upper limit of transformation frequency in the negative controls (untreated and solvent) is
0.6%.
x The positive control chemical should induce a biologically relevant and statistically significant
increase in morphological cell transformation compared to the solvent control.

Data interpretation criteria
60.
Although biological relevance of the results should be considered first, both statisticalsignificance and biological relevance of data are considered in the interpretation of the negative andpositive results. The level of concentration(s) increasing the MTF is carefully considered, taking into
account the range of cytotoxic/non-cytotoxic concentrations.
61.
Providing that all acceptability criteria are fulfilled, the following criteria are considered for theevaluation of results:
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ENV/JM/MONO(2015)18(1) the increase in MT colonies is concentration-related,(2) at least one of the test concentration exhibits a statistically significant increase comparedto the concurrent negative control,(3) the statistically significant result is outside the distribution range of the historical negative
control data (e.g. 95% confidence interval).
62.
A result can be considered clearly biologically relevant and a test chemical is considered a clear
positive if all the above criteria are met.
63.
met.

A test chemical is considered as a clear negative if none of the criteria above (paragraph 62) are

64.
Results are statistically analysed using the one-sided Fisher’s exact test to determine if anincrease in morphological transformation occurred at each concentration level compared to the concurrent
solvent control. A p<0.05 level of significance indicates a treatment related effect on MTF. The CochranArmitage trend test can be used to contribute to the evaluation of positive concentration-related response.
65.
When results do not meet the criteria for a clear positive or a clear negative call, the test chemical
should be evaluated by expert judgement and/or the experiment should be repeated. Modification of study
parameters over an extended or narrowed range of concentrations, as appropriate, should be considered infollow-up experiments. In rare cases, even after further investigations, the data set will preclude making aconclusion of positive or negative results, and will therefore be concluded as equivocal.
Test report
66.

The test report should include the following information:

Test chemical
Mono-constituent substance:-

physical appearance, water solubility, and additional relevant physicochemical properties;-

chemical identification, such as IUPAC or CAS name, CAS number, SMILES or InChI code,
structural formula, purity, chemical identity of impurities as appropriate and practically feasible,
etc.
Multi-constituent substance, UVBCs and mixtures:-characterised as far as possible by chemical identity (see above), quantitative occurrence andrelevant physicochemical properties of the constituents.

Solvent (if appropriate)-justification for choice of solvent
concentrations tested and preparation of the dosing solutions
signs of precipitation (absence or presence)

Cells
-

source of cells
number of cell subcultures
maintenance of cell culturesabsence of mycoplasms
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identification of serum (provider and batch number)

Test conditions
rationale for selection of concentrations, including cytotoxicity data and solubility limitations
composition of the media, CO2, pH
serum concentration, origin and quality
concentrations of test chemicals
volume of solvent and test chemical added
duration of treatment
incubation temperature
number of cells plated
positive and negative controls
criteria for scoring MT colonies
Results
cytotoxicity results
signs of precipitationpH, osmolality of culture media after addition of the test chemical
number of total scorable colonies
relative cloning efficiency
concurrent feeder cell control
dose-response relationship, where possible
statistical analyses
concurrent negative (solvent) and positive control data
historical negative (solvent) and positive control data, with ranges, means, standard deviation,and confidence interval (e.g. 95%)
67.
Data should be presented in tabular form. The following values should be presented for eachgroup (treated and untreated groups, solvent and positive controls):
i. total number, and average number per dish of scorable colonies for each groupii. plating and relative plating efficiency %iii. cloning efficiency
iv. colony size/density
v. number of transformed colonies
vi. morphological transformation frequency (MTF %)vii. Fisher’s exact test p-value (one-sided)
Discussion of the results
Conclusion

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ANNEX 1:
Culture medium, reagent and solutions used for cell preparation
The culture medium is DMEM: Dulbecco’s Modified Eagle’s Medium containing 1g/L glucose, 4 mM
glutamine and 110 mg/L sodium pyruvate, with or without phenol red. The media can be purchased
readymade from the vendors and should be stored according to the parameters (time, temperature)provided with the batch media.
If powder media is used, depending on the pH selected, the DMEM medium is adjusted to pH 7.0 with 1.5
g/L NaHCO3 or to pH 6.7 with 0.75 g/L NaHCO3 and sterilized by membrane filtration (0.1 μm porosity).
The culture medium can be stored at 4° C during a period not exceeding 2 weeks.
This culture medium serves to prepare the following media:- Complete growth medium (CGM)
The complete culture medium is prepared with addition of fetal bovine serum (FBS) at a concentration of
15% or 20% (v/v) for the SHE pH 7.0 and the SHE pH 6.7 CTAs, respectively.
- Cryopreservation medium
The cryopreservation medium is the pH-adjusted DMEM, added with 10% FBS and 10% DMSO orwith 20% FBS and 7.5% DMSO (recommended if the test is carried out at pH 6.7).
- Cell isolation medium (CIM)
The cell isolation medium is the pH-adjusted DMEM added with 15% FBS and antibiotics penicillin 100
U/mL and streptomycin 100 μg/mL).
The solutions used for cell preparation and assay protocol are as follows:- Buffered saline (e.g. calcium- and magnesium-free Hank’s balanced solution (CMF- HBSS) or calciumand magnesium-free phosphate buffered saline (CMF-PBS))- Colony staining solution: 10% (v/v) Giemsa in aqueous buffer- Cell staining solution (e.g. 0.4% to 0.5% (w/v) trypan blue in buffered saline)- Fixing solution: ethanol or methanol- Detachment solution (e.g. 0.25% (w/v) trypsin in buffered saline or [0.05% (w/v) trypsin + 0.02% (w/v)
Na2EDTA-H2O] in buffered saline)- Dissociation solution (e.g. dispase 2 U/mL in buffered saline or [1.25% (v/v) Enzar-T, 2.5% (v/v)
pancreatin with 200 U/mL of penicillin and 200 μg/mL streptomycin] in buffered saline)- Wash solution: buffered saline with 200 U/mL of penicillin and 200 μg/mL streptomycin

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