皮膚感作性試験代替法h-CLATの有用性の検討

1 Ann. Rep. Tokyo Metr. Inst. Pub. Health, 63, 131-136, 2012 a 169-0073 3-24-1 b 169-0073 3-24-1 c 169-0073 3-24-1 d 169-0073 3-24-1 e 169-0073 3-24-1 h-CLAT ac b a a c d e h-CLAT h-CLAT THP-1 CD86 CD54 in vitro in vivo LLNA OECD Test Guideline 429 HC 2 HB2 HC 1 HR1 h-CLAT h-CLAT THP-1 24 CD86 CD54 CD86

2 CD54 RFI 2 LLNA h-CLAT h-CLAT in vitro THP-1 CD86 CD54 22 2 1) OECD Local Lymph Node Assay LLNA OECD Test Guideline 429 2 human Cell Line Activation Test h-CLAT h-CLAT h-CLAT 1. Fig. 12) dendritic cell DC DC T DC T Direct Peptide Reactivity Assay DPRA h-CLAT DC LLNA T Guinea Pig Maximization Test GPMT LLNA GPMT DPRA h-CLAT 2.

3 H-CLAT THP-1 T CD86 CD54 TNF- IL-8 MHC Ann. Rep. Tokyo Metr. Inst. Pub. Health, 63, 2012 132 3,4) DC 2 THP-1 h-CLAT 5,6) h-CLAT COLIPA 5 7 ECVAM 7) 1. 1) HC 2 HB2 2,2 -{[4-(2-hydroxyethyl)amino-3-nitrophenyl ]imino}bisethanol Fig. 2(a) SCCP 8) LLNA 5% Aldrich DMSO 2) HC 1 HR1 4-amino-2-nitrodiphenylamine Fig. 2(b) SCCP 9) LLNA 5% GPMT 3% DMSO 2. 1-chloro-2,4-dinitrobenzene DNCB nickel(II) sulfate hexahydrate Ni SIGMA sodium dodecyl sulfate SLS SIGMA DNCB DMSO Ni SLS 3.

4 FITC CD86 Clone FUN-1 BD Pharmingen FITC CD54 Clone Human and animal test methods LLNA [7] GPMT [9] Human patch test [9]1. Epidermal absorption2. Peptide bindingChemical sensitizer3. Antigen presentation4. DC activation6. Antigen recognition5. Migration7. T cell proliferation8. Inflammatory cytokine productionChemical sensitizerLymphatic SystemVascular SystemLymph NodeNon-animal test methods Direct Peptide Reactivity Assay (DPRA) [2] h-CLAT [4]TcellTCRCD86CD54 SkinSkinDendritic cell DC MHC class II9. InflammationChallengeInductionHuman and animal test methods LLNA [7] GPMT [9] Human patch test [9]1. Epidermal absorption2. Peptide bindingChemical sensitizer3. Antigen presentation4. DC activation6. Antigen recognition5. Migration7. T cell proliferation8. Inflammatory cytokine productionChemical sensitizerLymphatic SystemVascular SystemLymph NodeNon-animal test methods Direct Peptide Reactivity Assay (DPRA) [2] h-CLAT [4]TcellTCRCD86CD54 SkinSkinDendritic cell DC MHC class II9.

5 InflammationChallengeInductionFig. 1. Mechanism and Test Methods on Skin Sensitization NHCH2CH2O2NO2N(CH2CH2OH)2(a)O2 NNH2NH(b)NHCH2CH2O2NO2N(CH2CH2OH)2(a)O2 NNH2NH(b)Fig. 2. Structural Formulae of HB2 (a) 63, 2012 133 0200400600800100012001400 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(a)0200400600800100012001400 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(a)(b)0200400600800100012001400 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(b)0200400600800100012001400 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)050100150200250300 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(c)050100150200250300 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(c) FITC IgG1 Clone DAK-GO1 Dako Propidium iodide PI PBS BSA SIGMA 4.

6 Cell Lab QuantaTM SC 5. 1) THP-1 AT C C 10% FBS MP Biomedicals 1% - GIBCO mM 2-mercaptoethanol RPMI1640 GIBCO 37 C 5% CO2 2 2 2) h-CLAT 6. h-CLAT THP-1 DNCB CD86 CD54 3 3 Fig. 3(a) Ni CD86 CD54 3 3 Fig. 3(b) SLS CD86 CD54 3 3 Fig. 3(c) 6. 10) 1) THP-1 DMSO 96 48 72 THP-1 105 /160 L/well 2 24 BSA PBS FACS bu ffer PI 75% CV75 2) 24 48 72 THP-1 1 106 /1 mL/well CV75 1 6 8 Fig.

7 3. RFI Values (%) of CD86 ( )/CD54 ( ) and Cell Viability (%) ( ) of DNCB (a), Ni (b), and SLS (c). The positive criteria are RFI value of 150% (CD86; ..) and 200% (CD54; ..), respectively. CV75 values ( g/mL) for DNCB, Ni, and SLS are , 55, and 48, respectively. The concentrations of each test are A = CV75 ; B = CV75 ; C = CV75 ; D = CV75 ; E = CV75 ; F = CV75 ; G = CV75; and H = CV75 24 FACS buffer Fc FITC CD86 FITC CD54 FITC Ann. Rep. Tokyo Metr. Inst. Pub. Health, 63, 2012 134 MFI MFI MFI MFI 0100200300400500600700800 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(a)0100200300400500600700800 ABCDEFGHABCDEFGHABCDEFGH1st2nd3rdTest concentrationsRFI (%)020406080100 Cell viability (%)(a)0100200300400500600700800 ABCDEFGHABCDEFGH1st2ndTest concentrationsRFI (%)020406080100 Cell viavility (%)(b)0100200300400500600700800 ABCDEFGHABCDEFGH1st2ndTest concentrationsRFI (%)020406080100 Cell viavility (%)(b)IgG1 3 FAC S buffer PI CD86 CD54 (geometric)

8 Mean fluorescence intensity MFI relative fluorescence intensity RFI 1 CD86 RFI 150% CD54 RFI 200% 2 3 2 50% 8 4 50% RFI % 100 HB2 3 Fig. 4(a) 3 50% CD86 3 RFI 150% CD54 2 RFI 200% HR1 2 Fig. 4(b) 2 50% CD86 2 RFI 150% CD54 2 RFI 200% LLNA HB2 HR1 h-CLAT h-CLAT h-CLAT LLNA 84% 10) 1 in vitro in vitro T in vitro 11) h-CLAT in vitro Fig.

9 4. RFI Values (%) of CD86 ( )/CD54 ( ) and Cell Viability (%) ( ) of HB2 (a) and HR1 (b). The positive criteria are RFI value of 150% (CD86; ..) and 200% (CD54; ..), respectively. CV75 values ( g/mL) for HB2 and HR1 are 700 and 96, respectively. The concentrations of each test are A = CV75 ; B = CV75 ; C = CV75 ; D = CV75 ; E = CV75 ; F = CV75 ; G = CV75; and H = CV75 1) 2011 46-50, 2011. 2) 44(9), 875-879, 2008. 3) Ashikaga, T., Hoya, M., Itagaki, H., et al.: Toxicology in Vitro, 16, 711-716, 2002. 4) Yoshida, Y., Sakaguchi, H., Ito, Y., et al.: Toxicology in Vitro, 17, 221-228, 2003. 5) Ashikaga, T., Yoshida, Y., Hirota, M., et al.: Toxicology in Vitro, 20, 767-773, 2006. 6) Sakaguchi, H., Ashikaga, T., Miyazawa, M., et al.: Toxicology in Vitro, 20, 774-784, 2006. 7) FRAGRANCE JOURNAL 39(12), 56-62, 63, 2012 135 2011.

10 8) SCCP: Opinion on HC Blue No. 2, Doc. No. 1035, 2006. 9) SCCP: Opinion on HC Red No. 1, Doc. No. 0981, 2006. 10) Ashikaga, T., Sakaguchi, H., Sono, S., et al.: ATLA, 38, 275-284, 2010. 11) Jowsey I. R., Basketter, D. A., Westmoreland, C., et al., J. Appl. Toxicol., 26, 341-350, 2006. Ann. Rep. Tokyo Metr. Inst. Pub. Health, 63, 2012 a Tokyo Metropolitan Institute of Public Health, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan b Tokyo Metropolitan Institute of Public Health, at the time when this work was carried out, 3-24-1, Hyakunin-cho, Shinjuku-ku, Tokyo 169-0073, Japan 136 Study on the Usefulness of the Alternative Method h-CLAT for Skin Sensitization Testing Yasushi ONOa, Atsumi YAMAGUCHIb, Yoshiaki NAKAMURAa, Masayuki KURITAa, Ichiro TAKANOa, Dai NAKAEa and Toshihiro NAGAYAMAa Due to the call to review animal testing in the name of animal welfare, development of the human Cell Line Activation Test (h-CLAT) as alternative method for skin sensitization testing has improved.