皮膚感作性試験の適用性拡大に関する研究

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(1)2020. 9.

(2) P-1 P-2 P-3 P-4. 1. LLNA:BrdU-ELISA. GHS. 1-1 1-2. GHS. 1-3. CBA/J. 1-4. LLNA. GHS. 1-5. 2 2-1 2-2. LLNA. 2-3. 1.

(3) 3. in vitro. 3-1 3-2. 3-3 3-4. 2.

(4) P-1. Fig. 1. 2005. T 4. 4 Fig. 2. 3.

(5) 2005. 4.

(6) P-2. Organisation for Economic Co-operation and Development. OECD. Adverse Outcome Pathway:. AOP. OECD, 2012 KE. AO. AOP Adverse Outcome. Fig. 3. KE-1: KE-2: KE-3: KE-4: AO: AO Guinea pig maximization test Buehler test 406. BT: Buehler, 1965. GPMT: Magunusson and Kligman, 1969 Fig. 4. OECD Test Guideline TG. OECD, 1992 AO. 5.

(7) Kimber et al. al., 1989; Basketter et al., 1996 Node Assay LLNA. Kimber et. Local Lymph. KE-4 Thymidine 3H-. TdR. Fig. 5 EC3 Kimber et al., 2003. LLNA. OECD TG429. OECD, 2010. 2013. EU. 2013. EU. TG 3. EU,. 2018 KE , 2018. 6.

(8) P-3. Globally Harmonized System of Classification and Labelling of Chemicals. GHS. Fig.. 6. UN 2019. GHS. 1 1A. OECD TG406. 1B. OECD TG429. in vitro 1. 7.

(9) 8.

(10) P-4. LLNA. LLNA. RI. RI. LLNA:BrdU-ELISA. Fig. 7: Takeyoshi et al., 2001. OECD. OECD, 2018. TG442B LLNA:BrdU-ELISA. GHS. GHS 1 Adjuvant and Strip (Sato et al. 1980). ICH. (Herber 1969). S10 (2013). 9.

(11) LLNA. OECD TG OECD. AOP. KE-1. KE-2. KE-3. 1. Integrated Approaches to Testing and Assessment OECD, 2019 KeratinoSensTM. IATA. OECD. KE-2. LuSens. OECD TG442D OECD 2018. LLNA. 10.

(12) BrdU-ELISA. GHS LLNA. in vitro. 11.

(13) Fig. 1 Allergic contact dermatitis caused by fragrancesmanifesting as cheilitis after application of lipstick (Salvador et al., 2017). 12.

(14) Fig. 2 Mechanism of skin sensitization (. 13. , 2005).

(15) Fig. 3 Skin sensitization Adverse Outcome Pathway (OECD, 2012). 14.

(16) Fig. 4 Outline of Guinea pig maximization test above, Magunusson and Kligman, 1969 and Buehler test below, Buehler, 1965. 15.

(17) Fig. 5 Outline of local lymph node assay (LLNA: Kimber et al., 1989; Basketter et al., 1996 ). 16.

(18) Fig. 6 GHS label for human health hazard (UN, 2019). 17.

(19) Fig. 7 Outline of LLNA:BrdU-ELISA (Takeyoshi et al., 2001). 18.

(20) 1. LLNA:BrdU-ELISA. GHS. 1-1. Kimber et al. 2002, 2011. Globally Harmonized System. GHS. United Nations, 2019 Organization of Economic Cooperation and Development (OECD) TG. Guinea pig maximization test. Buehler test. OECD TG406 Local Lymph Node Assay. 19. LLNA, OECD TG429:.

(21) LLNA-RI. GHS. 1. 3 GHS. 1A/1B. LLNA: BrdU-ELISA. RI. LLNA. OECD TG442B BrdU-ELISA. Takeyoshi et al. 2001, 2003, 2005. Japanese Center for Validation of Alternative Methods. Kojima et al. 2011. 2010 LLNA:. JaCVAM. Interagency Coordinating Committee on the Validation of. Alternative Methods ICCVAM. GHS. 1. GHS. LLNA BrdU-ELISA CBA/JN. CBA/J CBA/JN. CBA/JN. OECD TG442B CBA/JN. LLNA. CBA/JN. CBA/J. CBA/Ca. LLNA. 20.

(22) LLNA:BrdU-ELISA LLNA: BrdU-ELISA GHS CBA/J. LLNA:BrdU-ELISA. 21. GHS.

(23) 1-2. GHS. 1-2. ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node. Assay: BrdU-ELISA. ICCVAM, 2010a. GHS. Data source ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node Assay: BrdU-ELISA. ICCVAM, 2010a. LLNA BrdU-ELISA ICCVAM ICCVAM Test Method Evaluation Report on the Murine Local Lymph Node Assay: ICCVAM. BrdU-ELISA report. Table 1. LLNA-RI. 32 LLNA: BrdU-ELISA. 22.

(24) EC3. EC1.6. LLNA Basketter et al. 1999. ECx= c + {(X-d)/(b-d)} × (a-c). X=3 or 1.6 SI 3. a, b. SI. , SI. 1.6. ,. c, d. EC Kimber et al. 2003 32. ICCVAM report GHS. 1A. GHS. ICCVAM, 2010a. LLNA-RI EC3. 1B. GHS. United Nations,. 2019 GHS 13. Table 1-3 GHS. 1A EC3> 2%. GHS. GHS. 1B. 23. 1B 1A. 19.

(25) LLNA: BrdU-ELISA. GHS. GHS. %. 1%. 10% 1% LLNA-RI. %. =. %. %. LLNA: BrdU-ELISA. /LLNA-RI. x 100. %. =. LLNA: BrdU-ELISA. ) x 100. 24. / LLNA-RI.

(26) EC1.6 LLNA: BrdU-ELISA GHS 1B EC1.6. 1A. EC1.6. Table 1. EC1.6. 0.065%-12.907%. 0.862%-73.596%. LLNA: BrdU-ELISA. GHS %. / 6%. 1A. GHS. GHS. 1B. Table 2a 7%. GHS 92.3%. 84.2% 2-. Mercaptobenzothiazole. GHS. 1A. 2b. 25. 100%. Table.

(27) LLNA BrdU-ELISA. GHS. ICCVAM Test Method. Evaluation Report on the Murine Local Lymph Node Assay: BrdU-ELISA 2010a GHS 84.2%. EC1.6 1A. GHS. 1B. ICCVAM,. 6% 92.3%. 2-Mercaptobenzothiazole. 100%. 26. GHS. 1A.

(28) Table 1. Date set of chemicals used in this study Chemical name. GHS classification. RI-LLNA EC3 (%). LLNA BrdU-ELISA EC1.6 (%). 1A. 0.009. 0.065. 1A 1A 1A 1A 1A. 0.01 0.049 0.05 0.083 0.11 0.5 0.66 0.8 1.4 1.5 1.7 1.9 3.2 3.6 4.7 4.8 9 8.1 9.2 9.7 10.1 13.6 21 22.3 24 24 28 30 32.8 44 47.5. 0.15 0.032 0.45 0.115 0.285 0.163 0.316 1.081 1.53 5.156 12.907 4.808 2.99 8.049 0.862 1.027 11.029 13.334 7.143 12.92 8.851 16.954 24.091 41.496 13.636 49.545 31.751 27.596 33.333 9.44 73.596. 5-Chloro-2-methyl-4-isothiazolin-3-one solution p-Benzoquinone 2,4-Dinitrochlorobenzene Diphenylcyclopropenone Glutaraldehyde 4-phenylenediamine Formaldehyde Cobalt chloride 4-Methylaminophenol sulfate trans-Cinnnamaldehyde Isoeugenol 2-Mercaptobenzothiazole Cinnamic aldehyde 3-aminophenol Diethyl malate Trimellitic anhydride Nickel sulfate 4-Chloroaniline Sodium lauryl sulfate Citral Hexyl cinnamic aldehyde Eugenol Phenyl benzoate Cinnamic alchol Cyclamen aldehyde Hydroxycitronellal Imidazolidinyl urea Ethylene glycol dimethacrylate Linalool Ethyl acrylate Isopropyl myristate Aniline. 1A 1A 1A 1A 1A 1A 1A. 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B. 1B 1B 1B 1B. 27.

(29) Table 2a. Determination of the optimal cutoff (%) for GHS sub-categorization by LLNA: BrdU-ELISA GHS sub-category. Proposed Cutoff. 1A. 1B. (%). CORRECT. INCORRECT. 1. 61.5% (8/13). 38.5% (5/13). 94.7% (18/19). 5.3% (1/19). 2. 76.9% (10/13). 23.1% (3/13). 89.5% (17/19). 10.5% (2/19). 3. 76.9% (10/13). 23.1% (3/13). 84.2% (16/19). 15.8% (3/19). 4. 76.9% (10/13). 23.1% (3/13). 84.2% (16/19). 15.8% (3/19). 5. 84.6% (11/13). 15.4% (2/13). 84.2% (16/19). 15.8% (3/19). 6. 92.3% (12/13). 7.7% (1/13). 84.2% (16/19). 15.8% (3/19). 7. 92.3% (12/13). 7.7% (1/13). 84.2% (16/19). 15.8% (3/19). 8. 92.3% (12/13). 7.7% (1/13). 78.9% (15/19). 21.1% (4/19). 9. 92.3% (12/13). 7.7% (1/13). 73.7% (14/19). 26.3% (5/19). 10. 92.3% (12/13). 7.7% (1/13). 68.4% (13/19). 31.6% (6/19). CORRECT. INCORRECT. Bold type: Lower cutoff (%) showing the highest percentage of correct outcomes for GHS sub-categories 1A and 1B chemicals.. 28.

(30) Table 2b. Determination of the optimal cutoff (%) for GHS sub-categorization by LLNA: BrdU-ELISA GHS sub-category. Proposed Cutoff (%) 6. 1A* CORRECT 100% (12/12). 1B INCORRECT 0% (0/12). CORRECT 84.2% (16/19). INCORRECT 15.8% (3/19). *The correct/incorrect outcomes (%) for GHS category 1A chemicals were calculated by excluding 2mercaptobenzothiazole which may cause strain specific low response.. 29.

(31) 1-3. CBA/J. 1-3. GHS. 1-2. GHS. CBA/J. LLNA. LLNA. GHS. BrdU-ELISA. 15. Table 3 performance standard GHS. ICCVAM. 2009. UN, 2019. GHS. LLNA LLNA-RI. GHS GHS. 1B: EC3 >2%. Table 3. EC3. 8. CBA/J. Charles River Japan, Inc.. Japan 30. Kanagawa,.

(32) (215W×320D×150H mm 210W×320D×130H mm). 23±2°C. 55±15%. (Toyoriko Co., Ltd., Tokyo, Japan) (MF, Oriental Yeast Co., Tokyo, Japan) 10 1. 15. /h. 12 CERI. 2005. LLNA:BrdU-ELISA LLNA performance standard EC3. 2. ICCVAM,2009) 3. 3. 25µL (. (0. 5mg). 2. ) 4. BrdU. 5 -. (ELISA) 20°C. BrdU. ELISA (Merck KGaA Darmstadt 200. no. 11647229001). 31. ELISA Germany; Cat..

(33) SEMITEC Corp., Osaka, Japan) Corning Inc.. Falcon 352350. NY USA). 15ml. 100 µl. 1. 96. Costar 3595; Corning Inc. NY USA (3000g 10. 3. ). 200 l. Fix-Denat. 30. Fix-Denat. BrdU-POD included in ELISA kit) PBS. 3. 1 tetramethylbenzidine. 100 l. 15. ARVO X4, Perkin. Elmer, Inc. 940 Winter Street Waltham, MA 02451 USA. FLUOstar. OPTIMA, BMG LABTECH, Ortenberg, Germany 370 nm (SI). 492nm. BrdU BrdU. BrdU. EC1.6 EC1.6. Cobalt chloride. 32. SI.

(34) 1.6. EC1.6. EC1.6ex. B Ryan et al., 2007). EC1.6 = c + {(1.6-d) / (b-d)} × (a-c). A. EC1.6ex = 2^ {log2c + (1.6-d) (log2a-log2c)/(b-d)}. A. B. 1-3. B. SI. 1.6. SI. SI. a,b. SI. c,d). GHS 1-2 GHS. GHS. 1A. 1B 1B: EC1.6 >6% 1-2. GHS. 1B. 33. 1A. GHS.

(35) (%) = (LLNA: BrdU-ELISA LLNA-RI. :/ 100. ). (%) = (LLNA: BrdU-ELISA LLNA-RI. EC3. :/ 100. ). EC1.6. LLNA-RI. EC3. LLNA:BrdU-ELISA. EC1.6. (Graphpad PRISM version 6.02, Graphpad software Inc., CA, USA). 34.

(36) EC1.6 LLNA: BrdU-ELISA GHS. EC1.6. 1A. EC1.6 6.03%. 0.0095%. Table 4. 5.03% GHS. 75%. GHS LLNA-RI. EC3. CBA/J. GHS. 1A. LLNA:BrdU-ELISA. GHS. EC1.6. 1A GHS. GHS 1B. CBA/J. 1-2. 1B GHS. LLNA:BrdU-ELISA /. Table 5 GHS. 1-2. 1A. GHS. 100%. 35. GHS 1B. 1B.

(37) EC3. EC1.6 (r) EC3. CBA/J. 0.9076(p<0.0001) LLNA:BrdU-ELISA. EC1.6. (Fig. 8). CBA/J RI. LLNA-RI. LLNA :BrdU-ELISA EC3. EC1.6 GHS. 36. LLNACBA/J.

(38) Table 3. Chemicals used in this study Chemical name. a. CASRN. Manufacturerb. Purity. Vehicle for. LLNA. GHS. (%). BrdU-ELISAc. EC3 (%). Sub-categoryd. CMIa solution. 26172-55-4. Fluka. 1.3. DMF. 0.009. 1A. 2,4-Dinitrochlorobenzene. 97-00-7. Wako. 100.0. AOO. 0.049. 1A. 4-Phenylenediamine. 106-50-3. TCI. >97. DMF. 0.11. 1A. Cobalt chloride. 7646-79-9. Wako. 99.5. DMSO. 0.6. 1A. Isoeugenol. 97-54-1. Wako. 99.1. AOO. 1.5. 1A. 2-Mercaptobenzothiazole. 149-30-4. TCI. DMF. 1.7. 1A. Citral. 5392-40-5. Wako. 99.48. AOO. 9.2. 1B. Hexyl cinnamic aldehyde. 101-86-0. Wako. 98.0. AOO. 9.7. 1B. Eugenol. 97-53-0. TCI. 99.3. AOO. 10.1. 1B. Phenyl benzoate. 93-99-2. Wako. >99.0. AOO. 13.6. 1B. Cinnamic alcohol. 104-54-1. Wako. >97. AOO. 21. 1B. Imidazolidinyl urea. 39236-46-9. MP Biomedicals. 92.2. DMF. 24. 1B. Ethylene glycol dimethacrylate. 97-90-5. Wako. 99.3. MEK. 28. 1B. Methyl methacrylate. 80-62-6. Sigma Aldrich. 99. AOO. 90. 1B. Xylene. 1330-20-7. Wako. 85.2. AOO. 95.8. 1B. 5-chloro-2-methyl-4-isothiazolin-3-one. b c. AOO: acetone-olive oil(4:1), DMF: N,N-dimethylformamide, MEK: methyl ethyl ketone, DMSO: dimethyl sulfoxide.. d. The chemicals were allocated into GHS hazard sub-. based on the known EC3 values cited in the LLNA performance standard (ICCVAM, 2009).. 37.

(39) Table 4. The results of LLNA: BrdU-ELISA and GHS sub-categorization using newly proposed criterion. LLNA: BrdU-ELISA Chemicals. %Tested (SI value). LLNA-RI. EC1.6. GHS sub-category. GHS sub-category. 0.0095 0.029 0.22 0.10* 1.3. 1A 1A 1A 1A 1A. 1A 1A 1A 1A 1A. CMIa solution 2,4-Dinitrochlorobenzene 4-Phenylenediamine Cobalt chloride Isoeugenol. 0.005 (1.2), 0.05 (5.2) 0.01 (1.1), 0.1 (3.5) 0.05 (1.2), 0.3 (1.8) 0.2 (2.3), 2 (4.7) 1 (1.5), 10 (4.5). 2-Mercaptobenzothiazole Citral Hexyl cinnamic aldehyde Eugenol Phenyl benzoate. 2.5 (1.5), 5 (1.6), 10 (2.2) 5 (1.1), 25 (2.6) 5 (1.3), 25 (4.2) 5 (1.4), 25 (5.3) 3 (1.4), 10 (1.1), 30 (1.8). 5.03 11.7 7.07 6.03 24.3. 1A 1B 1B 1B 1B. 1A 1B 1B 1B 1B. Cinnamic alchol Imidazolidinyl urea Ethylene glycol dimethacrylate Methyl methacrylate Xylene. 10 (1.6), 50 (2.4) 5 (1.5), 25 (2.9) 10 (1.1), 50 (2.2) 25 (1.2), 100 (1.8) 10 (1.1), 50 (1.7). 10 6.43 28.18 75 43.3. 1B 1B 1B 1B 1B. 1B 1B 1B 1B 1B. a. 5-chloro-2-methyl-4-isothiazolin-3-one * EC1.6ex 38.

(40) Table 5. CORRECT/INCORRECT outcome (%) using newly proposed sub-categorization criterion for LLNA: BrdU-ELISA GHS sub-category 1A. 1B. CORRECT. INCORRECT. CORRECT. INCORRECT. 100% (6/6). 0% (0/6). 100% (9/9). 0% (0/9). 39.

(41) Fig. 8 Correlation between EC3 values obtained by the standard LLNA-RI and EC1.6 values obtained by LLNA: BrdU-ELISA (r=0.9076, p<0.0001). The dashed lines indicate cut-off values for sub-categorization by LLNA-RI and LLNA: BrdU-ELISA, and each separated field represents sub-category determined by LLNA-RI or LLNA: BrdU-ELISA.. 40.

(42) 1-4. LLNA. LLNA. CBA/J 1920. CBA/Ca. Bagg. DBA CBA/Ca. 1947. Carter. CBA/J. 1948. Jackson Laboratory. (The Jackson. Laboratory, 2002) CBA/J. CBA/Ca. (Green et al., 1965). LLNA. CBA/J. LLNA. CBA/Ca. LLNA CBA/Ca. CBA/J OECD TG429. 41. LLNA.

(43) CBA/J Japan. Charles River Laboratories Japan, INC., Kanagawa,. CBA/Ca 1. (Japan SLC, Inc., Shizuoka, Japan). 4 (215W 23. 2. C. 55. 320D 150H mm). 15%. (Toyoriko. Co., Ltd., Tokyo, Japan) 1. 10. 15. 12. (MF, Oriental Yeast Co., Tokyo, Japan) 8. 10. CERI. 2005. 42.

(44) 5 (Basketter et al, 2000). Human Allergy Class 1. Human Allergy Class. 5. 5. 2,4-Dinitrochlorobenzene (DNCB, Tokyo Chemical Industry Co., Ltd. Japan). Table 6 TCI , Tokyo,. Isoeugenol (Wako Pure Chemical Industries, Ltd., Osaka, Japan). -. Hexylcinnamic aldehyde (HCA, Wako Pure Chemical Industries, Ltd., Osaka, Japan) Propylene glycol (PG, Wako Pure Chemical Industries, Ltd., Osaka, Japan) Hexane (Kanto chemical CO., INC., Tokyo, Japan) Acetone (Wako Pure Chemical Industries, Ltd., Osaka, Japan)/Olive oil (Wako Pure Chemical Industries, Ltd., Osaka, Japan) mixture (4:1, v/v) (AOO). 43.

(45) LLNA LLNA. OECD. TG 429 OECD, 2010. 3 3. 3. 20 Ci. H-methyl thymidine. USA. 3H-TdR, Moravek Biochemicals, CA,. 3H-TdR. 5. PBS; MP Biomedicals, LLC., 200. CA, USA. (LNCs) LNC. 1mL. PBS. 2 1mL. PBS. 5% Trichloroacetic acid. TCA, Wako. 4 C. 18 TCA. 1mL. 10mL. (EcolumeTM, MP. Biomedicals) 3H-TdR. (Tri-. Carb 3110TR, Perkin Elmer, Inc., MA, USA) (DPM). DPM. Disintegrations Per Minute DPM. 44.

(46) (SI). OECD TG429. DPM. CBA/J. CBA/Ca. Unpaired t-test. (P < 0.05). EC3 LLNA. SI. 3. EC3. Ryan et al. 2007. a b SI. c d. LLNA. 3. SI SI. 45. 3.

(47) 3H-TdR DPM SE SE. CBA/J 487 ± 56. CBA/Ca. N = 20. 1202 ± 66. DPM N=8. ± Fig.9. P < 0.01 DPM DPM (0.08%). SE. Fig. 10. DNCB :652 ± 108 (0.016%) 2979 ± 211. 21536 ± 1281(0.4%) Isoeugenol :793 ± 194 (0.4%) 1735 ± 227 (2%). 15622 ± 2945 (10%). HCA:378 ± 38 (2.5%). 567(25%) PG:562 ± 86(2%) 499 ± 56(10%) 162 (25%) 770 ± 51 (50%). (0.016%) 8536 ± 954(0.08%). (100%). SE 32022 ± 1071(0.4%). 22718 ± 4492(10%). 11161 ± 2206(25%). 1115 ± 240 (50%). 2464 ±. 220 ± 36(50%) Hexane: 674 ±. DPM. (0.4%) 5016 ± 802(2%). 1408 ± 360 (10%). 1787 ± 185(100%). CBA/Ca. ± 364(10%). CBA/J. DNCB:1610 ± 166 Isoeugenol:3041 ± 603. HCA:2111 ± 249(2.5%). 5359. PG:1635 ± 382(2%) 1554 ± 236 (10%). Hexane:1531 ± 239(25%). Hexane 100%. 1587 ± 148(50%) (P < 0.05). 46. 1889 ± 458.

(48) LLNA SI. EC3. Table 7. CBA/J. SI. 5.9 ± 0.4 (0.08%). ± SE). (. 42.7 ± 2.5 (0.4%) Isoeugenol:1.7 ± 0.4 (0.4%) 3.7 ± 0.5 (2%). 33.5 ± 6.3(10%). HCA:1.0 ± 0.1(2.5%). PG :1.9 ± 0.2(2%) 1.7 ± 0.2(10%) 1.0 ± 0.1(50%). 3.9 ± 1.0(10%). 6.8 ± 1.6(25%). 0.7 ± 0.1(50%) Hexane:0.8 ± 0.2(25%). 2.2 ± 0.2(100%) DNCB Isoeugenol. CBA/Ca. SI. 7.4 ± 0.8(0.08%). HCA. Hexane. DNCB:1.4 ± 0.2(0.016%). Isoeugenol:2.5 ± 0.5(0.4%) 4.1 ± 0.7(2%). HCA:1.7 ± 0.2(2.5%). PG:1.3 ± 0.3(2%) 1.3 ± 0.2(10%). PG. ± SE). (. 27.4 ± 0.9(0.4%). 18.7 ± 3.7(10%). ± 0.1(50%). DNCB:1.3 ± 0.2 (0.016%). 4.4 ± 0.3(10%). 9.2 ± 1.8(25%). 0.9 ± 0.2(50%) Hexane:1.3 ± 0.3(25%) 1.3. 1.8 ± 0.6(100%) CBA/Ca PG. LLNA. DNCB. Hexane. 47. Isoeugenol. HCA.

(49) EC3 DNCB Isoeugenol LLNA. EC3 LLNA. HCA. 0.04 EC3. 1.4 0.03. 48. CBA/J 7.7. 0.9. CBA/Ca 6.0.

(50) Table 6 Chemicals used in this study and their human allergy classes Chemical 2,4-Dinitrochlorobenzene DNCB Isoeugenol -Hexylcinnamic aldehyde HCA Propyleneglycol PG Hexane (Hex). Supplier. CAS. Lot No.. Human Allergy Class*. TCI. 97-00-7. JXPYL-KE. 1. Wako. 97-54-1. ALR3319. 2. Wako. 101-86-0. LAP0946. 3. Wako. 57-55-6. TSJ4048. 4. KANTO CHEMICAL. 110-54-3. 411U1893. 5. * Human Allergy Class reported by Basketter, D.A. et al, (2000).. 49.

(51) Table 7 Summary of the results in LLNA using CBA/J and CBA/Ca mice. P:Positive, N: Negative. 50.

(52) Fig. 9 DPM values in vehicle control groups (AOO) in local lymph node assay. Data are represented as individual DPM values in each vehicle control group with CBA/J (N=20) or CBA/Ca (N=8). The horizontal bar shows Mean DPM value of each groups. DPM: Disintegration per minute, AOO (acetone/olive oil mixture=4:1 (v/v)) **: P<0.01 (Un-paired t-test). 51.

(53) Fig. 10 DPM values in chemical treated groups in local lymph node assay. Data are represented as individual DPM values in each chemical treated group with CBA/J (N=4) or CBA/Ca (N=4). The horizontal bar shows Mean DPM value of each group. DPM: Disintegration per minute, *: P<0.05, **: P<0.01, NS: not significant (Un-paired t-test). 52.

(54) LLNA. CBA/J CBA/J 1947. CBA/Ca CBA/Ca. 1948. LLNA LLNA. LLNA:BrdU-ELISA (Woolhiser et al., 2000;. Takeyoshi et al., 2004; Takeyoshi et al., 2006) CBA/CaOlaHsd. CBA/CaHsdRcc. CBA/Ca LLNA Anzai et al.,. 2010. OECD TG429. CBA/J. CBA/Ca. CBA/J. CBA/Ca. OECD TG429. LLNA. 53.

(55) DPM CBA/J. CBA/Ca. DPM. DPM. P < 0.01. 3H-. TdR CBA/Ca 100%. CBA/J. DPM. Hexane. DPM SI. DNCB. Isoeugenol. LLNA SI. /. EC3 Interagency Coordinating Committee on the. Validation of Alternative Methods (ICCVAM) Performance Standards for LLNA (ICCVAM, 2009). EC3 Human Allergy Class 1. 5. 54.

(56) 3H-TdR SI. LLNA. EC3. 55.

(57) 1-4. Globally Harmonized System. GHS. GHS 1 1. 1A/1B. OECD TG406 Buehler test. maximization test /. OECD TG429. LLNA BrdU-ELISA. 2010. LLNA-RI OECD TG442B GHS. LLNA BrdU-ELISA GHS LLNA. CBA/J. LLNA BrdU-ELISA LLNA BrdU-ELISA LLNA. 32 BrdU-ELISA. EC1.6. 10%. 1% EC3. 56.

(58) GHS. /. EC1.6. 6%. 12. 7%. 19. GHS. GHS. 13. GHS. 1A. 1B. 1A. 84.2%. 16. 92.3% GHS. 1B. 7%. 6% LLNA-RI. GHS. 2-Mercaptbenzothiazole. 1A. 1B. 2- Mercaptbenzothiazole. CBA / JN. EC1.6. 2- Mercaptbenzothiazole. EC. LLNA LLNA BrdU-ELISA. DA. LLNA DA. CBA / JN 2- Mercaptbenzothiazole. EC. CBA / JN ICCVAM. 2010b. ICCVAM report. EC3=1.7 1993). 2- Mercaptbenzothiazole CBA/Ca. Och et al.. Mercaptbenzothiazole. EC3. 2000. EC3. Basketter et al. BALB/c 9.669. LLNA-RI. 2GHS. 57.

(59) GHS. 1B. LLNA: BrdU-ELISA. CBA/J. EC1.6=5.0 GHS. 2- Mercaptbenzothiazole. 1A. LLNA 2- Mercaptbenzothiazole. GHS. 1A. 3. GHS. 1B. sulfate. GHS. 1A. 3- aminophenol. 100% 3- aminophenol. GHS. 3.2. Table1 2b. Trimellitic anhydride. 1B. LLNA-RI. EC3. Nickel. EC3. LLNA-RI 3- aminophenol. anhydride. Trimellitic. GHS. LLNA-RI. EC3. Trimellitic anhydride et al. 2003. 0.20. EC3. 9.2. EC3. 0.22 Kimber. Fukuyama et al. 2011. Trimellitic anhydride. GHS. 1B. GHS. 1A. LLNA: BrdU-ELISA LLNA-RI LLNA BrdU-ELISA. GHS GHS. 1B 1A. 58. Nickel sulfate LLNA-RI.

(60) LLNA: BrdU-ELISA. DMSO LLNA: BrdU-ELISA. EC1.6. 1.84, 0.57. ± 0.70. 0.67. ICCVAM 2010a. Ha et al. 2017. 1.03. Nickel sulfate. EC1.6. 2%. GHS. ELISA. ±. GHS. LLNA:BrdU-. 1A. 67.0% 32. 1A. Mercaptbenzothiazole. 92.7%. 31. 2-. 100% 1A LLNA. LLNA. GHS. CBA/J LLNA-RI. GHS. BrdU-ELISA. 1A. Performance Standard(ICCVAM, 2009) GHS. 1B EC1.6. 59. 15 Cobalt.

(61) chloride. SI. 1.6. EC1.6ex. 0.10%. (0.2%) Cobalt chloride. 0.2%(SI=2.3). 1.6. LLNA-RI GHS. Cobalt chloride. EC3. 1B GHS. GHS. GHS. CBA/J 1A. SI 1A. 1A. LLNA:BrdU-ELISA GHS. 1B. 100% EC3. CBA/J. LLNA: BrdU-ELISA. EC1.6 (r=0.9076, p<0.0001, Fig. 8) EC1.6. LLNA:BrdU-ELISA. LLNA-RI. EC3 CBA/J. GHS. CBA/J. TG429. LLNA. CBA / Ca LLNA-RI. CBA/J. CBA/Ca. LLNA-RI. 60.

(62) 3H-TdR. SI. LLNA-RI LLNA:BrdU-ELISA. LLNA-RI. CBA/Ca. LLNA:BrdU-ELISA GHS CBA/Ca. GHS. 1B:EC1.6>6%. LLNA:BrdU-ELISA. GHS. 61. GHS CBA/J. EC.

(63) 2. 2-1. IV. Adjuvant and Strip. (Sato et al. 1980). Herber. (Herber 1969). (Gerberick et al . 1990a 1990b) Local Lymph Node Assay(LLNA)(Ulrich et al. 1998). ICH. S10 (2013). 62.

(64) LLNA. 63.

(65) 2-2. LLNA. CBA/Ca. (Japan SLC, Inc., Shizuoka, Japan) 1. 2. 1. 4 (215W. 23. 2. C. 55. 320D 150H mm). 15%. (Toyoriko. Co., Ltd., Tokyo, Japan) 1. 10. 15. 12. (MF, Oriental Yeast Co., Tokyo, Japan) 8. 10 CERI. 2005. 64.

(66) 7 13. 6 Table. 8 Ketoprofen. Cardoso et al. 2009, Tokura 2009, EMCPPTS Taskforce. 2012, Onoue piroxicam Cardoso et al. 2009, Tokura 2009, EMCPPTS Taskforce.. et al.2017. 2012, Onoue et al.2017) al.2017. doxycycline hydrochloride. Tokura 2009, Onoue et. promethazine hydrochloride Cardoso et al. 2009, Scalf et al. 2009, Tokura. 2009, Onoue et al.2017), 8-methoxypsoralen al.2017) 3,3',4',5-tetrachlorosalicylanilide et al. 2014). Bonamonte et al.2010, Onoue et Matsuo et al.2002, Scalf et al. 2009, Gao. chlorpromazine hydrochloride. Scalf et al. 2009, Tokura 2009, Gao. et al. 2014, Onoue et al.2017) 7. Ethanol, propyleneglycol, 1,3-butyleneglycol, 2-propanol, 2-ethylhexyl methacrylate and minocycline hydrochloride. Minocycline hydrochloride. 6. UV. UV. Tokura et al.. 2001. 65.

(67) Methanol Industries, Ltd., Osaka, Japan. MeOH; Wako Pure Chemical. 1 N, N-Dimethylformamide DMF; Wako. 70% Ethanol Wako. 3. EtOH; Wako,. :EtOH = 3:7. UVA. 66. Methylethyl ketone. MEK;.

(68) UV 6. FL40S BLB, Toshiba Corporation, Tokyo,. Japan. 350~355nm UVA 320~400nm. UVB. UVB. UVA. 67.

(69) 1: UV Fig.11 290~400nm. UV. BioSpec. mini, Shimadzu, Kyoto, Japan ( ). = A/(c l) (A:. ;c:. ;l:. :. ). 1000 L mol 1 cm. ICH S10. 1. 1000 L mol 1 cm. 2 UV UV 3. 68. UV 1.

(70) 2: LLNA LLNA. OECD. TG 429 OECD 2010 OECD TG429. EtOH PG 1,3-BG IPA 2-EHMA KP Px DOC MIC PMZ 8-MOP 100. TCSA CPZ. 100 100. 25. % 10. 100. 100. 100. 100. 100. 0.02. 3 3. 20. Ci. 3. H-methyl thymidine 3H-TdR, Moravek Biochemicals, CA, USA 3H-TdR. 5. PBS; MP Biomedicals, LLC., 200. CA, USA. (LNCs) LNC. 1mL. PBS. 2 1mL. 5% Trichloroacetic acid. PBS TCA, Wako. 18. 69. 4 C.

(71) TCA. 1mL. (EcolumeTM, MP. 10mL. Biomedicals) 3H-TdR. (Tri-. Carb 3110TR, Perkin Elmer, Inc., MA, USA). Disintegrations Per Minute. DPM. (DPM) (SI). DPM. OECD TG429. LLNA. SI. 3. EC3. Ryan et al. 2007. a b SI. c d. LLNA. 3. SI. 3. SI. LLNA EC3 10J/cm2 3. 70. UVA.

(72) 0 0. 6 5%. 25%. 71.

(73) 3: UV. (UV+). UV. (UV-). 2. 2 NOAEL UV+ 2. UVA. 3. UV 3H-TdR. LLNA. pSI DPM. pSI. DPM. UV+ UV+. unpaired t-test. p< 0.05. 72. UV -. pSI.

(74) 1: UV UV. Table 9. 5. EtOH PG 1,3-BG IPA. 2-EHMA. 1000 L mol. 1. cm. 1. UV 8. (KP. Px. DOC. CPZ) 6570. MIC. PMZ 8-MOP 2200 20540. 13700 8260,. TCSA. 13490. 12910. 1000 L mol 1 cm. 4860. UV. 73. 1.

(75) 2: LLNA. Table 10. 4. CPZ). (2-EHMA EC3. 2.4. %. Px. TCSA. 6.5 0.7. 0.006. 9 Table 10. DOC. UV+. PMZ 8-MOP 0. 4 NOAELs. %. 5. 9. 74. 25. 0.025 0.1. CPZ 25%.

(76) 3:. 0.2 3.0 1.4. Table 11. UV-. 4.1 0.4 Px 0.25%. 1.8. 0.4. PMZ. 0.1. CPZ 0.1%. 3.9 1.3. 7. 25%. 1.3. (KP. 0.2 0.2. 0.6 TCSA 0.2. UV+. 0.006%. KP 1.0%. 0.6 DOC 5.0%. 12.7. 2.0. 8-MOP. 1.6. 0.3. 3.5. 1.9. 2.4 0.1. 0.025% 0.3. 13.3 ± 2.3. Px. DOC. p(p<0.05). PMZ 8-MOP. TCSA. CPZ). 6 7. TCSA. 3.6. pSI. (KP Px DOC PMZ 8-MOP. CPZ). 6. 75.

(77) Table 8. Chemicals used in this study and photoallergy information Abbreviation. Supplier. CASRN. Ethanol Propyleneglycol 1,3-Butyleneglycol 2-Propanol 2-Ethylhexyl Methacrylate Ketoprofen Piroxicam Doxycycline Hydrochloride Minocycline Hydrochloride. EtOH PG 1,3-BG IPA 2-EHMA KP Px DOC MIC. Wako Wako TCI Wako Wako Wako TCI MP Biomedicals, LLC TCI. 64-17-5 57-55-6 107-88-0 67-63-0 688-84-6 22071-15-4 36322-90-4 10592-13-9 13614-98-7. Promethazine Hydrochloride 8-Methoxypsoralen 3,3',4',5-Tetrachlorosalicylanilide. PMZ 8-MOP TCSA. Chlorpromazine Hydrochloride. CPZ. Photoallergy information. References. EN EN EN EN EN P P P EN. 1), 2), 3), 4) 1), 2), 3), 4) 2), 4) 5). TCI 58-33-3 Sigma-Aldrich Co. LLC 298-81-7 Kanto chemical 40743-51. P P P. 1), 2), 4), 6) 4), 7) 6), 8), 9). TCI. P. 2), 4), 6), 9). 69-09-0. EN: Excepted Negative, P: Positive, -: No information, TCI: Tokyo Chemical Industry Co., Ltd. 1) Cardoso et al. 2009, 2) Tokura 2009, 3) The European Multicentre Photopatch Test Study Taskforce. 2012, 4) Onoue et al.2017, 5) Tokura et al.2001, 6) Scalf et al. 2009, 7) Bonamonte et al. 2010, 8) Matsuo et al. 2002, 9) Gao et al. 2014.. 76.

(78) Table 9. Results of Step 1: UV absorption analysis UV absorption analysis Chemicals. MEC (Lmol-1cm-1). P/N. Ethanol Propyleneglycol 1,3-butyleneglycol 2-propanol 2-ethylhexyl Methacrylate Ketoprofen Piroxicam Doxycycline hydrochloride. 290 325 350. 2200 20540 13490. N N N N N P P P. Minocycline hydrochloride Promethazine hydrochloride 8-methoxypsoralen 3,3',4',5-tetrachlorosalicylanilide. 340 300 299 325. 12910 6570 13700 8260. P P P P. Chlorpromazine hydrochloride. 308. 4860. P. MEC: molar extinction coefficient : no UV absorption, P: Positive, N: Negative. 77.

(79) Table 10. Results of Step 2: Skin phototoxicity evaluation based on LLNA Skin phototoxicity. LLNA Chemical. P/N. EC3 (%). Concentration Increase rate of ear thickness (%) (%) Mean (Left) Mean (Right). P/N. Ethanol. N. Not calculated. 100. 2.7. 3.5. N. Propyleneglycol. N. Not calculated. 100. 0.9. 2.8. N. 1,3-Butyleneglycol. N. Not calculated. 100. 2.0. 4.4. N. 2-Propanol. N. Not calculated. 100. 0.2. 0.1. N. 2-Ethylhexyl Methacrylate. P. 6.5. 6.5. 3.1. 3.6. N. 0.1 0.25 0.5. 7.7 1.1 3.2. 6.0 10.4 13.8. 1. 10.9. 11.3. 0.1. 0.4 1.0. 2.1 0.3. -. -. 11.4 19.3 25.0. 9.3 17.3 25.1. Ketoprofen. Piroxicam. N. P. Not calculated. 0.25 0.5 0.7. 0.7. 2.5 Doxycycline Hydrochloride. N. Not calculated. 5 10. N: Negative, P: Positive,-: Data were excluded because of systemic toxicity or excessive irritation *: Concentration indicated in bold typed were selected concentration for Step 3. 78. N. N. P.

(80) Table 10 (Continued). Results of Step 2: Skin phototoxicity evaluation based on LLNA Skin phototoxicity. LLNA Chemical. P/N. EC3 (%). Minocycline Hydrochloride. N. Not calculated. Promethazine Hydrochloride. N. Not calculated. 8-Methoxypsoralen. 3,3',4',5-Tetrachlorosalicylanilide. Chlorpromazine Hydrochloride. N. P. P. Concentration Increase rate of ear thickness (%) (%) Mean (Left) Mean (Right) 2.5. Not calculated. 0.006. 2.4. 5. 8.0 6.4. 7.3 8.5. N. 25 40. 10.4 25.0. 12.1 25.3. P. 0.01 0.025 0.05 0.1. 20.7 24.3 41.8 -. 16.4 22.4 39.6 -. 1.0 10. -. -. 0.006. 1.8. 3.0. 0.1. 12.3. 9.2. 0.25. 27.4. 23.3. 0.5. 33.2. 30.7. 1.0. 55.4. 60.6. 2.4. -. -. N: Negative, P: Positive,-: Data were excluded because of excessive irritation *: Concentration indicated in bold typed was selected concentration for Step 3. 79. P/N. P. N. P.

(81) Table 11. Results of Step 3: Photoallergy evaluation Chemical. Concentration (%). Ethanol. 100. Propyleneglycol. 100. 1,3-Butyleneglycol. 100. 2-Propanol. 100. 2-Ethylhexyl Methacrylate. 6.5. Ketoprofen. 1. Piroxicam. 0.25. Doxycycline Hydrochloride. 5. Minocycline Hydrochloride. 5. Promethazine Hydrochloride. 25. UV +/. 1.0. 0.2. +. 0.7. 0.1. +. 1.0 0.7. 0.1 0.1. N. +. 0.8 0.7. 0.1 0.1. N. 3.2. 0.2. +. 3.7. 0.3. +. 2.4 4.1 **. 0.2 0.4. 1.8. 0.2. +. 3.6 **. 0.6. +. 1.9 3.0 **. 0.1 0.4. 1.8. 0.4. 1.5. 0.2. +. 3,3',4',5-Tetrachlorosalicylanilide. 0.006. SE 0.1 0.0. +. 0.025. Mean. Photoallergy P/N. 0.8 0.6. +. 8-Methoxypsoralen. pSI. + +. 1.3 12.7 **. 0.2 2.0. 1.4. 0.1. 3.9 **. 0.6. 1.6. 0.3. 3.5 **. 0.3. N N. N P P P N P P P. ** Significantly different from the concurrent UV(-) control group (Unpaired t-test, p<0.01) N: negative, P: positive. 80.

(82) Table 11 (Continued). Results of Step 3: Photoallergy evaluation Chemical. Chlorpromazine Hydrochloride. Concentration (%). UV +/. pSI Mean 1.3. 0.1. +. 13.3 **. SE 0.2 2.3. Photoallergy P/N P. ** Significantly different from the concurrent UV(-) control group (Unpaired t-test, p<0.01) N: negative, P: positive. 81.

(83) Table 12. Summary of results in three-step approach UV absorption. Skin sensitization. Ethanol Propyleneglycol 1,3-Butyleneglycol 2-Propanol. N N N N. N N N N. N N N N. N N N N. 2-Ethylhexyl Methacrylate. N. P. N. N. Ketoprofen. P. N. N. P. Piroxicam. P. P. N. P. Doxycycline Hydrochloride. P. N. P. P. Minocycline Hydrochloride. P. N. N. N. Promethazine Hydrochloride. P. N. P. P. 8-Methoxypsoralen. P. N. P. P. 3,3',4',5-Tetrachlorosalicylanilide. P. P. N. P. Chlorpromazine Hydrochloride. P. P. P. P. Chemicals. N:Negative,. P:Positive. 82. Skin Photoallergy phototoxicity.

(84) Fig. 11. Flow diagram for photoallergy evaluation. New method consists of three steps; UV spectrum analysis, skin phototoxicity evaluation based on LLNA to determine NOAEL, and photoallergy evaluation based on 1. cm 1 at any wavelength were considered. UV absorptionL mol 1 cm 1 at any wavelength were considered UV absorption-negative. The no observable adverse effect level (NOAEL) for skin sensitization and skin phototoxicity decided based on the results in Steps 2. An unpaired t-test was performed using pSI was considered as photoallergy positive if a significant difference was found between groups (p < 0.05) in Step 3.. 83.

(85) 2-3. 1. ICH S10. 2013 LLNA. 3 1. UV. LLNA. 2. NOAEL. LLNA 3 Table 12. 7 UV. 6 hydrochloride (DOC). ketoprofen promethazine hydrochloride. 5. 84. piroxicam. doxycycline. chlorpromazine hydrochloride.

(86) DOC. minocycline hydrochloride. MIC. DOC Tokura 2009, Onoue et al. 2017. MIC. UV Tokura 2001). MIC. LLNA. 85.

(87) 3. in vitro. 3-1. (Kimber et al,2002, 2011) OECD OECD, 1992. (OECD TG406). Guinea pig maximization test 2010 OECD, 2018. Buehler test. OECD TG429 OECD,. Local Lymph Node Assay LLNA). OECD TG442B. LLNA:BrdU-ELISA. EU (EU, 2013). 4. (Key event, KE). 86.

(88) (Adverse Outcome Pathway AOP). (OECD, 2012). KE-1: KE-2: KE-3: KE-4: T. OECD TG 4. KE. 1. Integrated Approaches to Testing and Assessment OECD TG LuSens. IATA. OECD. OECD, 2019. KE-2. KeratinoSens. OECD TG442D(OECD 2018). 87. OECD.

(89) Dual luciferase in vitro 1. Firefly luciferase(Photinus pyralis 2. (ARE)-Nrf2 ) ARE-Nrf2. ARE-Nrf2 -Sens. LLNA. Human data. 88. ). Renilla luciferase(Renilla reniformis.

(90) 3-2. 2u-globulin. AUG. 3-2. ARE-Nrf2. Table 13. MCDB153. 37. 5%CO2. 10%FBS-DMEM/F-12 5%CO2. 89. 37.

(91) pGL3 A. antioxidant response element. Promega KK., Wisconsin, US -TGGTCGCAAGGTGTG. CAAGCTGCTGAGTCACCCTGACTGCATCAACCCCAGGAGCTAUG TATA Box. Takeyoshi et al. 2003. 3. pA. Table 14. RE-AUG. Renilla luciferase. pRL-TK. Promega KK.,. Wisconsin, USA. OECD TG442D Cinnamic aldehyde. 10 Table 15 Dimethyl sulfoxide. DMSO, FUJIFILM Wako Pure. Chemical Corporation, Osaka, Japan. OECD TG442D. Cinnamic aldehyde. 90.

(92) 6cm 6 g. Lipofectamine LTX Thermo Fisher Scientific K.K.) 37. Cinnamic aldehyde. 5%CO2. 72. 20mM Cinnamic aldehyde. 2. DMSO. 50 1 104 cells/. 96 5. Cinnamic aldehyde DMSO. 24. Cinnamic aldehyde Dual-Glo® Reagent. 24 (Promega KK., Wisconsin, USA). 10. (ARVO X2, Perkin Elmer, Inc., Waltham, USA). Firefly Dual-Glo® Stop & Glo® Reagent. luciferase (Promega KK., Wisconsin, USA). 10. Renilla luciferase Fold induction. Firefly luciferase. RLU. 91.

(93) Fold induction =. luciferase. RLU /. Renilla luciferase. (%) = (. rLuc. rLuc. RLU. RLU. RLU /. 12 inducton. luciferase. rLuc. RLU) × 100. 3. Fold. SD OECD TG442D. Fold induction. Fold induction Fold induction. 1.5. 1.5. Imax. EC1.5. EC1.5 = c + {(1.5. a b induction. c d. Fold induction. 1.5. 1.5. Fold induction. 92. Fold.

(94) AUG Cinnamic aldehyde 3. PHK 16-0b. CHO. PHK 16-0b. CHO. pGL3-ARE-AUG. OECD TG442D Cinnamic aldehyde PHK 16-0b. Imax. EC1.5. ARE-AUG200. pGL3OECD. TG442D. Table 15. 10. EC1.5. 93.

(95) Table 16 PHK16-0b. EC1.5. Imax. 1.5µM. 12.8 CHO. 2. AUG AUG. Table 17. CHO 0b. PHK 16AUG200. Imax. EC1.5. 1.5µM. 12.8. OECD TG442D OECD TG442D Table 18. CHO. PHK 16-0b TG442D. 94. 6.

(96) CHO. EC1.5. 16-0b. 6. Reference range. PHK. Ethylene glycol dimethacrylate. Methyldibromo. gultaronitrile 4- Methylamino phenol sulfate EC1.5 Mercaptbenzothiazole. Reference range 2. 2,4-Dinitro chrolobenzene. 4. Cinnamiyl alchol. 2-. Reference range. 95.

(97) PHK 16-0b. ARE. AUG OECD TG442D. EC1.5. 96. 200.

(98) Table 13. List of host cell lines. SVIA6. JCRB Cell Bank. JCRB1092. SVIIA3. JCRB Cell Bank. JCRB1091. NCE16. JCRB Cell Bank. JCRB0140. PSVK1. JCRB Cell Bank. JCRB1093. PHK16-0b JCRB Cell Bank. JCRB0141. HeLa229 JCRB Cell Bank. JCRB9086. CHO. RIKEN Cell Bank RCB0285. 97.

(99) Table 14. List of plasmide. ARE-AUG100. 100bp. ARE-AUG150. 150bp. ARE-AUG200. 200bp. 98.

(100) Table 15 List of use chemicals in transient reporter gene assay TG429. CAS. TG442D. Isopropanol. 67-63-0. Non-sensitiser. Negative. Salicylic acid. 69-72-7. Non-sensitiser. Negative. Lactic acid. 50-21-5. Non-sensitiser. Negative. Glycerol. 56-81-5. Non-sensitiser. Negative. Cinnamyl alcohol. 104-54-1. Sensitiser. Positive. Ethylene glycol dimethacrylate. 97-90-5. Sensitiser. Positive. 2-Mercaptbenzothiazol. 149-30-4. Sensitiser. Positive. 35691-65-7. Sensitiser. Positive. 4-(Methylamino)phenol sulfate. 55-55-0. Sensitiser. Positive. 2,4-Dinitro-chlorobenzene. 97-00-7. Sensitiser. Positive. Methyldibromo glutaronitrile. 99.

(101) Table 16 Sammary of host cell line decision EC1.5( M. Imax Imax FL. SVIA6. NC. 1.5. 15. SVIIA3. 7.8. 2.9. 44. NCE16. 13.4. 3.9. 678. PSVK1. 3.5. 5.3. 77. PHK16-0b. 1.5. 12.8. 617. HeLa229. NC. NC. NC. CHO. 4.5. 5.6. 42806. 100.

(102) Table 17 Sammary of plasmid decision CHO Run. EC1.5. PHK 16-0b. M SD. ARE-AUG100. ARE-AUG150. ARE-AUG200. EC1.5. Imax. M. SD 5.0. Imax SD. 2.7. SD 4.5. 1. 2.0. 2. 3.1. 3. 7.8. 5.9. 1.4. 5.6. 1. 2.6. 4.6. 3.3. 5.2. 2. 7.8. 3. 2.7. 5.6. 3.4. 5.2. 1. 2.0. 5.2. 1.6. 8.9. 2. 3.6. 3. 7.8. 4.3. 4.4. 4.5. 3.1. 3.0. 3.0. 6.1. 4.9. 5.6 6.0. 5.7. 5.0. 5.6. 0.6. 0.5. 0.4. 1.3. 2.0. 1.2 1.8. 101. 1.8. 2.9. 1.5. 0.8. 0.8. 0.3. 5.5. 7.8. 15.7 13.8. 5.2. 0.6. 6.1. 1.5. 12.8. 3.5.

(103) Table 18 Summary of transient reporter gene assay using CHO cell line and PHK 16-0b cell line TG442D. CHO. EC1.5. Proficiency substances. PHK 16-0b. EC1.5. Reference range. EC1.5. Mean. SD. Mean. SD. Isopropanol. N. 1000. NC. N. NC. NC. N. NC. NC. Salicylic acid. N. 1000. NC. N. NC. NC. N. NC. NC. Lactic acid. N. 1000. NC. N. NC. NC. N. NC. NC. Glycerol. N. 1000. NC. N. NC. NC. N. NC. NC. Cinnamyl alcohol. P. 25-175. 123.6. P. 28.6. 13.7. P. 20.9. 6.2. Ethylene glycol dimethacrylate. P. 5-125. 57.4. P. 15.3. 6.7. P. 15.9. 10.7. 2-Mercaptbenzothiazol. P. 25-250. 48.1. P. 41.3. 18.7. P. 21.1. 1.1. Methyldibromo glutaronitrile. P. 20. 7.8. P. 1.8. 1. P. 3.7. 1.5. 4-(Methylamino)phenol sulfate. P. 12.5. 9.4. P. 1.5. 0.3. P. 1.4. 0.4. 2,4-Dinitro-chlorobenzene. P. 12.5. 2.5. P. 1.2. 0.5. P. 0.4. 0.1. 102.

(104) 3-3. 3-2 -Sens. Table 19 KeratinoSensTM 19. 9. DB-ALM, 2013 28. Dimethyl sulfoxide DMSO, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan. -Sens assay 3-2. PHK 16-0b JCRB cell bank,. Osaka, Japan HKGS (Thermo Fisher Scientific K. K., Massachusetts, USA) 103. Epi-Life.

(105) (Thermo Fisher Scientific K. K., Massachusetts, USA). 37. 5% CO2. ARE-Nrf2 3-2 AUG. 2U-globulin. Takeyoshi et al., 2003. antioxidant response element A. -TGGTCGCAAGGTGTGCAAGCTGCTGAGTCACCCTGACTGCATCAACC Firefly luciferase. CCAGGAGCT-. pLVSIN. Takara Bio Inc., Sh. pLVSIN-ARE-AUG-Luc. iga Japan. Thy midine kinase TK. Renilla luciferase. rLuc. pLVSIN. pLVSIN-TK-rLuc pLentiviral High Titer Packaging Mix (Takara Bio Inc., Shiga, Japan) pLVSIN-TK-rLuc. AUG-Luc. packaging mix. pLVSIN-ARETransIT®-293 Transfection. Lenti-XTM 293T. (Takara Bio Inc., Shiga, Japan). (Takara Bio Inc.,. Shiga, Japan) 37. 5% CO2. 48. DISMIC-25AS filter (0.44 µm, Toyo Roshi Kaisha Ltd., Tokyo, Japan). 104.

(106) ARE-AUG-Luc. TK-rLuc. PHK 16-0b. 50 µg/mL Hygromycin B Gold. InvivoGen, CA, USA. 2.5 µg/mL of puromycin. Thermo Fisher Scientific K. K., Massachusetts, USA. 7. Mitochondrial reductase. Renilla luciferase. OECD TG442D. Cinnamic aldehyde (FUJIFILM. Wako Pure Chemical Corporation, Osaka, Japan). LuSens. Mitochondrial reductase Renilla luciferase Mitochondrial reductase Methylthiazolyl tetrazolium MTT assay Cell Counting Kit-8 Soluble Tetrazolium salts-8 WST-8 Cinnamic aldehyde 50. CCK-8, Dojindo, Osaka, Japan. Water. assay. 20mM DMSO. 2 2 106 cells/mL. 96. 105.

(107) 100µL/ Cinnamic aldehyde. 20000 cells/well. 24. DMSO. 24. DMSO. Cinnamic aldehyde 5. CCK-8. 100µL. 37°C. 5% CO2. 1. ARVO X2, Perkin Elmer, Inc., Waltham, USA 450nm. (%) =. /. × 100. Renilla luciferase rLuc. rLuc. RLU. (%) = (. rLuc. RLU /. Mitochondrial reductase Sens. rLuc. RLU) × 100. -. Renilla luciferase IC50. (Graphpad PRISM. 106.

(108) version 6.02, Graphpad software Inc., CA, USA). -Sens assay 200mM. DMSO. 2. 12. 50 96. (#136102, Thermo Fisher Scientific K. K.,. Massachusetts, USA) cells/well. 2. 106 cells/mL. 100µL/. 24. 20000. DMSO 24. (. ). DMSO. Dual-Glo® Reagent (Promega KK., Wisconsin, 10. USA). Waltham, USA). (ARVO X2, Perkin Elmer, Inc., Firefly luciferase. Dual-Glo® Stop & Glo® Reagent (Promega KK., Wisconsin, USA) 10. Renilla luciferase. ARE-Nrf2. Firefly luciferase. 107.

(109) Relative Light Unit (RLU). Renilla luciferase. RLU. RLU. normalized RLU(nRLU) normalized ARE Activity (nAA). nAA =. nRLU /. Fold induction. nRLU. Firefly luciferase. Fold induction =. RLU. Firefly luciferase. RLU /. Firefly luciferase. RLU. Renilla luciferase. (%) = (. rLuc. 12. rLuc. RLU /. 3. RLU. rLuc. RLU) × 100. nAA. 108.

(110) SD. nAA. nAA+2SD. %. -Sens, KeratinoSensTM. LuSens. Cooper statistics (Cooper et al., 1979). -Sens. sensitivity (LLNA. -Sens -Sens. (LLNA accuracy (. ). KeratinoSensTM. ). 109. specificity ). LuSens.

(111) Mitochondrial reductase. Renilla luciferase. OECD TG442D. Cinnamic aldehyde. WST-8. rLuc Fig. 12 rLuc. IC50. 200µM. 61.9. 0.9%. WST-8. 200µM. 50%. IC50. -Sens assay -Sens. nAA nAA. Table 20. 0.85-1.32. Fig.13 nAA. 1.76-26.42. 110.

(112) Table 21 nAA (1.32) + 2SD (=1.57). 1.6 100%. 96.4 %. LLNA. 1.6. -Sens, KeratinoSensTM. LuSens. -Sens, KeratinoSensTM. LuSens. LLNA. Table 22 accuracy sensitivity. specificity. Table 23 LLNA 95.0%. -Sens. specificity. 100.0%. 89.3% 92.9% 89.5%. 96.4% 100%. KeratinoSensTM. sensitivity. accuracy sensitivity. LLNA. 85.0%. accuracy sensitivity. 100.0%. 100.0% specificity. specificity. LuSens LLNA 89.3% 92.9% 94.7%. 111. 90.0% 88.9%. 87.5%. accuracy.

(113) 3-2 -Sens. -Sens. ARE-Nrf2 normalized ARE Activity nAA accuracy sensitivity. specificity. LLNA 96.4%. 100% KeratinoSensTM. KE-2. LuSens KE-2. 112. 95.0%. 100.0%.

(114) Table 19 Chemicals used in this study. LLNA. Human data5. Chemical. CASRN. Source. Oxazolone. 15646-46-5. SIGMA. P(0.0028)3. P. 5-Chloro-2-methylisothiazolinone. 26172-55-4. FLUKA. P(0.01)3. P. Result (EC3). 2,4-Dinitrochlorobenzene. 97-00-7. TCI. P(0.049). P. 4-Nitrobenzylbromide. 100-11-8. TCI1. P(0.1)3. P. 4-Phenylenediamine. 106-50-3. 1. TCI. P(0.11). P. Glyoxal. 107-22-2. TCI1. P(0.7)3. P. 1. P(0.8). 3. P. P(0.9 /1.9)3. 1. 3. 3. 4-Methylaminophenolsulphate. 55-55-0. TCI. Methyldibromo glutaronitrile. 35691-65-7. FLUKA. P. Cinnamic aldehyde. 104-55-2. Wako. P(1.3). P. Isoeugenol. 97-54-1. TCI1. P(1.5)3. P. 3. P. 2. 3. 2-Mercaptobenzothiazole. 149-30-4. FLUKA. P(2.5). Tetramethylthiuramdisulfide. 137-26-8. FLUKA. P(3.1)3. P. 4. N. Sodium lauryl sulfate. 151-21-3. FLUKA. P(8.1). Hexyl cinnamic aldehyde. 101-86-0. Wako2. P(9.9)3. P. 3. P. 1. Citral. 5392-40-5. TCI. P(9.8). Eugenol. 97-53-0. TCI1. P(10.1)3. P. Phenyl benzoate. 93-99-2. 1. TCI. 3. P(13.6). P. Cinnamyl alcohol. 104-54-1. SIGMA. P(21.0)3. P. 3. P. Imidazolidinyl urea. 39236-46-9. SIGMA. P(24.0). Ethylene glycol dimethacrylate. 97-90-5. TCI1. P(32.9)3. P. -. N. 1. Chlorobenzene. 108-90-7. TCI. Diethyl phthalate. 84-66-2. TCI1. -. N. 2. Glycerol. 56-81-5. Wako. -. N. Isopropanol. 67-63-0. Wako2. -. N. Lactic acid. 50-21-5. 2. Wako. -. N. Methyl salicylate. 119-36-8. TCI1. -. N. 1. Salicylic acid. 69-72-7. TCI. -. N. Sulfanilamide. 63-74-1. TCI1. -. N. P: Positive, N: Negative 1:. Tokyo Chemical Industry Co., Ltd., 2: FUJIFILM Wako Pure Chemical Corporation, 3:DB-ALM,. 2013, 4: ICCVAM, 2009, 5: Tzutzuy et al., 2014. 113.

(115) Table 20 Summary of the results of -Sens. Chemical. Mean maximum. Cell viability. nAA ± SD. (%). Oxazolone. 2.35 ± 0.35. 76.5. 5-Chloro-2-methylisothiazolinone. 2.45 ± 0.29. 57.7. 5.7 ± 1.58. 52.6. 4-Nitrobenzylbromide. 1.75 ± 0.05. 71.2. 4-Phenylenediamine. 7.77 ± 1.46. 50.5. Glyoxal. 2.44 ± 0.3. 57.1. 4-Methylaminophenolsulphate. 4.92 ± 0.8. 58.4. Methyldibromo glutaronitrile. 3.41 ± 0.72. 76.5. 18.94 ± 2.26. 70.9. Isoeugenol. 4.21 ± 0.16. 65.1. 2-Mercaptobenzothiazole. 2.91 ± 0.17. 54.2. Tetramethylthiuramdisulfide. 2.62 ± 0.48. 80.9. Sodium lauryl sulfate. 1.06 ± 0.13. 87.1. Hexyl cinnamic aldehyde. 2.77 ± 0.19. 77.3. Citral. 5.46 ± 1.03. 77.2. 2.7 ± 0.52. 63. 2,4-Dinitrochlorobenzene. Cinnamic aldehyde. Eugenol Phenyl benzoate. 1.89 ± 0.1. 61.7. Cinnamyl alcohol. 8.08 ± 0.19. 54.3. Imidazolidinyl urea. 2.82 ± 0.44. 80. 26.42 ± 5.72. 56.1. Chlorobenzene. 0.85 ± 0.14. 102.6. Diethyl phthalate. 1.32 ± 0.13. 67.4. Glycerol. 1.15 ± 0.23. 102.1. Isopropanol. 1.07 ± 0.18. 104.5. Lactic acid. 1.18 ± 0.2. 83.5. Methyl salicylate. 1.31 ± 0.13. 105.3. Salicylic acid. 1.11 ± 0.18. 98.8. Sulfanilamide. 0.99 ± 0.12. 94.7. Ethylene glycol dimethacrylate. 114.

(116) Table 21 Determination of the optimal cutoff for -Sens. Correct outcome (%) obtained by the nAA cutoff value 1.2. 1.3. 1.4. vs. LLNA. 89.3. 89.3. 96.4. vs. Human data. 92.9. 92.9. 100.0. 1.5. 1.6. 1.7. 1.8. 1.9. 2.0. 96.4. 96.4. 96.4. 89.3. 82.1. 82.1. 100.0. 100.0. 100.0. 92.9. 89.3. 89.3. Bold type: Optimal cutoff criterion. 115.

(117) Table 22 Summary of the outcomes in the -Sens, KeratinoSensTM and LuSens KeratinoSensTM1. LuSens2. Oxazolone. P. P. P. P. P. 5-Chloro-2-methylisothiazolinone. P. P. P. P. P. 2,4-Dinitrochlorobenzene. P. P. P. P. P. 4-Nitrobenzylbromide. P. P. P. P. P. 4-Phenylenediamine. P. P. P. P. P. Glyoxal. P. P. P. P. P. 4-Methylaminophenolsulphate. P. P. P. P. P. Methyldibromo glutaronitrile. P. P. P. P. P. Cinnamic aldehyde. P. P. P. P. P. Isoeugenol. P. P. P. P. P. 2-Mercaptobenzothiazole. P. P. P. P. P. Tetramethylthiuramdisulfide. P. P. P. P. P. Sodium lauryl sulfate. P. N. N. N. N. Hexyl cinnamic aldehyde. P. P. P. P. P. Citral. P. P. P. P. P. Eugenol. P. N. P. P. P. Phenyl benzoate. P. N. N. P. P. Cinnamyl alcohol. P. P. P. P. P. Imidazolidinyl urea. P. P. P. P. P. Ethylene glycol dimethacrylate. P. P. P. P. P. Chlorobenzene. N. N. N. N. N. Diethyl phthalate. N. N. N. N. N. Glycerol. N. N. N. N. N. Isopropanol. N. N. N. N. N. Lactic acid. N. N. N. N. N. Methyl salicylate. N. N. P. N. N. Salicylic acid. N. N. N. N. N. Sulfanilamide. N. N. N. N. N. P: Positive, N: Negative 1. Human. LLNA1. Chemical. : DB-ALM, 2013, 2: Tzutzuy et al., 2014. 116. data2. -Sens.

(118) Table 23 Comparison of predictive performance of -Sens, KeratinoSensTM, and LuSens. vs. LLNA LLNA. KeratinoSensTM. -Sens. LuSens. Positive. Negative. Positive. Negative. Positive. Negative. Positive. 19. 1. 17. 3. 18. 2. Negative. 0. 8. 0. 8. 1. 7. Sensitivity (%). 95.0. 85.0. 90.0. Specificity (%). 100.0. 100.0. 87.5. Accuracy (%). 96.4. 89.3. 89.3. KeratinoSensTM. LuSens. vs. Human data Human. -Sens. Positive. Negative. Positive. Negative. Positive. Negative. Positive. 19. 0. 17. 2. 18. 1. Negative. 0. 9. 0. 9. 1. 8. Sensitivity (%). 100.0. 89.5. 94.7. Specificity (%). 100.0. 100.0. 88.9. Accuracy (%). 100.0. 92.9. 92.9. Sensitivity (%) = TP/(TP+FP) 100. Specificity (%) =TN/(TN+FN). 100.. Accuracy (%) =(TP+TN) / (TP+FP+TN+FN). 100.. TP: True positive, FP: False positive, TN: True negative, FN: False negative.. 117.

(119) Fig. 12 Comparison of the outcome of cytotoxicity tests of cinnamic aldehyde using the reporter gene assay and the mitochondrial reductase activity Open circle: cell viability based on mitochondrial reductase activity, closed circle: cell viability based on Renilla luciferase activity.. 118.

(120) Fig. 13 Dose-related changes in nAA of chemicals The -Sens cell line was treated with different concentrations of each chemical for 24h. Broken line: Cut off (nAA=1.6). 119.

(121) Fig. 14 Dose-related changes in fold induction, nAA and cell viability in -Sens The -Sens cell line was treated with different concentrations of phenyl benzoate, methyl salicylate (a), and methyldibromo glutaronitrile (b) for 24h. Open circle: cell viability, closed circle: fold induction of transcriptional activity (left) or nAA (right).. 120.

(122) 3-4. EU EU. EU 2013. OECD AOP. 4. KE. 1. KE-2. OECD TG 442D LuSens (OECD, 2018). Dual luciferase in vitro Firefly luciferase (Photinus pyralis. Renilla luciferase (Renilla reniformis. 121. ARE-Nrf2. ) ).

(123) OECD TG442D. Cinnamic aldehyde. mitochondrial reductase. Renilla luciferase. rLuc. rLuc IC50 0.9% 200µM. 200µM WST-8. 50%. IC50. Fig. 12 2 Mitochondrial reductase. luciferase ARE. luciferase. Mitochondrial reductase. Mertl et al (2019) luciferase. KE-2. KeratinoSensTM. assay. 122. LuSens. ARE-Nrf2 luciferase.

(124) WST-8. MTT assay. mitochondrial reductase. Mertl et al. (2019). ARE-Nrf2. NanoLuc. Firefly luciferase. Dual-. Luciferase. Firefly luciferase. Renilla luciferase. Dual-Luciferase. ARE. luciferase. Renilla luciferase ARE-Nrf2 ARE-Nrf2 Renilla luciferase nAA KeratinnoSensTM. 28 nAA nAA. 1.76-26.42. 0.85-1.32 Table 20. -Sens. 123.

(125) LLNA nAA+2SD. 1.57. 1.6 LLNA. 96.4 -Sens. 100.0%. KeratinoSensTM. (Table 21) LuSens. accuracy (%) sensitivity (%). specificity (%). LLNA (%). LLNA Table 23. accuracy (%). 96.4% 95.0%. sensitivity (%). specificity. 100%. 100% -Sens. KeratinoSensTM. nAA LuSens. (Fig. 14a). luciferase. Methyl salicylate Dual-Lucferase. Mertl et al.. Methyldibromoglutaronitril. (2019) (Fig. 14b). LuSens. -Sens. Methyldibromoglutaronitril. Fold inducton. nAA. KE-2 OECD TG. 124. Firefly.

(126) KeratinoSensTM. LuSens. ARE. Otake et al. in vitro. Isocyanates. h-CLAT. OECD TG442E. LLNA. OECD, 2016. Isocyanates. Otake et al., 2018 2. KEKE-2 Epi-Life. -Sens. 125. in vitro.

(127) Local Lymph Node Assay LLNA OECD TG429. Globally Harmonized System of Classification and Labelling of Chemicals. GHS. UN. 1. 2019. GHS. 1A 1B. OECD TG406. 126. OECD.

(128) TG429. in vitro 1. LLNA. LLNA. RI. RI. LLNA:BrdU-ELISA. Takeyoshi et al., 2001. OECD, 2018 LLNA:BrdU-ELISA. GHS. 1. ICH. 127. S10 (2013).

(129) LLNA. OECD TG OECD. AOP. KE-1. KE-2. KE-3. 1. Integrated Approaches to Testing and Assessment OECD, 2019 KeratinoSensTM. IATA. OECD. KE-2. LuSens. OECD TG442D OECD 2018. LLNA. 128.

(130) BrdU-ELISA. GHS LLNA. in vitro. LLNA BrdU-ELISA GHS. GHS EC1.6. 1A. 6%. LLNA. 1B. 92.3%. GHS. 84.2%. 1A 2-. Mercaptbenzothiazole. A. CBA/J. LLNA BrdU-ELISA LLNA. 1B. 100%. GHS. GHS. 100% GHS. EC1.6 1A. GHS. LLNA:BrdU-ELISA. GHS. GHS. 1B:EC1.6>6%. LLNA. UV 1. 2. 129. GHS.

(131) 3. 3. LLNA. /. Doxycycline. Minocycline. LLNA. KE-2. -Sens. -Sens. normalized ARE Activity nAA accuracy 100%. sensitivity. specificity. KE-2 KE-2 KE-2 -Sens. LLNA:BrdU-ELISA. 130. GHS.

(132) LLNA. GHS. GHS. LLNA. LLNA. EC3. -Sens. -Sens. 131.

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(134) 1 . 2012. CBA/J. CBA/Ca. LLNA. CBA/J. OECD TG429 TG442B. .. 25. LLNA .. . 2015. Murine local. 2. .. lymph node assay (LLNA) 42. .. 3 . 2017. Nrf2-keap1-ARE. in vitro .. 30. 4. .. . 2018. LLNA:BrdU-ELISA OECD TG442B. GHS 31. .. . . 2019. Dual luciferase. 5. ARE-Nrf2 based skin sensitization screening assay 32. .. 133. ..

(135) Globally Harmonized System of Classification and Labelling of Chemicals. GHS. GHS 1. 1A 1B. Local Lymph Node Assay LLNA. LLNA. RI RI. LLNA:BrdU-ELISA GHS. 134.

(136) 1. ICH. S10. OECD TG KeratinoSensTM. LuSens. OECD TG442D. LLNA. 135. BrdU-.

(137) ELISA. GHS. LLNA. in vitro. GHS GHS. EC1.6. 1A. 1B. 6%. LLNA 92.3%. CBA/J LLNA GHS. 100%. GHS. LLNA. LLNA. in vitro. EC1.6. GHS. LLNA. -Sens. 136. 84.2%.

(138) -Sens 3. 137.

(139) 138.

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