Research Progress on Alternative Methods of Skin Sensitization Test

doi:10.12300/j.issn.1674-5817.2021.173

Abstract

Abstract:

Allergic contact dermatitis is a type Ⅳ hypersensitivity reaction caused by repeated skin exposure to a substance and is a common public health problem. Traditional skin sensitization tests are based on animal experiments such as guinea pig maximum test and closed patch. In recent years, with the increasing attention of animal ethics and the development of science and technology, alternative methods of skin sensitization test have emerged. According to different principles, these alternative methods are divided into in vivo alternative methods, several in vitro alternative methods based on harmful outcome pathways, and genomic allergen rapid test, etc. In this paper, we reviewed the progress of these alternative methods of skin sensitization test, and several integrated testing and evaluation methods based on adverse outcome pathways.

Key words: Skin sensitization experiment, Allergic contact dermatitis, Alternative method

CLC Number:

R-332

Jingyi HUANG, Peining LI, Xiangmei LIU, Zhonghua LIU, Yufeng HUANG. Research Progress on Alternative Methods of Skin Sensitization Test[J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 313-321.

Figures/Tables 1

References 68

1	MORTZ C G, BINDSLEV-JENSEN C, ANDERSEN K E. Hand eczema in The Odense Adolescence Cohort Study on Atopic Diseases and Dermatitis (TOACS): prevalence, incidence and risk factors from adolescence to adulthood[J]. Br J Dermatol, 2014, 171(2):313-323. DOI:10.1111/bjd.12963 .
2	GERBERICK G F, RYAN C A, KERN P S, et al. Compilation of historical local lymph node data for evaluation of skin sensitization alternative methods[J]. Dermatitis, 2005, 16(4):157-202.
3	OECD. Test No. 429: Skin sensitisation: Local lymph node assay[M]. Paris: OECD, 2010. DOI:10.1787/9789264071100-en .
4	BASKETTER D A, LEA L J, COOPER K, et al. Threshold for classification as a skin sensitizer in the local lymph node assay: a statistical evaluation[J]. Food Chem Toxicol, 1999, 37(12):1167-1174. DOI:10.1016/S0278-6915(99)00112-X .
5	VOCANSON M, HENNINO A, ROZIÈRES A, et al. Effector and regulatory mechanisms in allergic contact dermatitis[J]. Allergy, 2009, 64(12):1699-1714. DOI:10.1111/j.1398-9995.2009. 02082.x .
6	BALL N, CAGEN S, CARRILLO J C, et al. Evaluating the sensitization potential of surfactants: integrating data from the local lymph node assay, Guinea pig maximization test, and in vitro methods in a weight-of-evidence approach[J]. Regul Toxicol Pharmacol, 2011, 60(3):389-400. DOI:10.1016/j.yrtph.2011.05.007 .
7	KREILING R, HOLLNAGEL H M, HARENG L, et al. Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the Guinea pig maximization test (GPMT)[J]. Food Chem Toxicol, 2008, 46(6):1896-1904. DOI:10.1016/j.fct.2008.01.019 .
8	TAKEYOSHI M, SAWAKI M, YAMASAKI K, et al. Assessment of statistic analysis in non-radioisotopic local lymph node assay (non-RI-LLNA) with α-hexylcinnamic aldehyde as an example[J]. Toxicology, 2003, 191(2-3):259-263. DOI:10.1016/S0300-483X(03)00255-5 .
9	胡培丽, 张露勇, 李波, 等. 两品系小鼠局部淋巴结试验结果比较[J]. 中国比较医学杂志, 2015, 25(5):54-57. DOI:10.3969/j.issn.1671.7856.2015.005.013 .
	HU P L, ZHANG L Y, LI B, et al. The comparative study of two strains on results of Local lymph node assays[J]. Chin J Comp Med, 2015, 25(5):54-57. DOI:10.3969/j.issn.1671.7856.2015.005.013 .
10	秦珩, 程洁, 靳苏香, 等. 小鼠局部淋巴结试验(BrdU-ELISA)改良法在2, 4-二硝基氯代苯皮肤致敏性检测中的应用[J]. 毒理学杂志, 2020, 34(2):138-142. DOI:10.16421/j.cnki.1002-3127.2020.02.010 .
	QIN H, CHENG J, JIN S X, et al. Application of modified local lymph node assay (BrdU-ELISA) in the detection of DNCB skin sensitization in mice[J]. J Toxicol, 2020, 34(2):138-142. DOI:10.16421/j.cnki.1002-3127.2020.02.010 .
11	阳晓燕, 赵康峰, 刘辉, 等. LLNA: BrdU-ELISA法与LLNA: DA法检测15种染发剂致敏性的对比研究[J]. 环境与健康杂志, 2018, 35(9):791-794. DOI:10.16241/j.cnki.1001-5914.2018.09.010 .
	YANG X Y, ZHAO K F, LIU H, et al. LLNA: BrdU-ELISA and LLNA: DA used in assessment of skin sensitization of fifteen hair dyes: a comparative study[J]. J Environ Health, 2018, 35(9):791-794. DOI:10.16241/j.cnki.1001-5914.2018.09.010 .
12	ZHANG H, SHI Y, WANG C, et al. An improvement of LLNA: DA to assess the skin sensitization potential of chemicals[J]. J Toxicol Sci, 2017, 42(2):129-136. DOI:10.2131/jts.42.129 .
13	OECD. Test No. 442A: Skin sensitization: Local lymph node assay: DA[M]. Paris: OECD, 2010. DOI:10.1787/9789264090972-en .
14	STEWART I, SEAWRIGHT A A, SCHLUTER P J, et al. Primary irritant and delayed-contact hypersensitivity reactions to the freshwater cyanobacterium Cylindrospermopsis raciborskii and its associated toxin cylindrospermopsin[J]. BMC Dermatol, 2006, 6:5. DOI:10.1186/1471-5945-6-5 .
15	瞿小婷, 程树军, 秦瑶, 等. 有害结局通路指南及毒性测试应用分析[J]. 日用化学工业, 2016, 46(8):473-478. DOI:10.13218/j.cnki.csdc.2016.08.010 .
	QU X T, CHENG S J, QIN Y, et al. Adverse outcome pathways guide and toxicity test applications[J]. China Surfactant Deterg ＆ Cosmet, 2016, 46(8):473-478. DOI:10.13218/j.cnki.csdc.2016.08.010 .
16	OECD. The adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins[M]. Paris: OECD, 2012.
17	GERBERICK G F, VASSALLO J D, FOERTSCH L M, et al. Quantification of chemical peptide reactivity for screening contact allergens: a classification tree model approach[J]. Toxicol Sci, 2007, 97(2):417-427. DOI:10.1093/toxsci/kfm064 .
18	杨婷婷, 易路遥, 王绎, 等. 采用DPRA替代方法评价3种香料的皮肤致敏性[J]. 日用化学工业, 2021, 51(5):438-442.
	YANG T T, YI L Y, WANG Y, et al. Evaluation of skin sensitization of three perfumes by using DPRA alternative method[J]. China Surfactant Deterg ＆ Cosmet, 2021, 51(5):438-442.
19	OECD. OECD guideline for the testing of chemicals. In chemico skin sensitisation: Direct peptide reactivity assay (DPRA)[M]. Paris: OECD, 2015.
20	GERBERICK G F, TROUTMAN J A, FOERTSCH L M, et al. Investigation of peptide reactivity of pro-hapten skin sensitizers using a peroxidase-peroxide oxidation system[J]. Toxicol Sci, 2009, 112(1):164-174. DOI:10.1093/toxsci/kfp192 .
21	WEI Z X, FANG Y H, GOSZTYLA M L, et al. A direct peptide reactivity assay using a high-throughput mass spectrometry screening platform for detection of skin sensitizers[J]. Toxicol Lett, 2021, 338:67-77. DOI:10.1016/j.toxlet.2020.12.002 .
22	WANIBUCHI S, YAMAMOTO Y, SATO A, et al. The amino acid derivative reactivity assay with fluorescence detection and its application to multi-constituent substances[J]. J Toxicol Sci, 2019, 44(12):821-832. DOI:10.2131/jts.44.821 .
23	FUJITA M, YAMAMOTO Y, TAHARA H, et al. Development of a prediction method for skin sensitization using novel cysteine and lysine derivatives[J]. J Pharmacol Toxicol Methods, 2014, 70(1):94-105. DOI:10.1016/j.vascn.2014.06.001 .
24	FUJITA M, YAMAMOTO Y, WATANABE S, et al. Cause of and countermeasures for oxidation of the cysteine-derived reagent used in the amino acid derivative reactivity assay[J]. J Appl Toxicol, 2019, 39(2):191-208. DOI:10.1002/jat.3707 .
25	FUJITA M, YAMAMOTO Y, WANIBUCHI S, et al. A newly developed means of HPLC-fluorescence analysis for predicting the skin sensitization potential of multi-constituent substances using ADRA[J]. Toxicol Vitro, 2019, 59:161-178. DOI:10.1016/j.tiv.2019.04.014 .
26	熊款款, 谭磊, 王爱兵, 等. Keap1-Nrf2/ARE信号通路抗氧化机制及抗氧化剂的研究进展[J]. 动物医学进展, 2021, 42(4):89-94. DOI:10.16437/j.cnki.1007-5038.2021.04.018 .
	XIONG K K, TAN L, WANG A B, et al. Progress on anti-oxidation mechanisms and antioxidants of the Keap1-Nrf2/ARE signaling pathway[J]. Prog Vet Med, 2021, 42(4):89-94. DOI:10.16437/j.cnki.1007-5038.2021.04.018 .
27	RAMIREZ T, MEHLING A, KOLLE S N, et al. LuSens: a keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification[J]. Toxicol Vitro, 2014, 28(8):1482-1497. DOI:10.1016/j.tiv.2014.08.002 .
28	OECD. Test No. 442D: In vitro skin sensitisation: ARE-Nrf2 luciferase test method[M]. Paris: OECD, 2022. DOI:10.1787/9789264229822-en .
29	SAKAGUCHI H, ASHIKAGA T, MIYAZAWA M, et al. The relationship between CD86/CD54 expression and THP-1 cell viability in an in vitro skin sensitization test - human cell line activation test (h-CLAT)[J]. Cell Biol Toxicol, 2009, 25(2):109-126. DOI:10.1007/s10565-008-9059-9 .
30	MIYAZAWA M, ITO Y, YOSHIDA Y, et al. Phenotypic alterations and cytokine production in THP-1 cells in response to allergens[J]. Toxicol Vitro, 2007, 21(3):428-437. DOI:10.1016/j.tiv.2006.10.005 .
31	OECD.Test No.442E: In vitro skin sensitisation[M]. Paris: OECD,2018. DOI:10.1787/9789264264359-en
32	袁园, 阳晓燕, 石莹, 等. 人细胞系活化试验用于皮肤致敏性测试的实验验证[J]. 环境卫生学杂志, 2019, 9(1):56-64. DOI:10.13421/j.cnki.hjwsxzz.2019.01.011 .
	YUAN Y, YANG X Y, SHI Y, et al. Experimental verification of human cell line activation test for skin allergenicity detection[J]. J Environ Hyg, 2019, 9(1):56-64. DOI:10.13421/j.cnki.hjwsxzz.2019.01.011 .
33	ASHIKAGA T, SAKAGUCHI H, SONO S, et al. A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA)[J]. Altern Lab Anim, 2010, 38(4):275-284. DOI:10.1177/026119291003800403 .
34	KARKHANIS A V, CHAN E C Y, REN E C. Preliminary discovery of novel markers for human cell line activation test (h-CLAT)[J]. Toxicol Vitro, 2021, 74:105154. DOI:10.1016/j.tiv.2021.105154 .
35	JOHANSSON H, LINDSTEDT M, ALBREKT A S, et al. A genomic biomarker signature can predict skin sensitizers using a cell-based in vitro alternative to animal tests[J]. BMC Genomics, 2011, 12:399. DOI:10.1186/1471-2164-12-399 .
36	STEVENSON M, CZEKALA L, SIMMS L, et al. The use of Genomic Allergen Rapid Detection (GARD) assays to predict the respiratory and skin sensitising potential of e-liquids[J]. Regul Toxicol Pharmacol, 2019, 103:158-165. DOI:10.1016/j.yrtph.2019.01.001 .
37	JOHANSSON H, ALBREKT A S, BORREBAECK C A K, et al. The GARD assay for assessment of chemical skin sensitizers[J]. Toxicol Vitro, 2013, 27(3):1163-1169. DOI:10.1016/j.tiv.2012.05.019 .
38	JOHANSSON H, RYDNERT F, KÜHNL J, et al. Genomic allergen rapid detection In-house validation—a proof of concept[J]. Toxicol Sci, 2014, 139(2):362-370. DOI:10.1093/toxsci/kfu046 .
39	MASINJA W, ELLIOTT C, MODI S, et al. Comparison of the predictive nature of the Genomic Allergen Rapid Detection (GARD) assay with mammalian assays in determining the skin sensitisation potential of agrochemical active ingredients[J]. Toxicol Vitro, 2021, 70:105017. DOI:10.1016/j.tiv.2020.105017 .
40	LOVELESS S E, LADIES G S, GERBERICK G F, et al. Further evaluation of the local lymph node assay in the final phase of an international collaborative trial[J]. Toxicology, 1996, 108(1-2):141-152. DOI:10.1016/0300-483X(95)03279-O .
41	MONTELIUS J, BOMAN A, WAHLKVIST H, et al. The murine local lymph node assay: search for an alternative, more adequate, vehicle than acetone/olive oil (4:1)[J]. Contact Dermatitis, 1996, 34(6):428-430. DOI:10.1111/j.1600-0536.1996.tb02249.x .
42	ANDERSON S E, SIEGEL P D, MEADE B J. The LLNA: a brief review of recent advances and limitations[J]. J Allergy (Cairo), 2011, 2011:424203. DOI:10.1155/2011/424203 .
43	HAYASHI D, NOZAKI Y, TAKAGI H, et al. Sensitivity comparison of 3 CBA mouse strains under the LLNA: BrdU-ELISA test method[J]. Altern Animal Test Exp, 2012, 17(2):63-68.
44	陈宁,黄泽愉,孙晓,等. 局部淋巴结试验(LLNA:BrdU-ELISA)对14种化学物刺激和致敏性的评价[J]. 毒理学杂志, 2022, 36(2) :157-162. DOI:10.16421/j.cnki.1002-3127.2022.02.018 .
	CHEN N, HUANG Z Y, SUN X, et al. Local lymph node assay( LLNA: BrdU-ELISA) for evaluating the irritation and sensitization of 14 chemicals[J].J Toxcol. 2022, 36(2) :157-162. DOI:10.16421/j.cnki.1002-3127.2022.02.018 .
45	冼静雯, 郭煜堂, 陈宁, 等. BALB/c小鼠LLNA: DA改良法在15种化学物皮肤致敏性评价中的应用[J]. 环境与健康杂志, 2017, 34(5):441-443. DOI:10.16241/j.cnki.1001-5914.2017.05.018 .
	XIAN J W, GUO Y T, CHEN N, et al. Performance of the LLNA: DA using BALB/c mice for 15 substances in skin-sensitivity evaluation[J]. J Environ Health, 2017, 34(5):441-443. DOI:10.16241/j.cnki.1001-5914.2017.05.018 .
46	丁诗璇, 李小林, 曲栗, 等. 直接多肽反应试验在化妆品检测中的应用[J]. 日用化学品科学, 2018, 41(11):19-24. DOI:10.13222/j.cnki.dc.2018.11.004 .
	DING S X, LI X L, QU L, et al. Application of direct peptide reactivity assay on cosmetics[J]. Deterg ＆ Cosmet, 2018, 41(11):19-24. DOI:10.13222/j.cnki.dc.2018.11.004 .
47	梅承翰, 庄慧敏, 刘师卜, 等. 直接肽反应试验及其研究进展[J]. 中国医药生物技术, 2018, 13(6):556-559. DOI:10.3969/j.issn.1673-713X.2018.06.014 .
	MEI C H, ZHUANG H M, LIU S B, et al. Direct peptide reactivity assay and its research progress[J]. Chin Med Biotechnol, 2018, 13(6):556-559. DOI:10.3969/j.issn.1673-713X.2018.06.014 .
48	FUJITA M, YAMAMOTO Y, WATANABE S, et al. The within- and between-laboratory reproducibility and predictive capacity of the in chemico amino acid derivative reactivity assay: results of validation study implemented in four participating laboratories[J]. J Appl Toxicol, 2019, 39(11):1492-1505. DOI:10.1002/jat.3834 .
49	陈虹, 黄元礼, 王涵, 等. KeratinoSens试验在医疗器械致敏性检测中的应用研究[J]. 癌变·畸变·突变, 2021, 33(6):455-460. DOI:10.3969/j.issn.1004-616x.2021.06.010 .
	CHEN H, HUANG Y L, WANG H, et al. Research on KeratinoSens assay applied to medical devices[J]. Carcinog Teratog Mutagen, 2021, 33(6):455-460. DOI:10.3969/j.issn.1004-616x.2021.06.010 .
50	BAUCH C, KOLLE S N, RAMIREZ T, et al. Putting the parts together: combining in vitro methods to test for skin sensitizing potentials[J]. Regul Toxicol Pharmacol, 2012, 63(3):489-504. DOI:10.1016/j.yrtph.2012.05.013 .
51	李晨, 沙毅杰, 霍倩, 等. 人源THP-1细胞系活化试验在化妆品皮肤致敏性评价中的应用研究[J]. 现代预防医学, 2022, 49(2):349-353, 379.
	LI C, SHA Y J, HUO Q, et al. Application of THP-1 human cell line activation test in skin sensitization evaluation of cosmetics[J]. Mod Prev Med, 2022, 49(2):349-353, 379.
52	所雅琼, 罗飞亚, 张凤兰, 等. 皮肤致敏替代方法和整合策略研究进展及思考[J]. 香料香精化妆品, 2021(5):96-102.
	SUO Y Q, LUO F Y, ZHANG F L, et al. Research progress and analysis of alternative methods and integration strategies for skin sensitization[J]. Flavour Fragr Cosmet, 2021(5):96-102.
53	FORRERYD A, JOHANSSON H, ALBREKT A S, et al. Prediction of chemical respiratory sensitizers using GARD, a novel in vitro assay based on a genomic biomarker signature[J]. PLoS One, 2015, 10(3): e0118808. DOI:10.1371/journal.pone.0118808 .
54	OECD. Guidance document on the reporting of defined approaches to be used within integrated approaches to testing and assessment[M]. Paris: OECD, 2017. DOI:10.1787/9789264274822-en .
55	罗飞亚, 苏哲. 中国化妆品安全评估中替代方法的现状[J]. 口腔护理用品工业, 2020, 30(5):62-64.
	LUO F Y, SU Z. Present situation of alternative methods in cosmetic safety assessment in China[J]. Oral Care Ind, 2020, 30(5):62-64.
56	CAO Y P, MA P C, LIU W D, et al. Evaluation of the skin sensitization potential of chemicals in THP-1/keratinocyte co-cultures[J]. Immunopharmacol Immunotoxicol, 2012, 34(2):196-204. DOI:10.3109/08923973.2011.591800 .
57	柯逸晖, 陈彧, 程树军, 等. 直接多肽结合试验组合人细胞系活化试验预测皮肤致敏物的探讨[J]. 中国实验动物学报, 2016, 24(6):611-617. DOI:10.3969/j.issn.1005-4847.2016.06.011 .
	KE Y H, CHEN Y, CHENG S J, et al. Preliminary study for integrating DPRA with h-CLAT to predict skin sensitizers[J]. Acta Lab Animalis Sci Sin, 2016, 24(6):611-617. DOI:10.3969/j.issn.1005-4847.2016.06.011 .
58	安丽英, 相玉红, 张卓勇, 等. 定量构效关系研究进展及其应用[J]. 首都师范大学学报(自然科学版), 2006, 27(3):52-57. DOI:10.19789/j.1004-9398.2006.03.014 .
	AN L Y, XIANG Y H, ZHANG Z Y, et al. The new advance and applications of quantitative structure-activity relationship[J]. J Cap Norm Univ Nat Sci Ed, 2006, 27(3):52-57. DOI:10.19789/j.1004-9398.2006.03.014 .
59	ALVES V M, MURATOV E, FOURCHES D, et al. Predicting chemically-induced skin reactions. Part II: QSAR models of skin permeability and the relationships between skin permeability and skin sensitization[J]. Toxicol Appl Pharmacol, 2015, 284(2):273-280. DOI:10.1016/j.taap.2014.12.013 .
60	汤欣, 赵文静, 于清. 医疗器械毒理学风险评估中QSAR的应用[J]. 中国医疗器械杂志, 2022, 46(2):200-205.
	TANG X, ZHAO W J, YU Q. Applications of QSAR in toxicological risk assessment of medical devices[J]. Chin J Med Instrum, 2022, 46(2):200-205.
61	王雪梅, 罗飞亚, 邢书霞, 等. 皮肤致敏整合测试与评估策略研究进展及思考[J]. 香料香精化妆品, 2020(5):79-85.
	WANG X M, LUO F Y, XING S X, et al. Research progress and analysis on integrated strategies for alternative tests of skin sensitization[J]. Flavour Fragr Cosmet, 2020(5):79-85.
62	URBISCH D, MEHLING A, GUTH K, et al. Assessing skin sensitization hazard in mice and men using non-animal test methods[J]. Regul Toxicol Pharmacol, 2015, 71(2):337-351. DOI:10.1016/j.yrtph.2014.12.008 .
63	JAWORSKA J, DANCIK Y, KERN P, et al. Bayesian integrated testing strategy to assess skin sensitization potency: from theory to practice[J]. J Appl Toxicol, 2013, 33(11):1353-1364. DOI:10.1002/jat.2869 .
64	JAWORSKA J S, NATSCH A, RYAN C, et al. Bayesian integrated testing strategy (ITS) for skin sensitization potency assessment: a decision support system for quantitative weight of evidence and adaptive testing strategy[J]. Arch Toxicol, 2015, 89(12):2355-2383. DOI:10.1007/s00204-015-1634-2 .
65	DEL BUFALO A, PAULOIN T, ALEPEE N, et al. Alternative integrated testing for skin sensitization: assuring consumer safety[J]. Appl Vitro Toxicol, 2018, 4(1):30-43. DOI:10.1089/aivt.2017.0023 .
66	TOURNEIX F, ALÉPÉE N, DETROYER A, et al. Skin sensitisation testing in practice: applying a stacking meta model to cosmetic ingredients[J]. Toxicol Vitro, 2020, 66:104831. DOI:10.1016/j.tiv.2020.104831 .
67	HIROTA M, FUKUI S, OKAMOTO K, et al. Evaluation of combinations of in vitro sensitization test descriptors for the artificial neural network-based risk assessment model of skin sensitization[J]. J Appl Toxicol, 2015, 35(11):1333-1347. DOI:10.1002/jat.3105 .
68	孙方卉, 宋肖洁, 霍刚. 皮肤致敏测试整合策略的现状与展望[J]. 日用化学工业, 2021, 51(8):782-788.
	SUN F H, SONG X J, HUO G. Current situation and prospect of integrated strategies for skin sensitization testing[J]. China Surfactant Deterg ＆ Cosmet, 2021, 51(8):782-788.

方法 Method	适用范围 Application scope	优点 Advantages	局限性 Limitation	检测终点 Detection endpoint	准确度 Accuracy
LLNA	化学品 (包括化妆品、农药等)、脂溶性小分子物质	减少实验时间和成本，动物用量少，体积小，灵敏度高	非致敏的皮肤刺激性化学物质^[40]；溶剂假阳性结果^[41]；对受试物溶解性要求高；需要使用实验动物、放射性物质	强致敏剂：SI≥3，EC3<0.1%	72%^[42]
LLNA: BrdU-ELISA	化学品 (包括化妆品、农药等)	综合了LLNA的优点，且未使用放射性物质	未能解决LLNA法中出现的假阳性问题^[43]	ELISA检测增殖淋巴细胞中BrdU的标记数	86%^[44]
LLNA: DA	化学品 (包括化妆品、农药等)	具有LLNA：BrdU-ELISA的全部优点	时间限制；假阳性；不适用于影响ATP产生的化合物	ATP数量、淋巴结质量、增殖的淋巴细胞数，以及SI和EC值	87%^[45]
DPRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高	缺乏代谢体系^[46]；溶解性、透皮性差的待测物会影响结果^[47]；待测物与亲核试剂共同洗脱导致准确性下降	液相色谱法测定被测化合物消耗半胱氨酸和赖氨酸多肽的情况	80%
ADRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高，对待测物浓度要求低	对待测物溶解性要求较高；评估多成分物质时无法避免待测物的免疫抑制	NAC/NAL的反应性	86.9%^[48]
KeratinoSens^TM/ LuSens	可溶并能形成稳定体系的待测物	灵敏度高，准确度高，不使用实验动物	选择性与赖氨酸残基反应的待测物会出现假阳性^[49]；如待测物需要酶的激活可能出现假阳性^[50]	荧光诱导倍数FI>1.5可判断受试物为致敏物	94%/83%
h-CLAT	可溶性化合物	不使用实验动物，细胞株易于获得且稳定，灵敏度高，特异性高	对待测物的溶解度要求较高；荧光干扰以及待测物细胞毒性引起的假阳性^[51]；弱致敏物质易产生假阴性结果^[52]	当CD86>150%且 CD54>200%时，可判定为致敏物	84%
GARD	可溶性固/液化合物	准确度高，不使用实验动物，稳定、高重复性，可检测呼吸道致敏物	成本高；不能涵盖完整的AOP通路	DC>0，为致敏剂；DC≤0为非致敏剂	84%^[53]

方法 Method	适用范围 Application scope	优点 Advantages	局限性 Limitation	检测终点 Detection endpoint	准确度 Accuracy
LLNA	化学品 (包括化妆品、农药等)、脂溶性小分子物质	减少实验时间和成本，动物用量少，体积小，灵敏度高	非致敏的皮肤刺激性化学物质^[40]；溶剂假阳性结果^[41]；对受试物溶解性要求高；需要使用实验动物、放射性物质	强致敏剂：SI≥3，EC3<0.1%	72%^[42]
LLNA: BrdU-ELISA	化学品 (包括化妆品、农药等)	综合了LLNA的优点，且未使用放射性物质	未能解决LLNA法中出现的假阳性问题^[43]	ELISA检测增殖淋巴细胞中BrdU的标记数	86%^[44]
LLNA: DA	化学品 (包括化妆品、农药等)	具有LLNA：BrdU-ELISA的全部优点	时间限制；假阳性；不适用于影响ATP产生的化合物	ATP数量、淋巴结质量、增殖的淋巴细胞数，以及SI和EC值	87%^[45]
DPRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高	缺乏代谢体系^[46]；溶解性、透皮性差的待测物会影响结果^[47]；待测物与亲核试剂共同洗脱导致准确性下降	液相色谱法测定被测化合物消耗半胱氨酸和赖氨酸多肽的情况	80%
ADRA	可溶性的单一化学物质以及化妆品原料	不使用实验动物，灵敏度和准确性高，对待测物浓度要求低	对待测物溶解性要求较高；评估多成分物质时无法避免待测物的免疫抑制	NAC/NAL的反应性	86.9%^[48]
KeratinoSens^TM/ LuSens	可溶并能形成稳定体系的待测物	灵敏度高，准确度高，不使用实验动物	选择性与赖氨酸残基反应的待测物会出现假阳性^[49]；如待测物需要酶的激活可能出现假阳性^[50]	荧光诱导倍数FI>1.5可判断受试物为致敏物	94%/83%
h-CLAT	可溶性化合物	不使用实验动物，细胞株易于获得且稳定，灵敏度高，特异性高	对待测物的溶解度要求较高；荧光干扰以及待测物细胞毒性引起的假阳性^[51]；弱致敏物质易产生假阴性结果^[52]	当CD86>150%且 CD54>200%时，可判定为致敏物	84%
GARD	可溶性固/液化合物	准确度高，不使用实验动物，稳定、高重复性，可检测呼吸道致敏物	成本高；不能涵盖完整的AOP通路	DC>0，为致敏剂；DC≤0为非致敏剂	84%^[53]

[1]	CHEN Xiu-juan, HUANG Yu-feng, LIU Xiang-mei, PANG Zeng-xiong, JIANG-Yi, LI Pei-ning, LI Guang-xian, CHEN Zi-ling, SUN Xia, WANG Qiang. Using Several Alternative Test Methods Strategically Combination to Assess Eye Irritation/Corrosion of 1,2-hexanediol [J]. Laboratory Animal and Comparative Medicine, 2018, 38(3): 217-221.
[2]	ZHOU Jing-jing1，YANG Ming2，MAO Yu-chang3，LU Jie-qi2，YANG You-ming3，HU Zhuo-han1,3. Validation of Alternative Method for Evaluating Effects of Drug Metabolism on Cytotoxicity [J]. , 2010, 30(2): 87-94.