皮肤致敏替代方法kDPRA的联合验证研究

doi:10.3969/j.issn.2097-2806.2026.10.013

Abstract

Abstract:

This study systematically investigates the applicability of the kinetic direct peptide reactivity assay （kDPRA）, an alternative method for skin sensitization assessment adopted by the Organisation for Economic Cooperation and Development （OECD） in 2022, in domestic laboratories. By examining the influence of key experimental factors such as reagents, the research combines internal laboratory validation with a ring test involving four external laboratories. The results show that different brands of monobromobimane reagent have no significant impact on the assay results, and commercially available versus laboratory-prepared neutral phosphate buffers yield consistent results under identical formulations. Inter-laboratory validation achieves 100% accuracy in the sensitization potency classification of 10 reference substances, fully aligning with OECD TG 442C requirements. For the 10 blind-coded samples, the predictive accuracy across the five participating laboratories ranges between 80% and 100%, meeting the validation acceptance criteria. Furthermore, kDPRA demonstrates reliable performance when applied within a defined approach for evaluating the sensitization potential of the yellow-colored compound berberine. These findings indicate that the kDPRA method exhibits good reproducibility and reliability in Chinese laboratories. It is suitable for domestic implementation and adoption, and can serve as a component of an Adverse Outcome Pathway （AOP） -based integrated testing strategy for skin sensitization assessment. This work provides scientific evidence and practical support for establishing national kDPRA standards, promoting the method, and improving China’s alternative testing system for animal experimentation.

Key words: sensitization, allergic contact dermatitis, kinetic direct peptide reactivity assay, OECD TG442C, berberine

CLC Number:

TQ658

Xue Geng, Ping Liu, Kaiji Wang, Ning Chen, Yanqin Song, Qingfen Zhu. Establishment and inter-laboratory validation of the kDPRA method for skin sensitization assessment[J].China Surfactant Detergent & Cosmetics, 2026, 56(10): 1403-1412.

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References 19

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序号	名称	CAS号	品牌	货号	批号	储存条件	致敏性分级
1	氯苯	108-90-7	1	C119461-100 mL	E2520340	室温	极强致敏剂
2	4-乙氧基亚甲基-2-苯基噁唑啉-5-酮	15646-46-5	1	E486138-200 mg	K2418269	2~8 ℃	极强致敏剂
3	4-甲氧基苯乙酮	100-06-1	1	M103875-25 g	G24122482	2~8 ℃	非致敏剂
4	2-甲基-4-异噻唑啉-3-酮	2682-20-4	2	M813550-5 g	C16365214	室温	极强致敏剂
5	庚炔羧酸甲酯	111-12-6	1	M107624-25 mL	K2110412	室温	强致敏剂
6	异丁香酚	97-54-1	3	17206-5 g	1003458343	室温	中等致敏剂
7	2, 3-丁二酮	431-03-8	1	B104601-100 mL	F23161023	2~8 ℃	弱致敏剂
8	4-甲氧基苯乙酮	100-06-1	3	11734-5 g	BCCF5045	室温	非致敏剂
9	二甲基丙烯酸乙二醇酯	97-90-5	1	E106223-100 mL	J2327543	2~8 ℃	弱致敏剂
10	2, 4-二硝基氯苯	97-00-7	4	C0162	CPSOG-NA	室温	极强致敏剂
11	咪唑烷基脲	39236-46-9	3	15133-25 g	102657137	2~8 ℃	弱致敏剂
12	4-苯基-3-丁烯-2-酮	122-57-6	3	147885-25 g	SHBL9127	室温	非致敏剂
13	二苯基环丙烯酮	886-38-4	1	A115107-5 g	B2302498	2~8 ℃	极强致敏剂

时间/min	lg k_max
	阿拉丁试剂（上海）有限公司		上海源叶生物科技有限公司		Millipore公司
	受试物	阳性对照	受试物	阳性对照	受试物	阳性对照
10	-0.27	-0.84	-0.28	—	-0.32	—
30	-0.50	-1.27	-0.52	-1.44	-0.52	-1.39
90	-0.63	-1.49	-0.88	-1.56	-0.65	-1.65
150	-1.28	-1.58	-1.07	-1.62	-1.13	-1.72
210	-1.37	-1.66	-1.38	-1.71	-1.31	-1.91
1 440	-2.41	-2.48	-2.48	-2.54	-2.45	-2.71
结果判定	GHS 1A类		GHS 1A类		GHS 1A类

	市售缓冲液1		自制缓冲液
	4-乙氧基亚甲基-2-苯基噁唑啉-5-酮	4-甲氧基苯乙酮	4-乙氧基亚甲基-2-苯基噁唑啉-5-酮	4-甲氧基苯乙酮
lg k_max	-0.27	n-r	-0.32	n-r
结果判定	GHS 1A类	非GHS 1A类	GHS 1A类	非GHS 1A类

名称	pH	配方组成
磷酸盐缓冲液1	7.5	0.1 mol/L NaH₂PO₄ 0.1 mol/L Na₂HPO₄
磷酸盐缓冲液2	7.4	2.0 mmol/L KH₂PO₄ 137 mmol/L NaCI 10.0 mmol/L Na₂HPO₄ 2.7 mmol/L KCl

序号	受试物名称	lg k_max值	试验是否成立	kDPRA试验结果	结果是否与OECD 442C一致
1	2-甲基-4-异噻唑啉-3-酮	-0.51	是	1A	是
2	庚炔羧酸甲酯	-1.13	是	1A	是
3	异丁香酚	-0.40	是	1A	是
4	2, 3-丁二酮	-2.03	是	非1A类	是
5	4-甲氧基苯乙酮	n-r	是	非1A类	是
6	二甲基丙烯酸乙二醇酯	-2.36	是	非1A类	是
7	2, 4-二硝基氯苯	-0.34	是	1A	是
8	氯苯	n-r	是	非1A类	是
9	4-乙氧基亚甲基-2-苯基噁唑啉-5-酮	-0.28	是	1A	是
10	4-甲氧基苯乙酮	n-r	是	非1A类	是

Establishment and inter-laboratory validation of the kDPRA method for skin sensitization assessment

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编号	样品名	lg k_max				致敏机制^[17]
编号	样品名	实验室1	实验室2	实验室3	实验室4	致敏机制^[17]
盲样1	2-甲基-4-异噻唑啉-3-酮	n-r	-0.26	-0.68	-0.47	双分子亲核取代反应
盲样2	庚炔羧酸甲酯	-1.92	-1.23	-0.30	-0.82	迈克尔加成
盲样3	异丁香酚	-1.62	-0.71	-0.13	-0.34	醌甲基化物/亚胺类
盲样4	2, 3-丁二酮	n-r	-2.40	n-r	-2.30	双分子亲核取代反应
盲样5	4-甲氧基苯乙酮	n-r	n-r	n-r	n-r	—
盲样6	二甲基丙烯酸乙二醇酯	-2.83	-2.17	n-r	-2.52	迈克尔加成
盲样7	2, 4-二硝基氯苯	-0.91	-0.17	-0.26	-0.31	酰基转移
盲样8	咪唑烷基脲	n-r	n-r	-1.94	-1.80	迈克尔加成
盲样9	4-苯基-3-丁烯-2-酮	-2.37	-1.80	-1.65	-1.84	苯环亲电取代反应（巯基反应）
盲样10	二苯基环丙烯酮	-1.67	-0.56	-0.62	-0.57	亲核加成

受试物	本实验室	实验室1	实验室2	实验室3	实验室4	人体试验GHS分类	kDPRA GHS 分类^[16]
盲样1	1A类	非1A类	1A类	1A类	1A类	1A	1A
盲样2	1A类	1A类	1A类	1A类	1A类	1A	1A
盲样3	1A类	1A类	1A类	1A类	1A类	1A	1A
盲样4	非1A类	非1A类	非1A类	非1A类	非1A类	1B	非1A类
盲样5	非1A类	非1A类	非1A类	非1A类	非1A类	NC	非1A类
盲样6	非1A类	非1A类	非1A类	非1A类	非1A类	1B	非1A类
盲样7	1A类	非1A类	非1A类	1A类	1A类	1B	1A类
盲样8	1A类	非1A类	1A类	1A类	1A类	1A	1A类
盲样9	1A类	1A类	1A类	1A类	1A类	1A	1A类
盲样10	1A类	1A类	1A类	1A类	1A类	1A	1A类

指标	本实验室	实验室1	实验室2	实验室3	实验室4
灵敏度	100% （6/6）	67% （4/6）	100% （6/6）	100% （6/6）	100% （6/6）
特异度	75% （3/4）	100% （4/4）	100% （4/4）	75% （3/4）	75% （3/4）
准确率	90% （9/10）	80% （8/10）	100% （10/10）	90% （9/10）	90% （9/10）

		lg k_{90 min}	lg k_{150 min}	lg k_max	分类结果
实验室1	试验1	—	-1.55	-1.55	GHS 1A
	试验2	-1.76	-2.05	-1.76	GHS 1A
	试验3	-1.69	-1.98	-1.69	GHS 1A
	试验4	-1.73	-2.32	-1.73	GHS 1A
	试验5	-1.75	-2.28	-1.75	GHS 1A
实验室2	试验1	-1.53	-1.62	-1.32	GHS 1A
	试验2	-1.56	-1.54	-1.20	GHS 1A
	试验3	-1.48	-1.57	-0.52	GHS 1A
	试验4	-1.37	-1.56	-1.11	GHS 1A
实验室3	试验1	-1.54	-1.61	-0.88	GHS 1A
	试验2	-1.49	-1.62	-1.03	GHS 1A
	试验3	-1.57	-1.69	-0.91	GHS 1A
	试验4	-1.50	-1.52	-0.92	GHS 1A
实验室4	试验1	-1.53	-1.62	-0.99	GHS 1A
	试验2	-1.56	-1.61	-0.93	GHS 1A
	试验3	-1.56	-1.61	-0.94	GHS 1A
	试验4	-1.50	-1.64	-1.14	GHS 1A
	试验5	-1.49	-1.62	-0.98	GHS 1A

组别	受试物终质量浓度/ （μg/mL）	同型对照组MFI	试验组MFI	RFI	结果
溶剂对照组	—	386.2	1 805.1	—	—
小檗碱1	26.96	17 491.7	20 481.3	210.7	阳性
小檗碱2	22.46	14 906.7	18 466.8	250.9	阳性
小檗碱3	18.72	12 924.9	15 568.5	186.3	阳性
小檗碱4	15.60	10 722.3	13 539.1	198.5	阳性
小檗碱5	13.00	9 362.7	11 662.5	162.1	阳性
小檗碱6	10.83	8 134.9	9 783.6	116.2	阴性
小檗碱7	9.03	7 007.5	8 232.3	86.3	阴性
小檗碱8	7.52	5 976.6	7 089.6	78.4	阴性

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