皮肤致敏体外方法：动力学直接多肽反应试验方法（kDPRA）的转化与验证

doi:10.3969/j.issn.2097-2806.2025.06.006

Abstract

Abstract:

The in vitro method kinetic direct peptide reactivity assay （kDPRA） for skin sensitisation is the first key event （KE1） for modelling the skin sensitisation adverse outcome pathway （AOP）: covalent binding of electrophilic substances to nucleophilic centres of skin proteins. Based on the reactivity of tested substance and cysteine, the logarithm of the maximum rate constant （lg k_max） of the tested substance was calculated by detecting the amount of peptide remaining after incubation with different concentrations of the tested substance at six time points, which is used to support the discrimination of UN GHS subcategory 1A skin sensitisers （GHS 1A）. This method has not yet been reported in China. The effect of different fluorescence efficacy monobromobimane on the test was examined. Seven compounds with known sensitising strengths were selected to validate the predictive accuracy of the kDPRA. The results show that the effect of different brands of monobromobimane on the prediction results can be accepted. The prediction of allergenicity for the 7 substances is consistent with the known classification, and that kDPRA can accurately identify the subcategory 1A skin sensitisers.

Key words: skin sensitisation, in vitro, kinetic direct peptide reactivity assay, validation

CLC Number:

TQ658

Ting Liu, Shibu Liu, Li Zhang, Hui Duo, Yong Lu, Luyong Zhang. Skin sensitisation in vitro method: kinetic direct peptide reactivity assay (kDPRA) transformation and validation[J].China Surfactant Detergent & Cosmetics, 2025, 55(6): 723-730.

Figures/Tables 6

Tab. 1

Tab. 2

Fig. 1

Fig. 2

Tab. 3

Tab. 4

Predicted sensitisation results of the kDPRA method for 7 compounds"

化合物	时间/min		10	30	90	150	210	1 440	预测结果
T1	相对肽消耗/%	5.00 mmol/L	53.79	93.36	99	99	99	99	1A
		2.50 mmol/L	21.9	72.05	94.46	98.32	98.49	99
		1.25 mmol/L	9.07	47.6	74.67	87.1	84.59	99
		0.63 mmol/L	4.54	20.67	48.96	66.07	51.58	99
		0.31 mmol/L	1.56	5.2	26.8	43.33	23.92	63.85
	相关系数R^#	0.985 6	0.999 6	0.990 6	0.999 9	0.999 5	1
	k_observedR²	0.981 4
	lg [k_t / （mol/L/s）]	-0.57	-0.5	-0.77	-0.75	-0.84	-0.88
T2	相对肽消耗/%	5.00 mmol/L	78.46	96.97	98.81	97.95	96.44	88.29	1A
		2.50 mmol/L	51.74	92.44	94.69	93.46	88.82	74.13
		1.25 mmol/L	26.08	72.87	84.67	81.03	73.87	59.46
		0.63 mmol/L	7.23	45.47	53.18	53.28	45.36	34.68
		0.31 mmol/L	1	27.41	33.27	35.5	29.77	31.04
	相关系数R^#	0.999 7	0.968 9	0.977 6	0.974 3	0.983 4	0.989 7
	k_observedR²	0.408 1
	lg [k_t / （mol/L/s）]	-0.26	-0.42	-0.81	-1.09	-1.3	-2.35
T3	相对肽消耗/%	5.00 mmol/L	4.1	22.38	26.14	50.66	53.03	94.51	1A
		2.50 mmol/L	1	11.78	16.99	32.81	28.85	80.37
		1.25 mmol/L	3.8	7.71	5.35	23.72	13.97	60.28
		0.63 mmol/L	2.5	1.89	1	7.67	5.46	36.62
		0.31 mmol/L	9.9	1	1	6.23	2.35	32.86
	相关系数R^#		0.994	0.985 5	0.988 2	0.999 3	0.998 3
	k_observedR²	0.964 3
	lg [k_t / （mol/L/s）]		-1.54	-1.91	-1.82	-1.9	-2.2
T4	相对肽消耗/%	5.00 mmol/L	87.86	70.89	63.72	59.12	66.15	78.65	1A
		2.50 mmol/L	80.63	56.56	59.45	57.22	64.51	77.01
		1.25 mmol/L	69.76	43.62	61.47	59.78	64.24	80.97
		0.63 mmol/L	55.86	41.95	58.84	53.46	62.21	80.68
		0.31 mmol/L	49.83	44.8	61.22	56.66	62.48	84.31
	相关系数R^#	0.972 6	0.979 6	0.667 8	0.522 7	0.941 9	0.684
	k_observedR²	-0.803 5
	lg [k_t / （mol/L/s）]	-0.3	-1.08			-2.75
T5	相对肽消耗/%	5.00 mmol/L	1	1	1	1	7.3	60.22	Non-1A
		2.50 mmol/L	1	1	1	1	3.1	31.08
		1.25 mmol/L	7.4	1	1	1	11	13.39
		0.63 mmol/L	3.9	1	1	1	8.2	5.59
		0.31 mmol/L	15.3	1	1	1	11.8	2.55
	相关系数R^#						0.996
	k_observedR²
	lg [k_t / （mol/L/s）]						-2.65
T6	相对肽消耗/%	5.00 mmol/L	1	1	1	1	1	1	Non-1A
		2.50 mmol/L	1.2	1	1	1.6	1	1
		1.25 mmol/L	1	1	1	1	1	1
		0.63 mmol/L	1	1	1	1.8	1	1
		0.31 mmol/L	3.8	1	2.4	3.2	1	1
	相关系数R^#
	k_observedR²
	lg [k_t / （mol/L/s）]	无反应
T7	相对肽消耗/%	5.00 mmol/L	1	1	1	1	1	1	Non-1A
		2.50 mmol/L	1	1	1	1	1	1
		1.25 mmol/L	1	1	1	1	1	2.3
		0.63 mmol/L	1	1	1	1	1	2
		0.31 mmol/L	2.3	3.6	1	6.2	1	3.3
	相关系数R^#
	k_observedR²
	lg [k_t / （mol/L/s）]	无反应

Tab. 4

References 17

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	E/（mmol/L）	T_PC1（m1）			T_PC2（m2）			T_PC3（m3）			RSD
VC		5.6×10⁸	5.5×10⁸	5.7×10⁸	7.4×10⁸	8.4×10⁸	7.2×10⁸	7.6×10⁸	8.1×10⁸	8.3×10⁸
BC		1.7×10⁷	1.8×10⁷	1.7×10⁷	5.4×10⁶	5.4×10⁶	5.0×10⁶	4.4×10⁶	4.8×10⁶	4.9×10⁶
T_PC校正剩余肽	0.310	4.4×10⁸	4.3×10⁸	5.5×10⁸	6.1×10⁸	7.0×10⁸	6.9×10⁸	7.9×10⁸	8.6×10⁸	8.7×10⁸
	0.63	4.2×10⁸	4.4×10⁸	4.8×10⁸	6.2×10⁸	6.7×10⁸	6.2×10⁸	8.1×10⁸	8.0×10⁸	8.4×10⁸
	1.25	3.7×10⁸	4.4×10⁸	4.7×10⁸	5.8×10⁸	7.5×10⁸	5.0×10⁸	6.4×10⁸	7.1×10⁸	6.1×10⁸
	2.50	3.5×10⁸	4.5×10⁸	3.8×10⁸	4.6×10⁸	5.7×10⁸	4.3×10⁸	6.5×10⁸	6.1×10⁸	6.7×10⁸
	5.00	2.8×10⁸	4.5×10⁸	4.3×10⁸	3.9×10⁸	4.6×10⁸	4.0×10⁸	5.4×10⁸	5.1×10⁸	5.3×10⁸
T_PC相对剩余肽	0.310	0.81	0.80	1.02	0.80	0.92	0.91	1.00	1.07	1.09	0.10
	0.63	0.78	0.81	0.89	0.81	0.88	0.81	1.02	1.00	1.06	0.11
	1.25	0.68	0.82	0.87	0.76	0.98	0.66	0.80	0.89	0.76	0.01
	2.50	0.64	0.83	0.70	0.61	0.75	0.57	0.82	0.77	0.84	0.08
	5.00	0.53	0.83	0.80	0.52	0.61	0.53	0.67	0.63	0.67	0.09

时间/min		10	30	90	150	210	1 440
相对肽消耗/%	5.00 mmol/L	1.00^*	19.38	43.34	60.67	63.44	50.11
	2.50 mmol/L	1.00^*	1	18.39	42.23	43.58	40.43
	1.25 mmol/L	1.00^*	3.64	15.38	27.85	26.7	29.38
	0.63 mmol/L	1.00^*	9.83	5.56	12.34	13.21	19.71
	0.31 mmol/L	1.00^*	2.92	3.05	5.24	4.37	8.64
VC的CV/%	10.75	11.27	10.78	12.03	11.96	11.32
相关系数R^#		0.712 8	0.983 2	0.991 3	0.995 2	0.957 6
k_observed的R²	0.955 5
lg [k_t / （mol/L/s）]		^△	-1.69	-1.69	-1.8	-2.86

化合物	物理状态	溶剂	UN GHS LLNA	UN GHS 人体
T1	固体	乙腈	1A	1A
T2	固体	乙腈	1A	无数据
T3	液体	乙腈	1A	1A
T4	固体	PB	1A	1A
T5	液体	PB	1B	1B
T6	固体	乙腈	未分类	未分类
T7	液体	乙腈	未分类	未分类

Skin sensitisation in vitro method: kinetic direct peptide reactivity assay (kDPRA) transformation and validation

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