氨基酸衍生物结合试验在化妆品检测中的应用

doi:10.3969/j.issn.2097-2806.2023.10.007

摘要/Abstract

摘要：

以OECD指南为基础，在实验室建立氨基酸衍生物结合试验（ADRA）方法并进行实验室内验证，同时评估该方法对测试化妆品配方致敏反应的可能性。依据OECD 442C指南，将受试物（包括化学品和化妆水配方）分别与半胱氨酸衍生物和赖氨酸衍生物以50∶1的比例避光孵育24 h，采用高效液相色谱法分析两种氨基酸衍生物的消耗，计算氨基酸衍生物消耗率并依据ADRA预测模型对受试物致敏性进行判定。同样条件下使用商业化ADRA试剂盒按照操作规程检测相同受试物的致敏性。由试验结果可知，实验室建立的ADRA方法可以准确区分13种化学品的致敏性，检测结果与OECD 442C指南及文献中小鼠淋巴结试验（LLNA）结果一致，也与商业化试剂盒检测结果一致；ADRA对5种化妆水配方检测结果显示4种为致敏物、1种为非致敏物。

关键词: 化妆品, 氨基酸衍生物结合试验, 皮肤致敏, 替代方法

Abstract:

A laboratory method of amino acid derivative reactivity assay （ADRA） was established and validated based on the OECD guideline. Meanwhile, the possibility of applying ADRA to predict skin sensitization in cosmetic products was also evaluated. According to the guidance of OECD 442C, test substances （including chemicals and toners） were incubated with cysteine and lysine derivatives at a ratio of 50∶1, respectively, in the dark for 24 h. The amino acid derivatives consumptions were analyzed by high performance liquid chromatography （HPLC）. The percent depletion was calculated, and each chemical was classified into sensitizers and non-sensitizers using the ADRA prediction model. The same test substances were applied to an ADRA commercial kit. The results show that, 13 chemicals are correctly classified by ADRA as sensitizers or non-sensitizers. The results obtained in the lab are consistent with LLNA data, OECD TG 442C and the results of the commercial kit. Four toners are identified by ADRA as sensitizers and one as non-sensitizer.

Key words: cosmetics, amino acid derivative reactivity assay （ADRA）, skin sensitization, alternative methods

中图分类号:

TQ658

丁诗璇, 李小林, 陈瑜, 于婕. 氨基酸衍生物结合试验在化妆品检测中的应用[J]. 日用化学工业（中英文）, 2023, 53(10): 1166-1172.

Ding Shixuan, Li Xiaolin, Chen Yu, Yu Jie. Application of amino acid derivative reactivity assay for skin sensitization prediction of cosmetics[J]. China Surfactant Detergent & Cosmetics, 2023, 53(10): 1166-1172.

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参考文献 16

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组分	w/%
组分	配方1	配方2	配方3	配方4	配方5
水	59.900	67.900	69.700	69.884	69.900
酒精	20.000	20.000	20.000	20.000	20.000
甘油	10.000	10.000	10.000	10.000	10.000
吐温80	0.100	0.100	0.100	0.100	0.100
肉桂醛	10.000	2.000	0.200	0.016	0.000

反应溶液		V/μL
反应溶液		NAC溶液	NAL溶液	pH 8.0磷酸盐缓冲液	pH 10.2磷酸盐缓冲液	受试物溶液	溶剂
样品	1∶50 NAC	150				50
样品	1∶50 NAL		150			50
空白对照	NAC	150					50
空白对照	NAL		150				50
共洗脱对照	NAC			150		50
共洗脱对照	NAL				150	50

两种氨基酸衍生物平均消耗率/%	NAC消耗率/%	ADRA预测结果
<4.9	<5.6	阴性
≥4.9	≥5.6	阳性

受试物	NAC消耗率/%	NAL消耗率/%	氨基酸衍生物平均消耗率/%	ADRA预测结果	LLNA
对苯醌	100.00±0.00	46.89±6.64	73.45	阳性	极强致敏物
二苯基环丙烯酮	27.39±6.37	7.84±3.21	17.62	阳性	强致敏物
2，4-二硝基氯苯	56.29±2.73	2.25±4.99	29.27	阳性	极强致敏物
2-甲基-4-异噻唑啉-3-酮	100.00±0.00	0.10±4.55	50.05	阳性	中致敏物
苯乙醛	28.95±2.59	81.70±4.28	55.33	阳性	中致敏物
反式肉桂醛	100.00±0.00	4.11±3.73	52.06	阳性	中致敏物
金合欢醛	20.04±3.03	3.48±1.04	11.76	阳性	低致敏物
咪唑烷基脲	35.36±3.91	6.08±1.94	20.72	阳性	低致敏物
间苯二甲酸二甲酯	4.81±4.51	1.45±0.43	3.13	阴性	非致敏物
乳酸	0.00±0.00	0.35±6.37	0.18	阴性	非致敏物
2-丙醇	4.67±5.58	0.62±4.27	2.65	阴性	非致敏物
对羟基苯甲酸丙酯	2.17±1.44	1.22±3.70	1.70	阴性	非致敏物
甘油	6.28±4.53	1.02±2.77	3.65	阴性	非致敏物

受试物	NAC消耗率/%	NAL消耗率/%	氨基酸衍生物平均消耗率/%	商业化ADRA 试剂盒预测结果	ADRA预测结果
对苯醌	100.00±0.00	48.94±5.93	74.47	阳性	阳性
二苯基环丙烯酮	20.57±2.35	1.43±3.23	11.00	阳性	阳性
2, 4-二硝基氯苯	63.22±2.69	0.00±0.00	31.61	阳性	阳性
2-甲基-4-异噻唑啉-3-酮	100.00±0.00	0.66±1.92	50.33	阳性	阳性
苯乙醛	20.66±7.03	79.98±3.27	50.32	阳性	阳性
反式肉桂醛	69.40±8.19	4.80±2.94	37.10	阳性	阳性
金合欢醛	22.65±3.43	6.73±5.45	14.69	阳性	阳性
咪唑烷基脲	34.70±5.01	1.73±3.65	18.22	阳性	阳性
间苯二甲酸二甲酯	5.68±1.28	0.96±2.34	3.32	阴性	阴性
乳酸	2.59±6.83	5.25±4.13	3.92	阴性	阴性
2-丙醇	0.34±0.92	3.85±2.64	2.10	阴性	阴性
对羟基苯甲酸丙酯	4.31±6.95	0.00±0.00	2.16	阴性	阴性
甘油	0.00±0.00	0.53±2.52	0.27	阴性	阴性