化妆品原料的过敏性体外方法——氨基酸衍生化反应法（ADRA）方法转化与验证

doi:10.3969/j.issn.1001-1803.2023.02.005

摘要/Abstract

摘要：

在皮肤致敏不良结果通路（AOP）的第一个关键事件中化学反应为亲电物质与皮肤蛋白中的亲核中心共价结合，即皮肤与物质接触后，可能与皮肤上的蛋白结合形成抗原，导致不良反应。采用半胱氨酸和赖氨酸衍生物替代皮肤组织，模拟皮肤蛋白与化合物结合，通过计算半胱氨酸和赖氨酸衍生物反应后耗竭值（百分比）将物质分类，以此区分皮肤敏化剂和非敏化剂，此种方法称之为氨基酸衍生化反应法（ADRA）。本实验选择10种致敏性已知的物质，探究ADRA方法用于化合物致敏性评价的准确度，并在10种物质中随机选取几种物质采用国内已转化验证过的方法直接反应多肽法（DPRA）对结果进行验证。结果表明，10种物质致敏性判定结果符合样品自身致敏性，认定ADRA致敏性判定准确度较好，可作为皮肤致敏AOP的第一个关键事件替代实验方法。

关键词: 皮肤致敏性, 替代实验, 氨基酸衍生化反应（ADRA）

Abstract:

A skin sensitizer refers to a substance that will lead to an allergic response following repeated skin contact as defined by the United Nations Globally Harmonized System of Classification and Labelling of Chemicals. The current knowledge of the chemical and biological mechanisms associated with skin sensitisation has been summarised as an Adverse Outcome Pathway （AOP） starting with a molecular initiating event through intermediate events to the adverse effect, namely allergic contact dermatitis. Using cysteine and lysine derivatives instead of skin tissue, simulating the reaction of skin proteins with compounds, classifying the substances by calculating the percentage of depletion value after the reaction of cysteine and lysine derivatives to distinguish skin sensitizers and desensitizers, this method is called Amino acid Derivative Reactivity Assay （ADRA）. In this study, ten known allergens were selected to explore the accuracy of the sensitivity evaluation of ADRA method, and verified accuracy by DPRA that several substances were randomly selected among the ten substances. The results show that the sensitivity determination results of 10 substances are consistent with the sensitivity of samples, and the accuracy of determination of ADRA sensitivity is good.

Key words: skin sensitisation, alternatives experiment, ADRA

中图分类号:

TQ658

沈立, 王雪梅, 张慧文, 所雅琼, 吴景, 邢书霞. 化妆品原料的过敏性体外方法——氨基酸衍生化反应法（ADRA）方法转化与验证[J]. 日用化学工业（中英文）, 2023, 53(2): 157-164.

Shen Li, Wang Xuemei, Zhang Huiwen, Suo Yaqiong, Wu Jing, Xing Shuxia. In chemico skin sensitisation: Amino acid Derivative Reactivity Assay （ADRA） transformation and validation[J]. China Surfactant Detergent & Cosmetics, 2023, 53(2): 157-164.

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参考文献 7

[1]	OECD (2012). Series on Testing and Assessment No. 168. The Adverse Outcome Pathway for Skin Sensitisation Initiated by Covalent Binding to Proteins. Part 1: Scientific Evidence[EB/OL]. Organisation for Economic Cooperation and Development, Paris, France: Organisation for Economic Cooperation and Development, 2012[2022-01-28]. http://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=ENV/JM/MONO(2012)10/PART1&docLanguage=En
[2]	Karlberg A T, Bergström M A, Börje A, et al. Allergic contact dermatitis—formation, structural requirements, and reactivity of skin sensitizers.[J]. Chemical Research in Toxicology, 2007.
[3]	OECD (2020). OECD Guidelines for the Testing of Chemicals, Section 4. Test No. 442C: In Chemico Skin Sensitisation. Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins. [EB/OL]. Paris, France: Organisation for Economic Cooperation and Development. 2021 [2022-01-20]. https://www.oecd-ilibrary.org/docserver/9789264229709-en.pdf?expires=1651130191&id=id&accname=guest&checksum=770259B159798FDB9A6AAA96767A7C8E
[4]	Yamamoto Y, Tahara H, Usami R, et al. A novel in chemico method to detect skin sensitizers in highly diluted reaction conditions[J]. Journal of Applied Toxicology, 2015, 35 (11):1348-1360. doi: 10.1002/jat.3139 pmid: 25809859
[5]	Fujita M, Yamamoto Y, Tahara H, et al. Development of a prediction method for skin sensitization using novel cysteine and lysine derivatives[J]. Journal of Pharmacological and Toxicological Methods, 2014, 70 (1):94-105. doi: 10.1016/j.vascn.2014.06.001 pmid: 24928155
[6]	Amino acid Rerivative Reactivity Assay (ADRA) JaCVAM Validation Study Report[R]. Version 1.2, July, 2018.
[7]	Kasahara K T. The underlying factors that explain why nucleophilic reagents rarely co-elute with test chemicals in the ADRA[J]. Journal of Pharmacological and Toxicological Methods, 2019, 96: 95-105. doi: S1056-8719(18)30768-8 pmid: 30776483

标准线性溶液和空白对照	稀释溶剂（std1）
	标准线性溶液剂量std2~std7
	对照A平行样品1~样品3
共洗脱对照	受试物1共洗脱对照
共洗脱对照	受试物2共洗脱对照等其他受试物共洗脱对照依次排列
对照	对照B平行样品1~样品3
重复进样第一次	对照C平行样品1
	阳性对照第一份平行样品
	各受试物的第一份平行样品
重复进样第二次	对照C平行样品2
	阳性对照第二份平行样品
	各受试物的第二份平行样品
重复进样第三次	对照C平行样品3
	阳性对照第三份平行样品
	各受试物的第三份平行样品
对照	对照B平行样品4~样品6

NAC和NAL消耗值的均值	预测结果
0%≤消耗值的均值<4.9%	阴性
4.9%≤消耗值的均值≤100%	阳性

NAC消耗值的均值	ADRA预测结果
0%≤消耗值的均值<5.6%	阴性
5.6%≤消耗值的均值≤100%	阳性

样品编号	阳性对照				对照A、B、C						样品		是否符合要求
	阳性对照				半胱氨酸衍生物			赖氨酸衍生物			样品
	半胱氨酸消耗值/%	消耗值SD/%	赖氨酸消耗值/%	消耗值SD/%	对照A平均浓度/（μmol/L）	对照B、C峰面积RSD/%	对照C平均浓度/（μmol/L）	对照A平均浓度/（μmol/L）	对照B、C峰面积RSD/%	对照C平均浓度/（μmol/L）	半胱氨酸消耗值SD/%	赖氨酸消耗值SD/%
T1	9.04	2.22	75.25	3.03	3.407	4.61	3.567	4.26	2.33	4.3	0	1.80	是
T2											1.40	0.57	是
T3											2.29	1.08	是
T4											2.08	7.41	是
T5											0.33	0.16	是
T6											2.79	2.14	是
T7											0.16	0.14	是
T8											0.17	1.85	是
T9											1.36	1.20	是
T10											0.75	5.05	是

样品编号	受试物名称	OECD准则氨基酸衍生物消耗值（参考）范围/%			本实验室氨基酸衍生物消耗值/%
样品编号	受试物名称	NAC	NAL	致敏性预测结果	NAC	NAL	致敏性预测结果
T1	对苯醌	90~100	40~70	阳性	100	74.28	阳性
T2	对羟基苯甲酸丙酯	≤7	≤7	阴性	2.69	1.89	阴性
T3	苯甲醇	≤7	≤7	阴性	0.64	1.25	阴性
T4	棕榈酰氯	≤10	50~100	阳性	3.11	42.23	阳性
T5	间苯二甲酸二甲酯	≤7	≤7	阴性	2.62	2.02	阴性
T6	甘油	≤7	≤7	阴性	-0.48	-0.5	阴性
T7	氯胺T	90~100	90~100	阳性	97.56	96.65	阳性
T8	肉桂醛	40~100	≤20	阳性	50.04	0.71	阳性
T9	咪唑烷基脲	10~45	≤10	阳性	14.53	-0.01	阳性
T10	金合欢醇	20~40	≤15	阳性	25.56	-3.06	阳性