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

Abstract

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

CLC Number:

TQ658

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.

Figures/Tables 9

Fig. 1

Tab. 1

Tab. 2

Tab. 3

Tab. 4

Tab. 5

Tab. 6

Tab. 7

Tab. 8

References 16

[1]	OECD. The adverse outcome pathway for skin sensitisation initiated by covalent binding to proteins. Part 2:use of the AOP to develop chemical categories and integrated assessment and testing approaches. Series on Testing and Assessment No.168[R]. Paris, 2012.
[2]	OECD. Test No. 442C: key event-based test guideline for in chemico skin sensitisation assays addressing the adverse outcome pathway key event on covalent binding to proteins[S]. Organization for Economic Cooperation and Development, Paris, 2021.
[3]	OECD. Test No. 442D: In vitro skin sensitisation: ARE-Nrf2 luciferase test method[S]. Organization for Economic Cooperation and Development, Paris, 2022.
[4]	OECD. Test No. 442E: In vitro skin sensitisation: in vitro skin sensitisation assays addressing the adverse outcome pathway key event on activation of dendritic cells[S]. Organization for Economic Cooperation and Development, Paris, 2022.
[5]	Yamamoto Y, Fujita M, Wanibuchi S, et al. Expanding the applicability of the amino acid derivative reactivity assay (ADRA): determining a weight concentration for preparation of test chemical solutions that yields a predictive capacity identical to the conventional method using molar concentration and demonstrating the capacity to detect sensitizers in liquid mixtures[J]. Journal of Pharmacological and Toxicological Methods, 2019, 97: 67-79. doi: S1056-8719(18)30714-7 pmid: 30796999
[6]	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]. Toxicology in Vitro, 2019, 59: 161-178. doi: 10.1016/j.tiv.2019.04.014
[7]	Wanibuchi S, Yamamoto Y, Sato A, et al. The amino acid derivative assay with fluorescence detection and its application to multi-constituent substances[J]. The Journal of Toxicological Science, 2019, 44: 821-832. doi: 10.2131/jts.44.821
[8]	Yamamoto Y, Fujita M, Wanibuchi S, et al. Applicability of amino acid derivative reactivity assay for prediction of skin sensitization by combining multiple alternative methods to evaluate key events[J]. Journal of Toxicological Sciences, 2019, 44: 585-600. doi: 10.2131/jts.44.585 pmid: 31474740
[9]	Imamura M, Wanibuchi S, Yamamoto Y, et al. Improving predictive capacity of the Amino acid Derivative Reactivity Assay test method for skin sensitization potential with an optimal molar concentration of test chemical solution[J]. Journal of Applied Toxicology, 2021, 41: 303-329. doi: 10.1002/jat.4082 pmid: 33124715
[10]	Divkovic M, Pease C K, Gerberick G F, et al. Hapten-protein binding: from theory to practical application in the in vitro prediction of skin sensitization[J]. Contact Dermatitis, 2005, 53: 189-200. pmid: 16191014
[11]	Ahlfors S R, Sterner O, Hansson C. Reactivity of contact allergenic haptens to amino acid residues in a model carrier peptide, and characterization of formed petide-hapten adducts[J]. Skin Pharmacol Appl Skin Physiol, 2003, 16: 59-68. pmid: 12566830
[12]	Wareing B, Urbisch D, Kolle S N, et al. Prediction of skin sensitization potency sub-categories using peptide reactivity data[J]. Toxicology in Vitro, 2017, 45: 134-145. doi: S0887-2333(17)30245-X pmid: 28882705
[13]	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: 94-105. doi: 10.1016/j.vascn.2014.06.001 pmid: 24928155
[14]	Yamamoto Y, Tahara H, Usami R, et al. A novel in chemico method to detect skin sensitisers in highly diluted reaction conditions[J]. Journal of Applied Toxicology, 2015, 35: 1348-1360. doi: 10.1002/jat.3139 pmid: 25809859
[15]	Urbisch D, Mehling A, Guth K, et al. Assessing skin sensitization hazard in mice and men using non-animal test methods[J]. Regulatory Toxicology & Pharmacology, 2015, 71: 337-351.
[16]	Fujita M, Yamamoto Y, Wanibuchi S, et al. 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

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

Application of amino acid derivative reactivity assay for skin sensitization prediction of cosmetics

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 9

References 16

Related Articles 15

Metrics

Comments

Recommended 10

时间/min	NAC		NAL
时间/min	φ（流动相A）/%	φ（流动相B）/%	φ（流动相A）/%	φ（流动相B）/%
0	70	30	80	20
9.5	45	55	55	45
10	0	100	10	100
13	0	100	10	100
13.5	70	30	80	20
20	70	30	80	20

化妆水配方	肉桂醛含量/%	ADRA				商业ADRA化试剂盒
化妆水配方	肉桂醛含量/%	NAC消耗率/ %	NAL消耗率/ %	平均消耗率/ %	预测结果	NAC消耗率/ %	NAL消耗率/ %	平均消耗率/ %	预测结果
1	10.000	100.00±0.00	37.36±5.25	68.68	阳性	100.00±0.00	39.25±3.61	69.63	阳性
2	2.000	100.00±0.00	28.60±2.71	64.30	阳性	100.00±0.00	14.11±4.56	57.06	阳性
3	0.200	100.00±0.00	0.00±0.00	50.00	阳性	100.00±0.00	0.00±0.00	50.00	阳性
4	0.016	29.24±4.34	0.00±0.00	14.62	阳性	28.76±3.60	0.00±0.00	14.38	阳性
5	0.000	1.91±1.98	0.00±0.00	0.96	阴性	0.00±0.00	0.00±0.00	0.00	阴性

[1]	Jingxuan Liu, Jianming Jin, Hua Wu. Botanical cosmetic ingredients （VII）Research and development of plant antifungal [J]. China Surfactant Detergent & Cosmetics, 2024, 54(3): 259-266.
[2]	Wu Bi, Xiaohong Pan, Xiaoqin Tu, Shuai Yin, Hui Sun. Analysis of the mechanism of anti-sensitive skin effect of cosmetic raw material Stephania tetrandra based on network pharmacology [J]. China Surfactant Detergent & Cosmetics, 2024, 54(3): 305-312.
[3]	Yaoyao Li. Study on the anti-aging and antioxidant effects of isosinensetin [J]. China Surfactant Detergent & Cosmetics, 2024, 54(3): 313-319.
[4]	Liyuan Zhang, Linqi Yan, Qiaoyuan Cheng, Lvye Qi, Rong Wang, Liuqian Huang. Determination of 14 kinds of α-hydroxy acids and hydroxy esters in cosmetics [J]. China Surfactant Detergent & Cosmetics, 2024, 54(3): 353-359.
[5]	Wei Xu, Po Zou, Changyu Li, Ming Yang, Yan Lu, Huiliang Li. Determination of 36 stimulants in cosmetics by ultra performance liquid chromatography-tandem mass spectrometry [J]. China Surfactant Detergent & Cosmetics, 2024, 54(3): 360-368.
[6]	Kangfu Zhou, Yixuan Zhi, Feifei Wang, Yazhuo Shang. New emulsion system and its application in cosmetics （VI）Microemulsion [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 139-148.
[7]	Hongling Zhang, Lin Cheng, Haiyan Wang, Feiya Luo, Huiliang Zhang, Lei Sun. Using DPRA alternative method to evaluate the skin sensitization of 3 coumarins [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 156-160.
[8]	Zhen Xie, Wei Huang, Jinsong Zhang, Shuhuai Chen, Linji Qu, Rong Kuang. Study on biomarkers of corneal injury in the evaluation of eye irritation of cosmetics [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 161-167.
[9]	Xiaohong Pan, Ziqi Gao, Zhen Chen, Shuai Yin, Haiping Huang, Bin Hu. Discussion on the current situation of research and management on the stability of cosmetic products in China [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 201-208.
[10]	Li Lu, Fang Fang, Youlong Feng, Ling Cao. Screening for illegal addition of sulfonamides in cosmetic products using ultra-performance liquid chromatographytriple quadrupoletandem mass spectrometry with precursor ion scanning [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 216-223.
[11]	Ren Wang, Yuanyang Wu, Jia Qiao, Linqi Yan, Cen Chen, Liyuan Zhang. Study on phenoxyethanol content in children’s cosmetics on the mark and preliminary risk assessment [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 224-230.
[12]	Yixiang Lu, Liting Wu, Jimin Jiang, Hailu Chen, Xuan Huang. Determination of tolnaftate and liranaftate in cosmetics by high performance liquid chromatography and verification by high performance liquid chromatography-tandem mass spectrometry [J]. China Surfactant Detergent & Cosmetics, 2024, 54(2): 231-238.
[13]	Liyuan Zhang, Qiaoyuan Cheng, Cen Chen, Zehua Li, Liuqian Huang, Lvye Qi. Determination of 3 kinds of α-hydroxy acids and their esters in cosmetics by high performance liquid chromatography [J]. China Surfactant Detergent & Cosmetics, 2024, 54(1): 102-106.
[14]	Linling Lu, Hui Lu, Chunyan Min, Yefei Qian. Determination of functional components of Glycyrrhizae, Ginseng and Scutellariae in facial masks by UHPLC-MS/MS [J]. China Surfactant Detergent & Cosmetics, 2024, 54(1): 107-113.
[15]	Xu Han, Jiajia Wu, Na Wu, Yazhuo Shang. New emulsion system and its application in cosmetics （V） Janus emulsion [J]. China Surfactant Detergent & Cosmetics, 2024, 54(1): 24-31.

化妆水配方	肉桂醛含量/%	半胱氨酸多肽消耗率/%	赖氨酸多肽消耗率/%	多肽平均消耗率/%	反应程度	DPRA预测结果	ADRA预测结果
1	10.000	100.00±0.00	100.00±0.00	100.00	高	阳性	阳性
2	2.000	82.60±2.23	77.02±0.75	79.81	高	阳性	阳性
3	0.200	32.11±0.34	11.43±1.39	21.77	低	阳性	阳性
4	0.016	1.11±0.16	0.00±0.00	0.56	无	阴性	阳性
5	0.000	0.00±0.00	0.00±0.00	0.00	无	阴性	阴性