基于in vitro和in silico技术的化学品眼刺激性评价替代方法研究

doi:10.3969/j.issn.1001-1803.2022.09.015

摘要/Abstract

摘要：

建立基于荧光素漏出试验（Fluorescein Leakage test,FL test）和计算机模拟评价（in silico）技术的化学品眼刺激性初步评估和筛选的替代方法,并探讨其替代整体动物眼刺激性试验（Draize test）的可能性。分别采用FL test和Toxtree软件对经济合作与发展组织（the Organization for Economic Co-operation and Development,OECD）推荐的8种实验室能力验证物质的眼刺激性进行检测,并对结果进行分级。同时,分别比较两种方法测定结果的一致性。FL试验在判断受试物是否为严重刺激性/腐蚀性时和在进一步判定物质是否为严重刺激性/腐蚀性、中度刺激性或轻刺激性/无刺激性时,结果均具有较好的一致性（Kappa值为1,P=0.005;Kappa值为0.579,P=0.035）。Toxtree软件预测结果显示其更适合对“轻刺激性/无刺激性”受试物进行初步筛选。FL test是一种有效的眼刺激性评价体外替代方法,具有良好的应用和推广价值,更适用于判定有严重眼刺激性/腐蚀性的化合物,或作为“自上而下”眼刺激性评价分层组合测试策略中首先开展的实验。Toxtree预测结果可作为眼刺激性综合评估的参考数据。

关键词: 眼刺激性, 替代方法, 体外试验, 定量构效关系, 荧光素漏出试验, Toxtree软件

Abstract:

The aim of the study was to establish the alternative method for preliminary assessing eye irritation of chemicals using Fluorescein Leakage test （FL test） and Quantitative Structure Activity Relationship （QSAR）, to detect and classify the ocular irritation of the recommended substances, and to explore the application value of different alternative methods. Using Toxtree models to predict eye irritation and irritation potency of 8 recommended substances for demonstrating technical proficiency. The test substances were prepared into series of concentrations and exposed with the Madin-Darby canine kidney cell （MDCK） for 1 min to test the percentage of fluorescein leakage （FL/%）. The results were categorized according to the classification criteria for assessing eye irritation. Then, the sensitivity, specificity, and accuracy of FL test and Toxtree models were evaluated. Furthermore, the consistency of FL test or Toxtree model was compared with UN-GHS （United Nationas Globally Harmonized System of Classification and Labelling of Chemicals） classification. When FL test is used to distinguish if the test substance belongs to severe irritation/corrosion （GHS Cat. 1）, the irritation potency of all test substances is concordant with that in GHS classification （Kappa=1, P=0.005）. When FL test is used to distinguish if the test substance is severe irritation/corrosion （GHS Cat. 1）, moderate irritation （GHS Cat. 2A） or mild/no irritation （GHS Cat. 2B/No Cat.） substances, the irritation potency of all test substances is concordant with GHS classification in the eye irritant potency （Kappa=0.579, P=0.035）. The results predicted by Toxtree software demonstrate that it is more suitable to firstly estimate the possible substances with mild/no ocular irritation. The FL test is an effective and valuable alternative method for evaluating eye irritation, and it is more applicable to identify chemicals with severe irritation/corrosion or to be the study firstly carried out using the “Top-Down approach” of the integrated decision-tree testing strategy for eye irritation. The results predicted by Toxtree software could be reference data for the integrated assessment on eye irritation.

Key words: eye irritation, alternative method, in vitro, quantitative structure-activity relationship, fluorescein leakage test, Toxtree model

中图分类号:

TQ658

林铌,罗飞亚,曹春然,胡宇驰. 基于in vitro和in silico技术的化学品眼刺激性评价替代方法研究[J]. 日用化学工业, 2022, 52(9): 1016-1022.

Lin Ni,Luo Feiya,Cao Chunran,Hu Yuchi. Study on the alternative method for assessing eye irritation of chemicals using in vitro and in silico approaches[J]. China Surfactant Detergent & Cosmetics, 2022, 52(9): 1016-1022.

图/表 5

表1

表2

表3

表4

表5

参考文献 19

[1]	Yrok M, Steiling W. A critical review of the assessment of eye irritation potential using the Draize rabbit eye test[J]. Journal of Applied Toxicoy: JAT, 1998, 18 (4) : 233-240.
[2]	Dholakiya S L, Barile F A. Alternative methods for ocular toxicology testing: validation, applications and troubleshooting[J]. Expert Opinion on Drug Metabolism & Toxicology, 2013, 9 (6) : 699-712.
[3]	Lotz C, Schmid F F, Rossi A, et al. Alternative methods for the replacement of eye irritation testing[J]. Altex, 2016, 33 (1) : 55-67.
[4]	Cheng S, Liu C, Ma L. An integrated decision-tree testing strategy for eye irritation of chemicals with respect to the requirements of the EU REACH legislation[J]. Chinese Journal of Health Laboratory Technology, 2012, 22 (4) : 4.
[5]	Gerner I, Liebsch M, Spielmann H. Assessment of the eye irritating properties of chemicals by applying alternatives to the Draize rabbit eye test: the use of QSARs and in vitro tests for the classification of eye irritation[J]. Alternatives to Laboratory Animals, 2005, 33 (3) : 215-237.
[6]	Qian Y, Wang R, Li H, et al. Research progress on in vitro alternative method of eye irritation evaluation[J]. Chinese Journal of New Drugs, 2020 (6) : 6.
[7]	Gartlon J C R. Fluorescein leakage assay background review document as an alternative method for eye irritation testing[EB/OL]. (2008) [2021-11-18]. http://158.199.139.163/files/effort/Fluorescein/Fluorescein_08.pdf.
[8]	NMPA. Technical specification for the safety of cosmetics[EB/OL]. (2015) [2021-11-18]. https://www.nmpa.gov.cn/hzhp/hzhpggtg/hzhpqtgg/20151223120001986.html.
[9]	Bagley D M, Gardner J R, Holland G, et al. Eye irritation: updated reference chemicals data bank[J]. Toxicology in Vitro: an International Journal Published in Association with BIBRA, 1999, 13 (3) : 505-510.
[10]	Espersen R J, Olsen P, Nicolaisen G M, et al. Assessment of recovery from ocular irritancy using a human tissue equivalent model[J]. Toxicology in Vitro: an International Journal Published in Association with BIBRA, 1997, 11(1/2): 81-88.
[11]	J rov D, Kejlova K, Janousek S, et al. Eye irritation hazard of chemicals and formulations assessed by methods in vitro[J]. Neuro Endocrinology Letters, 2014, 35: 133-140.
[12]	Schrage A, Gamer A O, Van Ravenzwaay B, et al. Experience with the HET-CAM method in the routine testing of a broad variety of chemicals and formulations[J]. Alternatives to Laboratory Animals: ATLA, 2010, 38 (1) : 39-52.
[13]	Bhhatarai B, Wilson D M, Parks A K, et al. Derek Nexus in silico models for ocular irritation and development of a knowledge-based framework to improve the prediction of severe irritation[J]. Chemical Research in Toxicology, 2016, 29 (5) : 810-822. doi: 10.1021/acs.chemrestox.5b00531 pmid: 27018716
[14]	Geerts L, Adriaens E, Alpen, et al. CON4EI: Evaluation of QSAR models for hazard identification and labelling of eye irritating chemicals[J]. Toxicology in Vitro: an International Journal Published in Association with BIBRA, 2017: 60-62.
[15]	Verheyen G R, Braeken E, Deunkv, et al. Evaluation of existing (Q)SAR models for skin and eye irritation and corrosion to use for REACH registration[J]. Toxicology Letters, 2016, 265: 47. doi: 10.1016/j.toxlet.2016.11.007
[16]	Wang Q, Peng J. Evaluation of the Draize eye irritation test[J]. Traditional Chinese Drug Research & Clinical Pharmacology, 2005, 16 (4) : 301-304.
[17]	Lotz C, Kiesewetter L, Schmid F F, et al. Replacing the Draize eye test: Impedance spectroscopy as a 3R method to discriminate between all GHS categories for eye irritation[J]. Scientific Reports, 2018, 8 (1).
[18]	Scott L, Eskes C, Hoffmann S, et al. A proposed eye irritation testing strategy to reduce and replace in vivo studies using Bottom-Up and Top-Down approaches[J]. Toxicology in Vitro, 2010, 24 (1) : 1-9. doi: 10.1016/j.tiv.2009.05.019
[19]	Gallegos A, Patlewicz G, Worth A. Review of literature-based models for skin and eye irritation and corrosion[EB/OL]. (2006)[2021-11-18]. https://www.researchgate.net/publication/254622024_Review_of_Literature-Based_Models_for_Skin_and_Eye_Irritation_and_Corrosion.

眼刺激程度分级	UN GHS C&L ^[11]	MMAS ^[12]	FL₂₀ ^[7]/（mg/mL）	Toxtree ^[13]
无刺激性	No Cat.	<2.5	>750	Nonirritant/not corrosive
轻刺激性	Cat. 2B	2.5~25.0
中度刺激性	Cat. 2A	25.0~50.0	100~750	Moderate reversible irritation to the eye
严重刺激性/腐蚀性	Cat. 1	>50.0	≤100	Serious lesions to the eye /corrosion

序号	受试物	CAS号	化学类别	理化性质
序号	受试物	CAS号	化学类别	物理状态	分子量	pK_a	熔点	c lg P^*
1	苯扎氯铵（5%）	8001-54-5	季铵盐,阳离子表面活性剂	液体	340	N/A	N/A	2.126
2	盐酸异丙嗪	58-33-3	胺/脒,杂环,有机硫化合物	固体	321	9.287	228.5	4.915
3	氢氧化钠（10%）	1310-73-2	碱	液体	40	N/A	323	0.467
4	十二烷基硫酸钠（15%）	151-21-3	羧酸盐	液体	288	N/A	205.5	4.106
5	4-醛基苯甲酸	619-66-9	羧酸,醛	固体	150	4.089	256	1.33
6	2-甲基乙酰乙酸乙酯	609-14-3	酮,酯	液体	144	N/A	187	0.924
7	月桂醇聚氧乙烯醚 Brij-35（30%）	9002-92-0	脂肪醇,醚,非离子型表面活性剂	液体	1 199.5	14.436	40~45	4.28
8	甘油	56-81-5	醇	液体	92.1	17.965	18	-1.88

序号	UN-GHS眼刺激性类别	Draize test		FL test		Toxtree
序号	UN-GHS眼刺激性类别	MMAS	分级	FL₂₀/ （mg/mL）	分级	结果	分级	警示结构
1	1	108	严重刺激性/腐蚀性	6.33	严重刺激性/腐蚀性	中度可逆性眼刺激 Moderate reversable irritation to the eye	中度刺激性	芳香盐铵 Aromatic ammonium salts
2	1	71.7	严重刺激性/腐蚀性	12.43	严重刺激性/腐蚀性	非皮肤腐蚀性 NOT skin corrosion R34 or R35	N/A
3	1	108	严重刺激性/腐蚀性	9.73	严重刺激性/腐蚀性	非皮肤腐蚀性 NOT skin corrosion R34 or R35	N/A
4	1	59.2	严重刺激性/腐蚀性	8.58	严重刺激性/腐蚀性	非皮肤腐蚀性 NOT skin corrosion R34 or R35	N/A
5	2A	50.0	中度刺激性	>750	轻刺激性/无刺激性	非刺激性Nonirritant	无刺激性
6	2B	18	轻刺激性	>750	轻刺激性/无刺激性	非刺激性Nonirritant	无刺激性
7	2B	16	轻刺激性	133.82	中度刺激性	未知分类Unknown	N/A
8	No Cat.	1.7	无刺激性	>750	轻刺激性/无刺激性	未知分类Unknown	N/A

眼刺激程度分级/结果		GHS结果		灵敏度/%	特异度/%	准确度/%
眼刺激程度分级/结果		阳性（4）	阴性（4）	灵敏度/%	特异度/%	准确度/%
FL test	阳性	4	0	100	100	100
FL test	阴性	0	4	100	100	100
Toxtree	阳性	0	0	0	50	25
Toxtree	阴性	1	2	0	50	25

眼刺激程度分级/结果		GHS分级			准确度/%
眼刺激程度分级/结果		严重刺激性/腐蚀性（4）	中度刺激性（1）	无刺激性/轻刺激（3）	准确度/%
FL test	严重刺激性/腐蚀性	4	0	0	75
	中度刺激性	0	0	1
	无刺激性/轻刺激性	0	1	2
Toxtree	严重刺激性/腐蚀性	0	0	0	12.5
	中度刺激性	1	0	0
	无刺激性/轻刺激性	0	1	1