2016年5月，AltTox（推进毒理学检测中使用非动物方法的网站）更新了《已经验证及已被接受的替代方法列表》，该表是AltTox.org网站点击量最高的页面之一。正如很多AltTox用户已了解的，该表提供了ICCVAM (美国)、 EURL ECVAM (欧盟)和/或 JaCVAM (日本)已支持的替代方法。除此之外， 表中也列入了OECD指导文件内容。自第一版列表于2007年12月在AltTox发布至今，该表仍是唯一可获得的整合了毒理方面三大权威替代方法验证中心信息的资料。我们最新的焦点文章对这一极具价值的资源做了简要介绍，并对表格中最重要并值得注意的更新内容做了总结。

（上接第25期续）

已被验证&接受的替代方法

类别	方法名称	测试方式1	科学有效性认可： ESAC Statements;EURL ECVAM Recommendations; ECVAM Protocols; ICCVAM Evaluations; ICCVAM Protocols; JaCVAM Evaluations; JaCVAM Statements		已接受的标准
			首要权威组织	随后的支持者	国际 (OECD链接:OECD TGs; Draft TGs; GDs; Draft GDs)	国家/地区2
药物研发临床前和非临床安全性评价研究 （通过合理指导可以减少动物的使用量）	M3(R2)药物进行临床试验和上市的非临床安全性研究指导原则	体内试验			ICH (2010)	FDA; EMA; PMDA
	生物技术药临床前安全评价, ICH S6(R1)	体内试验			ICH (1997; updated 2011)	FDA; EMA; PMDA
致热性	鲎阿米巴样细胞裂解物(LAL)试验	体外试验/体内试验				EU, US, and Japanese Pharmacopeia (2005); ICH harmonized text (2010)
	5种人细胞试验检测由于革兰氏阴性内毒素导致的致热原性：人全血IL-1，人全细胞IL-6，人冷藏保存的全细胞IL-1，外周血单核细胞IL-6和MM6细胞IL-6
		体外实验	ESAC (2006)	ICCVAM (2008)12		European Pharmacopeia;FDA
生殖&发育毒性	胚胎干细胞试验检测胚胎毒性17	体外试验	ESAC (2002)
	微团胚胎毒性试验	体外试验（但取自活体）	ESAC (2002)
	全鼠胚胎毒性试验	体外试验（但取自活体）	ESAC (2002)
	超出一代的生殖毒性研究	体内试验		ICCVAM16	Updated OECD TG 443 (2012)
皮肤腐蚀性	体外皮肤腐蚀性：重建人表皮实验（RHE）: Episkin™, Epiderm™, SkinEthic™, epiCS® (以前的EST-1000)	体外试验	ESAC (1998; 2000; 2006; 2009)	ICCVAM 2002	OECD TG 431 (2004; updated 2015)13a
	体外膜屏障试验检测皮肤腐蚀性：CORROSITEX™	体外试验	ICCVAM (1999)	ESAC (2000)	OECD TG 435 (2006; updated 2015)
	大鼠皮肤经皮电阻(TER)试验方法	体外试验（但取自活体）	ESAC (1998)	ICCVAM (2002)	OECD TG 430 (2004; updated 2015)
	Vitrolife-Skin™重建人表皮	体外试验	JaCVAM (2008)		JaCVAM (2008)
皮肤刺激性	重建人表皮方法：Episkin™, modified Epiderm™, SkinEthic™	体外试验	ESAC (2007; 2008; 2009)	JaCVAM (2010; 2012)	OECD TG 439 (2010; updated 2015)13b
	重建人表皮方法：LabCyte　EPI-MODEL24	体外试验	JaCVAM (2013)		OECD TG 439 (2010; updated 2015)13b
皮肤敏感反应	局部淋巴(LLNA)试验	体内试验	ICCVAM (1999; 2009)	ESAC (2000)	OECD TG 429 (2002; Updated 2010)
	减少的LLNA试验: rLLNA11	体内试验	ESAC (2007; 2008)	ICCVAM (2009) JaCVAM (2012)	OECD TG 429 (Updated 2010)
	非放射标记LLNA试验: DA	体内试验	ICCVAM (2010)	JaCVAM (2012)	OECD TG 442A (2010)
	非放射标记LLNA试验: BrdU-ELISA	体内试验	ICCVAM (2010)	JaCVAM (2012)	OECD TG 442B (2010)
	LLNA试验用于检测皮肤敏感性效价分类	体内试验	ICCVAM (2011)		UN GHS (2009)	Some US agencies (2012)
	ARE-Nrf2荧光素酶试验方法：KeratinoSens™20a	体外试验	EURL ECVAM(2014)		OECD TG 442D (2015)	REACH regulation
	直接肽反应试验(DPRA)20b	化学的	EURL ECVAM(2013) JaCVAM (2015)		OECD TG 442C (2015)	REACH regulation
	人细胞系激活试验(h-CLAT)20c	体外试验	EURL ECVAM(2015)		Draft OECD TG (2015)29	REACH regulation

 

1 Table content is limited to in vitro, ex vivo, in chemico and/or in silico test methods, and in vivo methods or test schemes proposed to reduce or refine animal use.

2 National and regional regulatory acceptance is not fully documented here.

3 Replaces animal use for initial dose setting, but in vivo test required to complete assessment.

4 In vitro cell transformation assays (CTAs) can detect both genotoxic and non-genotoxic carcinogens, which can be distinguished by parallel use of genotoxicity test(s). CTA results are recommended for use as part of a testing strategy and/or weight-of-evidence approach for the assessment of carcinogenic potential, and may reduce the use of in vivo testing.

5 The two reference test methods that provide the basis for this guideline are: the Stably Transfected TA (STTA) assay using the (h) ERα-HeLa-9903 cell line, and the BG1Luc ER TA assay. These methods are proposed for screening and prioritization purposes, but also provide mechanistic information that can be used in a weight of evidence approach.

6 BG1Luc ER TA test method has been adapted to a high throughput screening (HTS) format and incorporated into Tox21 screening

8 Can be used as screening test to distinguish water-soluble surfactant chemicals and certain types of surfactant-containing formulations that are not labeled as irritants (i.e., EPA Category IV, EU Not Labeled, FHSA Not Labeled) from all other hazard categories (i.e., EPA Category I, II, III; EU R41, R36; FHSA Irritant) for hazard classification and labeling under EPA, EU and FHSA classification systems; high false negative rate (24%-40%) for non-surfactant substances and formulations; high false positive rate (50% to 69%) for substances not labeled as irritants. Can be used as a screening test to identify water soluble substances as ocular corrosives and severe irritants (i.e., EPA Category I, EU R41, GHS Category 1) in tiered-testing strategy as part of weight-of-evidence approach; negative results need to be tested in another test method

9 Recommended for use as initial step within a Top-Down Approach to identify ocular corrosives and severe irritants (EU R41, GSH Category 1, and EPA Category I) for water-soluble chemicals; further refinement with respect to variability and applicability domain recommended

10 BCOP and ICE methods can be used to identify chemicals as either 1) causing “serious eye damage” (category 1 of GHS, or 2) not requiring classification for eye irritation or serious eye damage according to GHS (new to the 2013 TG updates).

11 rLLNA can be used for hazard classification when dose-response information is not needed

12 Subject to product-specific validation to demonstrate equivalence to the rabbit pyrogen test (RPT)

13a Four validated test methods using commercially available RhE models are included in TG 431: EpiSkin™ Standard Model (SM), EpiDerm™ Skin Corrosivity Test (SCT) (EPI-200), SkinEthic™ RHE, and epiCS® (previously named EST-1000).

13b Four validated test methods adhere to TG 439: EpiSkin™, EpiDerm™ SIT (EPI-200), SkinEthic™ RHE, and LabCyte EPI-MODEL24 SIT. “Depending on the regulatory framework and the classification system in use, this procedure may be used to determine the skin irritancy of test substances as a stand-alone replacement test for in vivo skin irritation testing, or as a partial replacement test, within a tiered testing strategy.”

14 Recommended as screening test or as part of sequential testing strategy; only positive test results accepted in the 2007 endorsement

15 Cell-based assay for botulinum toxin potency testing is limited to products produced using the Allergan method

16 Contributed to OECD TG development and/or review

17 Draft OECD GD 43, see page 18

18 EURL ECVAM reports that: “TG236 does not indicate whether the fish embryo acute toxicity test can be used as an alternative to the OECD TG203; however, several recently published papers demonstrate that the LC50 values produced with the fish embryo acute toxicity test correlate well with those observed in juvenile or adult fish (Lammer et al, 2009; Knoebel et al 2012; Belanger et al (2013).”

19 The assays using the thymidine kinase (TK) locus were originally contained in TG 476.

20 In vitro and in chemico methods endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests.

20a In vitro method endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests. The only ARE-Nrf2 luciferase test method covered currently by this TG, is the KeratinoSens™ test method.

20b In chemico method endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests.

20c In vitro method endorsed as valid for supporting the discrimination between skin sensitizers and non-sensitizers in accordance with the UN GHS. The guidelines explain it is likely that combinations of non-animal methods using integrated testing approaches will be needed to substitute for the animal tests.

21 This guideline describes assays providing the methodology for human recombinant in vitro assays to detect substances with estrogen receptor binding affinity, and comprises two similar reference test methods: Freyberger-Wilson (FW) in vitro estrogen receptor binding assay…, and Chemical Evaluation and Research Institute (CERI) in vitro estrogen receptor binding assay…; proposed for screening and prioritization purposes, and provide mechanistic information that can be used in a weight of evidence approach.

 

22 The only method currently covered by this TG is the EpiOcular™ Eye Irritation Test (EIT) developed using human skin cells.

23 ROS assay can be used in an integrated photosafety testing and decision strategy for drug research and development where negative results would not require further testing, while positive and inconclusive results would proceed to the next level of testing in an in vitro test system such as the 3T3 Phototoxicity Assay.

24 OECD Series on Testing and Assessment contains additional useful guidance and performance standards. For example, Guidance Document No. 211:Guidance for describing non-guideline in vitro test methods to facilitate their consideration in regulatory applications, and the draft Guidance Document on Revisions to OECD Genetic Toxicology Test Guidelines.

25 Also see: Draft Guidance Document on Revisions to OECD Genetic Toxicology Test Guidelines (2015) and Report on Statistical Issues Related to OECD Test Guidelines (TGs) on Genotoxicity (Series on Testing and Assessment, No. 198, 2014).

26 “The JaCVAM peer review panel concluded that the reproducibility and predictivity of the ROS assay is sufficient to support its use in an integrated photosafety testing and decision strategy for drug research and development…[where] negative results in the ROS assay would not require further testing in animals or other tests, while positive, weakly positive, and inconclusive results would proceed to the next level of testing in anin vitro test system such as the 3T3 Phototoxicity Assay (OECD Test Guideline 432).”

27 The in vitro pyrogenicity methods are not considered valid for use in Japan at this time.

28 Includes explanation of expanded use of BCOP for antimicrobial testing, and decision tree approach using 3 assays:

Bovine Corneal Opacity and Permeability test (BCOP)

EpiOcular assay

Cytosensor Microphysiometer assay.

29 hCLAT was approved by the OECD Working Group in April 2016

 

补充信息

A. EURL ECVAM: Test methods endorsed by EURL ECVAM and accepted by EU regulatory authorities; ESAC statements; DB-ALM (DataBase service on ALternative Methods); TSAR (Tracking System for Alternative test methods Review)

B. ICCVAM: Summary of test methods evaluated by ICCVAM; table showing validation and acceptance status of all methods reviewed; ICCVAM-recommended protocols

C. JaCVAM: Summary of test methods evaluated and accepted by Japan’s regulatory authorities

D. OECD Test Guidelines (TGs), Guidance Documents, Draft Test Guidelines

E. OECD Guidance Document for Describing Non-Guideline In Vitro Test Methods (No.211): intended to harmonize the way non-guideline in vitrotest methods are described

F. ICH test guidelines: technical requirements for medicinal products containing new drugs accepted by the regulatory bodies of EU, Japan, and USA

G. VICH test guidelines: EU-Japan-USA programme aimed at harmonising technical requirements for veterinary product registration

H. ISO: toxicity test guidelines for the biological evaluation of medical devices and ecotoxicity (soil quality, water quality, fire effluents, biocides used for anti-fouling systems on ships)

I. USDA APHIS Supplemental Assay Methods (SAMs) (updated March 17, 2016)

缩写

CFR: Code of Federal Regulations美国联邦法规

EMA: European Medicines Agency欧洲药品管理局

EPA: US Environmental Protection Agency美国环境保护署

ESAC: ECVAM Scientific Advisory Committee  ECVAM科学咨询委员会

EURL ECVAM: European Union Reference Laboratory for alternatives to animal testing欧盟动物实验替代品参比实验室

FDA: United States Food and Drug Administration美国食品与药品管理局

GD: OECD Guidance Document OECD指导文件

GHS: Globally Harmonized System of Classification and Labeling of Chemicals全球化学品统一分类和标签制度

ICCVAM: US Interagency Coordinating Committee on the Validation of Alternative Methods美国机构间替代方法验证协调委员会

ICH: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use人用药物注册技术要求国际协调会

ISO: International Organization for Standardization国际标准化组织

JaCVAM: Japanese Center for the Validation of Alternative Methods日本替代方法验证中心

OECD: Organisation for Economic Cooperation and Development经济与合作组织

OPPTS: US EPA’s Office of Prevention, Pesticides and Toxic Substances美国环境保护署污染、杀虫剂和有毒物质办公室

PMDA: Japan’s Pharmaceuticals and Medical Devices Agency日本药品与医疗器械管理局

TG: OECD Test Guideline OECD测试指南

USDA: United States Department of Agriculture美国农业部

VICH: International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products 兽药注册技术要求国际协调会

作者/贡献者：

Sherry L. Ward, PhD, MBA
AltTox Contributing Editor

AltTox 杂志审稿人：

Ian Kimber, PhD
Professor, University of Manchester

免责申明：

The information provided here is intended only as an overview, and is neither guidance or a comprehensive review of the laws, regulations, or toxicity tests that may be accepted by an individual agency. Countries/ regions and their regulatory authorities usually provide specific guidance on hazard/ toxicity testing requirements.