体外皮肤刺激模型的生物标志研究进展

皮肤刺激试验是人类健康相关产品危险性评价的常见项目,传统皮肤刺激试验采用实验动物进行,成本高周期长,给动物造成一定程度的痛苦。近年来,多种替代动物试验的体外模型被开发和应用。体外试验主要通过定量检测细胞活性和代谢变化的生物标志物预测体内的效应,应用最广泛的生物标志物是细胞活性、炎性因子、胞质酶等,在生物技术的推动下,新的特异性标志物被开发和验证。     

鸡胚尿囊膜（HET-CAM）眼刺激替代试验对产品及原料评价的比较

目的比较鸡胚尿囊膜试验（HET-CAM）作为一种眼刺激替代方法对产品或原料的评价。方法采用HET-CAM和兔眼Draize试验方法,对19种原料和23种化妆品及家用洗涤产品眼刺激性进行检测,对体内体外试验的结果进行统计比较。结果比较体内体外试验,原料和产品的kappa系数分别为0.826,0.531;灵敏度分别为100%,81.8%;特异度分别为85.7%,77.8%。结论 HET-CAM可作为Draize试验的替代试验,HET-CAM系统更适合对单纯化学品原料进行眼刺激试验。     

体外皮肤检测模型的组织工程研究进展

皮肤刺激试验是临床前安全评价的常见项目,由于3R原则的发展,现逐渐采用体外皮肤检测模型来替代传统的动物实验。然而在国内皮肤模型的研究还处于起步阶段,种子细胞和支架材料是目前研究的主要内容。原代细胞和永生化细胞是皮肤刺激试验模型的种子细胞中较常用的两类。支架材料应用较广的是天然材料、合成材料和复合材料,而国外批量生产的模型中支架材料现阶段还是以天然材料为主。随着科技发展纳米技术也被利用来制备支架材料,使得皮肤模型的研究进入更深的领域。     

GLP原则在体外毒理学评价实验室的应用

在动物福利运动的推动下,以减少、优化和替代动物试验为核心内容的体外试验系统已成为安全评价不可或缺的组成部分,在药品、化学品、化妆品毒理学评价中起到重要作用。体外试验系统不同于体内动物实验,体外毒理学实验室GLP原则的建立和运行应充分考虑体外试验系统的特殊性。目前我国专业的体外安全评价实验室的建设刚刚起步,还没有可供借鉴的成熟经验。本文从实验室组织、试验系统维护、人员职责、质量管理和运行几个方面,介绍了GLP原则在化妆品体外毒理学检验和评价实验室的应用。     

蜂胶提取物的安全性试验研究

目的 探讨蜂胶的乙醇提取物（EEP）的安全性。方法 用0.5％的EEP对家兔进行皮肤急性毒性试验、皮肤刺激试验以及对豚鼠的过敏试验。结果 EEP低剂量、高剂量皮肤破损组和对照组皮肤破损组各有一只家免,在24 h观察到皮肤破损处微红,48 h消失,其余各实验兔的皮肤、毛发、眼、粘膜、呼吸、中枢神经系统均无任何中毒表现;按照皮肤刺激反应强度,评价标准,完整皮肤组平均分为0,判为无刺激性;破损皮肤组平均分值为033＜0.50,亦判为无刺激性;A组空白对照组和B组EEP组动物均无红斑和水肿反应,判为无致敏性。结论 蜂胶提取物EEP在对实验动物体外寄生虫净化中安全、无毒、无刺激。     

化妆品眼刺激试验替代方法标准化与展望

眼刺激试验是化妆品安全评价的主要测试项目,目前正尝试建立代替传统动物实验的体外方法,这些方法处于研发、验证和认可的不同阶段。尽管还没有单一体外试验能完全替代兔眼实验,但系统分析替代方法建立的原理、科学相关性和标准化程度的不同,有助于合理选择运用体外方法指导化妆品眼刺激的标识分类、质量检验、产品开发和机制研究。     

抗菌肽在洗发液中防腐效能及安全性评价初探

防腐剂是化妆品中防止微生物污染的重要成分。为获得高效防腐且无毒副作用的洗发液防腐剂,实验选取4种抗菌肽进行MIC测定,筛选出针对个革兰氏阴性菌和阳性菌杀菌效果较好的两个抗菌肽N2和NZ2114。通过体外抗菌试验和细胞水平及小鼠皮肤刺激性实验对两种抗菌肽的防腐效果和安全性能进行测定和评估。实验结果表明,N2和NZ2114在低浓度(分别为40和20μg/mL)下即具有快速高效的杀菌效果,并且高浓度(128μg/mL)下,细胞存活率大于85%,具有低细胞毒性。ROS水平和小鼠皮肤刺激性实验证明,N2和NZ2114不会对皮肤细胞造成损伤,并且具有一定程度的抗氧化作用。本实验结果为抗菌肽作为高效安全的洗发液防腐剂提供参考依据。     

莲必治氯化钠注射液的安全性试验研究

目的：评价莲必治氯化钠注射液的安全性。方法：采用豚鼠全身主动过敏试验、主动皮肤过敏试验及被动皮肤过敏试验、体外溶血试验。兔血管刺激性试验和肌肉刺激性试验观察莲必治氯化钠注射液的安全性。结果：莲必治氯化钠注射液有轻微的过敏反应症状,无皮肤过敏反应。无溶血现象,对静脉血管、肌肉无刺激反应。结论：在该实验条件下莲必治氯化钠注射液,除有轻微的过敏反应症状外,是安全的。     

双斑东方鲀鱼皮胶原多肽的制备及其在化妆品中的功效与刺激性评价

河豚鱼皮中富含胶原蛋白,是一种优质的胶原多肽制备原料。鱼皮的精深加工不仅能减缓资源的浪费也能扩大我国河豚鱼的加工模式。本研究以双斑东方鲀鱼皮为实验对象,通过单因素实验和正交实验制备得到双斑东方鲀鱼皮多肽(Fugu bimaculatus collagen peptide,FBCP)并对其功效性及皮肤刺激性进行了研究。结果显示,碱性蛋白酶制备FBCP的最佳工艺条件为固液比1∶10、酶解温度50℃、加酶量8 000 U/g、酶解pH值9.0、酶解时间4 h。在此条件下,FBCP的肽得率为39.65%。利用超滤膜将胶原多肽提取液分离成不同分子量的四种组分,分别为FBCP1(Mw1 kDa)、FBCP2(1 kDaMw5 kDa)、FBCP3(5 kDaMw10 kDa)和FBCP4(Mw10 kDa)。吸湿保湿试验中,FBCP1组分显示出优于其它组分的吸湿和保湿性能。体外抗氧化实验结果表明,FBCP1自由基清除活性最好,其对超氧阴离子O■、羟基自由基·OH和DPPH的IC_(50)分别为1.659、5.582和1.801 mg/mL。多次皮肤刺激和急性眼刺激实验表明,涂抹高达50%(W/V)FBCP对受试兔皮肤及兔眼均无刺激性。综上所述,FBCP具有良好的抗氧化和保湿功效且对皮肤和眼睛无刺激性,可为其在化妆品中的应用提供基础参考。     

LLNA：BrdU-ELISA 改良法的建立及其在化妆品安全性评价中的应用

目的：建立可快速检测化学物致敏性和刺激性的局部淋巴结试验（ LLNA：BrdU-ELISA）改良法,并对化妆品产品进行评价。方法3种化学物（2,4二硝基氯苯（ DNCB）、丁子香酚、己基肉桂醛）和3种化妆品产品作为受试物,雌性BALB／c小鼠连续染毒3天,测量小鼠耳缘厚度,第5天腹腔注射BrdU,第6天称耳廓重,分离颌下淋巴结称重并制取单细胞悬液,用ELISA试剂盒检测淋巴细胞增殖。结果 DNCB（1.0％）、己基肉桂醛（25％、50％）和3号粉底霜引起耳肿胀和耳廓重显著增加（P ＜0.05或P ＜0.01）,可能为刺激物,其他均无刺激性;3种化学物和3号粉底霜致敏检测阳性,其他均为阴性。结论结合小鼠耳肿胀和耳廓重的LLNA：BrdU-ELISA改良法可较好地评价化学物和化妆品产品的致敏性和刺激性,有望在化妆品安全性评价中发挥重要作用。     

ECVAM's activities in validating alternative tests for skin corrosion and irritation

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commercially-produced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.     

The ECVAM international validation study on in vitro tests for acute skin irritation: report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, of which two employ reconstituted human epidermis models (EPISKIN, EpiDerm), and one, the skin integrity function test (SIFT), employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would correctly predict in vivo classifications according to the EU classification scheme, "R38" and "no label" (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identities by a single lead laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was judged to be inadequate. Since both the reconstituted skin models provided false predictions around the in vivo classification border (a rabbit Draize test score of 2), the release of a cytokine, interleukin-1alpha (IL-1alpha), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1alpha release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); with the combination of the MTT and IL-1alpha assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1alpha release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first use of in vitro toxicity testing to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.     

Integrated decision-tree testing strategies for skin corrosion and irritation with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME recently conducted a research project, sponsored by DEFRA, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on how to maximise the use of alternative methods (both in vitro and in silico) for skin corrosion and irritation testing within a tiered testing strategy. It considers the latest developments in in vitro testing, with particular reference to the reconstituted skin models which have now been now been successfully validated and independently endorsed as suitable for both skin corrosivity and irritancy testing within the EU.     

The in vitro skin irritation of chemicals: optimisation of the EPISKIN prediction model within the framework of the ECVAM validation process

In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: "no classification"). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol 15min-18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol 15min-42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1alpha and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a "decision-making" tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.     

Status and prospects of in vitro tests in risk assessment

According to the new chemicals policy of the European Union (EU), most chemicals, i.e. the 20,000 chemicals manufactured or imported at 1-10 tons annually, should be tested primarily by using in vitro methods. Also, for other chemicals, the use of in vitro methods is encouraged in the testing strategies given in the draft EU legislation. However, the validation and international acceptance of in vitro tests has been slow. Only recently has the OECD approved four new in vitro test methods, validated by the European Centre for the Validation of Alternative Methods. An analysis of ten randomly selected risk assessment reports of the EU Existing Chemicals Risk Assessment Programme showed that in vitro studies, for example, on cytotoxicity to different cell cultures, cell transformation, metabolism and skin penetration (a total of 115 studies) were used for the assessments. Key metabolic pathways and mechanisms of toxicity have been elucidated, for some chemicals, by using in vitro methods. On the other hand, the results of in vitro studies were regarded as secondary or unreliable in some cases. For several toxic endpoints, in vitro methods will probably serve as screening tools and for mechanistic studies, while target organ toxicity or physiologically regulated adverse effects caused by long-term exposure are difficult to observe without the use of animal models.     

Vaginal irritation models: the current status of available alternative and in vitro tests

Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are constantly exposed to bacteria and viruses, and are therefore potential sites of entry for numerous pathogens. The vaginal epithelium can be damaged mechanically, e.g. by the incorrect use of objects such as tampons, and by chemicals that are irritating or corrosive. Consequently, this can lead to an increase in susceptibility to further damage or infection. Pharmaceutical, cosmetic and personal care products that are specifically formulated for application onto human external mucosae can occasionally induce undesirable local or systemic side-effects. Therefore, the compatibility of applied materials with this mucosal surface represents a key issue to be addressed by manufacturers. The most frequently used method for assessing vaginal mucosal irritation is the in vivo rabbit vaginal irritation test. However, the current emphasis in the field of toxicology is to use alternative in vitro methods that reduce, refine, and replace the use of animals, and which model and predict human, not animal, responses. Such an approach is of particular interest to the personal care and cosmetic industries in their effort to comply with European legislative measures, such as the 7th Amendment to the EU Cosmetics Directive that does not permit the marketing of cosmetic products if they, or their ingredients, have been tested for irritation responses in animals. The focus of this review is to provide an overview of the alternative and in vitro tests that are currently available for vaginal mucosal irritation assessment, and which are already used, or may become useful, to establish the safety of newly-designed products for human use.     

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

Huggins has reported on the current situation relating to the development of alternatives to the Draize eye irritation test with rabbits, and an ECVAM Working Group have reviewed the efforts needed in order to replace this animal test within the next 10 years by using the results of non-animal assessment methods. Our report reviews regulatory experience gained over the last 20 years with the EU chemicals notification procedure with respect to the assessment of eye lesions observed in Draize tests. The nature of eye lesions and their importance for classification and labelling of possible hazards to human eyes are evaluated and discussed, with a view to promoting the development of specific in vitro assays which are able to discriminate between eye damage, moderate eye irritation, and minor irritation effects which are completely reversible within a few days. Structural alerts for the prediction of eye irritation/corrosion hazards to be classified and labelled according to international classification criteria, are presented, which should be validated in accordance with internationally agreed (OECD) principles for (Q)SAR system validation. Physicochemical limit values for prediction of the absence of any eye irritation potential relevant for human health can make available a definition of the applicability domains of alternative methods developed for the replacement of the Draize eye irritation test.