A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: Eye irritation

The need for alternative approaches to replace the in vivo rabbit Draize eye test for evaluation of eye irritation of cosmetic ingredients has been recognised by the cosmetics industry for many years. Extensive research has lead to the development of several assays, some of which have undergone formal validation. Even though, to date, no single in vitro assay has been validated as a full replacement for the rabbit Draize eye test, organotypic assays are accepted for specific and limited regulatory purposes. Although not formally validated, several other in vitro models have been used for over a decade by the cosmetics industry as valuable tools in a weight of evidence approach for the safety assessment of ingredients and finished products. In light of the deadlines established in the EU Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision-tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. Furthermore, recommendations are given on how remaining data gaps and research needs can be addressed.     

2种光源对体外3T3细胞光毒性试验结果的比较

目的 比较2种光源对体外3T3细胞光毒性试验结果的影响。方法 参照化学品体外3T3中性红摄取光毒性试验指导原则（OECD 432）,采用模拟阳光光源和紫外光源对6种参考物质进行试验。结果 6种参考物质在2种光源照射后其光刺激因子和平均光效应均接近OECD 432中的参考值,各个物质在无光和有光下的IC₅₀值也相近。结论 2种光源对体外3T3细胞光毒性试验结果基本无影响。     

Human health safety evaluation of cosmetics in the EU: A legally imposed challenge to science

As stated in the European legislation, cosmetic products present on the European market must be safe for the consumer. Safety evaluation of the products is carried out by a qualified safety assessor who needs to consider potential exposure scenarios next to the physicochemical and toxicological profiles of all composing ingredients. Whereas, until recently, the tools to determine the toxicological profile of cosmetic ingredients mainly consisted of animal experiments, they have now been narrowed down substantially by the legally imposed animal testing ban on cosmetic ingredients, taken up in the Cosmetic Products Directive (76/768/EEC). This Directive, however, is not a stand-alone piece of European legislation, since as well directly as indirectly it is influenced by a complex web of related legislations. Vertical legislations deal with different categories of chemicals, including dangerous substances, biocides, plant protection products, food additives, medicinal products, and of course also cosmetics. Horizontal legislative texts, on the contrary, cover more general fields such as protection of experimental animals, consumer product safety, misleading of consumers, specific provisions for aerosols, and others. Experience has learnt that having a general overview of these related legislations is necessary to understand their impact on the cosmetic world in general terms and on cosmetic safety evaluation in particular. This goes for a variety of concerned parties, including national and European regulators/agencies, contract laboratories, raw material suppliers, cosmetic companies, research and educational centers. They all deal with a number of aspects important for the quality and toxicity of cosmetics and their ingredients.This review summarises the most relevant points of the legislative texts of different types of product categories and emphasises their impact on the safety evaluation of cosmetics.     

A tiered approach to the use of alternatives to animal testing for the safety assessment of cosmetics: Skin irritation

Evaluation of the skin irritancy and corrosivity potential of an ingredient is a necessity in the safety assessment of cosmetic ingredients. To date, there are two formally validated alternatives to the rabbit Draize test for skin corrosivity in place, namely the rat skin transcutaneous electrical resistance (TER) assay and the Human Skin Model Test using EpiSkin™, EpiDerm™ and SkinEthic™ reconstructed human epidermal equivalents. For skin irritation, EpiSkin™, EpiDerm™ and SkinEthic™ are validated as stand-alone test replacements for the rabbit Draize test. Data from these tests are rarely considered in isolation and are evaluated in combination with other factors to establish the overall irritating or corrosive potential of an ingredient. In light of the deadlines established in the Cosmetics Directive for cessation of animal testing for cosmetic ingredients, a COLIPA scientific meeting was held in Brussels on 30th January, 2008 to review the use of alternative approaches and to set up a decision tree approach for their integration into tiered testing strategies for hazard and safety assessment of cosmetic ingredients and their use in products. In conclusion, the safety assessments for skin irritation/corrosion of new chemicals for use in cosmetics can be confidently accomplished using exclusively alternative methods.     

Guiding principles for the implementation of non-animal safety assessment approaches for cosmetics: Skin sensitisation

Characterisation of skin sensitisation potential is a key endpoint for the safety assessment of cosmetic ingredients especially when significant dermal exposure to an ingredient is expected. At present the mouse local lymph node assay (LLNA) remains the ‘gold standard’ test method for this purpose however non-animal test methods are under development that aim to replace the need for new animal test data. COLIPA (the European Cosmetics Association) funds an extensive programme of skin sensitisation research, method development and method evaluation and helped coordinate the early evaluation of the three test methods currently undergoing pre-validation. In May 2010, a COLIPA scientific meeting was held to analyse to what extent skin sensitisation safety assessments for cosmetic ingredients can be made in the absence of animal data. In order to propose guiding principles for the application and further development of non-animal safety assessment strategies it was evaluated how and when non-animal test methods, predictions based on physico-chemical properties (including in silico tools), threshold concepts and weight-of-evidence based hazard characterisation could be used to enable safety decisions. Generation and assessment of potency information from alternative tools which at present is predominantly derived from the LLNA is considered the future key research area.     

甲基硝基亚硝基胍对NIH3T3细胞转化作用

以ＭＮＮＧ为受试物，旨在探讨ＮＩＨ３Ｔ３细胞能否作为转化实验的细胞株。研究结果显示，ＭＮＮＧ在所设浓度下均能诱导ＮＩＨ３Ｔ３细胞出现典型的细胞转化灶，其转化率呈剂量－反应关系；对转化细胞的恶性鉴定结果表明，与正常细胞不同，转化细胞失去了接触性抑制，能在软琼脂内生长。因此，初步认为ＮＩＨ３Ｔ３细胞有可能作为评定化学物质转化作用的细胞株。     

The licorice root derived isoflavan glabridin increases the function of osteoblastic MC3T3-E1 cells

Glabridin, an isoflavan purified from licorice root, exhibits diverse biological activities, including estrogen-like activity. To investigate the bioactivities of glabridin, which act on bone metabolism, the effects of glabridin on the function of mouse osteoblastic cell line (MC3T3-E1) and the production of local factors in osteoblasts were studied. Glabridin (1-10microM) significantly increased the growth of MC3T3-E1 cells and caused a significant elevation of alkaline phosphatase (ALP) activity, collagen content and osteocalcin secretion in the cells (P<0.05). The effect of glabridin (10microM) in increasing ALP activity and collagen content was completely prevented by the presence of 10(-6)M cycloheximide and 10(-6)M tamoxifen, suggesting that glabridin's effect results from a newly synthesized protein component and might be partly involved in estrogen action. Then, the effects of glabridin on the TNF-alpha-induced apoptosis and production of prostaglandin E2 (PGE2) and nitric oxide (NO) in osteoblasts were examined. Treatment with glabridin (1-10microM) prevented apoptosis induced by TNF-alpha (10(-10)M) in osteoblastic cells. Moreover, glabridin (50microM) decreased the 10(-10)M TNF-alpha-induced production of PGE2 and NO in osteoblasts. Our data indicate that the enhancement of osteoblast function by glabridin may result in the prevention for osteoporosis and inflammatory bone diseases.     

The anti-angiogenic herbal extracts Ob-X from Morus alba,Melissa officinalis,and Artemisia capillaris suppresses adipogenesis in 3T3-L1 adipocytes

Context: Growing adipose tissue is thought to require adipogenesis, angiogenesis, and extracellular matrix (ECM) remodeling. Close examination of developing adipose tissue microvasculature reveals that angiogenesis often precedes adipogenesis. Since our previous study demonstrated that Ob-X, the anti-angiogenic herbal composition composed of Melissa officinalis L. (Labiatae), Morus alba L. (Moraceae), and Artemisia capillaris Thunb. (Compositae), reduced adipose tissue mass in obese mice, we hypothesized that adipogenesis can be inhibited by Ob-X.

Objective: To investigate the effects of the anti-angiogenic herbal extracts Ob-X on adipogenesis in 3T3-L1 adipocytes.

Materials and methods: After differentiated 3T3-L1 adipocytes were treated with Ob-X, we studied the effects of Ob-X on triglyceride accumulation and expression of genes involved in adipogenesis, angiogenesis, and ECM remodeling.

Results: Treatment of cells with Ob-X inhibited lipid accumulation and adipocyte-specific gene expression caused by troglitazone or monocyte differentiation-inducing (MDI) mix. Ob-X reduced mRNA levels of angiogenic factors (vascular endothelial growth factor-A, -B, -C, -D, and fibroblast growth factor-2) and matrix metalloproteinases (MMPs; MMP-2 and MMP-9), whereas it increased mRNA levels of angiogenic inhibitors [(thrombospondin-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2)] in differentiated cells. MMP-2 and MMP-9 activities were also decreased in Ob-X-treated cells.

Discussion and conclusion: These results suggest that the anti-angiogenic herbal composition Ob-X inhibits differentiation of preadipocytes into adipocytes. These events may be mediated by changes in the expression of genes involved in lipogenesis, angiogenesis, and the MMP system. Thus, by reducing adipogenesis, anti-angiogenic Ob-X provides a possible therapeutic approach for the prevention and treatment of human obesity and its related disorders.     

Legal issues of addiction assessment: the experience with hair testing in Greece

The purpose of this paper is to present Greek law and legislation for crimes and felonies regarding drugs of abuse and the interpretation of hair testing results with respect to Greek law. Details (such as the process, the decision and the competence of the Court, the police record, the indictment, the expert reports, the defendant's individuality, the crimes and the penal confrontal and many others) from legal cases related to toxicomany and its judicial verification were collected and analysed. Laboratory data of cases concerning the laboratory evaluation of toxicomany in addicts and also occasionally the legal course of cases with addict defendants are presented. In four representative cases segmental hair analysis proved that, for as long as the individuals were imprisoned, findings with drug substances corresponding to that period were lesser or practically absent compared with samples corresponding to the time out of prison, which showed increased drug abuse. Hair analysis provides information on chronic exposure rather than acute poisoning. Its detection window varies from some days to months or even years. The procedure that the law lays down in many cases is insufficient and in most cases impossible to abide by. When the medical examiner is not able to decide if the claim of toxicomany is real, segmental hair analysis may be the only way to prove it. In other cases where the medical examiner is able to diagnose the addiction, a segmental hair analysis is necessary because it can show long-term drug abuse.     

One new linear C14 polyacetylene glucoside with antiadipogenic activities on 3T3-L1 cells from the capitula of Coreopsis tinctoria

Based on characteristic UV spectrum of the ene-diyne chromophore, one new polyacetylene glucoside and three known polyacetylene glucosides have been isolated from the EtOH extract of Coreopsis tinctoria. Their chemical structures were determined by detailed spectroscopic analysis and by comparison with literature data. Compounds 1–2 were tested for their antiadipogenic effects on 3T3-L1 adipocytes, and both of them reduced lipid accumulation dose-dependently in 3T3-L1 adipocytes.     

慢性阻塞性肺疾病急性加重期低T3综合征及其临床意义

目的：了解低T3综合征在慢性阻塞性肺疾病急性加重患者中的发生情况及其与疾病严重程度的相关性和预后的关系。方法：选取AECOPD患者152例,根据动脉血气及临床表现分为无呼吸衰竭和右心功能衰竭组（A组）54例、有呼吸衰竭无右心功能衰竭组（B组）42例、无呼吸衰竭有右心功能衰竭组（C组）31例、有呼吸衰竭及右心功能衰竭组（D组）25例。应用化学发光法测定各组血清甲状腺激素水平。结果：低T3综合征发病率为18.42%;通过分类比较,B、C组分别和A组相比,D组分别与A组、B组、C组相比,TT3、FT3下降,差异有统计学意义;合并呼吸衰竭者（B组、D组,67例）较未合并呼吸衰竭者（A组、C组,85例）TT3、FT3下降,差异有统计学意义;152例患者,死亡9例,病情恶化自动出院12例,其余患者（131例）好转出院,与好转出院者相比,死亡组与自动出院组TT3、FT3下降,差异有统计学意义。以上所有比较结果TT4、FT4、TSH差异无统计学意义。结论：血清甲状腺激素水平在AECOPD患者中有不同程度的下降,监测甲状腺功能有助于判断AECOPD患者的病情危重程度及预测预后。     

小檗碱与梓醇及其配伍对胰岛素抵抗3T3-L1脂肪细胞Glut-4、IRS-1、IRS-1 Ser307磷酸化蛋白表达的影响

目的：观察梓醇与小檗碱及其配伍对胰岛素抵抗3T3-L1脂肪细胞葡萄糖消耗及这一过程中葡萄糖转运子-4（Glut-4）、胰岛素受体底物-1（IRS-1）和胰岛素受体底物-1丝氨酸307（IRS-1Ser307）磷酸化蛋白表达的影响。方法：采用高糖联合高胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗，分别给予小檗碱、梓醇、小檗碱＋梓醇、盐酸罗格列酮进行干预，以葡萄糖氧化酶法检测培养液中葡萄糖消耗量，以WesternBlot法检测蛋白的表达。结果：与模型组相比，小檗碱能增加培养液中葡萄糖的消耗（P〈0．01），但对Glut，4蛋白的表达无影响；梓醇、小檗碱＋梓醇均能显著增加培养液中葡萄糖的消耗（P〈0．01），并使细胞中Glut-4蛋白的表达增强（P〈0．05），且小檗碱＋梓醇组的效应优于梓醇组及小檗碱组；与模型组相比，小檗碱与梓醇及其配伍对IRS-1的表达没有显著性影响，但能降低IRS-1 Ser307磷酸化蛋白表达。结论：小檗碱、梓醇及其配伍能改善胰岛素抵抗3T3-L1脂肪细胞的胰岛素敏感性，其作用机制与罗格列酮不同。     

血液CD3＋CD4＋／CD3＋CD8＋T细胞水平对肺部真菌感染的影响

目的探讨血液CD3＋CD4＋／CD3＋CD8＋T淋巴细胞水平对非HIV感染患者发生侵袭性肺部真菌感染的影响。方法根据入选标准及诊治规范,入选IPFI组患者61例,非IPFI肺炎组患者47例及同期健康对照组体检者30例。收集记录上述患者的临床资料,观察3组病例CD3CD;T淋巴细胞百分比,CD3＋CD8＋T淋巴细胞百分比及CD3＋CD4＋／CD3＋CD8＋T淋巴细胞比值情况。结果白色念珠菌仍是最常见的致病真菌,占总检出菌株数的46．03％,同时本研究曲霉菌（30．16％）感染也占有相当高的比例。CD3＋CD4＋T淋巴细胞百分比及CD3＋CD4＋／CD3＋CD8＋T细胞比值水平,IPFI组较非IPFI肺炎组（t=5．910,P〈0．05;t=7．395,P〈0．05）及健康对照组（t=6．443,P〈0．05;t=7．428,P〈0．05）均明显下降。结论血液CD4＋／CD8＋T淋巴细胞水平测定有助于早期发现肺部真菌感染的发生。     

Assuring safety without animal testing: The case for the human testis in vitro

From 15 to 17 June 2011, a dedicated workshop was held on the subject of in vitro models for mammalian spermatogenesis and their applications in toxicological hazard and risk assessment. The workshop was sponsored by the Dutch ASAT initiative (Assuring Safety without Animal Testing), which aims at promoting innovative approaches toward toxicological hazard and risk assessment on the basis of human and in vitro data, and replacement of animal studies. Participants addressed the state of the art regarding human and animal evidence for compound mediated testicular toxicity, reviewed existing alternative assay models, and brainstormed about future approaches, specifically considering tissue engineering. The workshop recognized the specific complexity of testicular function exemplified by dedicated cell types with distinct functionalities, as well as different cell compartments in terms of microenvironment and extracellular matrix components. This complexity hampers quick results in the realm of alternative models. Nevertheless, progress has been achieved in recent years, and innovative approaches in tissue engineering may open new avenues for mimicking testicular function in vitro. Although feasible, significant investment is deemed essential to be able to bring new ideas into practice in the laboratory. For the advancement of in vitro testicular toxicity testing, one of the most sensitive end points in regulatory reproductive toxicity testing, such an investment is highly desirable.     

氯化钆对PKC的激活可促进成纤维细胞NIH3T3的存活及细胞周期的推进

本研究以氯化钆为代表性含稀土元素的化合物,对其在小鼠胚胎成纤维细胞NIH3T3中对蛋白激酶C家族蛋白的激活进行了研究。利用活细胞成像和共聚焦激光扫描技术可以观察到,在血清饥饿的条件下,50μM的氯化钆可以通过增强细胞粘附和细胞骨架重组促进细胞存活。使用蛋白质印迹技术发现蛋白激酶C家族蛋白在氯化钆作用不同时间后可以发生磷酸化,表明蛋白激酶C被激活。此外,双吲哚马来酰亚胺（bisindolylmaleimide,一种PKCpan的抑制剂）可以有效降低PKCpan磷酸化的水平（βⅡSer660）,同时也可以降低氯化钆引起的ERK的激活。以上结果表明,氯化钆激活的蛋白激酶C可以通过介导MAPK/ERK信号通路的激活,继而推动细胞周期和细胞存活。     

Antiobesity effects of quercetin-rich onion peel extract on the differentiation of 3T3-L1 preadipocytes and the adipogenesis in high fat-fed rats

The aim of the present study was to examine the effect of quercetin-rich onion peel extract (OPE) on anti-differentiation in 3T3-L1 preadipocytes and the antiobesity in high-fat fed rats. We found that lipid accumulations and TG contents in 3T3-L1 cells were markedly suppressed by OPE. The mRNA levels of activating protein (AP2) were down-regulated and those of carnitine palmitoyl transferase-1 α (CPT-1α) and fatty acid binding protein 4 (FABP4) were up-regulated by 75 and 100 μg/ml OPE. Body weight, retroperitoneal and mesenteric fat weights of SD rats were significantly lower in the 8 week high fat (HF) diet + 0.72% OPE group than in the HF group. Peroxisome proliferator-activated receptor (PPAR)γ mRNA levels were down-regulated in the epididymal fat of OPE than those of control and HF, and significant down-regulation of CCAAT/enhancer binding protein (C/EBP)α mRNA levels in OPE was also observed than the control. The mRNA levels of CPT-1α and uncoupling protein-1 (UCP-1) were up-regulated by the OPE, while those of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were down-regulated in HF and OPE groups compared to control group. These results suggest that quercentin-enriched OPE may have antiobesity effects by suppressing preadipocyte differentiation and inhibiting adipogenesis.     

Cytotoxicity and morphological transforming potential of cobalt nanoparticles,microparticles and ions in Balb/3T3 mouse fibroblasts: an in vitro model

We previously described the behaviour of different cobalt forms, i.e., cobalt nanoparticles (CoNP), cobalt microparticles (CoMP) and cobalt ions (Co²⁺), in culture medium (dissolution, interaction with medium components, bioavailability) as well as their uptake and intracellular distribution in Balb/3T3 mouse fibroblasts (Sabbioni, Nanotoxicology, 2012). Here, we assess the cytotoxicity and morphological transformation of CoNP compared not only to Co²⁺, but also to CoMP and to released Co products. Cytotoxicity reached maximum at 4-h exposure, with ranking CoMP > CoNP > Co²⁺. However, if we consider toxicity as a function of intracellular Co, toxicity of the ionic forms seems to prevail over the particles. Co forms other than Co²⁺ released from particles had toxicity intermediate between particles and ions. Alterations in concentrations of essential elements (Cu, Mg, Zn) in cells exposed to Co particles may contribute to toxicity. Both CoMP and CoNP (but not Co²⁺ and other released Co forms) induced morphological transformation (CoMP > CoNP). This was dependent on reactive oxygen species production and lipid peroxidation, as indicated by inhibition of type III foci with ascorbic acid. The present results suggest that the previously demonstrated massive mitochondrial and nuclear Co internalisation and DNA adduct formation by CoMP and CoNP (Sabbioni, Nanotoxicology, 2012) induce toxicity and transformation. On the contrary, the role of ions released by particles in culture medium is negligible. Thus, both the chemical and the physical properties of Co particles contribute to cytotoxicity and morphological transformation.     

宝华玉兰根皮挥发性化学成分的GC-MS分析及对NCI-3T3细胞体外生长抑制作用

分离鉴定宝华玉兰根皮乙醇总提取物(EERMZC)的挥发性成分,并考察其对小鼠成纤维细胞NCI-3T3的体外生长抑制作用。分析EERMZC的挥发性成分,用气-质联用(GC-MS)技术分析,并用峰面积归一化法测定各成分相对含量,采用MTT方法检测75,100,200,300,400μg/mL的EERMZC对NCI-3T3细胞体外生长抑制作用。气相色谱共分离出21个色谱峰,鉴定出其中14种化合物,占该挥发性成分总量的93.61%。EERMZC对NCI-3T3细胞的抑制率呈浓度依赖关系。在EERMZC的挥发性成分中,银胶菊素含量高达54.36%,其它含量较高的成分有非洲桧素(7.49%)、亚油酸乙酯(6.25%)、木香烯内酯(5.43%)等成分。EERMZC能明显抑制NCI-3T3细胞的生长。为进一步开发利用宝华玉兰药用价值提供科学依据。