ECVAM Technical Report on the Status of Alternative Methods for Cosmetics Testing (2008-2009)

The ECVAM technical report presents the progress made in the development and validation of alternative methods for the human health effects relevant to the Cosmetics Directive. It provides an update on the activities described by ECVAM in 2005 , 2006 and 2007 . The report intends to present the latest scientific and technical developments in the field during 2008-2009. As required by Directive 2003/15/EC, the seventh amendment to Directive 76/768/EEC, developments in refinement and reduction methods are also described (EU, 2003). Most successes in the development of alternative methods are in acute local toxicity and short-term testing, such as e.g. skin and eye irritation/corrosion, phototoxicity and skin penetration The test methods consuming a high number of animals, however, are in long-term testing and systemic toxicity, such as e.g. reproductive toxicity and repeated dose toxicity. In these complex fields, several research initiatives are ongoing. However full replacement approaches are still lacking.JRC.DG.I.3-In-vitro method

Beobachtung von Entwicklungstoxizität in vitro anhand muriner und menschlicher embryonalen Stammzellen

Die Europaeische Kommission publizierte die zukuenftige Chemikalienpolitik mit dem Titel Weissbuch: Strategie fuer eine zukuenftige Chemikalienpolitik . Um die Gefahren, die von Chemikalien ausgehen, die in Mengen zwischen einer und zehn Tonnen produziert werden, zu identifizieren, benoetigt die neue Chmikalienpolitik in vitro Methoden. Entwicklungstoxikologie ist ein Feld, in dem sehr grosse Mengen an Tieren fuer toxikolosche Studien verbraucht werden. Um Interspezies-Variationen in Entwicklungsstudien auszuschliessen, ist die Anwendung humaner embryonaler Stammzellen ein Muss. In dieser Studie wurden murine embryonale Stammzellen benutzt, um ein System zu entwickeln, dass in der Lage ist, teratogene Effekte von Substanzen auf Herzzellen sowie Osteoblasten zu detektieren, welche aus ES Zellen differenziert wurden. Zusaetzlich wurde dieses Maussystem eingesetzt, um durch Chemikalien verursachte wachstumsverzoegernde Effekte zu entdecken. Weiterhin wurde die Kapazitaet von humanen ES Zellen in funktionelle Kardiomyozyten zu differenzieren untersucht. Die murine Kardio-ES-Zelldifferenzierung wurde anhand der Gene Oct-4, Brachyury, Nkx2.5 and alpha-MHC untesucht, welche mit Hilfe von RT-PCR gemessen wurden. RA zeigte schon in der nicht zytotoxischen Konzentration einen Effekt auf die fruehe mesodermale Entwicklung anhand einer modifizierten Brachyury Expression. Zusaetzlich konnten wir eine starke Inhibition der kardialen Entwicklung anhand der verminderten Expression der Gene Nkx2.5 und alpha-MHC feststellen. Die Effekte auf die kardiale ES-Zellspezifikation wurden auch anhand der Verminderung der Aktivitaet von schlagenden Herzzellen identifiziert. Die Einwirkung von LiCl fuehrte zu einer verminderten kardiale Differenzierung. Eine reduzierte Expression kardiospezifischer Gene konnte beobachtet werden. Die murinen ES Zellen wurden waehrend der Kardio- und Osteoblastendifferenzierung mit Methotrexat (MTX) in der nicht zytotoxischen Konzentration behandelt. Die Expression von Schluesselgenen, die an der Kardiomyozytendifferenzierung und Osteoblastendifferenzierung beteiligt sind, wurden mit Hilfe von RT-PCR untersucht. Moegliche Effekte von MTX auf das EB-Wachstum wurden mit Hilfe von digital Image Analysis untersucht. Die MTX Behandlung resultierte nicht in einer veraenderten kardialen Genexpression. Eine verminderte Expression der Osteoblasten spezifischenen Gene konnte beobachtet werden. Da die Oct-4 und Brachyury Expression unveraendert blieb, konnte die spezifische Teratogenitaet von MTX auf die Knochenbildung bestaetigt werden. Die Kardiomyozytendifferenzierung der humanen ES Zellinie H1 wurde mit Hilfe von realtime PCR verfolgt. Viele humane Gene, die an der Kardiodifferenzierung beteiligt sind wurden analysiert. Um die Kardiomyozytenausbeute zu optimieren, wurden die ES Zellen in Kulturmedien mit verschiedenen Zusammensetzungen kultiviert. Zudem wurde mittels Genexpression die Entwicklung von Ectoderm und Endoderm untersucht. Die humanen ES Zellen zeigten die Faehigkeit, sich in alle drei Keimblaetter waehrend der in vitro Differenzierung entwickeln zu koennen. Die maximale Expression der mesodermspezifischen Gene wurde mit 20% Kaelberserum enthaltendem Medium erreicht. Die Kardiospezifikation erfolgte, deren Gene von Tag 18 bis Tag 25 der Differenzierung maximal exprimiert wurden. Heutzutage gibt es keine in vitro Systeme, die in der Lage sind, verzoegertes Wachstum festzustellen. Aus diesem Grund wurde ein System, das auf digital Image Analysis beruht, entwickelt. Das murine EB-Wachstum wurde ueber zehn Tage der Differenzierung beobachtet. Um die Verlaesslichkeit des Testes zu testen, wurden drei Substanzen Boric Acid (BA), 6-Amminonicotinamid (6-AN) und 5-Fluorouracil (5-FU) getestet, da sie verzoegertes Wachstum in vivo zu induzieren. Zusaetzlich wurde MTX als unbekannte Substanz und Saccharin (SAC) als negative Kontrolle getestet. Die IC-50 Werte der Wachstumsverzoegerung wurden mit denen des MTT-Testes verglichen. Die Effekte der Substanzen auf adultes Gewebe (Fibroblasten BALB/3T3) wurden zusaetzlich untersucht. Die Resultate demonstrieren einen signifikanten Unterschied zwischen genereller Zytotoxizitaet und spezifischen Effekten auf das EB-Wachstum fuer BA, 6-AN und 5-FU. Dieses Ergebnis bestaetigt die in vivo Daten und die Faehigkeit dieser Substanzen verzoegertes Wachstum hervorzurufen.MTX hat keinen Einfluss auf das EB-Wachstum, d.h. es konnten keine statistischen Unterschiede zwischen den IC-50 Werten festgestellt werden. Es liegen keine Literaturangaben vor, um diese Ergebnisse zu bestaetigen. Zieht man die verschiedenen Manifestationen der Entwicklungstoxikologie in Betracht, kann nur eine in vitro Testbatterie die noetigen Informationen fuer regulatorische entwicklungstoxikologische Zwecke liefern. Unsere Ergebnisse demonstrieren, dass die Verwendung von ES Zellen ein leistungsfaehiges Instrument darstellt, um in vitro Systeme zu entwickeln

The Effects of Solvents on Embryonic Stem Cell Differentiation

Dimethyl sulfoxidc (DMSO) and ethanol are common organic solvents used for dissolving lipophilic substances for in vitro testing. However, DMSO is known to induce differentiation in embryonic stem (ES) and embryonic leratocarcinoma (EC) cells. In order to clarify if solvents like DMSO and ethanol have an influence on in vitro developmental toxicity test systems, the presented study has evaluated their effects on differentiation by using different test systems. ES and EC cells were transfected with a construct containing the rnTert promoter combined with the green fluorescent protein gene (GFP). A down-regulation of mTert, a marker for un differentiated cells, results in a lower expression of GFP, which could be measured by flow cytomctry. Taking the specific characteristics of ES and EC cells into account this effect could be a hint for the interaction of DMSO with embryonic development. Additionally, the effects of the solvents ethanol and DMSO on Oct-4 expression, another marker for undifferentiated cells, were measured in wild-type ES cells. Beth selected molecular markers demonstrated an induction of differentiation after exposure to DMSO; in wild-type ES cells at a concentration of 0.125% and in transgenic EC cells at a concentration of 0.25% DMSO. All other differences from controls, including those which attained a level of statistical significance, were minor or not dosage related in degree, or were not consistent over time and are, therefore, considered to be of little lexicological importance. In addition, a cytotoxicity test demonstrated that the solvents affected the employed molecular markers in non-cytotoxic concen¬trations. The ES cells were the most sensitive towards the cytotoxic effects of the solvent DMSO while the EC cells were more sensitive when treated with the solvent ethanol.JRC.I.2-Validation of biomedical testing method

First Steps in Establishing a Developmental Toxicity Test Method Based on Human Embryonic Stem Cells

The embryonic stem cell test (EST) is currently one of the most promising in vitro developmental toxicity tests. However, it would be promising to adapt the EST to human embryonic stem (hES) cells to avoid false classification of substances due to inter-species variations. The EST consists of following endpoints: IC50 values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. In this study we established a cytotoxicity assay based on hES cells employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC50 values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability of several marker genes was tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes could serve as endpoints for a developmental toxicity test.JRC.I.2-Validation of Alternative Method

Monitoring of Teratogenic Effects in vitro by Analysing a Selected Gene Expression Pattern.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

Detection of the Embryotoxic Potential of Cyclophosphamide by Using a Combined System of Metabolic Competent Cells and Embryonic Stem Cells.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

Development of a Test Strategy for Detecting Embryotoxic Hazards of Chemicals in vitro by Using Embryonic Stemm Cell Models.

Abstract not availableJRC.I-Institute for Health and Consumer Protection (Ispra

Modulation of different Stress Pathways after Styrene and Styrene-7,8-oxide Exposure in HepG2 Cell Line and normal human Hepatocytes

Styrene is one of the most important monomers produced worldwide. IARC classified styrene as a possible carcinogen to humans (group 2B). Styrene-7,8-oxide (SO) is the main reactive metabolite of styrene, and it is found to be genotoxic in several in vitro test systems. Styrene and styrene-7,8-oxide (SO) toxicity to HepG2 cells was investigated by evaluating end-points such as heat shock proteins (Hsps), metallothioneins (MT), apoptosis-related proteins, accumulation of styrene within the cells and expression of two isoforms of cytochrome P450. The potential activity of styrene and styrene-7,8-oxide in modulating gene expression was also investigated. The results showed induction of Hsp70, metallothioneins, BclXS/L and c-myc expression and a decrease in Bax expression in HepG2 after treatments, confirming that these compounds activated protective mechanisms. Moreover, up-regulation of TGF β2 and TGF βRIII in HepG2 cells was found after exposure to styrene, while in human primary hepatocytes these genes were down-regulated after both treatments. Finally, it was found that styrene and SO treatments did not induce CYP1A2 and CYP2E1 protein expression. In conclusion, both compounds caused toxic stress in HepG2 cells, with SO being more toxic; in the meantime, a different effect of the two compounds in HepG2 cells and primary human hepatocytes was observed regarding their activity in gene modulation.JRC.I.2-Validation of biomedical testing method

In Vitro Tests for Detecting Chemicals Affecting the Embryo Implantation Process

This is the report of the xxxxxxx of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM' s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. The workshop on in vitro tests for detecting chemicals affecting the implantation process and placental toxicity was held in Ispra, Italy, on 2–3 October 2006, under the co-chairmanship of Susanne Bremer and Lennart Dencker. The workshop was built on the reports and recommendations of ECVAM Workshop 53 (1) and on the Research Area of Implantation-WP2 ReProTect Workshops held in Copenhagen on January 2005 and Uppsala on May 2005 (information not published). It was one of a series of strategic workshops within ReProTect, an integrated project aimed at furthering the development of in vitro methods in the area of reproductive toxicology, which consists of a consortium of 32 European partners and is financially supported by the European Commission with Euro 9.1 million. The ReProTect Research Area of “implantation” is aiming to develop tests and testing batteries that are able to detect chemical effects in various target cells/tissues and mechanisms involved in the preparation of the uterus for the implantation of the embryo as well as on placentation. The workshop, which was attended by 15 invited participants from six European Countries reviewed a number of possible subendpoints relevant for the process of implantation placental toxicity, and discussed and evaluated alternative methods that could mimic these subendpoints in vitro according to their test development status and their relevance. The modular approach was introduced and used as a template for the progression of each test system (2) In addition, the workshop proposes chemical classes and reference chemicals that should be selected for the development of ReProTect tests.JRC.I.2-Validation of biomedical testing method

The Value of Alternative Testing for Neurotoxicity in the Context of Regulatory Needs

Detection and characterization of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalized mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.JRC.I.2-Validation of biomedical testing method