Publications by authors named "Manfred Liebsch"

41 Publications

Evaluation of in vitro embryotoxicity tests for Chinese herbal medicines.

Reprod Toxicol 2019 10 20;89:45-53. Epub 2019 Jun 20.

College of Basic Medical Sciences, Zhejiang Chinese Medical University, Hangzhou, China; Department of Obstetrics & Gynaecology, The Chinese University of Hong Kong, Shatin, Hong Kong; School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong; Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong. Electronic address:

Chinese herbal medicines (CHMs) have been widely used during pregnancy, but feto-embryo safety tests are lacking. Here we evaluated in vitro embryotoxicity tests (IVTs) as alternative methods in assessing developmental toxicity of CHMs. Ten CHMs were selected and classified as strongly, weakly and non-embryotoxic. Three well validated IVTs and prediction models (PMs), including embryonic stem cell test (EST), micromass (MM) and whole embryo culture (WEC), were compared. All strongly embryotoxic CHMs were predicted by MM and WEC PM2. While all weakly embryotoxic CHMs were predicted by MM and WEC PM1. All non-embryotoxic CHMs were classified by EST, MM, but over-classified as weakly embryotoxic by WEC PM1. Overall predictivity, precision and accuracy of WEC determined by PM2 were better than EST and MM tests. Compared with validated chemicals, performance of IVTs for CHMs was comparable. So IVTs are adequate to identify and exclude embryotoxic potential of CHMs in this training set.
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http://dx.doi.org/10.1016/j.reprotox.2019.06.001DOI Listing
October 2019

Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility.

Mutat Res Genet Toxicol Environ Mutagen 2018 Mar 10;827:27-41. Epub 2018 Jan 10.

Procter & Gamble, 8700 Mason-Montgomery Road, Mason, OH 45040, United States.

Recently revised OECD Testing Guidelines highlight the importance of considering the first site-of-contact when investigating the genotoxic hazard. Thus far, only in vivo approaches are available to address the dermal route of exposure. The 3D Skin Comet and Reconstructed Skin Micronucleus (RSMN) assays intend to close this gap in the in vitro genotoxicity toolbox by investigating DNA damage after topical application. This represents the most relevant route of exposure for a variety of compounds found in household products, cosmetics, and industrial chemicals. The comet assay methodology is able to detect both chromosomal damage and DNA lesions that may give rise to gene mutations, thereby complementing the RSMN which detects only chromosomal damage. Here, the comet assay was adapted to two reconstructed full thickness human skin models: the EpiDerm™- and Phenion Full-Thickness Skin Models. First, tissue-specific protocols for the isolation of single cells and the general comet assay were transferred to European and US-American laboratories. After establishment of the assay, the protocol was then further optimized with appropriate cytotoxicity measurements and the use of aphidicolin, a DNA repair inhibitor, to improve the assay's sensitivity. In the first phase of an ongoing validation study eight chemicals were tested in three laboratories each using the Phenion Full-Thickness Skin Model, informing several validation modules. Ultimately, the 3D Skin Comet assay demonstrated a high predictive capacity and good intra- and inter-laboratory reproducibility with four laboratories reaching a 100% predictivity and the fifth yielding 70%. The data are intended to demonstrate the use of the 3D Skin Comet assay as a new in vitro tool for following up on positive findings from the standard in vitro genotoxicity test battery for dermally applied chemicals, ultimately helping to drive the regulatory acceptance of the assay. To expand the database, the validation will continue by testing an additional 22 chemicals.
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http://dx.doi.org/10.1016/j.mrgentox.2018.01.003DOI Listing
March 2018

Fetal Bovine Serum (FBS): Past - Present - Future.

ALTEX 2018 9;35(1):99-118. Epub 2017 Aug 9.

Division of Physiology, Medical University Innsbruck, Innsbruck, Austria.

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.
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http://dx.doi.org/10.14573/altex.1705101DOI Listing
August 2018

Influence of massage and occlusion on the ex vivo skin penetration of rigid liposomes and invasomes.

Eur J Pharm Biopharm 2014 Feb 16;86(2):301-6. Epub 2013 Nov 16.

Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Charité - Universitätsmedizin Berlin, Germany.

Liposomes are frequently described as drug delivery systems for dermal and transdermal applications. Recently, it has been shown that particulate substances penetrate effectively into hair follicles and that the follicular penetration depth can be increased by massaging the skin, which simulates the in vivo movement of hairs in the hair follicles. In the present study, massage was applied to skin mounted to Franz diffusion cells. By means of confocal laser scanning microscopy, the influence of massage and occlusion on the follicular penetration depths of rigid and flexible liposomes loaded with a hydrophilic and lipophilic dye was investigated. The application of massage increased follicular penetration significantly. Occlusion resulted in an increased follicular penetration depth only for rigid liposomes, whereas invasomes did not penetrate more effectively if occlusion was applied. The results confirm that massage is an important tool for increasing follicular penetration in ex vivo studies using Franz diffusion cells. Occlusion may reduce the efficacy of follicular penetration depending on the specific liposomal preparation. Rigidity in particular appears to be a relevant parameter.
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http://dx.doi.org/10.1016/j.ejpb.2013.11.004DOI Listing
February 2014

Statistical analysis of the hen's egg test for micronucleus induction (HET-MN assay).

Mutat Res 2013 Sep 26;757(1):68-78. Epub 2013 Jul 26.

Leibniz Universität Hannover, Herrenhäuser Straße 2, 30419 Hannover, Germany.

The HET-MN assay (hen's egg test for micronucleus induction) is different from other in vitro genotoxicity assays in that it includes toxicologically important features such as absorption, distribution, metabolic activation, and excretion of the test compound. As a promising follow-up to complement existing in vitro test batteries for genotoxicity, the HET-MN is currently undergoing a formal validation. To optimize the validation, the present study describes a critical analysis of previously obtained HET-MN data to check the experimental design and to identify the most appropriate statistical procedure to evaluate treatment effects. Six statistical challenges (I-VI) of general relevance were identified, and remedies were provided which can be transferred to similarly designed test methods: a Williams-type trend test is proposed for overdispersed counts (II) by means of a square-root transformation which is robust for small sample sizes (I), variance heterogeneity (III), and possible downturn effects at high doses (IV). Due to near-to-zero or even zero-count data occurring in the negative control (V), a conditional comparison of the treatment groups against the mean of the historical controls (VI) instead of the concurrent control was proposed, which is in accordance with US-FDA recommendations. For the modified Williams-type tests, the power can be estimated depending on the magnitude and shape of the trend, the number of dose groups, and the magnitude of the MN counts in the negative control. The experimental design used previously (i.e. six eggs per dose group, scoring of 1000 cells per egg) was confirmed. The proposed approaches are easily available in the statistical computing environment R, and the corresponding R-codes are provided.
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http://dx.doi.org/10.1016/j.mrgentox.2013.04.023DOI Listing
September 2013

Assessment of the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity test method to identify substances not classified for acute oral toxicity (LD50>2000 mg/kg): results of an ECVAM validation study.

Regul Toxicol Pharmacol 2013 Apr 13;65(3):344-65. Epub 2012 Dec 13.

Institute for Health and Consumer Protection, European Commission Joint Research Centre, 21027 Ispra, Italy.

Assessing chemicals for acute oral toxicity is a standard information requirement of regulatory testing. However, animal testing is now prohibited in the cosmetics sector in Europe, and strongly discouraged for industrial chemicals. Building on the results of a previous international validation study, a follow up study was organised to assess if the 3T3 Neutral Red Uptake cytotoxicity assay could identify substances not requiring classification as acute oral toxicants under the EU regulations. Fifty-six coded industrial chemicals were tested in three laboratories, each using one of the following protocols: the previously validated protocol, an abbreviated version of the protocol and the protocol adapted for an automation platform. Predictions were very similar among the three laboratories. The assay exhibited high sensitivity (92-96%) but relatively low specificity (40-44%). Three chemicals were under predicted. Assuming that most industrial chemicals are not likely to be acutely toxic, this test method could prove a valuable component of an integrated testing strategy, a read-across argument, or weight-of-evidence approach to identify non toxic chemicals (LD50>2000 mg/kg). However, it is likely to under predict chemicals acting via specific mechanisms of action not captured by the 3T3 test system, or which first require biotransformation in vivo.
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http://dx.doi.org/10.1016/j.yrtph.2012.11.013DOI Listing
April 2013

Metabolically competent human skin models: activation and genotoxicity of benzo[a]pyrene.

Toxicol Sci 2013 Feb 11;131(2):351-9. Epub 2012 Nov 11.

German Federal Institute for Risk Assessment (BfR), Department of Product Safety, 10589 Berlin, Germany.

The polycyclic aromatic hydrocarbon (PAH) benzo[a]pyrene (BP) is metabolized into a complex pattern of BP derivatives, among which the ultimate carcinogen (+)-anti-BP-7,8-diol-9,10-epoxide (BPDE) is formed to certain extents. Skin is frequently in contact with PAHs and data on the metabolic capacity of skin tissue toward these compounds are inconclusive. We compared BP metabolism in excised human skin, commercially available in vitro 3D skin models and primary 2D skin cell cultures, and analyzed the metabolically catalyzed occurrence of seven different BP follow-up products by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). All models investigated were competent to metabolize BP, and the metabolic profiles generated by ex vivo human skin and skin models were remarkably similar. Furthermore, the genotoxicity of BP and its derivatives was monitored in these models via comet assays. In a full-thickness skin, equivalent BP-mediated genotoxic stress was generated via keratinocytes. Cultured primary keratinocytes revealed a level of genotoxicity comparable with that of direct exposure to 50-100 nM of BPDE. Our data demonstrate that the metabolic capacity of human skin ex vivo, as well as organotypic human 3D skin models toward BP, is sufficient to cause significant genotoxic stress and thus cutaneous bioactivation may potentially contribute to mutations that ultimately lead to skin cancer.
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http://dx.doi.org/10.1093/toxsci/kfs316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3551429PMC
February 2013

Skin phototoxicity of cosmetic formulations containing photounstable and photostable UV-filters and vitamin A palmitate.

Toxicol In Vitro 2013 Feb 10;27(1):418-25. Epub 2012 Aug 10.

University of Sao Paulo, Faculty of Pharmaceutical Sciences of Ribeirao Preto, SP, Brazil.

The aim of this study was to evaluate the in vitro skin phototoxicity of cosmetic formulations containing photounstable and photostable UV-filters and vitamin A palmitate, assessed by two in vitro techniques: 3T3 Neutral Red Uptake Phototoxicity Test and Human 3-D Skin Model In Vitro Phototoxicity Test. For this, four different formulations containing vitamin A palmitate and different UV-filters combinations, two of them considered photostable and two of them considered photounstable, were prepared. Solutions of each UV-filter and vitamin under study and solutions of four different combinations under study were also prepared. The phototoxicity was assessed in vitro by the 3T3 NRU phototoxicity test (3T3-NRU-PT) and subsequently in a phototoxicity test on reconstructed human skin model (H3D-PT). Avobenzone presented a pronounced phototoxicity and vitamin A presented a tendency to a weak phototoxic potential. A synergistic effect of vitamin A palmitate on the phototoxicity of combinations containing avobenzone was observed. H3D-PT results did not confirm the positive 3T3-NRU-PT results. However, despite the four formulations studied did not present any acute phototoxicity potential, the combination 2 containing octyl methoxycinnamate (OMC), avobenzone (AVB) and 4-methylbenzilidene camphor (MBC) presented an indication of phototoxicity that should be better investigated in terms of the frequency of photoallergic or chronic phototoxicity in humans, once these tests are scientifically validated only to detect phototoxic potential with the aim of preventing phototoxic reactions in the general population, and positive results cannot predict the exact incidence of phototoxic reactions in humans.
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http://dx.doi.org/10.1016/j.tiv.2012.08.006DOI Listing
February 2013

Wind of change challenges toxicological regulators.

Environ Health Perspect 2012 Nov 7;120(11):1489-94. Epub 2012 Aug 7.

German Federal Institute for Risk Assessment (BfR), Center for Alternative Methods to Animal Experiments (ZEBET), Berlin, Germany.

Background: In biomedical research, the past two decades have seen the advent of in vitro model systems based on stem cells, humanized cell lines, and engineered organotypic tissues, as well as numerous cellular assays based on primarily established tumor-derived cell lines and their genetically modified derivatives.

Objective: There are high hopes that these systems might replace the need for animal testing in regulatory toxicology. However, despite increasing pressure in recent years to reduce animal testing, regulators are still reluctant to adopt in vitro approaches on a large scale. It thus seems appropriate to consider how we could realistically perform regulatory toxicity testing using in vitro assays only.

Discussion And Conclusion: Here, we suggest an in vitro-only approach for regulatory testing that will benefit consumers, industry, and regulators alike.
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http://dx.doi.org/10.1289/ehp.1104782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3556610PMC
November 2012

The 3T3 neutral red uptake phototoxicity test: practical experience and implications for phototoxicity testing--the report of an ECVAM-EFPIA workshop.

Regul Toxicol Pharmacol 2012 Aug 9;63(3):480-8. Epub 2012 Jun 9.

European Commission Joint Research Centre, Ispra, Italy.

This is the report from the "ECVAM-EFPIA workshop on 3T3 NRU Phototoxicity Test: Practical Experience and Implications for Phototoxicity Testing", jointly organized by ECVAM and EFPIA and held on the 25-27 October 2010 in Somma Lombardo, Italy. The European Centre for the Validation of Alternative Methods (ECVAM) was established in 1991 within the European Commission Joint Research, based on a Communication from the European Commission (1991). The main objective of ECVAM is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine and replace the use of laboratory animals. The European Federation of Pharmaceuticals Industries and Association (EFPIA) represent the pharmaceutical industry operating in Europe. Through its direct membership of 31 national associations and 40 leading pharmaceutical companies, EFPIA is the voice on the EU scene of 2200 companies committed to researching, developing and bringing to patients new medicines that improve health and the quality of life around the world. The workshop, co-chaired by Joachim Kreysa (ECVAM) and Phil Wilcox (GSK, EFPIA) involved thirty-five experts from academia, regulatory authorities and industry, invited to contribute with their experiences in the field of phototoxicology. The main objectives of the workshop were: -to present 'in use' experience of the pharmaceutical industry with the 3T3 Neutral Red Uptake Phototoxicity Test (3T3 NRU-PT), -to discuss why it differs from the results in the original validation exercise, -to discuss technical issues and consider ways to improve the usability of the 3T3 NRU-PT for (non-topical) pharmaceuticals, e.g., by modifying the threshold of chemical light absorption to trigger photo-toxicological testing, and by modifying technical aspects of the assay, or adjusting the criteria used to classify a positive response. During the workshop, the assay methodology was reviewed by comparing the OECD Test Guideline (TG 432) with the protocols used in testing laboratories, data from EFPIA and JPMA 'surveys' were presented and possible reasons for the outcomes were discussed. Experts from cosmetics and pharmaceutical industries reported on their experience with the 3T3 NRU-PT and evidence was presented for phototoxic clinical symptoms that could be linked to certain relevant molecules. Brainstorming sessions discussed if the 3T3 NRU-PT needed to be improved and whether alternatives to the 3T3 NRU-PT exist. Finally, the viewpoint from EU and US regulators was presented. In the final session, the conclusions of the meeting were summarized, with action points. It was concluded that the 3T3 NRU-PT identifies phototoxicological hazards with a 100% sensitivity, and thus is accepted as the tier one test that correctly identifies the absence of phototoxic potential. Consequently, positive results in the 3T3 NRU-PT often do not translate into a clinical phototoxicity risk. Possible ways to improve the practical use of this assay include: (i) adaptation of changed UV/vis-absorption criteria as a means to reduce the number of materials tested, (ii) reduction of the highest concentration to be tested, and (iii) consideration of modifying the threshold criteria for the prediction of a positive call in the test.
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http://dx.doi.org/10.1016/j.yrtph.2012.06.001DOI Listing
August 2012

Regulatory assessment of in vitro skin corrosion and irritation data within the European framework: Workshop recommendations.

Regul Toxicol Pharmacol 2012 Mar 6;62(2):393-403. Epub 2011 Nov 6.

Services & Consultation on Alternative Methods (SeCAM), Agno, Switzerland.

Validated in vitro methods for skin corrosion and irritation were adopted by the OECD and by the European Union during the last decade. In the EU, Switzerland and countries adopting the EU legislation, these assays may allow the full replacement of animal testing for identifying and classifying compounds as skin corrosives, skin irritants, and non irritants. In order to develop harmonised recommendations on the use of in vitro data for regulatory assessment purposes within the European framework, a workshop was organized by the Swiss Federal Office of Public Health together with ECVAM and the BfR. It comprised stakeholders from various European countries involved in the process from in vitro testing to the regulatory assessment of in vitro data. Discussions addressed the following questions: (1) the information requirements considered useful for regulatory assessment; (2) the applicability of in vitro skin corrosion data to assign the corrosive subcategories as implemented by the EU Classification, Labelling and Packaging Regulation; (3) the applicability of testing strategies for determining skin corrosion and irritation hazards; and (4) the applicability of the adopted in vitro assays to test mixtures, preparations and dilutions. Overall, a number of agreements and recommendations were achieved in order to clarify and facilitate the assessment and use of in vitro data from regulatory accepted methods, and ultimately help regulators and scientists facing with the new in vitro approaches to evaluate skin irritation and corrosion hazards and risks without animal data.
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http://dx.doi.org/10.1016/j.yrtph.2011.10.015DOI Listing
March 2012

Developmental toxicity testing in the 21st century: the sword of Damocles shattered by embryonic stem cell assays?

Arch Toxicol 2011 Nov 19;85(11):1361-72. Epub 2011 Oct 19.

Department of Experimental Toxicology and Center for Alternatives to Animal Testing, German Federal Institute for Risk Assessment (BfR), Thielallee 88-92, Berlin, Germany.

Modern society faces an inherent dilemma. In our globalized society, we are spoilt for choice by an ever-increasing number of products, many of which are made of new materials and compound mixtures. At the same time, as consumers we got accustomed to the idea of a life minimized for risk, including our own exposure to chemicals from the environment or to compounds present in and released from everyday products. Chemical safety testing bridges these obviously diverging interests, and the corresponding legislation has hence been tremendously extended (e.g., introduction of the European legislation REACH in 2007). However, the underlying regulatory toxicology still relies mainly on animal testing, which is relatively slow, expensive, and ethically arguable. Meanwhile, recent years have seen a surge in efforts to develop alternative testing systems and strategies. Expectations are particularly high for the applicability of stem cells as test systems especially for developmental toxicity testing in vitro. For the first time in history, test systems can be based on differentiating cells and tissue progenitors in culture, thus bringing the 'vision of toxicity testing in the 21st century' a step closer.
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http://dx.doi.org/10.1007/s00204-011-0767-1DOI Listing
November 2011

Alternatives to animal testing: current status and future perspectives.

Arch Toxicol 2011 Aug;85(8):841-58

On the occasion of the 20th anniversary of the Center for Alternative Methods to Animal Experiments (ZEBET), an international symposium was held at the German Federal Institute for Risk Assessment (BfR) in Berlin. At the same time, this symposium was meant to celebrate the 50th anniversary of the publication of the book "The Principles of Humane Experimental Technique" by Russell and Burch in 1959 in which the 3Rs principle (that is, Replacement, Reduction, and Refinement) has been coined and introduced to foster the development of alternative methods to animal testing. Another topic addressed by the symposium was the new vision on "Toxicology in the twenty-first Century", as proposed by the US-National Research Council, which aims at using human cells and tissues for toxicity testing in vitro rather than live animals. An overview of the achievements and current tasks, as well as a vision of the future to be addressed by ZEBET@BfR in the years to come is outlined in the present paper.
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http://dx.doi.org/10.1007/s00204-011-0718-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3149673PMC
August 2011

A knowledge-based search engine to navigate the information thicket of nanotoxicology.

Regul Toxicol Pharmacol 2011 Feb 17;59(1):47-52. Epub 2010 Sep 17.

Scientific Consultancy - Animal Welfare, Hallstattfeld 16, 85579 Neubiberg, Germany.

The risk assessment of nano-sized materials (NM) currently suffers from great uncertainties regarding their putative toxicity for humans and the environment. An extensive amount of the respective original research literature has to be evaluated before a targeted and hypothesis-driven Environmental and Health Safety research can be stipulated. Furthermore, to comply with the European animal protection legislation in vitro testing has to be preferred whenever possible. Against this background, there is the need for tools that enable producers of NM and risk assessors for a fast and comprehensive data retrieval, thereby linking the 3Rs principle to the hazard identification of NM. Here we report on the development of a knowledge-based search engine that is tailored to the particular needs of risk assessors in the area of NM. Comprehensive retrieval of data from studies utilising in vitro as well as in vivo methods relying on the PubMed database is presented exemplarily with a titanium dioxide case study. A fast, relevant and reliable information retrieval is of paramount importance for the scientific community dedicated to develop safe NM in various product areas, and for risk assessors obliged to identify data gaps, to define additional data requirements for approval of NM and to create strategies for integrated testing using alternative methods.
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http://dx.doi.org/10.1016/j.yrtph.2010.09.005DOI Listing
February 2011

Comparison of human skin irritation patch test data with in vitro skin irritation assays and animal data.

Contact Dermatitis 2010 Feb;62(2):109-16

National Institute of Public Health, Srobárova 48, Prague 10, Czech Republic.

Background: Efforts to replace the rabbit skin irritation test have been underway for many years, encouraged by the EU Cosmetics Directive and REACH. Recently various in vitro tests have been developed, evaluated and validated.

Objective: A key difficulty in confirming the validity of in vitro methods is that animal data are scarce and of limited utility for prediction of human effects, which adversely impacts their acceptance. This study examines whether in vivo or in vitro data most accurately predicted human effects.

Methods: Using the 4-hr human patch test (HPT) we examined a number of chemicals whose EU classification of skin irritancy is known to be borderline, or where in vitro methods provided conflicting results.

Results: Of the 16 chemicals classified as irritants in the rabbit, only five substances were found to be significantly irritating to human skin. Concordance of the rabbit test with the 4-hr HPT was only 56%, whereas concordance of human epidermis models with human data was 76% (EpiDerm) and 70% (EPISKIN).

Conclusions: The results confirm observations that rabbits overpredict skin effects in humans. Therefore, when validating in vitro methods, all available information, including human data, should be taken into account before making conclusions about their predictive capacity.
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http://dx.doi.org/10.1111/j.1600-0536.2009.01640.xDOI Listing
February 2010

Implementation and enforcement of the 3Rs principle in the field of transgenic animals used for scientific purposes. Report and recommendations of the BfR expert workshop, May 18-20, 2009, Berlin, Germany.

ALTEX 2010 ;27(2):117-34

Department Safety of Consumer Products, German Federal Institute for Risk Assessment, Berlin, Germany.

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals. These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or three-dimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified ("humanised") animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with "negative" outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use. The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.
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August 2011

Permeation of topically applied caffeine through human skin--a comparison of in vivo and in vitro data.

Br J Clin Pharmacol 2009 Aug;68(2):181-6

Department of Dermatology, Charité-Universitätsmedizin Berlin, Berlin 10117, Germany.

Aims: Due to ethical reasons, in vivo penetration studies are not applicable at all stages of development of new substances. Therefore, the development of appropriate in vitro methods is essential, as well as the comparison of the obtained in vivo and in vitro data, in order to identify their transferability. The aim of the present study was to investigate the follicular penetration of caffeine in vitro and to compare the data with the in vivo results determined previously under similar conditions.

Methods: The Follicular Closing Technique (FCT) represents a method to investigate the follicular penetration selectively. In the present study, FCT was combined with the Franz diffusion cell in order to differentiate between follicular and intercellular penetration of caffeine into the receptor medium in vitro. Subsequently, the results were compared with the data obtained in an earlier study investigating follicular and intercellular penetration of caffeine in vivo.

Results: The comparison of the data revealed that the in vitro experiments were valuable for the investigation of the follicular penetration pathway, which contributed in vivo as well as in vitro to approximately 50% of the total penetration, whereas the kinetics of caffeine penetration were shown to be significantly different.

Conclusions: The combination of FCT with the Franz diffusion cell represents a valuable method to investigate follicular penetration in vitro. Nevertheless, in vivo experiments should not be abandoned as in vitro, structural changes of skin occur and blood flow and metabolism are absent, probably accounting for reduced penetration rates in vitro.
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http://dx.doi.org/10.1111/j.1365-2125.2009.03463.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2767280PMC
August 2009

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study.

Altern Lab Anim 2008 May;36(2):161-87

Freie Universität Berlin, Institut für Pharmazie, Berlin, Germany.

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.
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http://dx.doi.org/10.1177/026119290803600207DOI Listing
May 2008

Successful validation of in vitro methods in toxicology by ZEBET, the National Centre for Alternatives in Germany at the BfR (Federal Institute for Risk Assessment).

Exp Toxicol Pathol 2008 Jun 28;60(2-3):225-33. Epub 2008 Apr 28.

ZEBET (National Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments) at the Federal Institute for Risk Assessment (BfR=Bundesinstitut für Risikobewertung), Berlin, Germany.

A short description of the history of the 3Rs concept is given, which was developed as the scientific concept to refine, reduce and replace animal experiments by Russel and Burch more than 40 years ago. In addition, the legal framework in Europe for developing alternatives to animal experiments is given and the current status of in vitro systems in pharmacology and toxicology is described including an update on metabolising systems. The decrease in experimental animal numbers during the past decade in Europe is illustrated by the situation in Germany and the contribution of international harmonisation of test guidelines on reducing animal numbers in regulatory testing is described. A review of the development of the principles of experimental validation is given and the 3T3 NRU in vitro phototoxicity test is used as an example for a successful validation study, which led to the acceptance of the first in vitro toxicity test for regulatory purposes by the OECD. Finally, the currently accepted alternative methods for standardisation and safety testing of drugs, biologicals and medical devices are summarised.
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http://dx.doi.org/10.1016/j.etp.2008.01.012DOI Listing
June 2008

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.

Altern Lab Anim 2007 Dec;35(6):559-601

National Centre for Alternative Methods (ZEBET), Berlin, Germany.

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.
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http://dx.doi.org/10.1177/026119290703500614DOI Listing
December 2007

Assessment of the skin irritation potential of chemicals by using the SkinEthic reconstructed human epidermal model and the common skin irritation protocol evaluated in the ECVAM skin irritation validation study.

Altern Lab Anim 2006 Aug;34(4):393-406

ZEBET, National Centre for the Documentation and Evaluation of Alternative Methods to Animal Experiments, Federal Institute for Risk assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany.

Currently, two reconstructed human skin models, EpiDerm and EPISKIN are being evaluated in an ECVAM skin irritation validation study. A common skin irritation protocol has been developed, differing only in minor technical details for the two models. A small-scale study, applying this common skin irritation protocol to the SkinEthic reconstructed human epidermis (RHE), was performed at ZEBET at the BfR, Berlin, Germany, to consider whether this protocol could be successfully transferred to another epidermal model. Twenty substances from Phase III of the ECVAM prevalidation study on skin irritation were tested with the SkinEthic RHE. After minor, model-specific adaptations for the SkinEthic RHE, almost identical results to those obtained with the EpiDerm and EPISKIN models were achieved. The overall accuracy of the method was more than 80%, indicating a reliable prediction of the skin irritation potential of the tested chemicals when compared to in vivo rabbit data. As a next step, inter laboratory reproducibility was assessed in a study conducted between ZEBET and the Department of Experimental Toxicology, Schering AG, Berlin, Germany. Six coded substances were tested in both laboratories, with three different batches of the SkinEthic model. The assay results showed good reproducibility and correct predictions of the skin irritation potential for all six test chemicals. The results obtained with the SkinEthic RHE and the common protocol were reproducible in both phases, and the overall outcome is very similar to that of earlier studies with the EPISKIN and EpiDerm models. Therefore, the SkinEthic skin irritation assay test protocol can now be evaluated in a formal "catch-up" validation study.
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http://dx.doi.org/10.1177/026119290603400407DOI Listing
August 2006

Reconstructed human epidermis for skin absorption testing: results of the German prevalidation study.

Altern Lab Anim 2006 Jun;34(3):283-94

Freie Universität Berlin, Institut für Pharmazie, Königin-Luise-Strasse 2-4, 14195 Berlin, Germany.

Exposure to chemicals absorbed by the skin can threaten human health. In order to standardise the predictive testing of percutaneous absorption for regulatory purposes, the OECD adopted guideline 428, which describes methods for assessing absorption by using human and animal skin. In this study, a protocol based on the OECD principles was developed and prevalidated by using reconstructed human epidermis (RHE). The permeation of the OECD standard compounds, caffeine and testosterone, through commercially available RHE models was compared to that of human epidermis and animal skin. In comparison to human epidermis, the permeation of the chemicals was overestimated when using RHE. The following ranking of the permeation coefficients for testosterone was obtained: SkinEthic > EpiDerm, EPISKIN > human epidermis, bovine udder skin, pig skin. The ranking for caffeine was: SkinEthic, EPISKIN > bovine udder skin, EpiDerm, pig skin, human epidermis. The inter-laboratory and intra-laboratory reproducibility was good. Long and variable lag times, which are a matter of concern when using human and pig skin, did not occur with RHE. Due to the successful transfer of the protocol, it is now in the validation process.
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http://dx.doi.org/10.1177/026119290603400312DOI Listing
June 2006

Assessment of the human epidermis model SkinEthic RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431.

Toxicol In Vitro 2006 Aug 7;20(5):547-59. Epub 2006 Feb 7.

ZEBET, Bundesinstitut für Risikobewertung, BfR, Diedersdorfer Weg 1, D-12277 Berlin, Germany.

Based on two successfully completed ECVAM validation studies for in vitro skin corrosion testing of chemicals, the National Co-ordinators of OECD Test Guideline Programme endorsed in 2002 two new test guidelines: TG 430 'Transcutaneous Electrical Resistance assay' and TG 431 'Human Skin Model Test'. To allow all suitable in vitro human reconstructed (dermal or epidermal) models to be used for skin corrosion testing, the OECD TG 431 defines general and functional conditions that the model must meet before it will be routinely used for skin corrosion testing. In addition, the guideline requires correct prediction of 12 reference chemicals and assessment of intra- and inter-laboratory variability. To show that the OECD TG 431 concept works, in 2003 ZEBET tested several chemicals from the ECVAM validation trials on the SkinEthic reconstituted human epidermal (RHE) model. Based on knowledge that reconstructed human skin models perform similarly in toxicological studies, it was decided to adopt the validated EpiDerm skin corrosion test protocol and prediction model to the SkinEthic model. After minor technical changes, classifications were obtained in concordance with those reported for the validated human skin models EPISKIN and EpiDerm. To allow adequate determination of inter-laboratory reproducibility, a blind trial was conducted in three laboratories -- ZEBET (D), Safepharm (UK) and BASF (D), in which the 12 endorsed reference chemicals were tested. Results obtained with the SkinEthic epidermal model were reproducible, both within and between laboratories, and over time. Concordance between the in vitro predictions of skin corrosivity potential obtained with the SkinEthic model and the predictions obtained with the accepted tests of OECD TG 430 and TG 431 was very good. The new test was able to distinguish between corrosive and non-corrosive reference chemicals with an accuracy of 93%.
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http://dx.doi.org/10.1016/j.tiv.2005.11.008DOI Listing
August 2006

UV-induced effects.

Altern Lab Anim 2005 Jul;33 Suppl 1:131-46

ZEBET BfR, Diedersdorfer Weg 1, 12277 Berlin, Germany.

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http://dx.doi.org/10.1177/026119290503301s14DOI Listing
July 2005

Eye irritation.

Altern Lab Anim 2005 Jul;33 Suppl 1:47-81

ECVAM, Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra (VA), Italy.

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http://dx.doi.org/10.1177/026119290503301s09DOI Listing
July 2005

Skin irritation and corrosion.

Altern Lab Anim 2005 Jul;33 Suppl 1:35-46

ECVAM, Institute for Health and Consumer Protection, European Commission Joint Research Centre, Ispra (VA), Italy.

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http://dx.doi.org/10.1177/026119290503301s08DOI Listing
July 2005

The EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests--an assessment of the performance of the optimised test.

Altern Lab Anim 2005 Aug;33(4):351-67

Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments (ZEBET), Bundesinstitut fur Risikobewertung (BfR), Diedersdorfer Weg 1, 12277 Berlin, Germany.

During the past decade, several validation studies have been conducted on in vitro methods for discriminating between skin irritating and non-irritating chemicals. The reconstructed human skin models, EpiDerm and EPISKIN, provided the most promising results. Based on experience of the similar performance of the two skin models, it was suggested that a common test protocol and prediction model should be developed for the prediction of skin irritation potential with the two models. When the EPISKIN protocol was applied with the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was low. In 2003, the EPISKIN protocol was further refined by extending the post-incubation period following exposure to test chemicals. This extension and additional technical improvements to the EpiDerm protocol were evaluated with 19 chemicals from the prevalidation study. With the new test design, high sensitivity (80%) and specificity (78%) were obtained. The statistical probability for correct classifications was high, so the test was considered to be ready for formal validation. However, since test optimisation had been conducted with the same test chemicals as were used in the ECVAM prevalidation study, it was decided that the optimisation of the protocol had to be verified with a new set of chemicals. Thus, in the current study, 26 additional chemicals (10 rabbit irritants and 16 non-irritants), which had previously been selected and tested by LOREAL with EPISKIN, were evaluated in three independent experiments with EpiDerm. With this unbalanced testing set, a specificity of 94%, and a sensitivity of 60% were obtained, while the positive and negative predictivity and accuracy remained almost unchanged (around 80%) in comparison to the in vivo rabbit data. Overall, 45 chemicals (20 irritants and 25 non-irritants) were tested according to the final protocol. The resulting high positive (82%) and negative predictive values (79%) confirmed the reliability (accuracy of 80%) of the improved test protocol of the EpiDerm model.
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http://dx.doi.org/10.1177/026119290503300408DOI Listing
August 2005