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The alkaline comet assay is frequently used as in vivo follow-up test within different regulatory environments to characterize the DNA-damaging potential of different test items. The corresponding OECD Test guideline 489 highlights the importance of statistical analyses and historical control data (HCD) but does not provide detailed procedures. Therefore, the working group "Statistics" of the German-speaking Society for Environmental Mutation Research (GUM) collected HCD from five laboratories and >200 comet assay studies and performed several statistical analyses. Key results included that (I) observed large inter-laboratory effects argue against the use of absolute quality thresholds, (II) > 50% zero values on a slide are considered problematic, due to their influence on slide or animal summary statistics, (III) the type of summarizing measure for single-cell data (e.g., median, arithmetic and geometric mean) may lead to extreme differences in resulting animal tail intensities and study outcome in the HCD. These summarizing values increase the reliability of analysis results by better meeting statistical model assumptions, but at the cost of information loss. Furthermore, the relation between negative and positive control groups in the data set was always satisfactorily (or sufficiently) based on ratio, difference and quantile analyses.
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Daño del ADN , Proyectos de Investigación , Animales , Ensayo Cometa/métodos , Reproducibilidad de los Resultados , MutaciónRESUMEN
Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group "Statistics" within the German-speaking "Gesellschaft für Umwelt-Mutationsforschung e.V." (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified "laboratory" and "strain", but not "gender" as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.
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Grupos Control , Pruebas de Micronúcleos/estadística & datos numéricos , Animales , Médula Ósea , Femenino , Masculino , Ratas Wistar , Valores de ReferenciaRESUMEN
Genotoxicity testing is an important part of standard safety testing strategies. Animal studies have always been a key component, either as a mandatory part of the regulatory test battery, or to follow-up questionable in vitro findings. The strengths and weaknesses of in vivo assays is a continuous matter of debate, including their capacity to predict (human) carcinogenicity. We have therefore analysed the sensitivity of five routinely used in vivo tests to determine, in addition to other aspects, which tests or combination of tests best identify 73 chemicals classified as IARC Group 1 and 2A carcinogens. The in vivo tests included the micronucleus (MN), unscheduled DNA synthesis (UDS), comet, Pig-a and transgenic rodent assays (TGR). The individual assays detect 74.2% (49/66, MN), 64.3% (9/14, UDS), 92.1% (35/38, comet), 82.4% (14/17, Pig-a) and 90.3% (28/31, TGR) of the probable and confirmed human carcinogens that were tested in these assays. Combining assays that cover different genotoxicity endpoints and multiple tissues, e.g. the bone marrow MN and the liver comet assays, increases the sensitivity further (to 94%). Correlations in terms of organ-specificity for these assays with human cancer target organs revealed only a limited correlation for the hematopoietic system but not for other organs. The data supports the use of the comet and TGR assays for detection of 'site-of-first-contact' genotoxicants, but these chemicals were generally also detected in assays that measure genotoxicity in tissues not directly exposed, e.g. liver and the hematopoietic system. In conclusion, our evaluation confirmed a high sensitivity of the five in vivo genotoxicity assays for prediction of human carcinogens, which can be further increased by combining assays. Moreover, the addition of the comet to the in vivo MN test would identify all DNA reactive human carcinogens. Importantly, integration of some of the study readouts into one experiment is an animal-saving alternative to performing separate experiments.
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Carcinógenos/toxicidad , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Hígado/efectos de los fármacos , Animales , Células de la Médula Ósea/efectos de los fármacos , Ensayo Cometa , Humanos , Ratones , Pruebas de Mutagenicidad , RatasRESUMEN
N-nitrosamines, a very heterogeneous class of chemicals, may enter humans in small amounts through various sources and are produced endogenously, too. Some are known to be mutagenic carcinogens and have recently been detected as impurities in several marketed pharmaceuticals. Despite their known mutagenic properties, the suitability of the bacterial reverse mutation (Ames) assay and in particular the use of induced rat liver S9 to detect their mutagenic potential, is often discussed. Recently, it could be demonstrated that induced rat liver S9 is capable of metabolizing small alkyl nitrosamines to exert their mutagenic potential (Bringezu & Simon, 2022). In this project, the mutagenic potential of nitrosamines in vitro under different S9 conditions applying the preincubation protocol and OECD 471-compliant standard Ames test recommendations was investigated. These conditions included various amounts of S9 fraction from hamster and rat, uninduced or induced with Aroclor 1254 or Phenobarbital/beta-Naphthoflavone (PB/NF). The findings indicated that in addition to induced S9, uninduced hamster S9 also demonstrated effectiveness. Moreover, both rat and hamster S9 fractions exhibited suitable responses in terms of mutation frequencies. Increasing the S9 content did not increase the sensitivity of the Ames test. However, above 20% S9, reduced mutation frequency was observed in the higher concentration range suggesting cytotoxicity to the bacteria. Thus, limiting the S9 content to 10% provides reliable results and relates to a lower number of animals required for S9 production which is in concordance with the 3R principles (reduce, refine, replace) for animal testing. In addition, results obtained show that uninduced and induced hamster S9 are similarly effective, doubting the requirement of pretreating animals with enzyme inducers. Further investigations to compare mutagenicity data and rat and hamster S9 proteome analyses are ongoing.
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Historical data from control groups in animal toxicity studies is currently mainly used for comparative purposes to assess validity and robustness of study results. Due to the highly controlled environment in which the studies are performed and the homogeneity of the animal collectives it has been proposed to use the historical data for building so-called virtual control groups, which could replace partly or entirely the concurrent control. This would constitute a substantial contribution to the reduction of animal use in safety studies. Before the concept can be implemented, the prerequisites regarding data collection, curation and statistical evaluation together with a validation strategy need to be identified to avoid any impairment of the study outcome and subsequent consequences for human risk assessment. To further assess and develop the concept of virtual control groups the transatlantic think tank for toxicology (t4) sponsored a workshop with stakeholders from the pharmaceutical and chemical industry, academia, FDA, pharmaceutical, contract research organizations (CROs), and non-governmental organizations in Washington, which took place in March 2023. This report summarizes the current efforts of a European initiative to share, collect and curate animal control data in a centralized database and the first approaches to identify optimal matching criteria between virtual controls and the treatment arms of a study as well as first reflections about strategies for a qualification procedure and potential pitfalls of the concept.
Animal safety studies are usually performed with three groups of animals where increasing amounts of the test chemical are given to the animals and one control group where the animals do not receive the test chemical. The design of such studies, the characteristics of the animals, and the measured parameters are often very similar from study to study. Therefore, it has been suggested that measurement data from the control groups could be reused from study to study to lower the total number of animals per study. This could reduce animal use by up to 25% for such standardized studies. A workshop was held to discuss the pros and cons of such a concept and what would have to be done to implement it without threatening the reliability of the study outcome or the resulting human risk assessment.
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Investigación , Animales , Grupos Control , Preparaciones FarmacéuticasRESUMEN
The ICH S1B carcinogenicity global testing guideline has been recently revised with a novel addendum that describes a comprehensive integrated Weight of Evidence (WoE) approach to determine the need for a 2-year rat carcinogenicity study. In the present work, experts from different organizations have joined efforts to standardize as much as possible a procedural framework for the integration of evidence associated with the different ICH S1B(R1) WoE criteria. The framework uses a pragmatic consensus procedure for carcinogenicity hazard assessment to facilitate transparent, consistent, and documented decision-making and it discusses best-practices both for the organization of studies and presentation of data in a format suitable for regulatory review. First, it is acknowledged that the six WoE factors described in the addendum form an integrated network of evidence within a holistic assessment framework that is used synergistically to analyze and explain safety signals. Second, the proposed standardized procedure builds upon different considerations related to the primary sources of evidence, mechanistic analysis, alternative methodologies and novel investigative approaches, metabolites, and reliability of the data and other acquired information. Each of the six WoE factors is described highlighting how they can contribute evidence for the overall WoE assessment. A suggested reporting format to summarize the cross-integration of evidence from the different WoE factors is also presented. This work also notes that even if a 2-year rat study is ultimately required, creating a WoE assessment is valuable in understanding the specific factors and levels of human carcinogenic risk better than have been identified previously with the 2-year rat bioassay alone.
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This paper presents an inventory of in silico screening tools to identify substance properties of concern under the European chemicals' legislation REACH. The objective is to support the selection and implementation of appropriate tools as building blocks within integrated testing strategies (ITS). The relevant concerns addressed are persistence, bioaccumulation potential, acute and long-term aquatic toxicity, PBT/vPvB properties ((very) persistent, (very) bioaccumulative, toxic), CMR (carcinogenicity, mutagenicity, reproductive toxicity), endocrine disruption and skin sensitisation. The inventory offers a comparative evaluation of methods with respect to the underlying algorithms (how does the method work?) and the applicability domains (when does the method work?) as well as their limitations (when does the method not work?). The inventory explicitly addresses the reliability of predictions of different in silico models for diverse chemicals by applicability domain considerations. The confidence in predictions can be greatly improved by consensus modelling that allows for taking conflicting results into account. The inventory is complemented by a brief discussion of socio-economic tools for assessing the potential efficiency gains of using in silico methods compared to traditional in vivo testing of chemical hazards.
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Política Ambiental , Contaminantes Ambientales , Sustancias Peligrosas , Modelos Teóricos , Pruebas de Toxicidad/métodos , Animales , Política Ambiental/legislación & jurisprudencia , Contaminantes Ambientales/química , Contaminantes Ambientales/toxicidad , Europa (Continente) , Programas de Gobierno , Regulación Gubernamental , Sustancias Peligrosas/química , Sustancias Peligrosas/toxicidad , Humanos , Valor Predictivo de las Pruebas , Relación Estructura-Actividad Cuantitativa , Pruebas de Toxicidad/normas , Pruebas de Toxicidad/estadística & datos numéricosRESUMEN
Humans are exposed to low levels of N-nitrosamines via different sources. N-Nitrosamines have recently been detected as impurities in various marketed drugs and they are known mutagenic carcinogens belonging to the cohort of concern as referred to in the ICH M7 guideline. Despite their well-known mutagenic properties, there is ongoing discussion on the suitability of the bacterial reverse mutation assay and using induced rat liver S9 as the external source of metabolism to detect their mutagenic potential. Therefore, we have investigated the mutagenic potential of N-nitrosodimethylamine, N-nitrosodiethylamine, N-nitrosodipropylamine, and N-nitrosodibutylamine in vitro under various conditions. Our work showed that the bacterial reverse mutation assay applying plate incorporation or preincubation protocols and using Salmonella typhimurium strains TA100 and TA1535 and E. coli WP2 uvrA is suitable to predict the mutagenicity of n-nitrosamines in the presence of phenobarbital/ß-naphthoflavone induced rat liver S9.
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The pharmaceutical industry would benefit from the collaboration with academic groups in the development of predictive safety models using the newest computational technologies. However, this collaboration is sometimes hampered by the handling of confidential proprietary information and different working practices in both environments. In this manuscript, we propose a strategy for facilitating this collaboration, based on the use of modeling frameworks developed for facilitating the use of sensitive data, as well as the development, interchange, hosting, and use of predictive models in production. The strategy is illustrated with a real example in which we used Flame, an open-source modeling framework developed in our group, for the development of an in silico eye irritation model. The model was based on bibliographic data, refined during the company-academic group collaboration, and enriched with the incorporation of confidential data, yielding a useful model that was validated experimentally.
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Industria Farmacéutica , Simulación por ComputadorRESUMEN
Understanding the reliability and relevance of a toxicological assessment is important for gauging the overall confidence and communicating the degree of uncertainty related to it. The process involved in assessing reliability and relevance is well defined for experimental data. Similar criteria need to be established for in silico predictions, as they become increasingly more important to fill data gaps and need to be reasonably integrated as additional lines of evidence. Thus, in silico assessments could be communicated with greater confidence and in a more harmonized manner. The current work expands on previous definitions of reliability, relevance, and confidence and establishes a conceptional framework to apply those to in silico data. The approach is used in two case studies: 1) phthalic anhydride, where experimental data are readily available and 2) 4-hydroxy-3-propoxybenzaldehyde, a data poor case which relies predominantly on in silico methods, showing that reliability, relevance, and confidence of in silico assessments can be effectively communicated within Integrated approaches to testing and assessment (IATA).
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eTRANSAFE is a research project funded within the Innovative Medicines Initiative (IMI), which aims at developing integrated databases and computational tools (the eTRANSAFE ToxHub) that support the translational safety assessment of new drugs by using legacy data provided by the pharmaceutical companies that participate in the project. The project objectives include the development of databases containing preclinical and clinical data, computational systems for translational analysis including tools for data query, analysis and visualization, as well as computational models to explain and predict drug safety events.
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Historically, identifying carcinogens has relied primarily on tumor studies in rodents, which require enormous resources in both money and time. In silico models have been developed for predicting rodent carcinogens but have not yet found general regulatory acceptance, in part due to the lack of a generally accepted protocol for performing such an assessment as well as limitations in predictive performance and scope. There remains a need for additional, improved in silico carcinogenicity models, especially ones that are more human-relevant, for use in research and regulatory decision-making. As part of an international effort to develop in silico toxicological protocols, a consortium of toxicologists, computational scientists, and regulatory scientists across several industries and governmental agencies evaluated the extent to which in silico models exist for each of the recently defined 10 key characteristics (KCs) of carcinogens. This position paper summarizes the current status of in silico tools for the assessment of each KC and identifies the data gaps that need to be addressed before a comprehensive in silico carcinogenicity protocol can be developed for regulatory use.
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Sharing legacy data from in vivo toxicity studies offers the opportunity to analyze the variability of control groups stratified for strain, age, duration of study, vehicle and other experimental conditions. Historical animal control group data may lead to a repository, which could be used to construct virtual control groups (VCGs) for toxicity studies. VCGs are an established concept in clinical trials, but the idea of replacing living beings with virtual data sets has so far not been introduced into the design of regulatory animal studies. The use of VCGs has the potential of a 25% reduction in animal use by replacing the control group animals with existing randomized data sets. Prerequisites for such an approach are the availability of large and well-structured control data sets as well as thorough statistical evaluations. the foundation of data sharing has been laid within the Innovative Medicines Initiatives projects eTOX and eTRANSAFE. For a proof of principle participating companies have started to collect control group data for subacute (4-week) GLP studies with Wistar rats (the strain preferentially used in Europe) and are characterizing these data for its variability. In a second step, the control group data will be shared among the companies and cross-company variability will be investigated. In a third step, a set of studies will be analyzed to assess whether the use of VCG data would have influenced the outcome of the study compared to the real control group.
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Bases de Datos Factuales , Evaluación Preclínica de Medicamentos/métodos , Difusión de la Información , Proyectos de Investigación , Pruebas de Toxicidad/métodos , Bases del ConocimientoRESUMEN
The interaction of the antimicrobial peptide dicynthaurin (ala) monomer with model membranes of zwitterionic and negatively charged lipids and mixtures thereof was studied by means of isothermal titration calorimetry (ITC), fluorescent leakage, and dynamic light scattering (DLS) measurements. For the ITC analysis, we have applied the surface partitioning equilibrium model which shows that the interaction is predominately driven by hydrophobic effects (Kb between 2 x 10(4) and 1 x 10(5) M(-1)). Under low salt conditions, the enhanced electrostatic interaction leads to larger peptide concentrations immediately above the vesicle surface, which initiates the insertion of the peptide into the bilayer more effectively. Fluorescent leakage measurements have shown a fast leakage of the fluorescent dye within seconds after peptide addition. The analysis of the leakage kinetics was performed in terms of an initial pore formation model (up to t = 1000 s) that takes the reversible surface aggregation of bound peptide monomers into account. From this analysis, a minimum aggregation number of n = 7 +/- 2 per pore is obtained.
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Calorimetría , Fluorescencia , Membrana Dobles de Lípidos/metabolismo , Péptidos/farmacocinética , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Luz , Membrana Dobles de Lípidos/química , Péptidos/química , Porosidad , Dispersión de Radiación , Electricidad Estática , VolumetríaRESUMEN
This paper reports the first study on the interaction of the antimicrobial peptide dicynthaurin with 1,2-dipalmitoyl-glycerophosphatidyl-glycerol investigated in monolayers at the air-liquid interface. The influence of the peptide on the two-dimensional phase behavior of the negatively charged lipid was elucidated by means of pressure-area isotherm measurements, fluorescence microscopy, and grazing incidence X-ray diffraction measurements. The pure peptide forms a stable monolayer at the air-liquid interface up to 30 mN/m as shown for both the monomeric and the dimeric cynthaurins. The peptide lipid interaction was monitored in isotherm measurements showing a strong adsorption of the peptide and stabilization at the interface promoted by the lipid monolayer. The X-ray diffraction measurements in agreement with fluorescence microscopy studies showed that the peptide destabilizes the condensed chain lattice, leading to a complete fluidization of the condensed lipid phase on physiological buffer. The adsorption of the peptide to the negatively charged lipid monolayer and the fluidization of the condensed chain lattice suggest a direct link to the peptides' ability to expand the bacterial membrane that would be relevant for the in vivo mode of action.
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Antibacterianos/química , Glicerol/análogos & derivados , Palmitatos/química , Péptidos/química , Fosfolípidos/química , Aire , Glicerol/química , Microscopía Fluorescente , Tamaño de la Partícula , Solventes/química , Propiedades de Superficie , Temperatura , Agua/químicaRESUMEN
The effect of nonsteroidal anti-inflammatory drugs (NSAIDs) on the phase transition and phase properties of 1,2-dipalmitoylphosphatidylcholine (DPPC) has been investigated in both 2D (monolayers at the air/water interface) and 3D (multilayers in lipid/water dispersions) model systems. The 2D membrane models have been characterized by means of pressure-area isotherms and grazing incidence X-ray diffraction (GIXD) measurements. Differential scanning calorimetry (DSC) and simultaneous small- and wide-angle X-ray diffraction have been applied to lipid aqueous dispersions. All NSAIDs studied altered the main transition temperature of the gel to liquid-crystalline phase transition, with the arylacetic acid derivatives (acemetacin and indomethacin) showing the largest effects. A comparison of the results reveals distinct structural features of the membrane models after interaction with the NSAID. All drugs induced perturbations in the lipid liquid-crystalline phase, suggesting a major change in the hydration behavior of DPPC. Again, the largest effects on the structural parameters are found for the arylacetic acid derivatives. The results obtained in the different model systems give indications of the role of the membrane/NSAID interactions that might also be important for NSAID gastric injury.
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1,2-Dipalmitoilfosfatidilcolina/química , Antiinflamatorios no Esteroideos/química , Termodinámica , Membrana Dobles de Lípidos/química , Estructura Molecular , Presión , Difracción de Rayos XRESUMEN
This work focuses on the adsorption kinetics of dicynthaurin with lipid monolayers, the effect of peptide adsorption on the structure of the lipid condensed chain lattice, peptide orientation and secondary structure in the adsorbed state. The studies with DPPG as model system revealed strong adsorption and massive incorporation of the peptide into the monolayer. Infrared reflection absorption spectroscopy (IRRAS) experiments showed that the secondary structure of the peptide is maintained upon adsorption. Specular X-ray reflectivity showed the destabilization of the condensed phase of the pure lipid monolayer and revealed a tilted orientation of the long axis of the peptide helix of about 40 degrees from the surface normal. Incorporation of the peptide was found to be pressure dependent, and at high pressure a "squeeze-out" was observed; however, the peptide remained localized to the interface, as suggested by infrared data. These findings were supported by optical fluorescence microscopy measurements which showed the squeeze-out of the peptide on water, but not under physiological conditions. The results suggest that dicynthaurin is able to adsorb to the phosphatidylglycerol-rich inner cytoplasmic membrane of bacteria and alter membrane integrity. To identify and interact with membrane motifs that are characteristic of microbes, but which are absent in eukaryotic cells, might be an intrinsic ability of peptide antibiotics.
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Antibacterianos/química , Péptidos/química , Fosfolípidos/química , 1,2-Dipalmitoilfosfatidilcolina/química , Antibacterianos/síntesis química , Hemólisis/efectos de los fármacos , Membranas Artificiales , Microscopía Fluorescente , Modelos Moleculares , Péptidos/síntesis química , Fosfatidiletanolaminas/química , Estructura Secundaria de Proteína , Espectrofotometría Infrarroja , Difracción de Rayos XRESUMEN
The adsorption of doubly lipidated full-length N-Ras protein on 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) monolayers was studied by lateral pressure analysis, grazing incidence X-ray diffraction (GIXD), and specular reflectivity (XR). N-Ras protein adsorbs to the DPPC monolayer (lateral pressure of 20 mN/m) from the subphase thereby increasing the lateral pressure in the monolayer by 4 mN/m. The protein insertion does not alter the tilt angle and structure of the lipid molecules at the air/water interface but influences the electron density profile of the monolayer. Further, electron density differences into the subphase were observed. The Fresnel normalized reflectivity could be reconstructed in the analysis using box models yielding electron density profiles of the DPPC monolayer in the absence and in the presence of N-Ras protein. The electron density profiles of the DPPC monolayer in the presence of Ras showed clear intensity variations in the headgroup/glycerol/upper chain region, the so-called interface region where previous bilayer studies had confirmed Ras binding.
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1,2-Dipalmitoilfosfatidilcolina/química , Membrana Dobles de Lípidos/química , Liposomas Unilamelares/química , Proteínas ras/química , Adsorción , Sitios de Unión , Unión Proteica , SolucionesRESUMEN
This paper is focused on the thermodynamics and the structural investigation of the interaction of the antimicrobial peptide dicynthaurin monomer with model lipid membranes composed of mixtures of 1-palmitoyl-2-oleyl-glycerophosphocholine and -glycerophosphoglycerol. The thermodynamic binding parameters as obtained by isothermal titration calorimetry reveal strong binding toward the lipid model system dominated by large chemical binding constants which exceeds the electrostatic binding effects and thus suggests insertion of the amphipathic alpha-helical peptide into the hydrophobic membrane core. Circular dichroism study shows that the peptide exhibits trans-membrane alpha-helix secondary structure. Neutron diffraction measurements using partially deuterated sequences were successfully applied to determine the orientation of the peptide thus proving insertion into the hydrophobic membrane core. This insertion and the formation of higher order porelike aggregates is assumed to be the most relevant event in microbial membrane perturbation that in vivo finally leads to bacterial cell death on a fast time scale.