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Manfred Liebsch

Publications and source records attributed to Manfred Liebsch.

18 recordsLinked to original sources
PubMed2006

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

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%.

Caustics↗
PubMed2006

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

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.

Adult↗
PubMed2006

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.

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.

Cells, Cultured↗
PubMed2005

Assessment of the eye irritating properties of chemicals by applying alternatives to the Draize rabbit eye test: the use of QSARs and in vitro tests for the classification of eye irritation.

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

Animal Testing Alternatives↗
PubMed2005

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.

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.

Animal Testing Alternatives↗
PubMed2005

Eye irritation.

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Animal Testing Alternatives↗
PubMed2005

UV-induced effects.

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Animal Testing Alternatives↗
PubMed2004

Animal testing and alternative approaches for the human health risk assessment under the proposed new European chemicals regulation.

During the past 20 years the EU legislation for the notification of chemicals has focussed on new chemicals and at the same time failed to cover the evaluation of existing chemicals in Europe. Therefore, in a new EU chemicals policy (REACH, Registration, Evaluation and Authorization of Chemicals) the European Commission proposes to evaluate 30,000 chemicals within a period of 15 years. We are providing estimates of the testing requirements based on our personal experiences during the past 20 years. A realistic scenario based on an in-depth discussion of potential toxicological developments and an optimised "tailor-made" testing strategy shows that to meet the goals of the REACH policy, animal numbers may be significantly reduced below 10 million if industry would use in-house data from toxicity testing, which are confidential, if non-animal tests would be used, and if information from quantitative structure activity relationships (QSARs) would be applied in substance-tailored testing schemes. The procedures for evaluating the reproductive toxicity of chemicals have the strongest impact on the total number of animals bred for testing under REACH. We are assuming both an active collaboration with our colleagues in industry and substantial funding of the development and validation of advanced non-animal methods by the EU Commission, specifically in reproductive and developmental toxicity.

Animal Testing Alternatives↗
PubMed2004

Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests.

An ECVAM-funded prevalidation study (PV) was conducted during 1999 and 2000 to identify in vitro tests capable of reliably distinguishing between skin irritants (I) and non-irritants (NI) according to European Union risk phrases ("R38" or no classification). The tests evaluated were EpiDerm, EPISKIN, PREDISKIN, the non-perfused pig ear method, and the mouse skin integrity function test (SIFT). Whereas reproducibility of the two human skin model tests and SIFT was acceptable, none of the methods was deemed ready to enter a formal validation study due to their low predictivity. The ECVAM Skin Irritation Task Force therefore suggested improvements of protocols and prediction models for these tests. Furthermore, it was agreed that experience gained with the two human-skin models be shared, and a common protocol should be developed for EpiDerm and EPISKIN (Zuang et al., 2002). When we applied an improved EPISKIN protocol (Portes et al., 2002) to the EpiDerm model, an acceptable specificity (80%) was achieved, whereas the sensitivity (60%) was far too low. In 2003, the EPISKIN protocol was further refined by extension of the post-incubation period following chemical exposure. In the current study, we evaluated this EPISKIN refinement by applying it to EpiDerm. In addition, we developed technical improvements for the application of the test chemicals and rinsing procedure, which reduced the variability of results and increased the percentage of correct predictions. A set of twenty non-coded reference substances from the ECVAM prevalidation study phase III (Fentem et al., 2001) was tested with the final protocol in three independent runs. Both high sensitivity (80%) and high specificity (78%) were achieved, and the statistical probability of correct classifications was high, so that the test is now regarded ready for formal validation.

Animal Testing Alternatives↗
PubMed2002

Currently available in vitro methods used in the regulatory toxicology.

About two decades ago in vitro mutagenicity tests were adopted as the first in vitro methods in regulatory toxicology. For reasons of animal welfare and better science, many validation studies of various in vitro methods were performed between 1985 and 1995 for their potential to replace the Draize eye irritation test. Albeit the fact that four in vitro methods (HET-CAM, BCOP, IRE, and ECE) have gained regulatory tolerance in Europe for the classification of severe eye irritants, the lessons learned mainly from these validation studies led to the definition of internationally harmonised OECD validation principles and acceptance criteria (Final Report of the OECD Workshop on Harmonization of Validation and Acceptance Criteria for Alternative Toxicological Tests Methods, 1996. OECD Publication Office, Paris, France). Application of these principles to prevalidation and validation studies, and additional special studies performed in Europe with the support of ECVAM, ended in the scientific validation of new in vitro methods for the prediction of skin corrosivity and phototoxicity. They were accepted for regulatory use in Europe and adopted on 8 June 2000 as test methods B. 40 and B. 41 of Annex V of Directive 67/548/EEC. In quite a different approach, European industry has submitted in-house validation data to support a Draft OECD Test Guideline for in vitro dermal absorption testing, which-after peer review and a long lasting international discussion-will now be adopted by the OECD. The increasing importance of regulatory measures derived from quantitative cytotoxicity tests, and some examples of regulatory accepted in vitro methods, where the specific purpose is restricted to a specific regulation (e.g. testing of medicinal products, or medical devices), are also addressed.

Animal Testing Alternatives↗
PubMed2002

Validation successes: chemicals.

The ECVAM validation concept, which was defined at two validation workshops held in Amden (Switzerland) in 1990 and 1994, and which takes into account the essential elements of prevalidation and biostatistically defined prediction models, has been officially accepted by European Union (EU) Member States and by the Federal regulatory agencies of the USA and the OECD. The ECVAM validation concept was introduced into the ongoing ECVAM/COLIPA validation study of in vitro phototoxicity tests, which ended successfully in 1998. The 3T3 neutral red uptake in vitro phototoxicity test was the first experimentally validated in vitro toxicity test recommended for regulatory purposes by the ECVAM Scientific Advisory Committee (ESAC). It was accepted by the EU into the legislation for chemicals in the year 2000. From 1996 to 1998, two in vitro skin corrosivity tests were successfully validated by ECVAM, and they were also officially accepted into the EU regulations for chemicals in the year 2000. Meanwhile, in 2002, the OECD Test Guidelines Programme is considering the worldwide acceptance of the validated in vitro phototoxicity and corrosivity tests. Finally, from 1997 to 2000, an ECVAM validation study on three in vitro embryotoxicity tests was successfully completed. Therefore, the three in vitro embryotoxicity tests, the whole embryo culture (WEC) test on rat embryos, the micromass (MM) test on limb bud cells of mouse embryos, and the embryonic stem cell test (EST) including a permanent embryonic mouse stem cell line, are considered for routine use in laboratories of the European pharmaceutical and chemicals industries.

3T3 Cells↗
PubMed1998

[ERGATT/ECVAM Workshop on Acceptance of Validated Alternative Methods: Amden III]

Since validated in vitro toxicity tests have so far not been accepted by regulators the third international ERGATT/ECVAM validation workshop was held from January 26-30, 1998, in Amden/Switzerland. Scientific and political reasons for a delay of the acceptance of alternative methods were identified in the areas of phototoxicity, skin penetration and skin corrosivity and strategies to promote the acceptance were suggested. The OECD will harmonise the formal of criteria for the regulatory acceptance of new experimentally validated tests and of established tests from industry for which in-house and literature data exist. In vitro tests for phototoxicity and skin penetration will be accepted shortly by the OECD. For skin corrosivity two in vitro tests will be put forward to the OECD later this year. The Amden III workshop suggests that, the same acceptance criteria must be used for animal and in vitro tests. Finally, the OECD should remove tests that cause pain and distress to test animals from the official OECD list of official test guidelines if validated alternative methods exist. When obsolete animal tests are taken from the list of official OECD test guidelines, they will not any longer be accepted at the international level. Thus, the third Amden workshop has contributed new concepts for the regulatory acceptance of alternative toxicity tests which are supported by regulators in Europe, the USA and the OECD.

Journal Article↗
PubMed1997

[Reduction of the numbers of animals used for the classification of the acute oral toxicity of chemicals by taking into account cytotoxicity data from the Registry of Cytotoxicity]

Using cytotoxicity data a new classification procedure is introduced which will allow to allocate chemicals to the four toxicity classes for acute oral toxicity according to EU regulation. Simultaneously, the new procedure allows to reduce animals in experiments for determination these four toxicity classes. The cytotoxicity data estimated from in vitro cultivated mammalian cell lines were taken from the "Registry of Cytotoxicity" (RC) in which the mean IC50 (IC50x) of 347 chemicals are stored as well as the acute oral toxicity data (LD50) for rats and mice taken from NIOSH registry. As we have been used to calculate a standard regression line for predicting the dosage range of acute oral toxicity in the two species. The maximum, mean and minimum dosages of oral toxicity were predicted from the RC data and furthermore, these dosages were the basis for allocating chemicals into the four classes of acute oral toxicity defined by the EU. The accuracy for predicting the toxicity classes of the 347 chemicals registered in the RC in comparison to the toxicity classes of the corresponding NIOSH LD50 values amounts 80 percent to 88 percent. We have developed a tier testing strategy for the classification of chemicals into EU toxicity classes which takes into account the cytotoxicity data as predicted mean LD50 in connection with the protocol steps described in Acute Toxic Class (ATC) method. Compared with the ATC method the new combined RC-ATC procedure will allow to reduce animal numbers for allocating chemicals to the EU toxicity classes by about 30 percent. We suggest to validate the RC-ATC procedure in order to achieve regulatory acceptance at the international level.

Journal Article↗
PubMed1997

[Development of a new in vitro test for dermal phototoxicity using a model of reconstituted human epidermis]

Results are reported using the human epidermis model (EpiDermtrade mark) in a test protocol that was adopted from the method developed by ZEBET for phototoxicity testing with the model Skin2. In the new phototoxicity test the cytotoxicity of test materials applied topically to EpiDermtrade mark is determined at five concentrations in the absence and presence of a non-cytotoxic dose of UVA and visible light (sun simulation). Cytotoxicity is determined one day after irradiation in the MTT assay. Experiments performed to optimise test conditions are presented. Using appropriate phototoxic and non-phototoxic test chemicals our results demonstrate that a reconstructed human epidermal model, e.g. EpiDermtrade mark, can be used in phototoxicity testing in the same way as a full skin model.

Journal Article↗
PubMed1995

[History of the LAL-test: validation and regulatory acceptance]

Bacterial endotoxins gram-negative bacteria are the most relevant substances inducing hyperthermia in humans (pyrogens). Since endotoxins may contaminate pharmaceutical preparations during production process, purity is assured by monitoring the increase in body temperature of rabbits exposed to such preparations. This bioassay can be replaced by the LAL-test in which the clotting reaction of blood cells of the horseshoe crab limulus polyphemus is measured after contact with bacterial endotoxins. Although this reaction is significantly more sensitive to endotoxins than hyperthermia in rabbits, the LAL-test had to undergo 25-30 years of validation to achieve regulatory acceptance. Although endotoxin induced blood clotting in Limulus is quite similar to the same reaction in humans, acceptance of the assay as an alternative to testing in rabbits was delayed, since chemicals present in pharmaceutical preparations may interfere with the LAL clotting reaction. In addition, fever can be induced also by substances other than endotoxins. Therefore it has to be proven for each new preparation that the LAL-test can replace the rabbit pyrogen test in a case by case validation according to guidelines, as e.g. the German guideline, which was released in 1993. During the past decade some laboratories of the pharmaceutical industry in Germany were able to replace 95% of the rabbit pyrogen tests by the LAL-test without increasing the risk for humans.

Journal Article↗
PubMed1994

[First results of an EC/COLIPA validation project of in vitro phototoxicity testing methods]

In a joint project six laboratories from the European cosmetics industry (COLIPA) as well as from FRAME (England) and ZEBET (Germany) are validating in vitro methods to be incorporated into new international guidelines for photoirritancy testing. During the first stage of the study it was attempted to select the most promising in vitro photoirritancy tests for further validation. Twenty chemicals with known photoirritation properties (12 phototoxins (PT), 4 non-PTs and 4 UV absorbing non-PTs) were tested under identical UV exposure conditions (5 J/cm2, UV-A sun simulator) in a standardized cytotoxicty assay using 3T3 fibroblasts (endpoint): Neutral red uptake, NRU). The chemicals were also tested with in vitro phototoxicity assays established in industrial laboratories, e.g. the photohaemolyses (Pape et al, 1993), histine oxidation, candida albicans (Johnson et al, 1986), and, furthermore two commercial tests (SOLATEX PItrade mark and Skin2trade mark). Data from the 3T3 NRU photoirritancy test, the red blood cell photohaemolysis test and the Skin2trade mark assay showed a better overall correlation to human in vivo data than results from the other tests. These simple assays therefore, seem very promising for further validation under blind conditions. The protocols of the other tests have to be improved and standardized to permit better interlaboratory comparison.

Journal Article↗
PubMedDate not supplied

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2.

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.

Animal Testing Alternatives↗