Embryonic stem cell test remastered: comparison between the validated EST and the new molecular FACS-EST for assessing developmental toxicity in vitro.

The embryonic stem cell test (EST) represents a reliable, scientifically validated in vitro system for the detection and classification of compounds according to their teratogenic potency. However, some serious issues were frequently raised against the widespread implementation and practicability of the EST in its original version. Most importantly, the evaluation of the morphological endpoint of beating cell agglomerates requires extensive experimental experience and is prone to misjudgment. Also, the testing period of 10 days is too long and costly to be attractive for industries interested in high-throughput screening of potential drug candidates. These drawbacks prompted us to work out a new molecular approach based on analysis of the expression of certain marker proteins specific for developing heart tissue. We have previously reported that quantitative flow cytometry of marker proteins (i.e., sarcomeric myosin heavy chain and alpha-actinin) can be performed at day 7 in embryonic stem cells from mice and combined with concurrent cell viability analysis. In the present study, extensive investigations were performed in order to explore the predictive power and validity of the newly established EST, subsequently referred to as molecular fluorescence activated cell sorting (FACS)-EST, by applying and comparing a set of 10 well-known embryotoxicants that encompasses the full range of chemical inherent embryotoxic potencies possible. While the molecular FACS-EST offered the same sensitivity compared to the validated EST protocol, the test duration could be significantly reduced. Due to significant improvements, this new molecular method holds promise as a sensitive, more rapid and reproducible screen highly suited to predict developmental toxicity in vivo from in vitro data.

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.

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%.

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.

Oral acute toxic class method: a successful alternative to the oral LD50 test.

The oral acute toxic class method (ATC method) was developed as an alternative to replace the oral LD50 test. The ATC method is a sequential testing procedure using only three animals of one sex per step at any of the defined dose levels. Depending on the mortality rate three but never more than six animals are used per dose level. This approach results in the reduction of numbers of animals used in comparison to the LD50 test by 40-70%. The principle of the oral ATC method is based on the Probit model and it was first evaluated on a biometric basis before a national and subsequently an international ring study were conducted. The results demonstrated an excellent agreement between the toxicity and the animal numbers predicted biometrically and observed in the validation studies. The oral ATC method was adopted as an official test guideline by OECD in 1996 and was slightly amended in 2001. The ATC method has been successfully used in Germany and in 2003 >85% of all tests on acute oral toxicity testing was conducted as oral ATC tests. In member states of the European Union the ATC method is used in the range of 50% of all tests conducted. Meanwhile the oral LD50 test has been deleted by OECD, by the European Union and by the USA, making the use of alternatives to the oral LD50 test mandatory.

Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests.

A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.

Validation of the rat limb bud micromass test in the international ECVAM validation study on three in vitro embryotoxicity tests.

A detailed report is presented on the performance of the rat limb bud micromass (MM) test in a European Centre for the Evaluation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects on animals and/or humans, were tested in four laboratories. The outcome showed that the MM test is an experimentally validated test, which holds promise for use for identifying strongly embryotoxic chemicals, but which needs to be improved before it can be recommended for use for regulatory purposes.

Validation of the postimplantation rat whole-embryo culture test in the international ECVAM validation study on three in vitro embryotoxicity tests.

A detailed report is presented on the performance of the postimplantation rat whole-embryo culture (WEC) test in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the WEC test can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.

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.

Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test.

The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of "beating areas" and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and alpha-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and alpha-actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, alpha-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.

Trends in improving the embryonic stem cell test (EST): an overview.

The embryonic stem cell test (EST) is an in vitro assay that has been developed to assess the teratogenic and embryotoxic potential of drugs and chemicals. It is based on the capacity of murine ES cells (cell line D3) to differentiate into contracting myocardial cells under specific cell culture conditions. The appearance of beating cardiomyocytes in embryoid body (EB) outgrowths is used as a toxicological endpoint to assess the embryotoxic potential of a test substance. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study the EST predicted the embryotoxic potential of chemicals and drugs with the same reliability as two other in vitro embryotoxicity tests, which employed embryonic cells and tissues from pregnant animals. In a joint research project with German pharmaceutical companies we have successfully improved the EST by establishing molecular endpoints of differentiation in cultured ES cells. The quantification of cardiac-specific protein expression by intracellular flow cytometry has been studied in the presence of chemicals of different embryotoxic potential. The results obtained using molecular endpoints specific for differentiated cardiomyocytes employing FACS (fluorescence-activated cell sorting) analysis will be presented in comparison to the validated endpoint - the microscopic analysis of beating areas. FACS analysis provides a more objective endpoint for predicting the embryotoxic potential of chemicals than the validated method. Furthermore, flow cytometry promises to be suitable for high-throughput screening systems (HTS). In addition, our partners from the joint project have improved the EST by developing protocols that stimulate differentiation of ES cells into neural and endothelial cells, chondrocytes and osteoblasts, because some substances might have embryotoxic effects on specific cell-types other than cardiomyocytes. These protocols have been successfully established at ZEBET and in the participating laboratories. Additionally, molecular endpoints have been established for the detection of specific differentiation pathways. Furthermore, new prediction models (PMs) have been developed using single endpoints of the EST.

Considering the test performance for three class data using linear discriminant analysis: a case study.

The results of a European Centre for the Validation of Alternative Methods validation study on the embryonic stem (ES) cell test were used as an example for the test performance for "three-class" data using linear discriminant analysis (LDA). Applying LDA, a biostatistical prediction model was developed to assign test chemicals to three classes of embryotoxicity. Three endpoint values were identified to classify the embryotoxic potential of chemicals: a) the inhibition of differentiation of ES cells into cardiomyocytes (ID50), b) the decrease of viability of adult 3T3 cells (IC503T3), and c) the decrease of viability of ES cells (IC50D3) in an MTT cytotoxicity test. It could be demonstrated that an objective statistical method was required in order to minimise the probability of misclassification. The management team of the validation study defined performance criteria, taking into consideration the hit rate for three classes obtained just by chance. An additional purpose of the study reported here was to calculate a hypothetical prevalence and evaluate its influence on the classification results.

Comparison of broth microdilution, E Test, and agar dilution methods for antibiotic susceptibility testing of Campylobacter jejuni and Campylobacter coli.

A standardized broth microdilution method was compared to the E test and an agar dilution method for the antimicrobial susceptibility testing of Campylobacter jejuni and C. coli isolates. A group of 47 human clinical isolates, 37 isolates from retail poultry, and 29 isolates from living turkeys (total, 113 isolates) was included in the study. These encompassed 92 C. jejuni and 21 C. coli strains. The MICs of six antimicrobial agents were determined by the broth microdilution and E test methods, and the strains of human origin were additionally tested by the agar dilution method. In general, broth microdilution MICs agreed within 1 log(2) MIC increment with 90.0% of E test results and 78.7% of agar dilution test results. The agar dilution method gave much lower gentamicin MICs than the broth microdilution method, but the data were significantly (P < 0.01) correlated and there was 100% agreement in the sensitivities and specificities in the comparison of the tests. The broth microdilution method had the highest sensitivity for analysis of the susceptibilities of Campylobacter to nalidixic acid and trimethoprim-sulfamethoxazole. The MICs of ciprofloxacin and erythromycin complied numerically by all three methods. The classification of the results and the correlation of the data demonstrated a high degree of agreement. All methods were equally suitable for the testing of the sensitivity of Campylobacter to tetracycline. Thus, the broth microdilution method appears to be an easy and reliable method for determination of the MICs of antibiotics for C. jejuni and C. coli, and it may offer an interesting alternative to MIC determination by the agar dilution technique or the E test.

[Improving the embryonic stem cell test (EST) by establishing molecular endpoints of tissue specific development using murine embryonic stem cells (D3 cells)]. / Etablierung molekularer Endpunkte zur Weiterentwicklung des Embryonalen Stammzelltests (EST) mit embryonalen Stammzellen der Maus (Zelllinie D3).

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. In the embryonic stem cell test (EST) the capacity of ES cells of the mouse cell line D3 to differentiate into contracting cardiomyocytes is used to assess the embryotoxic potential of test compounds and in addition, the effects on the viability of ES cells and differentiated mouse fibroblasts (cell line 3T3) are compared. The three endpoints are used to classify the embryotoxic potential of chemicals after 10 days of exposure: (i) the inhibition of differentiation of ES cells into cardiomyocytes (ID50) and (ii) the decrease of viability of 3T3 cells (IC503T3) and (iii) ES cells (IC50D3) in a MTT cytotoxicity test. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed to assign test chemicals to three classes of embryotoxicity. In an international validation study funded by ECVAM it could be demonstrated that the EST can predict the embryotoxic potential of a test compound as good as frequently used mammalian systems based on pregnant animals. In a joint project with major German pharmaceutical companies we are attempting to improve the EST by establishing molecular endpoints of differentiation (e.g. cardiac, neuronal, chondrogenic) in cultured ES cells. We have studied the expression of tissue specific proteins in ES cell cultures in the presence of embryotoxic chemicals by immunofluorescent antibody techniques, e.g. FACS analysis. The other groups are focusing on endogenous gene expression in early development by RT-PCR methods or the DNA microarray technique. The results obtained recently using molecular markers specific for cardiac differentiation and employing intracellular flow cytometry for quantification will be presented. Molecular endpoints will allow improvement of the EST by measuring gene expression patterns in a small number of murine ES cells.

The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods.

From 1996 to 2000, ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, Germany) coordinated the European Centre for the Validation of Alternative Methods (ECVAM) prevalidation and validation study on three embryotoxicity tests: a) a test employing embryonic stem cell lines (EST); b) the micromass (MM) test; and c) the postimplantation rat whole-embryo culture assay (WEC test). The main objectives of the study were to assess the performance of these three in vitro tests in discriminating between non- embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. Phase I of the study (1997) was designed as a prevalidation phase, for test protocol optimisation, and for the establishment of a comprehensive database of in vivo and in vitro data on embryotoxic compounds. Phase II (1998-2000) involved a formal validation trial, conducted under blind conditions on 20 test compounds selected from the database, which were coded and distributed to the participating laboratories. In the preliminary phase of the validation study, six chemicals out of the 20, which showed embryotoxic potential, were tested. These results were used to define new biostatistically based prediction models (PMs) for the MM and WEC tests, and to evaluate those developed previously for the EST. As a next step, the PMs were evaluated by using the results for the remaining 14 chemicals of the definitive phase of the validation study. The three in vitro embryotoxicity tests proved to be applicable to testing a diverse group of chemicals with different embryotoxic potentials (non-embryotoxic, weakly embryotoxic, and strongly embryotoxic). The reproducibility of the three in vitro embryotoxicity tests were acceptable according to the acceptance criteria defined by the Management Team. The concordances between the embryotoxic potentials derived from the in vitro data and from the in vivo data were good for the EST and the WEC (PM2) test, and sufficient for the MM test and the WEC (PM1) tests according to the performance criteria defined by the Management Team before the formal validation study. When applying the PM of the EST to the in vitro data obtained in the definitive phase of the formal validation study, chemicals were classified correctly in 78% of the experiments. For the MM and the WEC tests, the PMs provided 70% and 80% (PM2) correct classifications, respectively. And, very importantly, an excellent predictivity (100%, except for PM1 of the WEC test, with 79%, considered as good) was obtained with strong embryotoxic chemicals in each of the three in vitro tests.