The need for a formal invalidation process for animal and non-animal tests

A plethora of regulations require that many chemicals and chemical products are tested for efficacy and/or toxicity. When permitted to operate effectively and without bias, the ECVAM/ICCVAM/OECD validation process can be used to independently establish that new animal and non-animal test procedures are sufficiently relevant and reliable for their stated purposes and should be considered for regulatory use. However, the validation process is under threat because of vested interests of various kinds, and it is clear that many currently-accepted animal tests and candidate animal and non-animal tests do not, and could never, meet the agreed criteria for necessity, test development, prevalidation, validation and acceptance. We therefore need an invalidation process to parallel and protect the validation process, so that such methods could be independently reviewed and declared irrelevant and/or unreliable for their claimed purposes. An additional advantage of such a process would be that valuable resources would no longer be wasted in attempts to secure the acceptance of inherently inadequate tests.     

ECVAM's activities in validating alternative tests for skin corrosion and irritation

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commercially-produced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.     

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.     

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.     

Selection of test chemicals for the ECVAM international validation study on in vitro embryotoxicity tests. European Centre for the Validation of Alternative Methods

The European Centre for the Validation of Alternative Methods (ECVAM) has sponsored a large international prevalidation and validation study of three embryotoxicity tests, involving embryonic stem cells, limb bud micromass cultures, and post-implantation whole-embryo cultures. The main objective of the study was to assess the performance of these in vitro tests in discriminating between non-embryotoxic, weakly embryotoxic and strongly embryotoxic compounds. An initial part of the study was to select 20 test substances for the formal validation trial, conducted under blind conditions. A database of in vivo and in vitro developmental toxicity test results was complied on 310 chemicals that had been used in previous validation studies, or suggested for such use, or that had good quality "segment II"-type in vivo data, or for which there were human data. From this database, a shortlist of about 30 candidates was constructed. Because the ECVAM study would not include metabolic activation, chemicals known to require activation for their developmental effects were excluded as candidates, although some known stable metabolites were included. Attempts were made: to include substances of diverse mechanism; to avoid overemphasis on pharmaceuticals; to avoid biologically inert substances as non-embryotoxicants; and to make the list different from those used previously. The candidates were of three categories: Class 3, strongly embryotoxic, was defined as developmentally toxic in all species tested, inducing multiple developmental effects, and with a high A/D ratio. Class 1, non-embryotoxic, was defined as not developmentally toxic at maternally toxic exposures, but which may show some minor embryo/fetal toxicity, which cannot be separated from maternal toxicity. Class 2, weakly embryotoxic, were chemicals of intermediate activity. From this candidate list, chemicals of known receptor (androgen, oestrogen, glucocorticoid, aryl hydrocarbon) mechanisms were excluded, on the basis that simple tests for such activity are already available. In addition, chemicals not freely available were excluded, and an emphasis on human data was applied. The final list of 20 chemicals was: Class 3--6-aminonicotinamide, 5-bromo- 2'-deoxyuridine, hydroxyurea, methylmercury chloride, methotrexate, all-trans-retinoic acid; Class 2--boric acid, dimethadione, lithium chloride, methoxyacetic acid, valproic acid (VPA), 2-propyl-4-pentynoic acid (4-yn-VPA), salicylic acid sodium salt; and Class 1--acrylamide, D-(+)-camphor, dimethyl phthalate, diphenhydramine hydrochloride, 2-ethyl-4- methylpentanoic acid (isobutyl-ethyl-VPA), Penicillin G sodium salt, saccharin sodium hydrate.     

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.     

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

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.     

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.     

Current practices and prospects for standardization of the hematopoietic colony-forming unit assay: a report by the cellular therapy team of the Biomedical Excellence for Safer Transfusion (BEST) Collaborative

Background aimsWide acceptance of the colony-forming unit (CFU) assay as a reliable potency test for stem cell products is hindered by poor inter-laboratory reproducibility. The goal of this study was to ascertain current laboratory practices for performing the CFU assay with an eye towards identifying practices that could be standardized to improve overall reproducibility.MethodsA survey to evaluate current laboratory practices for performing CFU assays was designed and internationally distributed.ResultsThere were 105 respondents to the survey, of whom 68% performed CFU assays. Most survey recipients specified that an automated rather than a manual cell count was performed on pre-diluted aliquots of stem cell products. Viability testing methods employed various stains, and when multiple sites used the same viability stain, the methods differed. Cell phenotype used to prepare working cell suspensions for inoculating the CFU assay differed among sites. Most respondents scored CFU assays at 14–16 days of incubation, but culture plates were read with various microscopes. Of 57 respondents, 42% had not performed a validation study or established assay linearity. Only 63% of laboratories had criteria for determining if a plate was overgrown with colonies.ConclusionsSurvey results revealed inconsistent inter-laboratory practices for performing the CFU assay. The relatively low number of centers with validated CFU assays raises concerns about assay accuracy and emphasizes a need to establish central standards. The survey results shed light on numerous steps of the methodology that could be targeted for standardization across laboratories.     

Report of the EPAA-ECVAM workshop on the validation of Integrated Testing Strategies (ITS)

The use of Integrated Testing Strategies (ITS) permits the combination of diverse types of chemical and toxicological data for the purposes of hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with the European Centre for the Validation of Alternative Methods (ECVAM), held a workshop on Overcoming Barriers to Validation of Non-animal Partial Replacement Methods/Integrated Testing Strategies, in Ispra, Italy, to discuss the extent to which current ECVAM approaches to validation can be used to evaluate partial replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusions of these discussions were that formal validation was only considered necessary for regulatory purposes (e.g. the replacement of a test guideline), and that current ECVAM approaches to validation should be adapted to accommodate such test methods. With these conclusions in mind, a follow-up EPAA-ECVAM workshop was held in October 2009, to discuss the extent to which existing validation principles are applicable to the validation of ITS test methods, and to develop a draft approach for the validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions that started with a review of the current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity), before covering the definition of ITS and their components, including their validation and regulatory acceptance. The following main conclusions/recommendations were made: that the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity; that, in the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, especially if the individual test methods had a high biological relevance; that ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of clearly defined ITS would be feasible, although practically quite difficult; and that test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed as parts of testing strategies. The added value from the formal validation of testing strategies, and the requirements needed in view of regulatory acceptance of the data, require further informed discussion within the EPAA forum on the basis of case studies provided by industry.     

A critical assessment of the OECD collaborative study to validate the uterotrophic assay for the detection of oestrogenic and anti-oestrogenic chemicals

The design and execution of a recently completed validation study on the rat uterotrophic assay for detecting oestrogens and anti-oestrogens, managed by the OECD, are critically assessed with respect to internationally agreed criteria for the validation of new in vitro and in vivo toxicity test methods. It is concluded that, while the design of the study did not take account of several important criteria for validation, the uterotrophic assay appears to reliably detect the strong and weak oestrogenic substances used in the study, which act via binding to the oestrogen receptor in vivo. However, the reliability of the assay has not been substantiated for detecting anti-oestrogens that act as antagonists, due to the involvement of an insufficient number of experiments and test chemicals. Moreover, the data do not permit an assessment of the accuracy of the prediction of oestrogenicity, and the protocols have not been sufficiently optimised with regard to controlling variables. This problem has been exacerbated by a wish to introduce as much flexibility as possible into the protocols during the formal validation phase of the study, rather than during a separate prevalidation stage. In addition, the choice between surgically treated and/or immature animals, and details of housing and husbandry conditions that are necessary for increasing the sensitivity and efficiency of the assay, need to be clarified. The assay also lacks a well-defined prediction model by which the overall relevance of the data to toxicity, and especially to human hazard, can be assessed, and no performance criteria have been established. The results of this analysis of the study indicate that it would be premature to produce an OECD test guideline for the uterotrophic assay at this time, before some of the above issues have been satisfactorily resolved.     

Peer review of validation studies: an assessment of the role of the OECD by reference to the validation of the uterotrophic assay for endocrine disruptors

The involvement of the OECD in managing the validation of the rat uterotrophic assay for endocrine disruptors, and in organising the peer review of the results of this study, has been assessed and compared with the many conclusions and recommendations in several published reports of international workshops on validation, and information in guidance documents, produced by the European Centre for the Validation of Alternative Methods (ECVAM), the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the OECD itself. It is concluded that the OECD has not followed the recommendations for full transparency and independence of the peer-review process. This is based on the fact that it has published a draft guidance document that differs from the report of a recent OECD workshop on validation, in such a way as to give the OECD the flexibility to fully control the peer-review process and, in so doing, to avoid full transparency. Comparison of the timing of the organisation of workshops by the OECD and the progression of the validation study, together with the fact that a draft test guideline for the assay was written before completion of the peer review, suggest that the OECD has given a higher priority to the expedition of the validation and regulatory acceptance of the uterotrophic assay than it has to good scientific and logistical practice. This severely undermines its credibility in the validation process, so, in order for the OECD to be rightly perceived as an honest broker, it is recommended that the OECD should play no role in the validation of new or revised tests, until after they have been successfully validated, peer reviewed, and endorsed by the appropriate authorities, and are ready for test guideline development. With regard to the on-going OECD validation studies of other in vivo assays for endocrine disruptors, the OECD should take immediate steps to ensure full independence and transparency of their peer review.     

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

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.     

In-house validation of the EpiOcular(TM) eye irritation test and its combination with the bovine corneal opacity and permeability test for the assessment of ocular irritation

In 2009, the Bovine Corneal Opacity and Permeability (BCOP) test was accepted by the regulatory bodies for the identification of corrosive and severe ocular irritants (Global Harmonised System [GHS] Category 1). However, no in vitro test is currently accepted for the differentiation of ocular irritants (GHS Category 2) and non-irritants (GHS No Category). Human reconstructed tissue models have been suggested for incorporation into a tiered testing strategy to ultimately replace the Draize rabbit eye irritation test (OECD TG 405). The purpose of this study was to evaluate whether the EpiOcular(TM) reconstructed cornea-like tissue model and the COLIPA pre-validated EpiOcular Eye Irritation Test (EpiOcular-EIT) could be used as suitable components of this testing strategy. The in-house validation of the EpiOcular-EIT was performed by using 60 test substances, including a broad variety of chemicals and formulations for which in vivo data (from the Draize rabbit eye irritation test) were available. The test substances fell into the following categories: 18 severe irritants/corrosives (Category 1), 21 irritants (Category 2), and 21 non-irritants (No Category). Test substances that decreased tissue viability to ≤ 60% (compared to the negative control tissue) were considered to be eye irritants (Category 1/2). Test substances resulting in tissue viability of > 60% were considered to be non-irritants (No Category). For the assessed dataset and the classification cut-off of 60% viability, the EpiOcular-EIT provided 98% and 84% sensitivity, 64% and 90% specificity, and 85% and 86% overall accuracy for the literature reference and BASF proprietary substances, respectively. Applying a 50% tissue viability cut-off to distinguish between irritants and non-irritants resulted in 93% and 82% sensitivity, 68% and 100% specificity, and 84% and 88% accuracy for the literature reference and BASF proprietary substances, respectively. Further, in the EpiOcular-EIT (60% cut-off), 100% of severely irritating substances under-predicted by the BCOP assay were classified as Category 1/2. The results obtained in this study, based on 60 test substances, indicate that the EpiOcular-EIT and the BCOP assay can be combined in a testing strategy to identify strong/severe eye irritants (Category 1), moderate and mild eye irritants (Category 2), and non-irritants (No Category) in routine testing. In particular, when the bottom-up strategy with the 60% viability cut-off was employed, none of the severely irritating substances (Category 1) were under-predicted to be non-irritant. Sensitivity for Category 1/2 substances was 100% for literature reference substances and 89% for BASF SE proprietary substances.     

Antroquinonol inhibits NSCLC proliferation by altering PI3K/mTOR proteins and miRNA expression profiles

The European Cosmetic Toiletry and Perfumery Association (COLIPA), along with contributions from the European Centre for the Validation of Alternative Methods (ECVAM), initiated a multi-lab international prevalidation project on the reconstructed skin micronucleus (RSMN) assay in EpiDerm? for the assessment of the genotoxicity of dermally applied chemicals. The first step of this project was to standardize the protocol and transfer it to laboratories that had not performed the assay before. Here we describe in detail the protocol for the RSMN assay in EpiDerm? and the harmonized guidelines for scoring, with an atlas of cell images. We also describe factors that can influence the performance of the assay. Use of these methods will help new laboratories to conduct the assay, thereby further increasing the database for this promising new in vitro genotoxicity test.     

Bioassays for risk assessment of coal conversion products

Traditional as well as biotechnological processing of coal leads to complex mixtures of products. Besides chemical and physical characterization, which provides the information for product application, there is a need for bioassays to monitor properties that are probably toxic, mutagenic or cancerogenic. Investigations carried out focused on the selection, adaptation and validation of bioassays for the sensitive estimation of toxic effects. Organisms like bacteria, Daphnia magna and Scenedesmus subspicatus, representing different complexities in the biosphere, were selected as test systems for ecotoxicological and mutagenicity studies. The results obtained indicate that bioassays are, in principle, suitable tools for characterization and evaluation of coal-derived substances and bioconversion products. Using coal products, coal-relevant model compounds and bioconversion products, data for risk assessment are presented. Received: 17 June 1998 / Received revison: 21 October 1998 / Accepted: 24 October 1998     

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.     

Use of substances of animal origin in pharmaceutics and compliance with the TSE-risk guideline -- a market survey.

The risk of infection with transmissible spongiform encephalopathy (TSE) from the use of animal-derived substances in drug manufacturing was assessed [Swissmedic. TSE guideline to the ordinance. 2001; http://www.swissmedic.ch/files/pdf/Anleitung_TSE.pdf; The European Directorate for the Quality of Medicines. Minimising the risk of transmitting animal spongiform encephalopathy agents via medicinal products. In: European Pharmacopoeia 2002; General chapter 5.2.8. http://www.pheur.org/medias/download/50208E.pdf]. In addition, the compliance of the Swiss Market Authorisation holders to implement the Swissmedic TSE guidelines was examined. To assess the compliance with the TSE guideline for products on the Swiss market a representative number of drugs were selected based on a statistical approach. The documents for the biological, anatomical and geographical origin of the animal substances used for drug manufacturing as well as for the manufacturing processes were requested to be provided within a short response period and were subsequently reviewed. A total of 438 drugs with 655 substances of animal origin were assessed during the survey. The documentation provided by the Market Authorisation holders showed, that in general the measures described in the TSE guidelines were implemented well. Therefore, the risk of these pharmaceutics to transmit TSE was considered minimized. However, the TSE documentation provided by drug companies was incomplete for approximately two-third of the drugs at the beginning of the survey. In particular for those containing excipients such as tallow derivatives or gelatine, numerous clarifications were needed prior assessment. The efforts of the drug companies to correspond to regulatory actions and to implement authoritative guidelines should be improved.     

Novel cytotoxicity test based on menadione-catalyzed H2O2 productivity for food safety evaluation

Menadione-catalyzed H₂O₂ production by viable cells was proportional to viable cell number, and the assay of this H₂O₂ production was applied to the cytotoxicity test of 17 substances which were used for international validation of fixed-dose procedure as an alternative to the classical LD₅₀ test. The cytotoxicity of substances tested was observed 4 h after the incubation with animal cells, and the viability was determined in 10 min according to menadione-catalyzed H₂O₂ production assay. IC₅₀ of each substance required for 50% inhibition of menadione-catalyzed H₂O₂ production was similar among HepG2, HuH-6KK, HUVE, Vero, Intestine407, NIH/3T3 and Neuro-2a cells. Twelve substances, 3 substances and 2 substances showed the difference of one, two and three orders in the magnitude between LD₅₀ and IC₅₀, respectively. These results show that menadione-catalyzed H₂O₂ production assay is useful for the rapid detection of toxic compounds having the basal cytotoxicity common to various cells, but is unfit for the detection of organ-specific toxic compounds. This revised version was published online in August 2006 with corrections to the Cover Date.     

Comparison and validation of novel pyrogen tests based on the human fever reaction

The Limulus amoebocyte lysate (LAL) test has replaced about 80% of the use of the rabbit pyrogen test. Ideally, human-based in vitro tests are needed, to replace the remaining use of the rabbit test and the use of the LAL test. The progress of an EU-funded project is described, in which a number of in vitro tests, based on the human fever reaction are passing through a prevalidation study on the way to evaluation in a formal validation study.