Metabolism-mediated neurotoxicity: the significance of genetically engineered cell lines and new three-dimensional cell cultures

Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process. Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.     

MEIC—A new international multicenter project to evaluate the relevance to human toxicity of in vitro cytotoxicity tests

A new international project to evaluate the relevance for human systemic and local toxicity of in vitro tests of general toxicity of chemicals has been organized by the Scandinavian Society of Cell Toxicology under the title Multicenter Evaluation of In Vitro Cytotoxicity (MEIC). The basic assumptions underlying the project, as well as the practical goals and the design of the program are outlined. The list of the first 50 reference chemicals is presented. The chemicals are an otherwise unbiased selection of compounds with known human acutely lethal dosage and blood concentrations, including LD50-values in the rat or mouse. Most agents also have other data on human toxicity and toxicokinetics, including more extensive animal toxicity data. International laboratories already using or developing in vitro tests of various partial aspects of general toxicity are invited to test the substances, the results of which will be evaluated by us. The predictivity of the in vitro results for both partial and gross human toxicity data will be determined with combined use of univariate regression analysis and soft multivariate modeling. The predictivity of the in vitro results will be compared with the predictivity of conventional animal tests for the same chemicals. Finally, batteries of tests with optimal prediction power for various types of human toxicity will be selected. The need for and possible uses of such batteries are discussed.     

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.     

Conformational changes in proteins in vitro as a means of predicting the acute toxicities of chemicals

Experimental data are presented on ovalbumin denaturation (OD, EC10) and human acetylcholine esterase (AChE) inhibition (IC50) in vitro, following exposure to the chemicals used in the international Multicentre Evaluation of In vitro Cytotoxicity (MEIC) programme. Data were obtained for 40 (OD test) and 43 (AChE test) of the 50 MEIC chemicals. These data were compared with similar data from other methods used in the MEIC programme, and good correlations (R2) were obtained with data from MEIC studies on cell lines: 0.80 for human, 0.81 for other animal, and 0.78 for fish cell line IC50 values and AChE values, and 0.76 for human, 0.69 other animal and 0.75 for fish cell line IC50 values and OD values. The correlation increased substantially, if chemicals which freely cross the blood-brain barrier were solely considered, with R2 = 0.90 for human, 0.90 for other animal, and 0.82 for fish cell line IC50 values and AchE values, and 0.87 for human, 0.86 for other animal, and 0.92 for fish cell line IC50 values and OD values, in this case. Such chemicals are the main cause of non-specific depression of the central nervous system (CNS). The AChE IC50 permits a good prediction of human acute toxicity, similar to the IC50 values obtained with human cell lines and the same MEIC chemicals. These results confirm the basal toxicity hypothesis formulated by Bj?rn Ekwall. It is concluded that in vitro methods based on the disruption of the functions of the proteins vital for body operation can be used as an alternative to the cell culture methods, when non-specific toxic effects of chemicals on humans and animals are evaluated.     

Proteomic analysis and in vitro developmental toxicity tests for mechanism-based safety evaluation of chemicals

Mechanism-based safety evaluation and reduction of animal use are important issues in recent developmental toxicology. In vitro developmental toxicity tests with proteomic analysis are the most promising solution to these issues. Groebe et al. systematically applied proteomic analysis to the embryonic stem cell test, a validated in vitro developmental toxicity test, and found protein-expression changes induced by model test chemicals selected from various categories of toxicity. Cluster analysis of all the proteins with expression changes classified the test chemicals into two groups: highly embryotoxic chemicals and non- or weakly embryotoxic chemicals. In addition, some protein biomarker candidates that were known to be involved in normal development were identified. Although further mechanistic investigations are needed, the use of in vitro developmental toxicity tests with proteomic analysis will contribute to mechanism-based safety evaluation with minimal use of animals.     

Toxicity testing in the 21 century: defining new risk assessment approaches based on perturbation of intracellular toxicity pathways

The approaches to quantitatively assessing the health risks of chemical exposure have not changed appreciably in the past 50 to 80 years, the focus remaining on high-dose studies that measure adverse outcomes in homogeneous animal populations. This expensive, low-throughput approach relies on conservative extrapolations to relate animal studies to much lower-dose human exposures and is of questionable relevance to predicting risks to humans at their typical low exposures. It makes little use of a mechanistic understanding of the mode of action by which chemicals perturb biological processes in human cells and tissues. An alternative vision, proposed by the U.S. National Research Council (NRC) report Toxicity Testing in the 21(st) Century: A Vision and a Strategy, called for moving away from traditional high-dose animal studies to an approach based on perturbation of cellular responses using well-designed in vitro assays. Central to this vision are (a) "toxicity pathways" (the innate cellular pathways that may be perturbed by chemicals) and (b) the determination of chemical concentration ranges where those perturbations are likely to be excessive, thereby leading to adverse health effects if present for a prolonged duration in an intact organism. In this paper we briefly review the original NRC report and responses to that report over the past 3 years, and discuss how the change in testing might be achieved in the U.S. and in the European Union (EU). EU initiatives in developing alternatives to animal testing of cosmetic ingredients have run very much in parallel with the NRC report. Moving from current practice to the NRC vision would require using prototype toxicity pathways to develop case studies showing the new vision in action. In this vein, we also discuss how the proposed strategy for toxicity testing might be applied to the toxicity pathways associated with DNA damage and repair.     

Trends in Risk Assessment of Chemicals in the European Union

Current legislation in the European Union (EU) requires a risk assessment for industrial chemicals. The underlying procedures and paradigms of such EU risk assessment for new and existing chemicals are explained. The risk assessment is performed according to a harmonised methodology, laid down in the Technical Guidance Documents (TGD). Important new, technical risk assessment aspects covered in a recent revision round of the TGD are highlighted. The most prominent change in the environmental TGD part is the addition of the marine risk assessment, including a Persistent Bioaccumulation and Toxicity (PBT) assessment. In the human health part a significant change is the new data requirement for reproductive toxicity. The performance of both the risk assessment and the risk reduction phase of EU existing chemicals have been evaluated. An important conclusion was that our a priori knowledge on possible risks of chemicals is poor. The European Commission has recently launched a proposal (REACH) for drastically changing the risk management process of industrial chemicals in the EU. Major changes are a shift in responsibility from authorities to industry (including downstream users) for the safe use of chemicals, an acceleration of data collection for ‘non-assessed’ chemicals, and an authorization step for substances of very high concern.     

In vitro phototoxicity testing: development and validation of a new concentration response analysis software and biostatistical analyses related to the use of various prediction models

As demonstrated in several validation studies, the dermal phototoxic potential of chemicals in humans can be effectively assessed by in vitro methods. The core of these methods is to monitor dose-response curves of a chemical in the absence and presence of light, to quantify the difference between these two curves by appropriate measures (either the photo-irritancy factor [PIF], or the mean photo effect [MPE]), and to use these measures as predictors of in vivo phototoxicity. We present new concentration-response analysis software for in vitro phototoxicity testing, which runs on current personal computers, and takes into account all the limitations identified when using a former program. We also demonstrate the validity and robustness of this new software by applying it retrospectively to all data available from two phases of the EU/COLIPA validation trial for the 3T3 neutral red update in vitro phototoxicity test. Some frequently raised questions pertaining to the use of prediction models in phototoxicity testing are addressed, including: the necessity of using prediction models based on a cut-off; whether it is justifiable to use sharp prediction cut-off values; whether there is a biostatistical justification for the highest concentration of the test chemical; and whether repeated testing of a chemical is required.     

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.     

Integrated decision-tree testing strategies for skin corrosion and irritation with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME recently conducted a research project, sponsored by DEFRA, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on how to maximise the use of alternative methods (both in vitro and in silico) for skin corrosion and irritation testing within a tiered testing strategy. It considers the latest developments in in vitro testing, with particular reference to the reconstituted skin models which have now been now been successfully validated and independently endorsed as suitable for both skin corrosivity and irritancy testing within the EU.     

The tenth anniversary of the Björn Ekwall memorial foundation

The Bj?rn Ekwall Memorial Foundation (BEMF) was initiated by the Scandinavian Society for Cell Toxicology in 2001, to honour the memory of Dr Bj?rn Ekwall (1940-2000) and to establish a prize, the Bj?rn Ekwall Memorial Award. The prize is awarded to scientists who have significantly contributed to the field of cell toxicology, and whose work is contributing toward the replacement of animal experiments by alternative toxicity tests. Over the past 10 years, the Bj?rn Ekwall Memorial Award has been presented annually. Bj?rn Ekwall, an outstanding Swedish cell toxicologist, was one of the pioneers in the development and application of alternative methods to animal tests in toxicology. All his scientific work was devoted to in vitro toxicology, and in particular, to the use of cultured human cells for the screening of toxic chemicals. In the middle of the 1980s, he initiated the international Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) project, to evaluate the usefulness of in vitro tests for the estimation of human acute systemic toxicity. To prove his "basal cytotoxicity concept", he established the MEMO database, in which data on the acutely toxic human blood concentrations of drugs and chemicals were collated from the literature and from clinical studies. He also initiated another project, Evaluation-Guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). The ideas from the EDIT project, together with those from the MEIC project, became the basis for today's international EU projects, e.g. ACuteTox, Sens-it-iv and ReProTect. In this article, 10 years after the start of the BEMF, the scientific achievements of each of the award winners in the field of in vitro toxicology are presented, together with a brief synopsis of their careers.     

Biostatistical methods for the validation of alternative methods for in vitro toxicity testing

Statistical methods for the validation of toxicological in vitro test assays are developed and applied. Validation is performed either in comparison with in vivo assays or in comparison with other in vitro assays of established validity. Biostatistical methods are presented which are of potential use and benefit for the validation of alternative methods for the risk assessment of chemicals, providing at least an equivalent level of protection through in vitro toxicity testing to that obtained through the use of current in vivo methods. Characteristic indices are developed and determined. Qualitative outcomes are characterised by the rates of false-positive and false-negative predictions, sensitivity and specificity, and predictive values. Quantitative outcomes are characterised by regression coefficients derived from predictive models. The receiver operating characteristics (ROC) technique, applicable when a continuum of cut-off values is considered, is discussed in detail, in relation to its use for statistical modelling and statistical inference. The methods presented are examined for their use for the proof of safety and for toxicity detection and testing. We emphasise that the final validation of toxicity testing is human toxicity, and that the in vivo test itself is only a predictor with an inherent uncertainty. Therefore, the validation of the in vitro test has to account for the vagueness and uncertainty of the "gold standard" in vivo test. We address model selection and model validation, and a four-step scheme is proposed for the conduct of validation studies. Gaps and research needs are formulated to improve the validation of alternative methods for in vitro toxicity testing.     

Workshop summary: Top concentration for in vitro mammalian cell genotoxicity assays; and report from working group on toxicity measures and top concentration for in vitro cytogenetics assays (chromosome aberrations and micronucleus)

The selection of maximum concentrations for in vitro mammalian cell genotoxicity assays was reviewed at the 5th International Workshop on Genotoxicity Testing (IWGT), 2009. Currently, the top concentration recommended when toxicity is not limiting is 10mM or 5mg/ml, whichever is lower. The discussion was whether to reduce the limit, and if so whether the 1mM limit proposed for human pharmaceuticals was appropriate for testing other chemicals. The consensus was that there was reason to consider reducing the 10mM limit, and many, but not all, attendees favored a reduction to 1mM. Several proposals are described here for the concentration limit. The in vitro cytogenetics expert working group also discussed appropriate measures and level of cytotoxicity. Data were reviewed from a multi-laboratory trial of the in vitro micronucleus (MN) assay with multiple cell types and several types of toxicity measurements. The group agreed on a preference for toxicity measures that take cell proliferation after the beginning of treatment into account (relative increase in cell counts, relative population doubling, cytokinesis block proliferation index or replicative index), and that this applies both to in vitro MN assays and to in vitro chromosome aberration assays. Since relative cell counts (RCC) underestimate toxicity, many group members favored making a recommendation against the use of RCC as a toxicity measure for concentration selection. All 14 chemicals assayed for MN induction in the multi-laboratory trial were detected without exceeding 50% toxicity by any measure, but some were positive only at concentrations with toxicity quite close to 50%. The expert working group agreed to accept the cytotoxicity range recommended by OECD guideline 487 (55±5% toxicity at the top concentration scored). This also reinforces the original intent of the guidance for the in vitro chromosome aberration assay, where ">50%" was intended to target the range close to 50% toxicity.     

Integrated decision-tree testing strategies for acute systemic toxicity and toxicokinetics with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME conducted a joint research project, sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for acute systemic toxicity and toxicokinetic testing. The paper reviews in vitro tests based on basal cytotoxicity and target organ toxicity, along with QSAR models and expert systems available for this endpoint. The use of PBPK modelling for the prediction of ADME properties is also discussed. These tests are then incorporated into a decision-tree style, integrated testing strategy, which also includes the use of refined in vivo acute toxicity tests, as a last resort. The implementation of the strategy is intended to minimise the use of animals in the testing of acute systemic toxicity and toxicokinetics, whilst satisfying the scientific and logistical demands of the EU REACH legislation.     

The case for taking account of metabolism when testing for potential endocrine disruptors in vitro

Legislation in the USA, Europe and Japan will require that chemicals are tested for their ability to disrupt the hormonal systems of mammals. Such chemicals are known as endocrine disruptors (EDs), and will require extensive testing as part of the new European Union Registration, Evaluation and Authorisation of Chemicals (REACH) system for the risk assessment of chemicals. Both in vivo and in vitro tests are proposed for this purpose, and there has been much discussion and action concerning the development and validation of such tests. However, to date, little interest has been shown in incorporating metabolism into in vitro tests for EDs, in sharp contrast to other areas of toxicity testing, such as genotoxicity, and, ironically, such in vitro tests are criticised for not modelling in vivo metabolism. This is despite the existence of much information showing that endogenous and exogenous steroids are extensively metabolised by Phase I and Phase II enzymes both in the liver and in hormonally active tissues. Such metabolism can lead to the activation or detoxification of steroids and EDs. The absence of metabolism from these tests could give rise to false-positive data (due to lack of detoxification) or false-negative data (lack of activation). This paper aims to explain why in vitro assays for EDs should incorporate mammalian metabolising systems. The background to ED testing, the test methods available, and the role of mammalian metabolism in the activation and detoxification of both endogenous and exogenous steroids, are described. The available types of metabolising systems are compared, and the potential problems in incorporating metabolising systems into in vitro tests for EDs, and how these might be overcome, are discussed. It is recommended that there should be: a) an assessment of the intrinsic metabolising capacity of cell systems used in tests for EDs; b) an investigation into the relevance of using the prostaglandin H synthase system for metabolising EDs; and c) a feasibility study into the generation of genetically engineered mammalian cell lines expressing specific metabolising enzymes, which could also be used to detect EDs.     

Spiked Sediment Toxicity Testing of Hydrophobic Organic Chemicals: Bioavailability,Technical Considerations,and Applications

Many evaluations estimating safe levels of hydrophobic organic chemicals in sediments do not account for confounding factors such as physical habitat quality or covariance among chemicals. Controlled experiments demonstrating cause and effect can be conducted with spiked sediment toxicity tests, but application of this methodology has been limited in part by concerns about chemical bioavailability and challenges in achieving target concentrations. Relevant literature was reviewed to assess the utility of standardizing sediment equilibration times; hydrophobicity, complex sediment characteristics, and temperature were identified as potentially equally important factors. Disequilibrium appears likely following limited equilibration time but should yield conservative toxicity test results relative to aged field sediments. Nominal and measured concentrations in over 20 published studies were compared to assess spiked chemical recovery (i.e., measured concentration/nominal concentration). Recovery varied substantially among studies and was not readily predictable based on spiking or extraction method, chemical properties, or measured sediment characteristics, although unmeasured differences between sediments appeared to be important. Factors affecting specific studies included chemical adsorption to glassware, biodegradation, and volatilization. Pre- and post-toxicity test analyses are recommended to confirm exposure concentrations. Studies with 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD) and hexachlorobenzene (HCB) exemplify the utility of verifying results of field studies using spiked sediment tests. Sediments spiked with these chemicals at concentrations greatly exceeding those in associated field studies caused no adverse effects in test organisms, demonstrating that other chemicals co-occurring in test sediment samples caused toxicity initially attributed to 2,3,7,8-TCDD and HCB in the field studies. Another key application of spiked sediment tests has been the investigation of TOC as the primary factor affecting bioavailability of hydrophobic organic chemicals. A review of LC50s for nine chemicals reported in 12 studies shows that comparable LC50s derived in different sediments generally agree within a factor of five when concentrations are normalized to a constant TOC. Additionally, use of spiked sediment toxicity testing to investigate toxicological interactions among chemicals provides a promising approach to improving the ability to predict sediment toxicity in the field.     

An integrated decision-tree testing strategy for repeat dose toxicity with respect to the requirements of the EU REACH legislation

This paper presents some results of a joint research project conducted by FRAME and Liverpool John Moores University, and sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with REACH. This paper focuses on the use of alternative (non-animal) methods (both in vitro and in silico) for repeat dose (sub-acute, sub-chronic and chronic) toxicity testing. It reviews the limited number of in silico and in vitro tests available for this endpoint, and outlines new technologies which could be used in the future, e.g. the use of biomarkers and the 'omics' technologies. An integrated testing strategy is proposed, which makes use of as much non-animal data as possible, before any essential in vivo studies are performed. Although none of the non-animal tests are currently undergoing validation, their results could help to reduce the number of animals required for testing for repeat dose toxicity.     

Reduction of misleading ("false") positive results in mammalian cell genotoxicity assays. II. Importance of accurate toxicity measurement

In a previous publication, Fowler et al. [4] demonstrated that the seemingly high rate of false or misleading positive results obtained in in vitro cytogenesis assays for genotoxicity - when compared with in vivo genotoxicity or rodent carcinogenicity data - was greater when rodent cell lines were used that were also reported to have mutant or non-functional p53. As part of a larger project for improvement of in vitro mammalian cell assays, we have investigated the impact of different toxicity measures, commonly used in in vitro cytogenetic assays, on the occurrence of misleading positive results. From a list of 19 chemicals that produce "false" positive results in in vitro mammalian cell assays [10], six substances that had given positive responses in CHO, CHL and TK6 cells [4], were evaluated for micronucleus induction in vitro, with different measures of toxicity for selection of the top concentration. The data show that estimating toxicity by relative cell count (RCC) or replication index (RI) consistently underestimates the toxicity observed by other measures (Relative Population Doubling, RPD, or Relative Increase in Cell Count, RICC). RCC and RI are more likely to lead to selection of concentrations for micronucleus scoring that are highly cytotoxic and thus could potentially lead to artefacts of toxicity being scored (elevated levels of apoptosis and necrosis), generating misleading positive results. These results suggest that a further reduction in the frequency of misleading positive results in in vitro cytogenetic assays can be achieved with this set of chemicals, by avoiding the use of toxicity measures that underestimate the level of toxicity induced.