Integrated decision-tree testing strategies for developmental and reproductive toxicity with respect to the requirements of the EU REACH legislation

Liverpool John Moores University and FRAME 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 paper focuses on the prospects for the use of alternative methods (both in vitro and in silico) in developmental and reproductive toxicity testing. It considers many tests based on primary cells and cell lines, and the available expert systems and QSARs for developmental and reproductive toxicity, and also covers tests for endocrine disruption. Ways in which reduction and refinement measures can be used are also discussed, particularly the use of an enhanced one-generation reproductive study, which could potentially replace the two-generation study, and therefore considerably reduce the number of animals required in reproductive toxicity. Decision-tree style integrated testing strategies are also proposed for developmental and reproductive toxicity and for endocrine disruption, followed by a number of recommendations for the future facilitation of developmental and reproductive toxicity testing, with respect to human risk assessment.     

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.     

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.     

Integrated testing strategies for use in the EU REACH system

Integrated testing strategies have been proposed to facilitate the process of chemicals risk assessment to fulfil the requirements of the proposed EU REACH system. Here, we present individual, decision-tree style, strategies for the eleven major toxicity endpoints of the REACH system, including human health effects and ecotoxicity. These strategies make maximum use of non-animal approaches to hazard identification, before resorting to traditional animal test methods. Each scheme: a) comprises a mixture of validated and non-validated assays (distinguished in the schemes); and b) decision points at key stages to allow the cessation of further testing, should it be possible to use the available information to classify and label and/or undertake risk assessment. The rationale and scientific justification for each of the schemes, with respect to the validation status of the tests involved and their individual advantages and limitations, will be discussed in detail in a series of future publications.     

Introduction to the EU REACH legislation

FRAME initiatives on the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals, first proposed as a White Paper in 2001, are summarised. These initiatives considered the scientific and animal welfare issues raised by the REACH proposals, and resulted in a number of suggestions for improvement, many of which seem to have been adopted during the current progress of the legislation through the European Council and European Parliament.     

Development of the EpiOcular(TM) eye irritation test for hazard identification and labelling of eye irritating chemicals in response to the requirements of the EU cosmetics directive and REACH legislation

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular(TM) eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients (r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.     

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.     

An overall strategy for the testing of chemicals for human hazard and risk assessment under the EU REACH system

In its White Paper, Strategy for a Future Chemicals Policy, published in 2001, the European Commission (EC) proposed the REACH (Registration, Evaluation and Authorisation of CHemicals) system to deal with both existing and new chemical substances. This system is based on a top-down approach to toxicity testing, in which the degree of toxicity information required is dictated primarily by production volume (tonnage). If testing is to be based on traditional methods, very large numbers of laboratory animals could be needed in response to the REACH system, causing ethical, scientific and logistical problems that would be incompatible with the time-schedule envisaged for testing. The EC has emphasised the need to minimise animal use, but has failed to produce a comprehensive strategy for doing so. The present document provides an overall scheme for predictive toxicity testing, whereby the non-animal methods identified and discussed in a recent and comprehensive ECVAM document, could be used in a tiered approach to provide a rapid and scientifically justified basis for the risk assessment of chemicals for their toxic effects in humans. The scheme starts with a preliminary risk assessment process (involving available information on hazard and exposure), followed by testing, based on physicochemical properties and (Q)SAR approaches. (Q)SAR analyses are used in conjunction with expert system and biokinetic modelling, and information on metabolism and identification of the principal metabolites in humans. The resulting information is then combined with production levels and patterns of use to assess potential human exposure. The nature and extent of any further testing should be based strictly on the need to fill essential information gaps in order to generate adequate risk assessments, and should rely on non-animal methods, as far as possible. The scheme also includes a feedback loop, so that new information is used to improve the predictivity of computational expert systems. Several recommendations are made, the most important of which is that the European Union (EU) should actively promote the improvement and validation of (Q)SAR models and expert systems, and computer-based methods for biokinetic modelling, since these offer the most realistic and most economical solution to the need to test large numbers of chemicals.     

An overall strategy for the testing of chemicals for human hazard and risk assessment under the EU REACH system

In its White Paper, "Strategy for a Future Chemicals Policy," published in 2001, the European Commission (EC) proposed the REACH (Registration, Evaluation and Authorisation of CHemicals) system to deal with both existing and new chemical substances. This system is based on a top-down approach to toxicity testing, in which the degree of toxicity information required is dictated primarily by production volume (tonnage). If testing is to be based on traditional methods, very large numbers of laboratory animals could be needed in response to the REACH system, causing ethical, scientific and logistical problems that would be incompatible with the time-schedule envisaged for testing. The EC has emphasised the need to minimise animal use, but has failed to produce a comprehensive strategy for doing so. The present document provides an overall scheme for predictive toxicity testing, whereby the non-animal methods identified and discussed in a recent and comprehensive ECVAM document, could be used in a tiered approach to provide a rapid and scientifically justified basis for the risk assessment of chemicals for their toxic effects in humans. The scheme starts with a preliminary risk assessment process (involving available information on hazard and exposure), followed by testing, based on physicochemical properties and (Q)SAR approaches. (Q)SAR analyses are used in conjunction with expert system and biokinetic modelling, and information on metabolism and identification of the principal metabolites in humans. The resulting information is then combined with production levels and patterns of use to assess potential human exposure. The nature and extent of any further testing should be based strictly on the need to fill essential information gaps in order to generate adequate risk assessments, and should rely on non-animal methods, as far as possible. The scheme also includes a feedback loop, so that new information is used to improve the predictivity of computational expert systems. Several recommendations are made, the most important of which is that the European Union (EU) should actively promote the improvement and validation of (Q)SAR models and expert systems, and computer-based methods for biokinetic modelling, since these offer the most realistic and most economical solution to the need to test large numbers of chemicals.     

Co-occurring words and retrieval efficiency: finding information about alternatives to animal testing in relation to skin irritation testing

Words appearing in abstracts of scientific articles are often useful as search terms, particularly those words and word patterns that are unique to the relevant field of endeavour. In view of the heightened interest in obtaining information about alternatives to animal testing, efforts directed toward enhancing retrieval of pertinent references from the biomedical literature are warranted. Words and phrases, and word-phrase co-occurrences describing methods of experimentation in abstracts about alternatives to skin-irritation testing in animals, were evaluated with regard to retrieval efficiency in the National Library of Medicine database, Toxline(. Precision of retrieval was defined as the number of pertinent references found in the total number of citations retrieved. Retrieval precision values ranged from 0.25% to 100%.     

Integrated testing strategies for toxicity employing new and existing technologies

We have developed individual, integrated testing strategies (ITS) for predicting the toxicity of general chemicals, cosmetics, pharmaceuticals, inhaled chemicals, and nanoparticles. These ITS are based on published schemes developed previously for the risk assessment of chemicals to fulfil the requirements of REACH, which have been updated to take account of the latest developments in advanced in chemico modelling and in vitro technologies. In addition, we propose an ITS for neurotoxicity, based on the same principles, for incorporation in the other ITS. The technologies are deployed in a step-wise manner, as a basis for decision-tree approaches, incorporating weight-of-evidence stages. This means that testing can be stopped at the point where a risk assessment and/or classification can be performed, with labelling in accordance with the requirements of the regulatory authority concerned, rather than following a checklist approach to hazard identification. In addition, the strategies are intelligent, in that they are based on the fundamental premise that there is no hazard in the absence of exposure - which is why pharmacokinetic modelling plays a key role in each ITS. The new technologies include the use of complex, three-dimensional human cell tissue culture systems with in vivo-like structural, physiological and biochemical features, as well as dosing conditions. In this way, problems of inter-species extrapolation and in vitro/in vivo extrapolation are minimised. This is reflected in the ITS placing more emphasis on the use of volunteers at the whole organism testing stage, rather than on existing animal testing, which is the current situation.     

Why 'suitable' in vitro methods, as defined in the final EU REACH legislation, are an inappropriate basis for risk assessment

The final text of the REACH legislation, that has recently been published by the European Parliament, and which will come into force in the middle of 2007, implies that results obtained from so-called 'suitable' in vitro methods could be used for testing. It is also stated that a 'suitable' test is one that has been deemed to be ready to enter a validation study - for example, according to criteria specified by ECVAM. It is argued that, because the wording of the legal text is too vague and imprecise, it is totally unsatisfactory, mainly because it seems to endorse the application of non-validated methods for regulatory risk assessment. While their use might be suitable for providing information for classification in a weight-of-evidence approach, setting safe dose levels should only ever be based on the use of data from a properly validated test method. This concern, and other problems raised by the final legislation, is discussed, and recommendations are made which could avoid the premature use of potentially unsuitable methods for risk assessment, whilst ensuring that potentially suitable tests can be used for this purpose, once they have been formally validated.     

Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles

An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural requirements of mitochondrial D-beta-hydroxybutyrate dehydrogenase with respect to its coenzyme

The purpose of this work was to test structural analogs of NAD+ in order to know enzyme requirements of chemical structure of coenzyme to get catalytic activity and, in a other hand to see which chemical parts of the coenzyme were involved in the coenzyme binding to the active site. The binding of the coenzyme analog at the catalytic site requires an adenosine diphosphoribose structure without any additional phosphate group on the ribose linked to adenine.     

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.     

A critical evaluation of the 2011 ECHA reports on compliance with the REACH and CLP regulations and on the use of alternatives to testing on animals for compliance with the REACH regulation

On 30 June 2011, the European Chemicals Agency published two reports, one on the functioning of the REACH system, the other on the use of alternatives to animal testing in compliance with that system. The data presented are based on information gained during the first registration period under the REACH system, which included high production volume chemicals and substances of very high concern, which have the most extensive information requirements. A total of 25,460 registration dossiers were received, covering 3,400 existing, so-called 'phase-in', substances, and 900 new, so-called 'non-phase-in', substances. Data sharing and the joint submission of data are reported to have worked successfully. In the registration dossiers for these substances, results from new animal tests were included for less than 1% of all the endpoints; testing proposals (required for 'higher-tier' information requirements) were submitted for 711 in vivo tests involving vertebrate animals. The registrants mainly used old, existing experimental data, or options for the adaptation (waiving) of information requirements, before collecting new information. For predicting substance toxicity, 'read-across' was the second most-used approach, followed by 'weight-of-evidence'. In vitro toxicity tests played a minor role, and were only used when the respective test methods had gained the status of regulatory acceptance. All in all, a successful start to the REACH programme was reported, particularly since, in contrast to most predictions, it did not contribute to a significant increase in toxicity testing in animals.