EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity Assay for Acute Oral Toxicity Testing

Acute oral toxicity is currently being assessed by a suite of refinement test methods based on the traditional LD50 lethality test and is, besides skin sensitisation, the only remaining animal test required under REACH Annex VII. In view of assessing the use of alternatives for this health endpoint, EURL ECVAM conducted a study on the 3T3 Neutral Red Uptake cytotoxicity test method addressing the method's capacity to support specifically the identification substances not requiring classification as acute toxicants. Following independent scientific peer review of this study by EURL ECVAM's scientific advisory committee (ESAC) and having considered input from regulators, stakeholders, international partners and the general public, EURL ECVAM concludes that the 3T3 NRU test method may prove a valuable component of a WoE or ITS approach for supporting hazard identification and safety assessment in agreement with the EU CLP Regulation and international regulatory schemes implementing the upper threshold of UN GHS Category 4 as the cut-off for non-classification of substances. In particular, data from the 3T3 NRU assay may constitute an information source within a WoE approach under the provisions of the REACH regulation (Annex XI, 1.2) potentially supporting conclusions on absence of acute oral toxicity of industrial chemicals.JRC.I.5-Systems Toxicolog

EURL ECVAM strategy to replace, reduce and refine the use of animals in the assessment of acute mammalian systemic toxicity

Information on acute systemic toxicity represents a standard requirement within several pieces of chemicals legislation in the EU. One of the main drivers of conducting the test is classification and labelling. Currently, only in vivo tests are accepted by regulatory bodies and most of the standard tests use lethality as endpoint. Based on an assessment of the regulatory needs and the scientific state-of-the art in the area, EURL ECVAM considers that efforts should be directed towards a) the reduction and replacement of animal tests for the identification and classification of acute systemic toxicity, and b) the refinement of in vivo studies. Consideration should be given to collecting, organising and applying mechanistic knowledge related to this endpoint, to provide a strong mechanistic basis for the design and validation of integrated prediction models. EURL ECVAM proposes to evaluate promising components of integrated approaches for testing and assessment (IATA), including the better use of existing alternative methods, such as mechanistically relevant in vitro assays. Information on repeated dose toxicity might also be useful in supporting classification and labelling for acute systemic toxicity. One clear target is minimising animal use for satisfying information requirements for acute systemic toxicity in relation to the 2018 REACH registration deadline. The aims and objectives underpinning the EURL ECVAM strategy can only be achieved through the coordinated and concerted efforts of all stakeholders.JRC.I.5-Systems Toxicolog

Application of the Virtual Cell Based Assay for Simulation of in vitro Chemical fate following Acute Exposure

In order to reliably assess the risk of adverse systemic effects of chemicals by using in vitro methods, there is a need to simulate their absorption, distribution, metabolism, and excretion (ADME) in vivo to determine the target organ bioavailable concentration, and to compare this predicted internal concentration with an effective internal concentration. The effective concentration derived from in vitro toxicity studies should ideally take into account the fate of chemicals in the in vitro test system, since there can be significant differences between the applied nominal concentration and the in vitro bioavailable concentration. Whereas PBK models have been developed to simulate ADME properties in vivo, the Virtual Cell Based Assay (VCBA) has been developed to simulate in vitro fate. In this project, the VCBA model in R code, was applied to better interpret previously obtained in vitro acute toxicity data and study how they can be compared to results from acute toxicity in vivo. For 178 chemicals previously tested in vitro with the 3T3 BALB/c cell line using the Neutral Red Uptake cytotoxicity assay, physicochemical parameters were retrieved and curated. Of these chemicals, 83 were run in the VCBA to simulate a 96-well microplate set up with 5% serum supplementation, and their no effect concentration (NEC) and killing rate (Kr) optimized against the experimental data. Analyses of results of partitioning of the chemicals show a strong relation with their lipophilicity, expressed here as the logarithm of the octanol/water partitioning coefficient, with highly lipophilic chemicals binding mostly to medium lipid. Among the chemicals analysed, only benzene and xylene were modelled to evaporate by more than 10 %, and these were also the chemicals with highest degradation rates during the 48 hours assay. Chemical degradation is dependent not only on the air and water degradation rates but also on the extent of binding of the chemical. Due to the strong binding of some chemicals to medium lipids and proteins we analysed the impact of different serum supplementations (0%, 5% and 10%) on the chemical dissolved concentrations. As expected, for the more lipophilic chemicals, different serum levels result in different dissolved concentrations, with lipid and protein binding reducing chemical loss by evaporation. Still the lack of saturation modelling might mislead the 0 % supplementation since the lipids coming solely from cells exudates are able to sequester chemical to a large extent, eg. after 48 hours, 63% (1.2E-5 M) of dimethyldioctadecylammonium chloride was bound to lipid from the cells. Although highly lipophilic chemicals have a very small bioavailable fraction, cellular uptake rate is also dependent on logKow, which compensates for this lack of bioavailability to some extent. Based on the relevance of lipophilicity on in vitro chemical bioavailability, we have developed an alert system based on logKow, creating four classes of chemicals for the experimental condition with 10% serum supplementation: logKow 5- 10 (A), logKow <5 (B), logKow <2.5 (C), and logKow <2 (D). New chemicals from Classes A and B, which will in the future be tested in vitro, were run first on the VCBA, without considering toxicity (NEC and Kr set to 0). VCBA simulations indicated that these chemicals are more than 50% bound to medium proteins, lipids and plastic. Therefore, for chemicals with logKow falling in these classes, special care should be taken when extrapolating the obtained in vitro toxic concentrations to in vivo relevant doses. A comparison of the VCBA-predicted dissolved concentrations corresponding to nominal IC50 values with the available rat oral LD50 values did not improve the previously obtained correlations. This is probably because other in vivo kinetic processes play an important role but were not considered in this in vitro-in vivo extrapolation. The comparison of the VCBA predicted IC50 dissolved concentrations with the available rat oral LD50 values, did not improve the previously obtained correlations. Nevertheless, other in vivo kinetic processes that are not modelled may play an important role. They should be considered in the in vitro-in vivo extrapolations. A local sensitivity analysis showed the relative low impact of Molar Volume and Molecular Diffusion Volume on the final dissolved concentration, supporting the use of approximated values obtained through the herein created QSARs. The logkow and Henry Law Constant showed, as expected, a high impact in partitioning. Killing rate was shown to also have a relative low impact in the final chemical concentration, indicating that although its optimization is important, finding the Kr that leads to the absolute best correlation between experimental and predicted concentration-viability curves, is not imperative. The VCBA can be applied to virtually any chemical as long as the physicochemical data (for the fate model) and the experimental toxicity data (that include cell growth/death) are available. However, being such a generic model, several assumptions had to be made: i) no distinction of chemical classes (inorganic, polar organic chemicals), ii) no consideration of metabolism, iii) saturation kinetics and iv) external in vitro conditions. The advantages of having a generic model are that the VCBA can fit several experimental set ups and should be used in an exploratory manner, to help refinement of experimental conditions. The herein obtained VCBA results should be double checked experimentally the partition with a set of chemical compounds to better understand to what extent VCBA represents chemicals of different properties. In future developments, it would be important to reduce the uncertainties of the model such as binding-saturation and consider inclusion of other endpoints such as metabolic activity.JRC.F.3-Chemicals Safety and Alternative Method

ECVAM Technical Report on the Status of Alternative Methods for Cosmetics Testing (2008-2009)

The ECVAM technical report presents the progress made in the development and validation of alternative methods for the human health effects relevant to the Cosmetics Directive. It provides an update on the activities described by ECVAM in 2005 , 2006 and 2007 . The report intends to present the latest scientific and technical developments in the field during 2008-2009. As required by Directive 2003/15/EC, the seventh amendment to Directive 76/768/EEC, developments in refinement and reduction methods are also described (EU, 2003). Most successes in the development of alternative methods are in acute local toxicity and short-term testing, such as e.g. skin and eye irritation/corrosion, phototoxicity and skin penetration The test methods consuming a high number of animals, however, are in long-term testing and systemic toxicity, such as e.g. reproductive toxicity and repeated dose toxicity. In these complex fields, several research initiatives are ongoing. However full replacement approaches are still lacking.JRC.DG.I.3-In-vitro method

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2013-April 2014)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches since the last report published in April 2013. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and international acceptance of alternative methods and approaches. R&D activities are ongoing for the complex endpoints where the toxicological processes and the mechanistic understanding have not been sufficiently elucidated yet and for which 3Rs solutions are more difficult to find. On the other hand, good progress In the validation and regulatory acceptance is made in areas where non-animal alternative methods have been developed and validated and where the focus lies in an intelligent combination/ integration of the various non-animal approaches.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2015)

The EURL ECVAM status report provides an update on the progress made in the development, validation and regulatory acceptance of alternative methods and approaches and their dissemination since the last report published in June 2014. It is informing on ongoing research and development activities, validation studies, peer reviews, recommendations, strategies and regulatory/international acceptance of alternative methods and approaches and dissemination activities. R&D activities within large European or International consortia continued in toxicity areas where 3Rs solutions are more difficult to find due to the underlying complexity of the area. On the other hand, toxicity areas where promising non-animal approaches have been developed, their validation and regulatory acceptance/international adoption could be progressed. Particular emphasis was given to the best and most intelligent combination and integration of these different non-animal approaches to ultimately obtain the required information without resorting to animal testing.JRC.I.5-Systems Toxicolog

Alternative methods for regulatory toxicology – a state-of-the-art review

This state-of-the art review is based on the final report of a project carried out by the European Commission’s Joint Research Centre (JRC) for the European Chemicals Agency (ECHA). The aim of the project was to review the state of the science of non-standard methods that are available for assessing the toxicological and ecotoxicological properties of chemicals. Non-standard methods refer to alternatives to animal experiments, such as in vitro tests and computational models, as well as animal methods that are not covered by current regulatory guidelines. This report therefore reviews the current scientific status of non-standard methods for a range of human health and ecotoxicological endpoints, and provides a commentary on the mechanistic basis and regulatory applicability of these methods. For completeness, and to provide context, currently accepted (standard) methods are also summarised. In particular, the following human health endpoints are covered: a) skin irritation and corrosion; b) serious eye damage and eye irritation; c) skin sensitisation; d) acute systemic toxicity; e) repeat dose toxicity; f) genotoxicity and mutagenicity; g) carcinogenicity; h) reproductive toxicity (including effects on development and fertility); i) endocrine disruption relevant to human health; and j) toxicokinetics. In relation to ecotoxicological endpoints, the report focuses on non-standard methods for acute and chronic fish toxicity. While specific reference is made to the information needs of REACH, the Biocidal Products Regulation and the Classification, Labelling and Packaging Regulation, this review is also expected to be informative in relation to the possible use of alternative and non-standard methods in other sectors, such as cosmetics and plant protection products.JRC.I.5-Systems Toxicolog

EURL ECVAM Status Report on the Development, Validation and Regulatory Acceptance of Alternative Methods and Approaches (2016)

Replacement, Reduction and Refinement of animal testing is anchored in EU legislation. Alternative non-animal approaches facilitate a shift away from animal testing. Cell-based methods and computational technologies are integrated to translate molecular mechanistic understanding of toxicity into safety testing strategies.JRC.F.3-Chemicals Safety and Alternative Method

t4 Workshop Report: Integrated Testing Strategies (ITS) for Safety Assessment

Integrated testing strategies (ITS), as opposed to single definitive tests or fixed batteries of tests, are expected to efficiently combine different information sources in a quantifiable fashion to satisfy an information need, in this case for regulatory safety assessments. With increasing awareness of the limitations of each individual tool and the development of highly targeted tests and predictions, the need for combining pieces of evidence increases. The discussions that took place during this workshop, which brought together a group of experts coming from different related areas, illustrate the current state of the art of ITS, as well as promising developments and identifiable challenges. The case of skin sensitization was taken as an example to understand how possible ITS can be constructed, optimized and validated. This will require embracing and developing new concepts such as adverse outcome pathways (AOP), advanced statistical learning algorithms and machine learning, mechanistic validation and “Good ITS Practices”.JRC.I.5-Systems Toxicolog

Short commentary to "Human in vivo database now on ACuteTox home page" [Toxicology In Vitro 27 (2013) 2350–2351]

This short note concerns a statement written by Ada Kolman and Cecilia Clemedson in their publication "Human in vivo database now on ACuteTox home page" [Toxicology In Vitro 27 (2013) 2350–2351], saying that animal and in vitro data from the ACuteTox project are not yet publicly available. Having been actively involved in the ACuteTox project throughout its whole duration, as project partners and leaders of two major work-packages, we would like express our disagreement with this statement. In fact, properly evaluated and quality checked in vivo oral LD50 and in vitro data from this EU FP6 project have been available to the public before the article of Kolman and Clemedson. The acute oral toxicity animal in vivo data relevant to the 97 reference chemicals selected for the project were available in the publication by Hoffmann et al. (2010) where summaries of oral rodent LD50 data collected are provided in tables. The in vitro data generated during the course of the ACuteTox project were published in 2013 in a dedicated Special Issue of Toxicology In Vitro. All the IC50 summary values from the relevant in vitro endpoints are available in the supplementary material provided in two publications, Kinsner Ovaskainen et al., 2013 and Prieto et al., 2013 (both accessible online already in 2012).JRC.I.5-Systems Toxicolog