- Email: [email protected]
ECVAM’s ongoing activities in the area of acute oral toxicity
Toxicology in Vitro 23 (2009) 1535–1540
Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
ECVAM’s ongoing activities in the area of acute oral toxicity Agnieszka Kinsner-Ovaskainen a,*, Anna Bulgheroni a, Thomas Hartung b, Pilar Prieto a a
In-Vitro Methods Unit, European Centre for the Validation of Alternative Methods (ECVAM), Institute for Health and Consumer Protection (IHCP), European Commission Joint Research Centre, Ispra, Italy b Center for Alternatives to Animal Testing, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, United States
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Article history: Received 15 October 2008 Accepted 6 July 2009 Available online 8 July 2009 Keywords: Acute toxicity Cytotoxicity Validation 3Rs
a b s t r a c t The 7th Amendment of the Cosmetics Directive (2003/15/EC) set up timelines for banning animal testing and marketing of cosmetic products and their ingredients tested on animals. For most of the human health effects, including acute toxicity, the deadline for these bans was in March 2009. Moreover, the new Regulation EC 1907/2006 on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) provided a strong impetus towards the application of alternative approaches to reduce the number of animals used for toxicological testing. Therefore, the European Centre for the Validation of Alternative Methods (ECVAM) is currently putting considerable effort into developing and validating alternative methods in the field of acute toxicity. The main activities in this area include: (1) the Integrated Project ACuteTox, funded by the European Commission’s 6th Framework Programme in 2005 with the aim to develop and pre-validate a testing strategy to fully replace acute oral toxicity testing in vivo; (2) a follow-up validation study to assess the predictive capacity of the validated BALB/3T3 Neutral Red Uptake cytotoxicity assay to discriminate between toxic/hazardous (LD50 < 2000 mg/kg) substances and substances not classified for acute toxicity (LD50 > 2000 mg/kg); (3) an approach to identify compounds with LD50 > 2000 mg/kg using information from 28-days repeated dose toxicity studies. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The 7th Amendment of the European Council’s Cosmetics Directive (76/768/EEC), adopted in 2004 (Anon, 2003), introduced new provisions related to non-animal testing of cosmetic products and ingredients, and was a major breakthrough that significantly moved forward research in the field of alternative methods in the European Union (EU) (Eskes and Zuang, 2005). Furthermore, the Regulation EC 1907/2006 on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), adopted in 2007 (Anon, 2007), requires information on the toxicological properties for safety assessment of new, but also of all the existing substances (i.e. those placed on the market before 1981). Conducting traditional toxicity testing for all of these chemicals would be economically impractical, time-consuming and would require the use of very large numbers of animals. Thus, REACH also provides a strong impetus towards the development, acceptance and use of alternative methods that could reduce the number of animals used for toxicological testing (Anon, 2005). Considering the demands of
* Corresponding author. Address: European Commission Joint Research Centre, Institute for Health and Consumer Protection, In-Vitro Methods Unit, European Centre for Validation of Alternative Methods (ECVAM), Via E. Fermi 2749, TP 580, I21027 Ispra (VA), Italy. Tel.: +39 0332 789246; fax: +39 0332 786297. E-mail address: [email protected] (A. Kinsner-Ovaskainen). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.07.004
these new legislations ECVAM, responsible for coordinating the validation of alternative test methods at the level of the EU, is currently putting much effort into the validation and, in some areas, also in the development and optimization of alternative methods for all the human health-related areas of concern to the Cosmetics Directive (Eskes and Zuang, 2005) and the REACH Regulation. From the 11th March 2009 the Cosmetics Directive introduced a ban of animal testing of cosmetic ingredients and cosmetic finished products in all human health endpoints, as well as a ban for marketing of products that include ingredients tested on animals in several human health endpoints (including acute toxicity). Thus, acute oral toxicity testing in vivo is one of the endpoints that urgently needs to be replaced with alternative, non-animal approaches. The current regulatory accepted methods for determination of acute oral toxicity are exclusively based on in vivo experimentation. They include three approved refinement and reduction alternative methods (modifications of the classical LD50 test) described in the Organisation for Economical Cooperation and Development (OECD) Test Guidelines TG 420 (Fixed Dose Procedure), TG 423 (Acute Toxic Class Method) and TG 425 (Up and Down Procedure) (OECD, 2001a,b,c). Over the last decades the in vitro cytotoxicity tests have been intensively assessed as potential alternatives for in vivo acute oral toxicity testing in several studies, such as the Multicentre
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Evaluation of In Vitro Cytotoxicity (MEIC) study (Ekwall et al., 1998; Clemedson and Ekwall, 1999), the Halle Registry of Cytotoxicity (Halle, 2003), and the International Validation Study of In Vitro Cytotoxicity Test Methods, organised jointly by the National Toxicology Programme Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) in the United States and ECVAM in Europe (Anon, 2006). The results of these studies have shown that there is a relatively good correlation of around 50–60% between in vitro cytotoxic concentrations (IC50) and the rat oral lethal doses (LD50 values). However, it was clear that the in vitro test methods are not sufficiently accurate to completely replace animals for regulatory purposes (hazard identification, classification and labelling). The peer review panel of the NICEATM/ ECVAM in vitro basal cytotoxicity validation study, which was finalised in 2006, concluded that the Neutral Red Uptake (NRU) assay performed in BALB/3T3 mice fibroblast cell line and in primary Normal Human Keratinocytes (NHK) may be used in a weight-ofevidence process to determine the starting dose for the acute oral in vivo toxicity studies (Anon, 2008). The OECD Guidance Document 24 on Acute Oral Toxicity Testing also recommends the use, among others, of results from in vitro toxicity tests to assist in selecting the starting dose, particularly in cases where minimal prior information on the substance is available (OECD, 2001d). It is expected that the use of in vitro methods will reduce the number of animals required for each toxicity test. However, the cytotoxicity assays so far are recognised only as additional tests that can be used for estimating the initial doses for acute oral systemic toxicity tests in vivo. Taking into consideration the complexity of biological processes that may lead to acute toxicity, a full replacement of the in vivo endpoint is a demanding task. Many factors can impair the prediction of in vivo toxicity from basal cytotoxicity, such as local and/or target-organ specific effects, the bioavailability of the compound (absorption, tissue distribution and elimination) and its metabolism (both bioactivation and detoxification) (reviewed by Gennari et al., 2004; Combes et al., 2008. Indeed, incorporation of chemical properties of a given compound (such as pH, lipophilicity, protein binding) and the use of physiologically based kinetic models can improve the extrapolation of in vitro data to a toxic dose in vivo (Gundert-Remy and Sonich-Mullin, 2002; Blaauboer, 2003; Forsby and Blaauboer, 2007). However, most of the complex biological processes are difficult to mimic using in vitro methods and significantly impair the development and acceptance of alternative methods the field of systemic toxicity. ECVAM has initiated and actively participates in several projects aimed to replace or reduce animal testing for acute oral systemic toxicity. Three activities are of particular importance and will be discussed more in detail in the following sections: (1) the FP6 Integrated Project ACuteTox (http://www.acutetox.org); (2) the follow-up of the NICEATM/ECVAM Validation study aiming to assess the predictive capacity of the validated BALB/3T3 NRU assay to discriminate between toxic/hazardous (LD50 < 2000 mg/kg) and not classified (LD50 > 2000 mg/kg) substances; (3) estimation of acute oral toxicity using the No Observed Adverse Effect Level (NOAEL) from 28-days repeated-dose studies in rats.
2. The ACuteTox project An international workshop, organised in 2000 by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to review the status of in vitro methods for assessing acute toxicity, recognised the need to consider parameters such as metabolism, kinetics and target-organs to improve the predictive capacity of cytotoxicity in vitro methods (Anon, 2001). In view of the new EU legislations (e.g. REACH and Cosmet-
ics Directive) an ECVAM Workshop on ‘‘Strategies to Replace In Vivo Acute Systemic Toxicity” discussed the possibilities to build an animal-free testing strategy that takes into consideration all the factors which could improve prediction of acute toxicity in comparison to simple cytotoxicity tests only. The workshop report (Gennari et al., 2004) served as the basis for the large Integrated Project ‘‘ACuteTox” that received in 2005 from the European Commission’s 6th Framework Programme a funding of nine million Euro to develop and pre-validate a testing strategy to fully replace the acute oral toxicity testing in vivo (Clemedson et al., 2006; Clemedson, 2008). Thirty five participants including academia, industry and governmental research institutes from 13 European countries contribute to this large collaborative effort. The main objectives of the project include the compilation, evaluation and generation of high quality in vitro and in vivo data for comparative analyses, and the identification of factors that influence the correlation between in vitro (concentration) and in vivo (dose) toxicity, particularly taking into consideration biokinetics, metabolism and organ toxicity (liver, central nervous system, kidney). Moreover, innovative tools (e.g. cytomics) and new cellular systems for anticipating animal and human toxicity are explored. Ultimately, the goal is to design a simple, robust and reliable in vitro test strategy amendable for robotic testing, associated with the prediction models for acute oral toxicity. The project is divided into 10 Work Packages (WPs) (Fig. 1). ECVAM contributes in particular to Work Package 1 (selection of reference chemicals, generation of the animal and human in vivo databases), Work Package 3 (development of an internet-based database for central management of all project data; adaptation of promising in vitro methods to robotic screening platforms; statistical analysis – identification of outliers and design of the preliminary algorithm/prediction model), Work Package 7.1 (in vitro assays for neurotoxicity) and Work Package 8 (construction and optimization of the in vitro testing strategy). In the last year of the project, ECVAM will be responsible for the work in Work Package 9 dealing with the organisation and management of the prevalidation of the in vitro testing strategy. Besides, ECVAM is actively involved in the Steering Committee, Management Board and Advisory Board of the project. In the first part of the project, 97 reference chemicals were selected and tested using 6 basal cytotoxicity assays (Clothier et al., 2008). Moreover, human and animal in vivo data for these substances were collected from literature. This allowed to identify the outliers from the in vitro/in vivo correlations, both using calculated human lethal concentrations (LC50) (Sjöström et al., 2008) and rat LD50 data (Clothier et al., 2008). These outliers, as well as a balanced number of non-outliers from the reference set, were then tested in several in vitro and in silico models. An overview of all the in vitro assays assessed in the project is available in the publication by Kinsner-Ovaskainen et al. (2009). By the end of 2007, fifty seven reference chemicals were tested in a number of functional tests covering absorption, distribution, excretion, metabolism and specific organ- and system-toxicity, such as haemato-, neuro-, nephro- and hepatotoxicity. The data generated were stored in a novel internet-based database (AcutoxBase) developed within the project (Kinsner-Ovaskainen et al., 2009). A Partial Least Square Analysis (PLS) was performed on all data generated and the best combinations of in vitro tests that gave a relatively good correlation with in vivo (rat and human) data were identified (data not shown). However, these combinations allowed only a slight improvement of the correlations as compared to the validated in vitro cytotoxicity NRU assay alone. Moreover, the analyses performed so far did not enable to select the promising methods that will be part of the testing strategy (Sjöström, personal communication). Further data analysis and data mining are needed before the construction of the testing strategy can be initiated. These activi-
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Fig. 1. The general structure of the ACuteTox project.
ties are now largely coordinated by ECVAM. In addition, the contribution of kinetics (e.g. oral absorption and volume of distribution) in the estimation of LD50 values from the in vitro cytotoxicity data is under evaluation and will be taken into consideration for the final design of the testing strategy (Blaauboer, personal communication). In the last phase of the project, the selected methods will be challenged with additional compounds under blind conditions, and the new data generated will allow a preliminary assessment of the predictive capacity of the testing strategy. This project represents the first attempt to create an integrated testing strategy based solely on in vitro and in silico methods, with the purpose of replacing animal testing for predicting human acute systemic toxicity. 3. The follow-up validation study of the BALB/3T3 neutral red uptake assay The complete validation of a fully developed in vitro testing strategy for acute toxicity will probably take a few additional years. Thus, ECVAM is looking for alternative approaches that would enable to immediately reduce the need for animal testing, especially of cosmetic ingredients. Recently, based on an analysis performed on dossiers from New Chemical Database (NCD) maintained at the Institute for Health and Consumer Protection (JRC, Ispra) (http://ecb.jrc.ec.europa.eu), we have shown that most of the industrial chemicals in the EU fall into two categories of the GHS classification system, i.e. harmful (9.4% – 300 < LD50 < 2000 mg/kg) and category 5 and not classified (86.6% – LD50 > 2000 mg/kg) (Bulgheroni et al., 2009). The results of the NICEATM/ECVAM Validation study have shown that the overall accuracy of the BALB/3T3 NRU cytotoxicity assay for correctly predicting each of the GSH acute oral toxicity classification category was low (around 30%), however, substances in the ranges 300 < LD50 < 2000 mg/kg b.w. were better predicted,
with 81% accuracy (Anon, 2006). This is supported by the data from the Register of Cytotoxicity (Halle, 2003), which has shown on a much larger number of compounds that the precision of prediction from cytotoxicity data of low systemic toxicity is much better than the prediction of high systemic toxicity. Thus, we assumed that the BALB/3T3 NRU cytotoxicity assay may allow to discriminate between toxic/harmful and non-toxic substances, according to the EU and GHS classification systems. Taking into account also the high prevalence of ‘‘non-toxic” chemicals in the whole population of chemicals registered (i.e. the proportion of chemicals falling into a category 5 and non-classified (LD50 > 2000 mg/kg b.w.), the use of this approach could significantly reduce in vivo testing for acute oral toxicity. However, to properly prove this, it is necessary to include in the analysis a larger number of chemical substances with and LD50 > 2000 mg/kg b.w., tested in the BALB/3T3 NRU cytotoxicity assay. For this purpose ECVAM funded in 2008 a follow-up validation study of the NRU cytotoxicity assay in BALB/3T3 cells. The main goal of the study is to evaluate the predictive capacity of the assay to give a simple ‘‘yes/no” answer in order to discriminate between chemicals with LD50 > 2000 mg/kg b.w. (GHS category 5 and not classified) and those falling in the other four GHS categories (i.e. GHS 1–4 with LD50 < 2000 mg/kg b.w.), as defined by the Globally Harmonised System (GHS) for the classification and labelling of acute oral toxicity (UN, 2007). According to a proposal of a regulation by the European Parliament and Council, the same categories will be soon applicable also in Europe (E.C., 2007). The predictive capacity (i.e. sensitivity, specificity, concordance) and the applicability domain of the BALB/3T3 NRU test used for this new purpose, will be assessed by testing coded reference substances using the already validated Standard Operating Procedure (SOP). In this case, according to the ECVAM’s modular approach (Hartung et al., 2004), only one laboratory is required to assess the predictive capacity and applicability domain (modules 5 and
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6), since the test definition, within- and between-laboratory reproducibility and transferability (modules 1–4) have been already evaluated in the NICEATM/ECVAM validation study, and the test has proven to be reliable (Anon, 2006). This approach allows for a significant saving in terms of time and costs of the validation studies. Fig. 2 illustrates the organisation of the study with respect to the tests included and the participating laboratories, as well as the responsibilities for management of the project, selection, coding and supply of test chemicals and the data analysis. The study is coordinated by the Management Team including two ECVAM staff members. ICCVAM is involved in the study as an observer. The Chemical Selection Committee has identified a total set of 56 industrial chemicals (drugs and pesticides were excluded) for distinguishing substances with LD50 > 2000 mg/kg b.w. (GHS category 5 and not classified) from those falling into GHS categories 1– 4. The test substances include a variety of molecular structures, balanced between two physical states (liquid and solid), as well as a wide range of physico-chemical properties. According to the database CosIng, hosted on the website of E.C. DG Enterprise (available at http://ec.europa.eu/enterprise/cosmetics/cosing), 60% of the selected compounds are also used as cosmetic ingredients (including hair dyes, surfactants, UV filters, masking agents, antioxidants, preservatives and others). Chemicals used in previous studies i.e. the NICEATM/ECVAM validation study and the ACuteTox project were not included. All 56 chemicals, which have been coded by ECVAM and distributed by Sigma Aldrich (Italy), will be tested in the Health and Safety Laboratory (HSL, UK) following the test method described in detail in the SOPs validated during the NICEATM/ECVAM validation study (Anon, 2006). The results from this laboratory will be used to establish the predictive capacity, according to module 5 (Hartung et al., 2004). Additionally, two variants of the validated procedure will be evaluated in two different laboratories using the same set of 56
substances. The Institute for Health and Consumer Protection (IHCP, JRC, Italy) will use an automated version of the protocol adapted for a robotic testing platform, while the Institute for In Vitro Sciences (IIVS, US) will test the chemicals with an abbreviated procedure, less expensive and time-consuming and thus more practical for industry. The results obtained with the two different protocols will be then compared with the data from the validated protocol. The NRU assay will be performed to analyse the in vitro toxicity of the selected industrial chemicals and in particular to determine the IC50 values for comparison with the rat oral acute toxicity data (LD50 values) collected from available literature and from databases of final risk assessment reports (e.g. ORATS). These data will be used to evaluate the predictive capacity of the assay. The study is foreseen to be completed in the second semester of 2009. It is expected that the proposed approach will allow identifying non-toxic substances, which are most relevant for use in cosmetic products, and thus enable to reduce animal testing for acute oral toxicity by more than 80% in this sector. 4. Estimation of acute oral toxicity using the NOAEL values form 28-days repeated dose studies in rats Despite enormous efforts undertaken in the area of systemic toxicity, none of the non-animal replacement methods (tests or testing strategies) for the prediction of acute oral toxicity, that are currently under development and evaluation, are ready, validated and regulatory accepted, although in vivo testing of cosmetic products and ingredients is already banned in Europe since the 11 March 2009. ECVAM recently performed an investigation (Bulgheroni et al., 2009) to assess whether it would be possible to extrapolate the toxicological information on acute oral toxicity of chemicals and cosmetic ingredients from the results of in vivo repeated dose toxicity studies. According to the 7th Amendment to the Cosmetics
Fig. 2. The organisation of the BALB/3T3 NRU cytotoxicity follow-up validation study. Laboratory 1 is the main laboratory performing the BALB/3T3 NRU validated protocol (assessment of Module 5); the results from laboratories 2 (automated protocol) and 3 (abbreviated protocol) will be evaluated in comparison to results from laboratory 1. Acronyms used in the Figure indicate: ECVAM (European Centre for the Validation of Alternative Methods), ECB (European Chemicals Bureau) [ECB was the official name of the unit January 2008. Currently most of the activities of this Unit have been handed over to other units of the Institute of Health and Consumer Protection (Ispra, Italy) and/or to the European Chemicals Agency (Helsinki, Finland).], ZEBET (Centre for Documentation and Evaluation of Alternatives to Animal Experiments), HSL (Heath and Safety Laboratory, UK), IHCP (Institute for Health and Consumer Protection, Ispra, Italy), IIVS (Institute for in Vitro Sciences, US), ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods.
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Directive, information derived from these studies (performed outside the EU) can still be used, at least until 2013. In the proposed approach, the compounds which have an LD50 > 2000 mg/kg (i.e. in the EU are not classified as acutely toxic via the oral route) will be identified using information from 28-days repeated dose toxicity studies. The evaluation was based on data retrieved from dossiers from the New Chemicals Database. Substances for which both the NOAEL values obtained from 28-days repeated dose studies in rats and the oral LD50 values were available in the submission files (1791 in total), were selected and included in the analysis. The substances were then grouped according to the GHS categories and from the obtained distribution the NOAEL P 200 mg/kg was identified as the best threshold that could discriminate ‘‘nontoxic” substances (LD50 > 2000 mg/kg b.w.) from the rest. This threshold allowed to correctly categorise 63% of the ‘‘non-toxic” compounds. Less than 1% of compounds were misclassified as ‘‘non-toxic” and all of them were falling in the harmful category (category 4). None of the toxic or very toxic substances were misclassified as ‘‘non-toxic”. The Negative Predictive Value (NPV) and the Positive Predictive Value (PPV) of this approach were 97% and 26.5%, respectively (for details of the analysis see Bulgheroni et al., 2009). The proposed approach could have an immediate impact for the testing of cosmetic ingredients, since, unlike data from in vivo acute toxicity studies, the results from repeated dose toxicity studies (performed outside the EU) can be still used until 2013. Taking into consideration the very high prevalence (87%) of substances categorised in EU as ‘‘non-toxic” after the acute oral exposure, we concluded that with this approach it is feasible to classify more than half of all the new chemical substances. Currently we are also evaluating whether in vitro cytotoxicity could be used to derive the starting dose for the 28-days repeated dose studies (as already demonstrated for acute oral toxicity), in order to replace the use of data from in vivo acute toxicity studies. 5. Conclusions The FP6 Integrated Project ACuteTox for the first time aims to provide an integrated testing strategy fully based on non-animal methods for a complete animal replacement for acute oral toxicity testing. However, even if the project will be fully successful, it will take few additional years to complete the scientific validation of the testing strategy and to achieve its regulatory acceptance. In the meantime results on the predictive capacity of cytotoxicity test to filter the ‘‘non-toxic” substances (LD50 > 2000 mg/kg b.w.) are expected in 2009. This approach could have an immediate impact especially in the context of the REACH legislation, given that 90% of the industrial chemicals are not classified as acutely toxic via the oral route (Bulgheroni et al., 2009). Moreover, the use of NOAEL values from 28-days repeated dose toxicity studies to identify with a high predictivity the ‘‘non-toxic” substances could be of major interest for the cosmetic industry since according to the Cosmetics Directive, although animal testing is already banned for all human health endpoint, however data coming from the repeated-dose studies in vivo can still be used until 2013. Acknowledgements The ACuteTox project is supported by an EU-FP6 Grant (FP6LIFESCIHEALTH-2004-512051). References Anon, 2001. Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity. NIH Publication No. 01-4499.
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Contents lists available at ScienceDirect
Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit
ECVAM’s ongoing activities in the area of acute oral toxicity Agnieszka Kinsner-Ovaskainen a,*, Anna Bulgheroni a, Thomas Hartung b, Pilar Prieto a a
In-Vitro Methods Unit, European Centre for the Validation of Alternative Methods (ECVAM), Institute for Health and Consumer Protection (IHCP), European Commission Joint Research Centre, Ispra, Italy b Center for Alternatives to Animal Testing, Johns Hopkins University, Bloomberg School of Public Health, Baltimore, Maryland, United States
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Article history: Received 15 October 2008 Accepted 6 July 2009 Available online 8 July 2009 Keywords: Acute toxicity Cytotoxicity Validation 3Rs
a b s t r a c t The 7th Amendment of the Cosmetics Directive (2003/15/EC) set up timelines for banning animal testing and marketing of cosmetic products and their ingredients tested on animals. For most of the human health effects, including acute toxicity, the deadline for these bans was in March 2009. Moreover, the new Regulation EC 1907/2006 on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) provided a strong impetus towards the application of alternative approaches to reduce the number of animals used for toxicological testing. Therefore, the European Centre for the Validation of Alternative Methods (ECVAM) is currently putting considerable effort into developing and validating alternative methods in the field of acute toxicity. The main activities in this area include: (1) the Integrated Project ACuteTox, funded by the European Commission’s 6th Framework Programme in 2005 with the aim to develop and pre-validate a testing strategy to fully replace acute oral toxicity testing in vivo; (2) a follow-up validation study to assess the predictive capacity of the validated BALB/3T3 Neutral Red Uptake cytotoxicity assay to discriminate between toxic/hazardous (LD50 < 2000 mg/kg) substances and substances not classified for acute toxicity (LD50 > 2000 mg/kg); (3) an approach to identify compounds with LD50 > 2000 mg/kg using information from 28-days repeated dose toxicity studies. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The 7th Amendment of the European Council’s Cosmetics Directive (76/768/EEC), adopted in 2004 (Anon, 2003), introduced new provisions related to non-animal testing of cosmetic products and ingredients, and was a major breakthrough that significantly moved forward research in the field of alternative methods in the European Union (EU) (Eskes and Zuang, 2005). Furthermore, the Regulation EC 1907/2006 on Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), adopted in 2007 (Anon, 2007), requires information on the toxicological properties for safety assessment of new, but also of all the existing substances (i.e. those placed on the market before 1981). Conducting traditional toxicity testing for all of these chemicals would be economically impractical, time-consuming and would require the use of very large numbers of animals. Thus, REACH also provides a strong impetus towards the development, acceptance and use of alternative methods that could reduce the number of animals used for toxicological testing (Anon, 2005). Considering the demands of
* Corresponding author. Address: European Commission Joint Research Centre, Institute for Health and Consumer Protection, In-Vitro Methods Unit, European Centre for Validation of Alternative Methods (ECVAM), Via E. Fermi 2749, TP 580, I21027 Ispra (VA), Italy. Tel.: +39 0332 789246; fax: +39 0332 786297. E-mail address: [email protected] (A. Kinsner-Ovaskainen). 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.07.004
these new legislations ECVAM, responsible for coordinating the validation of alternative test methods at the level of the EU, is currently putting much effort into the validation and, in some areas, also in the development and optimization of alternative methods for all the human health-related areas of concern to the Cosmetics Directive (Eskes and Zuang, 2005) and the REACH Regulation. From the 11th March 2009 the Cosmetics Directive introduced a ban of animal testing of cosmetic ingredients and cosmetic finished products in all human health endpoints, as well as a ban for marketing of products that include ingredients tested on animals in several human health endpoints (including acute toxicity). Thus, acute oral toxicity testing in vivo is one of the endpoints that urgently needs to be replaced with alternative, non-animal approaches. The current regulatory accepted methods for determination of acute oral toxicity are exclusively based on in vivo experimentation. They include three approved refinement and reduction alternative methods (modifications of the classical LD50 test) described in the Organisation for Economical Cooperation and Development (OECD) Test Guidelines TG 420 (Fixed Dose Procedure), TG 423 (Acute Toxic Class Method) and TG 425 (Up and Down Procedure) (OECD, 2001a,b,c). Over the last decades the in vitro cytotoxicity tests have been intensively assessed as potential alternatives for in vivo acute oral toxicity testing in several studies, such as the Multicentre
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Evaluation of In Vitro Cytotoxicity (MEIC) study (Ekwall et al., 1998; Clemedson and Ekwall, 1999), the Halle Registry of Cytotoxicity (Halle, 2003), and the International Validation Study of In Vitro Cytotoxicity Test Methods, organised jointly by the National Toxicology Programme Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) in the United States and ECVAM in Europe (Anon, 2006). The results of these studies have shown that there is a relatively good correlation of around 50–60% between in vitro cytotoxic concentrations (IC50) and the rat oral lethal doses (LD50 values). However, it was clear that the in vitro test methods are not sufficiently accurate to completely replace animals for regulatory purposes (hazard identification, classification and labelling). The peer review panel of the NICEATM/ ECVAM in vitro basal cytotoxicity validation study, which was finalised in 2006, concluded that the Neutral Red Uptake (NRU) assay performed in BALB/3T3 mice fibroblast cell line and in primary Normal Human Keratinocytes (NHK) may be used in a weight-ofevidence process to determine the starting dose for the acute oral in vivo toxicity studies (Anon, 2008). The OECD Guidance Document 24 on Acute Oral Toxicity Testing also recommends the use, among others, of results from in vitro toxicity tests to assist in selecting the starting dose, particularly in cases where minimal prior information on the substance is available (OECD, 2001d). It is expected that the use of in vitro methods will reduce the number of animals required for each toxicity test. However, the cytotoxicity assays so far are recognised only as additional tests that can be used for estimating the initial doses for acute oral systemic toxicity tests in vivo. Taking into consideration the complexity of biological processes that may lead to acute toxicity, a full replacement of the in vivo endpoint is a demanding task. Many factors can impair the prediction of in vivo toxicity from basal cytotoxicity, such as local and/or target-organ specific effects, the bioavailability of the compound (absorption, tissue distribution and elimination) and its metabolism (both bioactivation and detoxification) (reviewed by Gennari et al., 2004; Combes et al., 2008. Indeed, incorporation of chemical properties of a given compound (such as pH, lipophilicity, protein binding) and the use of physiologically based kinetic models can improve the extrapolation of in vitro data to a toxic dose in vivo (Gundert-Remy and Sonich-Mullin, 2002; Blaauboer, 2003; Forsby and Blaauboer, 2007). However, most of the complex biological processes are difficult to mimic using in vitro methods and significantly impair the development and acceptance of alternative methods the field of systemic toxicity. ECVAM has initiated and actively participates in several projects aimed to replace or reduce animal testing for acute oral systemic toxicity. Three activities are of particular importance and will be discussed more in detail in the following sections: (1) the FP6 Integrated Project ACuteTox (http://www.acutetox.org); (2) the follow-up of the NICEATM/ECVAM Validation study aiming to assess the predictive capacity of the validated BALB/3T3 NRU assay to discriminate between toxic/hazardous (LD50 < 2000 mg/kg) and not classified (LD50 > 2000 mg/kg) substances; (3) estimation of acute oral toxicity using the No Observed Adverse Effect Level (NOAEL) from 28-days repeated-dose studies in rats.
2. The ACuteTox project An international workshop, organised in 2000 by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) to review the status of in vitro methods for assessing acute toxicity, recognised the need to consider parameters such as metabolism, kinetics and target-organs to improve the predictive capacity of cytotoxicity in vitro methods (Anon, 2001). In view of the new EU legislations (e.g. REACH and Cosmet-
ics Directive) an ECVAM Workshop on ‘‘Strategies to Replace In Vivo Acute Systemic Toxicity” discussed the possibilities to build an animal-free testing strategy that takes into consideration all the factors which could improve prediction of acute toxicity in comparison to simple cytotoxicity tests only. The workshop report (Gennari et al., 2004) served as the basis for the large Integrated Project ‘‘ACuteTox” that received in 2005 from the European Commission’s 6th Framework Programme a funding of nine million Euro to develop and pre-validate a testing strategy to fully replace the acute oral toxicity testing in vivo (Clemedson et al., 2006; Clemedson, 2008). Thirty five participants including academia, industry and governmental research institutes from 13 European countries contribute to this large collaborative effort. The main objectives of the project include the compilation, evaluation and generation of high quality in vitro and in vivo data for comparative analyses, and the identification of factors that influence the correlation between in vitro (concentration) and in vivo (dose) toxicity, particularly taking into consideration biokinetics, metabolism and organ toxicity (liver, central nervous system, kidney). Moreover, innovative tools (e.g. cytomics) and new cellular systems for anticipating animal and human toxicity are explored. Ultimately, the goal is to design a simple, robust and reliable in vitro test strategy amendable for robotic testing, associated with the prediction models for acute oral toxicity. The project is divided into 10 Work Packages (WPs) (Fig. 1). ECVAM contributes in particular to Work Package 1 (selection of reference chemicals, generation of the animal and human in vivo databases), Work Package 3 (development of an internet-based database for central management of all project data; adaptation of promising in vitro methods to robotic screening platforms; statistical analysis – identification of outliers and design of the preliminary algorithm/prediction model), Work Package 7.1 (in vitro assays for neurotoxicity) and Work Package 8 (construction and optimization of the in vitro testing strategy). In the last year of the project, ECVAM will be responsible for the work in Work Package 9 dealing with the organisation and management of the prevalidation of the in vitro testing strategy. Besides, ECVAM is actively involved in the Steering Committee, Management Board and Advisory Board of the project. In the first part of the project, 97 reference chemicals were selected and tested using 6 basal cytotoxicity assays (Clothier et al., 2008). Moreover, human and animal in vivo data for these substances were collected from literature. This allowed to identify the outliers from the in vitro/in vivo correlations, both using calculated human lethal concentrations (LC50) (Sjöström et al., 2008) and rat LD50 data (Clothier et al., 2008). These outliers, as well as a balanced number of non-outliers from the reference set, were then tested in several in vitro and in silico models. An overview of all the in vitro assays assessed in the project is available in the publication by Kinsner-Ovaskainen et al. (2009). By the end of 2007, fifty seven reference chemicals were tested in a number of functional tests covering absorption, distribution, excretion, metabolism and specific organ- and system-toxicity, such as haemato-, neuro-, nephro- and hepatotoxicity. The data generated were stored in a novel internet-based database (AcutoxBase) developed within the project (Kinsner-Ovaskainen et al., 2009). A Partial Least Square Analysis (PLS) was performed on all data generated and the best combinations of in vitro tests that gave a relatively good correlation with in vivo (rat and human) data were identified (data not shown). However, these combinations allowed only a slight improvement of the correlations as compared to the validated in vitro cytotoxicity NRU assay alone. Moreover, the analyses performed so far did not enable to select the promising methods that will be part of the testing strategy (Sjöström, personal communication). Further data analysis and data mining are needed before the construction of the testing strategy can be initiated. These activi-
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Fig. 1. The general structure of the ACuteTox project.
ties are now largely coordinated by ECVAM. In addition, the contribution of kinetics (e.g. oral absorption and volume of distribution) in the estimation of LD50 values from the in vitro cytotoxicity data is under evaluation and will be taken into consideration for the final design of the testing strategy (Blaauboer, personal communication). In the last phase of the project, the selected methods will be challenged with additional compounds under blind conditions, and the new data generated will allow a preliminary assessment of the predictive capacity of the testing strategy. This project represents the first attempt to create an integrated testing strategy based solely on in vitro and in silico methods, with the purpose of replacing animal testing for predicting human acute systemic toxicity. 3. The follow-up validation study of the BALB/3T3 neutral red uptake assay The complete validation of a fully developed in vitro testing strategy for acute toxicity will probably take a few additional years. Thus, ECVAM is looking for alternative approaches that would enable to immediately reduce the need for animal testing, especially of cosmetic ingredients. Recently, based on an analysis performed on dossiers from New Chemical Database (NCD) maintained at the Institute for Health and Consumer Protection (JRC, Ispra) (http://ecb.jrc.ec.europa.eu), we have shown that most of the industrial chemicals in the EU fall into two categories of the GHS classification system, i.e. harmful (9.4% – 300 < LD50 < 2000 mg/kg) and category 5 and not classified (86.6% – LD50 > 2000 mg/kg) (Bulgheroni et al., 2009). The results of the NICEATM/ECVAM Validation study have shown that the overall accuracy of the BALB/3T3 NRU cytotoxicity assay for correctly predicting each of the GSH acute oral toxicity classification category was low (around 30%), however, substances in the ranges 300 < LD50 < 2000 mg/kg b.w. were better predicted,
with 81% accuracy (Anon, 2006). This is supported by the data from the Register of Cytotoxicity (Halle, 2003), which has shown on a much larger number of compounds that the precision of prediction from cytotoxicity data of low systemic toxicity is much better than the prediction of high systemic toxicity. Thus, we assumed that the BALB/3T3 NRU cytotoxicity assay may allow to discriminate between toxic/harmful and non-toxic substances, according to the EU and GHS classification systems. Taking into account also the high prevalence of ‘‘non-toxic” chemicals in the whole population of chemicals registered (i.e. the proportion of chemicals falling into a category 5 and non-classified (LD50 > 2000 mg/kg b.w.), the use of this approach could significantly reduce in vivo testing for acute oral toxicity. However, to properly prove this, it is necessary to include in the analysis a larger number of chemical substances with and LD50 > 2000 mg/kg b.w., tested in the BALB/3T3 NRU cytotoxicity assay. For this purpose ECVAM funded in 2008 a follow-up validation study of the NRU cytotoxicity assay in BALB/3T3 cells. The main goal of the study is to evaluate the predictive capacity of the assay to give a simple ‘‘yes/no” answer in order to discriminate between chemicals with LD50 > 2000 mg/kg b.w. (GHS category 5 and not classified) and those falling in the other four GHS categories (i.e. GHS 1–4 with LD50 < 2000 mg/kg b.w.), as defined by the Globally Harmonised System (GHS) for the classification and labelling of acute oral toxicity (UN, 2007). According to a proposal of a regulation by the European Parliament and Council, the same categories will be soon applicable also in Europe (E.C., 2007). The predictive capacity (i.e. sensitivity, specificity, concordance) and the applicability domain of the BALB/3T3 NRU test used for this new purpose, will be assessed by testing coded reference substances using the already validated Standard Operating Procedure (SOP). In this case, according to the ECVAM’s modular approach (Hartung et al., 2004), only one laboratory is required to assess the predictive capacity and applicability domain (modules 5 and
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6), since the test definition, within- and between-laboratory reproducibility and transferability (modules 1–4) have been already evaluated in the NICEATM/ECVAM validation study, and the test has proven to be reliable (Anon, 2006). This approach allows for a significant saving in terms of time and costs of the validation studies. Fig. 2 illustrates the organisation of the study with respect to the tests included and the participating laboratories, as well as the responsibilities for management of the project, selection, coding and supply of test chemicals and the data analysis. The study is coordinated by the Management Team including two ECVAM staff members. ICCVAM is involved in the study as an observer. The Chemical Selection Committee has identified a total set of 56 industrial chemicals (drugs and pesticides were excluded) for distinguishing substances with LD50 > 2000 mg/kg b.w. (GHS category 5 and not classified) from those falling into GHS categories 1– 4. The test substances include a variety of molecular structures, balanced between two physical states (liquid and solid), as well as a wide range of physico-chemical properties. According to the database CosIng, hosted on the website of E.C. DG Enterprise (available at http://ec.europa.eu/enterprise/cosmetics/cosing), 60% of the selected compounds are also used as cosmetic ingredients (including hair dyes, surfactants, UV filters, masking agents, antioxidants, preservatives and others). Chemicals used in previous studies i.e. the NICEATM/ECVAM validation study and the ACuteTox project were not included. All 56 chemicals, which have been coded by ECVAM and distributed by Sigma Aldrich (Italy), will be tested in the Health and Safety Laboratory (HSL, UK) following the test method described in detail in the SOPs validated during the NICEATM/ECVAM validation study (Anon, 2006). The results from this laboratory will be used to establish the predictive capacity, according to module 5 (Hartung et al., 2004). Additionally, two variants of the validated procedure will be evaluated in two different laboratories using the same set of 56
substances. The Institute for Health and Consumer Protection (IHCP, JRC, Italy) will use an automated version of the protocol adapted for a robotic testing platform, while the Institute for In Vitro Sciences (IIVS, US) will test the chemicals with an abbreviated procedure, less expensive and time-consuming and thus more practical for industry. The results obtained with the two different protocols will be then compared with the data from the validated protocol. The NRU assay will be performed to analyse the in vitro toxicity of the selected industrial chemicals and in particular to determine the IC50 values for comparison with the rat oral acute toxicity data (LD50 values) collected from available literature and from databases of final risk assessment reports (e.g. ORATS). These data will be used to evaluate the predictive capacity of the assay. The study is foreseen to be completed in the second semester of 2009. It is expected that the proposed approach will allow identifying non-toxic substances, which are most relevant for use in cosmetic products, and thus enable to reduce animal testing for acute oral toxicity by more than 80% in this sector. 4. Estimation of acute oral toxicity using the NOAEL values form 28-days repeated dose studies in rats Despite enormous efforts undertaken in the area of systemic toxicity, none of the non-animal replacement methods (tests or testing strategies) for the prediction of acute oral toxicity, that are currently under development and evaluation, are ready, validated and regulatory accepted, although in vivo testing of cosmetic products and ingredients is already banned in Europe since the 11 March 2009. ECVAM recently performed an investigation (Bulgheroni et al., 2009) to assess whether it would be possible to extrapolate the toxicological information on acute oral toxicity of chemicals and cosmetic ingredients from the results of in vivo repeated dose toxicity studies. According to the 7th Amendment to the Cosmetics
Fig. 2. The organisation of the BALB/3T3 NRU cytotoxicity follow-up validation study. Laboratory 1 is the main laboratory performing the BALB/3T3 NRU validated protocol (assessment of Module 5); the results from laboratories 2 (automated protocol) and 3 (abbreviated protocol) will be evaluated in comparison to results from laboratory 1. Acronyms used in the Figure indicate: ECVAM (European Centre for the Validation of Alternative Methods), ECB (European Chemicals Bureau) [ECB was the official name of the unit January 2008. Currently most of the activities of this Unit have been handed over to other units of the Institute of Health and Consumer Protection (Ispra, Italy) and/or to the European Chemicals Agency (Helsinki, Finland).], ZEBET (Centre for Documentation and Evaluation of Alternatives to Animal Experiments), HSL (Heath and Safety Laboratory, UK), IHCP (Institute for Health and Consumer Protection, Ispra, Italy), IIVS (Institute for in Vitro Sciences, US), ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods.
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Directive, information derived from these studies (performed outside the EU) can still be used, at least until 2013. In the proposed approach, the compounds which have an LD50 > 2000 mg/kg (i.e. in the EU are not classified as acutely toxic via the oral route) will be identified using information from 28-days repeated dose toxicity studies. The evaluation was based on data retrieved from dossiers from the New Chemicals Database. Substances for which both the NOAEL values obtained from 28-days repeated dose studies in rats and the oral LD50 values were available in the submission files (1791 in total), were selected and included in the analysis. The substances were then grouped according to the GHS categories and from the obtained distribution the NOAEL P 200 mg/kg was identified as the best threshold that could discriminate ‘‘nontoxic” substances (LD50 > 2000 mg/kg b.w.) from the rest. This threshold allowed to correctly categorise 63% of the ‘‘non-toxic” compounds. Less than 1% of compounds were misclassified as ‘‘non-toxic” and all of them were falling in the harmful category (category 4). None of the toxic or very toxic substances were misclassified as ‘‘non-toxic”. The Negative Predictive Value (NPV) and the Positive Predictive Value (PPV) of this approach were 97% and 26.5%, respectively (for details of the analysis see Bulgheroni et al., 2009). The proposed approach could have an immediate impact for the testing of cosmetic ingredients, since, unlike data from in vivo acute toxicity studies, the results from repeated dose toxicity studies (performed outside the EU) can be still used until 2013. Taking into consideration the very high prevalence (87%) of substances categorised in EU as ‘‘non-toxic” after the acute oral exposure, we concluded that with this approach it is feasible to classify more than half of all the new chemical substances. Currently we are also evaluating whether in vitro cytotoxicity could be used to derive the starting dose for the 28-days repeated dose studies (as already demonstrated for acute oral toxicity), in order to replace the use of data from in vivo acute toxicity studies. 5. Conclusions The FP6 Integrated Project ACuteTox for the first time aims to provide an integrated testing strategy fully based on non-animal methods for a complete animal replacement for acute oral toxicity testing. However, even if the project will be fully successful, it will take few additional years to complete the scientific validation of the testing strategy and to achieve its regulatory acceptance. In the meantime results on the predictive capacity of cytotoxicity test to filter the ‘‘non-toxic” substances (LD50 > 2000 mg/kg b.w.) are expected in 2009. This approach could have an immediate impact especially in the context of the REACH legislation, given that 90% of the industrial chemicals are not classified as acutely toxic via the oral route (Bulgheroni et al., 2009). Moreover, the use of NOAEL values from 28-days repeated dose toxicity studies to identify with a high predictivity the ‘‘non-toxic” substances could be of major interest for the cosmetic industry since according to the Cosmetics Directive, although animal testing is already banned for all human health endpoint, however data coming from the repeated-dose studies in vivo can still be used until 2013. Acknowledgements The ACuteTox project is supported by an EU-FP6 Grant (FP6LIFESCIHEALTH-2004-512051). References Anon, 2001. Report of the International Workshop on In Vitro Methods for Assessing Acute Systemic Toxicity. NIH Publication No. 01-4499.
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iccvam.niehs.nih.gov/methods/acutetox/inv_cyto_wksp.htm> (accessed June 2009). Anon, 2003. Directive 2003/15/EC of the European Parliament and the Council of 27 February amending Directive 76/786/EEC on the approximation of the laws of the member States relating to cosmetic products. Official Journal of the European Communities L66, 26–35. Office for Official Publications of the European Communities, Luxembourg. Anon, 2005. REACH and the need for intelligent testing strategies. Ispra, Italy, Institute for Health and Consumer Protection, E.C. Joint Research Centre [EUR 21554 EN], 1–32. Anon, 2006. Background Review Document (BRD): Validation of Neutral Red Uptake Test Methods. NIH Publication No. 07-4518.
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Organisation for Economic Cooperation and Development, 2001a. Guideline for Testing of Chemicals Number 420 on Acute Oral Toxicity – Fixed Dose Procedure. Paris, France. Organisation for Economic Cooperation and Development, 2001b. Guideline for Testing of Chemicals Number 423 on Acute Oral Toxicity – Acute Toxic Class Method. Paris, France. Organisation for Economic Cooperation and Development 2001 (c). Guideline for Testing of Chemicals Number 425 on Acute Oral Toxicity – Up- And Down Procedure. Paris, France.
Organisation for Economic Cooperation and Development, 2001d. Series on Testing and Assessment Number 24: Guidance Document on Oral Toxicity Testing, ENV/ JM/MONO(2001)4. Paris, France. Sjöström, M., Kolman, A., Clemedson, C., Clothier, R., 2008. Estimation of human blood LC50 values for use in modeling of in vitro–in vivo data of the ACuteTox project. Toxicology In Vitro 22, 1405–1411. United Nations, 2007. Globally Harmonized System of Classification and Labelling of Chemicals (GHS), ST/SG/AC.10/30/Rev.2.