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Physiologically based kinetic (PBK) models and the virtual cell based assay can be linked to form so called physiologically based dynamic (PBD) models. This study illustrates the development and application of a PBK model for prediction of estragole-induced DNA adduct formation and hepatotoxicity in humans. To address the hepatotoxicity, HepaRG cells were used as a surrogate for liver cells, with cell viability being used as the in vitro toxicological endpoint. Information on DNA adduct formation was taken from the literature. Since estragole induced cell damage is not directly caused by the parent compound, but by a reactive metabolite, information on the metabolic pathway was incorporated into the model. In addition, a user-friendly tool was developed by implementing the PBK/D model into a KNIME workflow. This workflow can be used to perform in vitro to in vivo extrapolation and forward as backward dosimetry in support of chemical risk assessment., Graphical abstract Image 1, Highlights • VCBA is coupled with PBK model linking intracellular concentration to external dose. • The integrated model is applied to perform IVIVE of estragole-induced cell damage. • The IVIVE process is automated by means of a KNIME workflow.

Hepatic metabolic clearance is one of the most important factors driving the overall kinetics of chemicals including substances used in various product categories such as pesticides, biocides, pharmaceuticals, and cosmetics. A large number of in vitro systems from purified isozymes and subcellular organelles to hepatocytes in simple cultures and in complex scaffold setups are available for measuring hepatic metabolic clearance for different applications. However, there is currently no approach for systematically characterising and comparing these in vitro methods in terms of their design, applicability and performance. To address this, existing knowledge in the field of in vitro human hepatic metabolic clearance methods was gathered and analysed in order to establish a framework to systematically characterise methods based on a set of relevant components. An analogous framework would be also applicable for non-human in vitro systems. The components are associated with the biological test systems used (e.g. subcellular or cells), the in vitro method (e.g. number of cells, test item solubility), related analytical techniques, data interpretation methods (based on substrate depletion/metabolite formation), and performance assessments (precision and accuracy of clearance measurements). To facilitate the regulatory acceptance of this class of methods, it is intended that the framework provide the basis of harmonisation work within the OECD.

Introduction Physiologically based pharmacokinetic (PBPK) models may be useful in emergency risk assessment, after acute exposure to chemicals, such as dichloromethane (DCM). We evaluated the applicability of three PBPK models for human risk assessment following a single exposure to DCM: one model is specifically developed for DCM (Bos) and the two others are semi-generic ones (Mumtaz and Jongeneelen). Materials and methods We assessed the accuracy of the models’ predictions by simulating exposure data from a previous healthy volunteer study, in which six subjects had been exposed to DCM for 1 h. The time-course of both the blood DCM concentration and percentage of carboxyhemoglobin (HbCO) were simulated. Results With all models, the shape of the simulated time course resembled the shape of the experimental data. For the end of the exposure, the predicted DCM blood concentration ranged between 1.52–4.19 mg/L with the Bos model, 1.42–4.04 mg/L with the Mumtaz model, and 1.81–4.31 mg/L with the Jongeneelen model compared to 0.27–5.44 mg/L in the experimental data. % HbCO could be predicted only with the Bos model. The maximum predicted % HbCO ranged between 3.1 and 4.2% compared to 0.4–2.3% in the experimental data. The % HbCO predictions were more in line with the experimental data after adjustment of the Bos model for the endogenous HbCO levels. Conclusions The Bos Mumtaz and Jongeneelen PBPK models were able to simulate experimental DCM blood concentrations reasonably well. The Bos model appears to be useful for calculating HbCO concentrations in emergency risk assessment.

Highlights • The concept of Margin of Internal Exposure (MOIE) is proposed for route-to-route extrapolation. • It is an extension of the Margin of Exposure (MOE) approach for cosmetics in the EU. • PBK modelling integrates in vitro and in silico predictions of ADME-properties. • The MOIE approach is transparent and facilitates to make uncertainties explicit. • The MOIE can be extended to include in vitro toxicity data in animal-free risk assessment., Route-to-route extrapolation is a common part of human risk assessment. Data from oral animal toxicity studies are commonly used to assess the safety of various but specific human dermal exposure scenarios. Using theoretical examples of various user scenarios, it was concluded that delineation of a generally applicable human dermal limit value is not a practicable approach, due to the wide variety of possible human exposure scenarios, including its consequences for internal exposure. This paper uses physiologically based kinetic (PBK) modelling approaches to predict animal as well as human internal exposure dose metrics and for the first time, introduces the concept of Margin of Internal Exposure (MOIE) based on these internal dose metrics. Caffeine was chosen to illustrate this approach. It is a substance that is often found in cosmetics and for which oral repeated dose toxicity data were available. A rat PBK model was constructed in order to convert the oral NOAEL to rat internal exposure dose metrics, i.e. the area under the curve (AUC) and the maximum concentration (Cmax), both in plasma. A human oral PBK model was constructed and calibrated using human volunteer data and adapted to accommodate dermal absorption following human dermal exposure. Use of the MOIE approach based on internal dose metrics predictions provides excellent opportunities to investigate the consequences of variations in human dermal exposure scenarios. It can accommodate within-day variation in plasma concentrations and is scientifically more robust than assuming just an exposure in mg/kg bw/day.

In toxicity testing the oral route is in general the first choice. Often, appropriate inhalation and dermal toxicity data are absent. Risk assessment for these latter routes usually has to rely on route-to-route extrapolation starting from oral toxicity data. Although it is generally recognized that the uncertainties involved are (too) large, route-to-route extrapolation is applied in many cases because of a strong need of an assessment of risks linked to a given exposure scenario. For an adequate route-to-route extrapolation the availability of at least some basic toxicokinetic data is a pre-requisite. These toxicokinetic data include all phases of kinetics, from absorption (both absorbed fraction and absorption rate for both the starting route and route of interest) via distribution and biotransformation to excretion. However, in practice only differences in absorption between the different routes are accounted for. The present paper demonstrates the necessity of route-specific absorption data by showing the impact of its absence on the uncertainty of the human health risk assessment using route-to-route extrapolation. Quantification of the absorption (by in vivo, in vitro or in silico methods), particularly for the starting route, is considered essential.

Information on toxicokinetics is critical for animal-free human risk assessment. Human external exposure must be translated into human tissue doses and compared with in vitro actual cell exposure associated to effects (in vitro-in vivo comparison). Data on absorption, distribution, metabolism and excretion in humans (ADME) could be generated using in vitro and QSAR tools. Physiologically-based toxicokinetic (PBTK) computer modelling could serve to integrate disparate in vitro and in silico findings. However, there are only few freely-available PBTK platforms currently available. And although some ADME parameters can be reasonably estimated in vitro or in silico, important gaps exist. Examples include unknown or limited applicability domains and lack of (high-throughput) tools to measure penetration of barriers, partitioning between blood and tissues and metabolic clearance. This paper is based on a joint EPAA--EURL ECVAM expert meeting. It provides a state-of-the-art overview of the availability of PBTK platforms as well as the in vitro and in silico methods to parameterise basic (Tier 1) PBTK models. Five high-priority issues are presented that provide the prerequisites for wider use of non-animal based PBTK modelling for animal-free chemical risk assessment.

International audience; Toxicokinetics (TK) is the endpoint that informs about the penetration into and fate within the body of a toxic substance, including the possible emergence of metabolites. Traditionally, the data needed to understand those phenomena have been obtained in vivo. Currently, with a drive towards non-animal testing approaches, TK has been identified as a key element to integrate the results from in silico, in vitro and already available in vivo studies. TK is needed to estimate the range of target organ doses that can be expected from realistic human external exposure scenarios. This information is crucial for determining the dose/concentration range that should be used for in vitro testing. Vice versa, TK is necessary to convert the in vitro results, generated at tissue/cell or sub-cellular level, into dose response or potency information relating to the entire target organism, i.e. the human body (in vitro-in vivo extrapolation, IVIVE). Physiologically based toxicokinetic modelling (PBTK) is currently regarded as the most adequate approach to simulate human TK and extrapolate between in vitro and in vivo contexts. The fact that PBTK models are mechanism-based which allows them to be 'generic' to a certain extent (various extrapolations possible) has been critical for their success so far. The need for high-quality in vitro and in silica data on absorption, distribution, metabolism as well as excretion (ADME) as input for PBTK models to predict human dose-response curves is currently a bottleneck for integrative risk assessment. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

The relatively high experimental animal use in developmental toxicity testing has stimulated the search for alternatives that are less animal intensive. Three widely studied alternative assays are the mouse Embryonic Stem cell Test (EST), the Zebrafish Embryotoxicity Test (ZET) and the rat postimplantation Whole Embryo Culture (WEC). The goal of this study was to determine their efficacy in assessing the relative developmental toxicity of six 1,2,4-triazole compounds,(1) flusilazole, hexaconazole, cyproconazole, triadimefon, myclobutanil and triticonazole. For this purpose, we analyzed effects and relative potencies of the compounds in and among the alternative assays and compared the findings to their known in vivo developmental toxicity. Triazoles are antifungal agents used in agriculture and medicine, some of which are known to induce craniofacial and limb abnormalities in rodents. The WEC showed a general pattern of teratogenic effects, typical of exposure to triazoles, mainly consisting of reduction and fusion of the first and second branchial arches, which are in accordance with the craniofacial malformations reported after in vivo exposure. In the EST all triazole compounds inhibited cardiomyocyte differentiation concentration-dependently. Overall, the ZET gave the best correlation with the relative in vivo developmental toxicities of the tested compounds, closely followed by the EST. The relative potencies observed in the WEC showed the lowest correlation with the in vivo developmental toxicity data. These differences in the efficacy between the test systems might be due to differences in compound kinetics, in developmental stages represented and in the relative complexity of the alternative assays.

The 7th amendment to the EU Cosmetics Directive prohibits to put animal-tested cosmetics on the market in Europe after 2013. In that context, the European Commission invited stakeholder bodies (industry, non-governmental organisations, EU Member States, and the Commissions Scientific Committee on Consumer Safety) to identify scientific experts in five toxicological areas, i.e. toxicokinetics, repeated dose toxicity, carcinogenicity, skin sensitisation, and reproductive toxicity for which the Directive foresees that the 2013 deadline could be further extended in case alternative and validated methods would not be available in time. The selected experts were asked to analyse the status and prospects of alternative methods and to provide a scientifically sound estimate of the time necessary to achieve full replacement of animal testing. In summary, the experts confirmed that it will take at least another 79 years for the replacement of the current in vivo animal tests used for the safety assessment of cosmetic ingredients for skin sensitisation. However, the experts were also of the opinion that alternative methods may be able to give hazard information, i.e. to differentiate between sensitisers and non-sensitisers, ahead of 2017. This would, however, not provide the complete picture of what is a safe exposure because the relative potency of a sensitiser would not be known. For toxicokinetics, the timeframe was 57 years to develop the models still lacking to predict lung absorption and renal/biliary excretion, and even longer to integrate the methods to fully replace the animal toxicokinetic models. For the systemic toxicological endpoints of repeated dose toxicity, carcinogenicity and reproductive toxicity, the time horizon for full replacement could not be estimated.

The health risks of inhalation exposure to volatile organic solvents may not only depend on the total external dose, but also on the pattern of exposure. It has been suggested that exposure to regularly occurring peak concentrations may have a stronger impact on the brain than constant exposure at the same average level. Recent animal experimental studies conducted in our laboratory using relatively high concentrations of toluene have shown different effects on discrimination performance and motor activity during and after exposure, depending on the exposure scenario. Relevance of these findings for man was evaluated in a volunteer study in which 11 healthy men (age 20-49 years) were exposed by inhalation for 4 h to either a constant concentration of 40 ppm toluene or to three 30-min exposure peaks at 110 ppm during this 4 h period. Selected tests from the Neurobehavioural Evaluation System (NES) were performed repeatedly during and after exposure. Blood concentrations of toluene as well as urinary o-cresol excretion were measured at relevant time points. The results show that toluene concentration in blood increased during constant exposure and fluctuated during occupationally relevant peak exposures. Presumably, brain concentrations showed similar qualitative patterns. No clear changes were observed on neurobehavioural measures of motor performance, attention, perceptual coding and memory, or on measures of mood and affect. The exposure conditions do not seem to induce significant acute changes in central nervous system function similar to those observed at much higher concentrations in animals, although a statistical correlation was found between one motor performance test (Finger Tapping Test with alternating hands) and blood toluene concentrations. Urinary o-cresol excretion appeared to be significantly higher during the first 2 h after exposure. © 2005 Elsevier B.V. All rights reserved.

The subacute oral and inhalation toxicity of furfural vapour was studied in Fischer 344 rats to investigate whether route-to-route extrapolation could be employed to derive the limit value for inhalation exposure from oral toxicity data. Groups of 5 rats per sex were treated by gavage daily for 28 days at dose levels of 6-192 mg/kg bw/day, or exposed by inhalation to concentrations of 20-1280 mg/m3 (6 h/day, 5 days/week) or 160-1280 mg/m3 (3 h/day, 5 days/week) for 28 days. Controls received vehicle (corn oil) or were exposed to clean air. Daily oral treatment with the highest dose of furfural (initially 192 mg/kg bw/day, later reduced to 144 mg/kg bw/day and finally to 120 mg/kg bw/day) resulted in mortality, and in increases in absolute and relative kidney and liver weight in surviving females of this group. Exposure of rats by inhalation for 6 h/day, 5 days/week for 28 days induced mortality at concentrations of 640 mg/m3 and above within 1-8 days. At 640 mg/m3 (3 h/day) and at 320 mg/m3 (3 and 6 h/day) and below, however, exposure was tolerated without serious clinical effects. In contrast, histopathological nasal changes were seen even at the lowest concentration of 20 mg/m3. With increasing exposure concentration, the nasal effects increased in incidence and severity and also expanded from the anterior part to the posterior part, including the olfactory epithelium. It was concluded that the no-observed-adverse-effect level (NOAEL) for oral toxicity was 96 mg/kg bw/day. The NOAEL for systemic inhalation toxicity was comparable, i.e. 92 mg/kg bw/day (corresponding to 320 mg/m3 (6 h/day) or 640 mg/m3 (3 h/day)) assuming 100% absorption. The presence of the histopathological nasal changes at the lowest tested concentration of 20 mg/m3 (corresponding to 6 mg/kg bw/day) proves that for locally acting substances like furfural extrapolation from the oral to the inhalation route is not valid. © 2004 Elsevier Ltd. All rights reserved.

An overview is presented on the molecular aspects of toxicity due to paracetamol (acetaminophen) and structural analogues. The emphasis is on four main topics, that is, bioactivation, detoxication, chemoprevention, and chemoprotection. In addition, some pharmacological and clinical aspects are discussed briefly. A general introduction is presented on the biokinetics, biotransformation, and structural modification of paracetamol. Phase II biotransformation in relation to marked species differences and interorgan transport of metabolites are described in detail, as are bioactivation by cytochrome P450 and peroxidases, two important phase I enzyme families. Hepatotoxicity is described in depth, as it is the most frequent clinical observation after paracetamol-intoxication. In this context, covalent protein binding and oxidative stress are two important initial (Stage I) events highlighted. In addition, the more recently reported nuclear effects are discussed as well as secondary events (Stage II) that spread over the whole liver and may be relevant targets for clinical treatment. The second most frequent clinical observation, renal toxicity, is described with respect to the involvement of prostaglandin synthase, N-deacetylase, cytochrome P450 and glutathione S-transferase. Lastly, mechanism-based developments of chemoprotective agents and progress in the development of structural analogues with an improved therapeutic index are outlined.

As part of a health-hazard survey on the health risk of hospital cleaning workers from exposure to Lyorthol, a hazard assessment of o-benzo-p-chlorophenol, one of the constituents of Lyorthol, has been prepared. In this paper, the physical and chemical characteristics, kinetics and effects of o-benzochlorophenol are described and discussed, and an overall, summarizing hazard evaluation is presented.

1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5-disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) on incubation of PAR and 3,5-diCH3-, 3,5-diC2H5-, 3,5-ditC4H9-, 3,5-diOCH3-, 3,5-diSCH3-, 3,5-diF-, 3,5-diCl- and 3,5-diBr-substituted analogues of PAR with horseradish peroxidase in the presence of hydrogen peroxide (H2O2). Initial analysis of the observed ESR spectra revealed all radical species to be phenoxy radicals, based on the absence of dominant nitrogen hyperfine splittings. No radicals were detected in rat liver cytochrome P450-containing microsomal or reconstituted systems. 2. To rationalize the observed ESR spectra, hydrogen atom abstraction of PAR and four of the 3,5-disubstituted analogues (3,5-diCH3-, 3,5-diOCH3-, 3,5-diF- and 3,5-diCl-PAR) was calculated using ab initio calculations, and a singlet oxygen atom was used as the oxidizing species. The calculations indicated that for all compounds studied an initial hydrogen atom abstraction from the phenolic hydroxyl group is favoured by approximately 125 kJ/mol over an initial hydrogen atom abstraction from the acetylamino nitrogen atom, and that after hydrogen abstraction from the phenolic hydroxyl group, the unpaired electron remains predominantly localised at the phenoxy oxygen atom (+/-85%). 3. The experimental finding of phenoxy radicals in horseradish peroxidase/H2O2 incubations paralleled these theoretical findings. The failure to detect experimentally phenoxy radicals in cytochrome P450-catalysed oxidation of any of the eight 3,5-disubstituted PAR analogues is more likely due to the reducing effects that agents like NADPH and protein thiol groups have on phenoxy radicals rather than on the physical instability of the respective substrate radicals.

In the current EU legislative frameworks on chemicals safety, the requirements with respect to information on general kinetic parameters (absorption, distribution, metabolism and excretion or ADME) or integrated toxicokinetic parameters (TK, i.e. plasma concentration-time curve, area under the curve etcetera) in humans and experimental animals vary widely. For agrochemicals and cosmetics, there are regulatory requirements whereas for other frameworks, such as food ingredients, biocides, consumer products and high production volume chemicals (REACH) there are very little or no requirements. This paper presents case studies that illustrate the importance of ADME and TK data in regulatory risk characterisations. The examples were collected by interviewing regulatory risk assessors from various chemicals (non-pharmaceutical) frameworks. The case studies illustrate how (1) applying ADME/TK in an early phase of toxicity testing can be used to improve study design and support the 3R-goals and how (2) increased use of ADME/TK data can improve the final risk assessment.

Hazard characterisation is largely based on an approach of (statistically) comparing dose groups with the controls in order to derive points of departure such as no-observed-adverse-effect levels (NOAELs) or lowest-observed-adverse-effect levels (LOAELs). This approach suggests the absence of any relevant effect at the NOAEL. The NOAEL approach has been debated for decades. A recent Scientific Opinion by the European Food Safety Authority (EFSA) concluded that the Benchmark Dose (BMD) approach should be preferred over the NOAEL approach for deriving human (health-based) limit or guidance values. Nonetheless, the BMD approach is used infrequently within European regulatory frameworks. The reason for this may lie in legislation or guidelines requiring the use of the NOAEL approach. In this context, various EU regulatory frameworks were examined on such demands. Interestingly, no single legislation was identified containing statutory requirements in conflict with the use of the BMD approach.

1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -C(H)3, -C2H5, -iC3H7) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K(s,app) varied from 0.14 mM for 3,5-diiC3H7-paracetamol to 2.8 mM for paracetamol. 2. All seven analogues underwent rat liver microsomal cytochrome P450-dependent oxidation, as reflected by the formation of GSSG in the presence of GSH. The GSSG-formation was increased in all cases upon pretreatment of rats by β-naphthoflavone (βNF) and was generally decreased upon pretreatment by phenobarbital (PB). 3. Rat liver microsomal cytochrome P450 as well as horseradish peroxidase catalysed the formation of 3,5-disubstituted NAPQI analogues from the corresponding parent compounds, as identified by UV-spectrophotometry of the NAPQI analogues and by GC/MS detection of the following GSH-conjugates: 2-glutathione-S-yl-3,5-dimethyl-1,4-dihydroxybenzene, 2-glutathione-S-yl-3,5-dichloro-paracetamol, and 2-glutathione-S-yl-3,5-dibromo-paracetamol. 4. In liver microsomal (βNF-induced) incubations, apparent K(m) values, as determined for the cytochrome P450 catalysis-dependent oxidation of GSH, for seven 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -CH3, -C2H5, iC3H7) varied from 0.07 to 0.64 mM. Paracetamol exhibited an apparent K(m) of 0.73 mM. Apparent V(max) values for the cytochrome P450 catalysis dependent oxidation of GSH varied from 0.66 nmol min-1 mg-1 protein for paracetamol to 3.0 nmol min-1 mg-1 protein for 3,5-dimethyl-paracetamol. Chemicals/CAS: Acetaminophen, 103-90-2; Benzoquinones; beta-Naphthoflavone, 6051-87-2; Cytochrome P-450 Enzyme System, 9035-51-2; Glutathione, 70-18-8; Imines; N-acetyl-4-benzoquinoneimine, 50700-49-7

Seven 3,5-disubstituted analogues of paracetamol were synthesised in order to compare their physicochemical, pharmacological and toxicological properties with those of paracetamol (4'-hydroxyacetanilide, acetaminophen). Oxidation of the phenolic structure is likely involved in the analgesic action of paracetamol as well as in its toxification by cytochrome P450. The effect of disubstitution adjacent to the phenolic hydroxyl group was studied in order to establish possible structure-activity relationships. 3,5-Substituents with electron-donating capacities (R = -CH3, -OCH3, -SCH3) decreased the half-wave oxidation potential substantially by 0.07 V to 0.16 V, whereas electron-withdrawing substituents (R = -F, -Cl, -Br, or -I) increased the oxidation potential by 0.04 V to 0.06 V when compared to paracetamol. Electron-donating substituents (R = -CH3, -OCH3, -SCH3) increased the mouse brain cyclooxygenase inhibiting capacity of paracetamol. Electron-withdrawing halogen substituents (R = -F, -Cl, -Br or -I) decreased this inhibiting capacity. In agreement with this, the in vivo analgesic activity of the 3,5-dihalogenated analogues was lower when compared to paracetamol. Electron-donating substituents (R = -CH3, -OCH3, -SCH3) decreased the cytotoxicity of paracetamol, when measured as leakage of lactate dehydrogenase from freshly isolated rat hepatocytes, almost completely. Most 3,5-dihalogen substituents (R = -F, -Cl or -Br) diminished it slightly. The fourth electron-withdrawing substituent (R = -I) strongly lowered the cytotoxicity of paracetamol in this test system. In conclusion, a higher cycylooxygenase inhibitory potency of 3,5-disubstituted analogues of paracetamol seemed to correlate with a lower cytotoxicity. 3,5-Disubstituted analogues with electron-donating substituents might therefore be safer analgesics than paracetamol itself. The opposite probably applies to analogues of paracetamol with electron-withdrawing substituents at the 3- and 5- positions of the aromatic nucleus.

The World Health Organization (WHO) International Programme on Chemical Safety (IPCS) Guidance on Characterization and Application of Physiologically Based Pharmacokinetic Models in Risk Assessment (IPCS, 2010) describes key principles for risk assessors and model developers. In the WHO Guidance, a template for model documentation was developed and a case study included. Here the WHO Guidance, including the template, is summarized and an additional case study is presented to illustrate its application, based upon an existing risk assessment for 2-butoxyethanol (CAS NO. 111-76-2). The goal of the WHO Guidance and the current paper is to increase regulatory acceptance of complex biologically descriptive pharmacokinetic (or toxicokinetic) models, such as PBPK models, by facilitating communication and successful interaction between modelers and risk assessors.

The chemical legislation of the EU, Registration, Evaluation, and Authorization of Chemicals (REACH), stipulates that about 30 000 chemical substances are to be assessed on their possible risks. Toxicological evaluation of these compounds will at least partly be based on animal testing. In particular, the assessment of reproductive toxicity is a very complicated, time-consuming and animal-demanding process. Introducing microarray-based technologies can potentially refine in vivo toxicity testing. If compounds of a distinct chemical class induce reproducible gene-expression responses with a recognizable overlap, these gene-expression signatures may indicate intrinsic features of certain compounds, including specific toxicity. In the present study, we have set out the first steps towards this approach for the reproductive toxicity of phthalates. Male rats were treated with a single dose of either reprotoxic or non-reprotoxic phthalates, and were analyzed 24 h afterwards. Subsequently, histopathological and gene-expression profiling analyses were performed. Despite ambiguous histopathological observations, we were able to identify genes with differential expression profiles between the reprotoxic phthalates and the non-reprotoxic counterparts. This shows that differences in gene-expression profiles, indicative of the type of exposure, may be detected earlier, or at lower doses, than classical pathological endpoints. These findings are promising for ‘early warning’ biomarker analyses and for using toxicogenomics in a category approach. Ultimately, this could lead to a more cost-effective approach for prioritizing the toxicity testing of large numbers of chemicals in a short period of time in hazard assessment of chemicals, which is one of the objectives of the REACH chemical legislation.

Hepatic systems toxicology is the integrative analysis of toxicogenomic technologies, e.g., transcriptomics, proteomics, and metabolomics, in combination with traditional toxicology measures to improve the understanding of mechanisms of hepatotoxic action. Hepatic toxicology studies that have employed toxicogenomic technologies to date have already provided a proof of principle for the value of hepatic systems toxicology in hazard identification. In the present review, acetaminophen is used as a model compound to discuss the application of toxicogenomics in hepatic systems toxicology for its potential role in the risk assessment process, to progress from hazard identification towards hazard characterization. The toxicogenomics-based parallelogram is used to identify current achievements and limitations of acetaminophen toxicogenomic in vivo and in vitro studies for in vitro-to-in vivo and interspecies comparisons, with the ultimate aim to extrapolate animal studies to humans in vivo. This article provides a model for comparison of more species and more in vitro models enhancing the robustness of common toxicogenomic responses and their relevance to human risk assessment. To progress to quantitative dose-response analysis needed for hazard characterization, in hepatic systems toxicology studies, generation of toxicogenomic data of multiple doses/concentrations and time points is required. Newly developed bioinformatics tools for quantitative analysis of toxicogenomic data can aid in the elucidation of dose-responsive effects. The challenge herein is to assess which toxicogenomic responses are relevant for induction of the apical effect and whether perturbations are sufficient for the induction of downstream events, eventually causing toxicity.

There are now numerous in vitro and in silico ADME alternatives to in vivo assays but how do different industries incorporate them into their decision tree approaches for risk assessment, bearing in mind that the chemicals tested are intended for widely varying purposes? The extent of the use of animal tests is mainly driven by regulations or by the lack of a suitable in vitro model. Therefore, what considerations are needed for alternative models and how can they be improved so that they can be used as part of the risk assessment process? To address these issues, the European Partnership for Alternative Approaches to Animal Testing (EPAA) working group on prioritisation, promotion and implementation of the 3Rs research held a workshop in November, 2008 in Duesseldorf, Germany. Participants included different industry sectors such as pharmaceuticals, cosmetics, industrial- and agro-chemicals. This report describes the outcome of the discussions and recommendations (a) to reduce the number of animals used for determining the ADME properties of chemicals and (b) for considerations and actions regarding in vitro and in silico assays. These included: standardisation and promotion of in vitro assays so that they may become accepted by regulators; increased availability of industry in vivo kinetic data for a central database to increase the power of in silico predictions; expansion of the applicability domains of in vitro and in silico tools (which are not necessarily more applicable or even exclusive to one particular sector) and continued collaborations between regulators, academia and industry. A recommended immediate course of action was to establish an expert panel of users, developers and regulators to define the testing scope of models for different chemical classes. It was agreed by all participants that improvement and harmonization of alternative approaches is needed for all sectors and this will most effectively be achieved by stakeholders from different sectors sharing data.

Both 1,2-dihydronaphthalene and 1,2-dihydroanthracene were hydroxylated at the benzylic (1-) or the allylic (2-) position by rat liver microsomes and purified cytochrome P-450 enzymes to yield "arene hydrates". Two other classes of metabolites were formed, the dehydrogenation products naphthalene and anthracene, and trans-1,2-dihydroxy-1,2,3,4-tetrahydronaphthalene and its anthracene analog as products of the classical expoxide pathway. Regioselectivity (hydroxylation at benzylic or allylic positions) and stereoselectivity (hydroxylation at pro-R or pro-S hydrogen atoms) during metabolism of dihydroarenes to yield arene hydrates were found to be dependent upon the nature of the inducing agents used during pretreatment of the rats and thus the level of particular P-450 enzymes. This selectivity was more pronounced for anthracene than for naphthalene. Naphthalene and anthracene were formed enzymatically by direct dehydrogenation of the dihydro compounds rather than by dehydration of the arene hydrate metabolites. A general mechanism involving the intermediacy of benzylic and resonance-stabilized allylic carbon radicals can account for the formation of both enzyme-catalyzed hydroxylation (arene hydrate) and dehydrogenation (arene) metabolites of dihydroarene substrates.

The new regulatory framework REACH (Registration, Evaluation, and Authorisation of Chemicals) foresees the use of non-testing approaches, such as read-across, chemical categories, structure–activity relationships (SARs) and quantitative structure–activity relationships (QSARs). Although information on skin absorption data are not a formal requirement under REACH, data on dermal absorption are an integral part of risk assessment of substances/products to which man is predominantly exposed via the dermal route. In this study, we assess the present applicability of publicly available QSARs on skin absorption for risk assessment purposes. We explicitly did not aim to give scientific judgments on individual QSARs. A total of 33 QSARs selected from the public domain were evaluated using the OECD (Organisation for Economic Co-operation and Development) Principles for the Validation of (Q)SAR Models. Additionally, several pragmatic criteria were formulated to select QSARs that are most suitable for their use in regulatory risk assessment. Based on these criteria, four QSARs were selected. The predictivity of these QSARs was evaluated by comparing their outcomes with experimentally derived skin absorption data (for 62 compounds). The predictivity was low for three of four QSARs, whereas one model gave reasonable predictions. Several suggestions are made to increase the applicability of QSARs for skin absorption for risk assessment purposes.

The increasing use of tissue dosimetry estimated using pharmacokinetic models in chemical risk assessments in various jurisdictions necessitates the development of internationally recognized good modelling practice (GMP). These practices would facilitate sharing of models and model evaluations and consistent applications in risk assessments. Clear descriptions of good practices for (1) model development i.e., research and analysis activities, (2) model characterization i.e., methods to describe how consistent the model is with biology and the strengths and limitations of available models and data, such as sensitivity analyses, (3) model documentation, and (4) model evaluation i.e., independent review that will assist risk assessors in their decisions of whether and how to use the models, and also model developers to understand expectations for various purposes e.g., research versus application in risk assessment. Next steps in the development of guidance for GMP and research to improve the scientific basis of the models are described based on a review of the current status of the application of physiologically based pharmacokinetic (PBPK) models in risk assessments in Europe, Canada, and the United States at the International Workshop on the Development of GMP for PBPK Models in Greece on April 27–29, 2007. Crown copyright © 2008 Published by Elsevier Inc. All rights reserved.

If in vitro data are to be used as a basis for hazard characterization, a translation of an in vitro concentration toward an in vivo dose must be made. In this study we examined the correlation between dose descriptors from the in vitro Whole Embryo Culture (WEC) test and in vivo developmental toxicity tests. We applied the Benchmark Dose (BMD) approach to estimate equipotent in vitro concentrations (Benchmark Concentrations [BMCs]) and equipotent in vivo doses (BMDs). Using the data generated in an European Center for the Validation of Alternative Methods validation study we found that the BMCs were highly reproducible among laboratories. The three endpoints analyzed (head length, crown-rump length, and total morphological score) were strongly correlated. A clear in vitro-in vivo correlation was found between BMCs and BMDs. However, a considerable uncertainty would remain if the BMDs were estimated from the BMC using this correlation: the confidence interval of such an in vivo dose estimate would span various orders of magnitude. Differences in toxicokinetic properties among the compounds explained at least part of the scatter of the in vitro-in vivo correlation. But also heterogeneity in the design of the available in vivo studies underlies much of the scatter, and this puts a limit on validating in vitro data as predictors of in vivo data. Further analysis of the in vitro-in vivo correlation would therefore require high-quality in vivo data, generated by appropriate (and similar) study designs.

Information on human toxicokinetics plays an important role in the safety assessment of chemicals, even though there are few data requirements in the EU regulatory framework. While existing EU test methods and OECD test guidelines are mostly based on animal procedures, there are increasing opportunities to achieve a 3Rs impact in this area by exploiting modern developments. For example, whole-body toxicokinetic information can be obtained by using physiologically-based toxicokinetic (PBTK) models that integrate data generated by in vitro methods for absorption, distribution, metabolism and excretion (ADME). The development of an infrastructure providing access to such models and their underlying data needs to be accompanied by the establishment of standards for human in vitro ADME methods, the development of guidance on the development and application of such models and the creation of regulatory incentives. Taking these needs into account, this report describes the EURL ECVAM strategy to achieve a 3Rs impact in the area of toxicokinetics and systemic toxicity. The proposed activities are expected to lay the foundation for a risk assessment approach that is increasingly based on human data. Implementation of the strategy will rely on the coordinated efforts of multiple stakeholders. EURL ECVAM strategy for achieving 3Rs impact in the assessment of toxicokinetics and systemic toxicity Jos Bessems, Sandra Coecke, Varvara Gouliarmou, Maurice Whelan, Andrew Worth

Due to a lack of route-specific toxicity data, the health risks resulting from occupational exposure are frequently assessed by route-to-route (RtR) extrapolation based on oral toxicity data. Insight into the conditions for and the uncertainties connected with the application of RtR extrapolation has not been clearly described in a systematic manner. In our opinion, for a reliable occupational health risk assessment, it is necessary to have insight into the accuracy of the routinely applied RtR extrapolation and, if possible, to give a (semi-)quantitative estimate of the possible error introduced. Therefore, experimentally established no-observed-adverse-effect-levels for inhalation studies were compared to no-adverse-effect-levels predicted from oral toxicity studies by RtR extrapolation. From our database analysis it can be concluded that the widely used RtR extrapolation methodology based on correction for differences in (estimates of) absorption is not generally reliable and certainly not valid for substances inducing local effects. More experimental data are required (from unpublished data or new experiments) to get insight into the reliability of RtR extrapolation and the possibility to derive an assessment factor to account for the uncertainties. Moreover, validated screening methods to predict/exclude the occurrence of local effects after repeated exposure are warranted. Especially, in cases where chemical exposure by inhalation or skin contact cannot be excluded route-specific toxicity studies should be considered to prevent from inadequate estimates of human health risks. © 2003 Elsevier Inc. All rights reserved.

The effect of 3,5-dihalogenation of paracetamol (PAR) on the cytotoxicity in rat hepatocytes isolated from β-naphthoflavone pretreated, non-fasted rats, and the role of cytochrome P-450 in this regard, were studied. On incubation, 3,5-difluoro-PAR, 3,5-dichloro-PAR and 3,5-dibromo-PAR, as well as PAR, caused severe leakage of lactate dehydrogenase (LDH) which was preceded by a rapid concentration- and time-dependent depletion of intracellular glutathione (GSH). IC50 values, representing the concentration of compound that caused 50% GSH depletion after 30 min of incubation, varied from 0.1 to 0.5 mM. This LDH leakage and GSH depletion could be inhibited by 1-ethynylpyrene. In hepatocytes from uninduced rats, GSH depletion was much less prominent and the concomitant LDH leakage almost completely absent. HPLC analysis of soluble metabolites and gas chromatography-mass spectrometry analysis, after alkaline peralkylation of the protein fraction, revealed (a) that 3,5-dihalogenated PAR analogues were liable to structure-related detoxification by glucuronidation, and (b) analogous to PAR, a substantial amount of each 3,5-dihalogenated PAR analogue was bioactivated by cytochrome P-450, ultimately leading to GSH-conjugates as well as (for 3,5-dichloro-PAR and 3,5-dibromo-PAR), protein adducts at regio-specific aromatic positions.

The protective effect of ebselen (PZ 51), an anti-inflammatory agent, on paracetamol-induced (1 mM) cytotoxicity in hepatocytes freshly isolated from beta-naphthoflavone-pretreated rats was studied. At a concentration of 50 microM added simultaneously with paracetamol, ebselen prevented paracetamol-induced leakage of lactate dehydrogenase (LDH) almost completely and lipid peroxidation (LPO) and depletion of glutathione (GSH) substantially. These protective effects were even more pronounced at 100 microM concentration of ebselen. When added to the hepatocytes 1 hr before paracetamol, 50 microM of ebselen also prevented LDH leakage, LPO and GSH depletion. Reverse addition of paracetamol and ebselen did not result in protection. Simultaneous incubation of 100 microM ebselen and paracetamol inhibited GSH conjugation of paracetamol by more than 50%, however, without any effect on glucuronidation and sulfation of paracetamol. Ebselen was shown not to react directly with paracetamol nor to inhibit cytochrome P450 activity measured as 7-ethoxycoumarin O-deethylase (ECD) activity in the hepatocytes. At mixing, synthetic ebselen selenol and synthetic N-acetyl-p-benzoquinone imine (NAPQI) were shown to form paracetamol and ebselen diselenide. No indication was found for the formation of an ebselen-paracetamol conjugate upon reacting synthetic NAPQI and synthetic ebselen selenol. Reduction of NAPQI, the reactive metabolite of paracetamol, by ebselen selenol is discussed in terms of the mechanism of cytoprotection.

(1992). Molecular Aspects of Paracetamol-Induced Hepatotoxicity and its Mechanism-Based Prevention. Drug Metabolism Reviews: Vol. 24, No. 3, pp. 367-407.

The relation between the major toxic effect of hexachlorobenzene, hepatic porphyria, and its oxidative biotransformation was studied in vivo, by observing the effect of modulating its biotransformation on the expression of porphyria. This modulation was achieved by selective in vivo inhibition of the major cytochrome P450 isoenzyme involved in both the hydroxylation of hexachlorobenzene and its primary oxidative metabolite, pentachlorophenol. The involvement of this isoenzyme, cytochrome P450p, was established by in vitro biotransformation studies using microsomes derived from rats treated with various inducers of cytochrome P450 isoenzymes and selective in vitro inactivation of cytochrome P450p by triacetyloleandomycin (TAO), resulting in a strong inhibition of the microsomal conversion of hexachlorobenzene and pentachlorophenol. In vivo inactivation of cytochrome P450p was achieved by coadministration of hexachlorobenzene and TAO. Female rats which were treated with this diet for 10 weeks showed a strongly diminished urinary excretion of the major oxidative metabolites, pentachlorophenol and tetrachloro-1,4-hydroquinone, as compared to rats treated with hexachlorobenzene alone. The TAO coadministration was found to result in complexation of 70% of the total amount of hepatic microsomal cytochrome P450. The group treated with hexachlorobenzene alone displayed a 600-fold increase in the amount of hepatic porphyrins, whereas an almost complete absence of hepatic porphyrins was observed after administration of hexachlorobenzene together with TAO. The urinary excretion of porphyrins was also significantly lowered by cotreatment with TAO. A strong correlation was found to exist between the amount of porphyrins excreted and the amount of oxidative metabolites excreted, as a function of exposure time. Glucuronidation of pentachlorophenol was observed to an average extent of 30%. This percentage was not influenced by either TAO or phenobarbital. These results suggest that oxidative biotransformation, and thus the formation of the very reactive tetrachloro-1,4-benzoquinone, is directly related to the porphyrinogenic action of hexachlorobenzene.