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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.

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.

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.

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 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.

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.

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 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.