Report from the EPAA workshop: In vitro ADME in safety testing used by EPAA industry sectors

AbstractThere 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

Toxicological profile for o-benzyl-p-chlorophenol

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

Oral-to-inhalation route extrapolation in occupational health risk assessment: A critical assessment

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

Cytotoxicity of paracetamol and 3,5-dihalgenated analogues: Role of cytochrome P450 and formation of GSH conjugates and protein adducts.

The effect of 3,5-dihalogenation of paracetamol (PAR) on the cytotoxicity in rat hepatocytes isolated from 13-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). I

Subacute (28-day) toxicity of furfural in Fischer 344 rats: A comparison of the oral and inhalation route

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

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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

Development of a QSAR for worst case estimates of acute toxicity of chemically reactive compounds

Future EU legislations enforce a fast hazard and risk assessment of thousands of existing chemicals. If conducted by means of present data requirements, this assessment will use a huge number of test animals and will be neither cost nor time effective. The purpose of the current research was to develop methods to increase the acceptability of in vitro data for classification and labelling regarding acute toxicity. For this purpose, a large existing database containing in vitro and in vivo data was analysed. For more than 300 compounds in the database, relations between in vitro cytotoxicity and rat or mouse intravenous and oral in vivo LD50 values were re-evaluated and the possibilities for definition of mechanism based chemical subclasses were investigated. A high in vitro¿in vivo correlation was found for chemicals classified as irritants. This can be explained by a shared unspecific cytotoxicity of these compounds which will act as the predominant mode of action for both endpoints, irritation and acute toxicity. For this subclass, which covered almost 40% of all compounds in the database, the LD50 values after intravenous dosing could be predicted with high accuracy. A somewhat lower accuracy was found for the prediction of oral LD50 values based on in vitro cytotoxicity data. Based on this successful correlation, a classification and labelling scheme was developed, that includes a hazard based definition of the applicability domain (irritants) and a prediction of the labelling of compounds for their acute iv and oral toxicity. The scheme was tested by an external validation