Assessing the transferability and reproducibility of 3D in vitro liver models from primary human multi-cellular microtissues to cell-line based HepG2 spheroids

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 μg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.Toxicolog

Anticoagulant rodenticides in predatory birds 2010: a Predatory Bird Monitoring Scheme (PBMS) report

The Predatory Bird Monitoring Scheme (PBMS; http://pbms.ceh.ac.uk/) is the umbrella project that encompasses the Centre for Ecology & Hydrology’s National Capability contaminant monitoring and surveillance work on avian predators. By monitoring sentinel vertebrate species, the PBMS aims to detect and quantify current and emerging chemical threats to the environment and in particular to vertebrate wildlife. Anticoagulant rodenticides, and in particular second generation anticoagulant rodenticides (SGARs), can be toxic to all mammals and birds. Predators that feed upon rodents are particularly likely to be exposed to these compounds. The PBMS, together with other studies, have shown that there is widespread exposure to SGARs of a diverse range of predators in Britain and that some mortalities occur as a result. This report summarises the PBMS monitoring for anticoagulant rodenticides in barn owls (Tyto alba), kestrel (Falco tinnunculus) and red kites (Milvus milvus) that were found dead in 2010 and presents long term trend analysis for barn owls. SGARs were detected in 91% of 53 barn owls analysed and the most prevalent compounds were difenacoum and bromadiolone. The majority of the residues were low and not diagnosed as directly causing mortality. The livers from 24 red kites analysed in 2010. Most (92%) had detectable liver SGAR concentrations, again mainly difenacoum and bromadiolone, although brodifacoum was also detected in over half the birds. Five of the red kites analysed showed signs of haemorrhaging thought possibly to be associated with rodenticide poisoning. However, only one of these birds had a sum SGAR liver concentration greater than 0.05 μg/g wet weight and the contribution of SGARs, if any, to the death of the other four birds is uncertain. SGARs were detected in 8 of 10 kestrels analysed. The most prevalent rodenticides detected in kestrel livers were those approved for outdoor use while the co-occurrence of multiple residues was also prevalent with 6 out of 10 kestrels having more than one SGAR present in their liver. Difethialone, a SGAR marketed since late 2011, was not detected in any of the three species tested in 2010. This represents a base-line against which future results can be compared. SGARs have been monitored in barn owls since 1983. Data on long-term trends have been adjusted to account for changes over time in sensitivity of analytical methods. This has meant that very low residues (<0.025 μg/g wet weight), which are now detectable, are not included in the time trend analysis. Overall, the proportion of barn owls with detectable liver concentrations of one or more SGAR has increased significantly over the course of monitoring. The highest value was recorded in 2008 but this was approximately twice that for the previous three years. The value for 2010 (23%) was lower than 2008 and is the lowest recorded since 2005. The proportion of barn owls with detectable SGAR residues over the period 1990-2010 was twofold higher in England than in Scotland and Wales. Within England, the proportion of owls with datable residues was higher in the Defra Eastern region than elsewhere

Anticoagulant rodenticides in predatory birds 2011: a Predatory Bird Monitoring Scheme (PBMS) report

The Predatory Bird Monitoring Scheme (PBMS; http://pbms.ceh.ac.uk/) is the umbrella project that encompasses the Centre for Ecology & Hydrology’s National Capability contaminant monitoring and surveillance work on avian predators. By monitoring sentinel vertebrate species, the PBMS aims to detect and quantify current and emerging chemical threats to the environment and in particular to vertebrate wildlife. Anticoagulant rodenticides, and in particular second generation anticoagulant rodenticides (SGARs), can be toxic to all mammals and birds. Predators that feed upon rodents are particularly likely to be exposed to these compounds. The PBMS, together with other studies, have shown that there is widespread exposure to SGARs of a diverse range of predators in Britain and that some mortalities occur as a result. This report summarises the PBMS monitoring for anticoagulant rodenticides in barn owls (Tyto alba), kestrel (Falco tinnunculus) and red kites (Milvus milvus) that were found dead in 2011 and presents long term trend analysis for barn owls. During this year’s analysis, a change to the analytical methods used by the PBMS was trialed where the use of matrix-matched standards was compared to solvent-matched standards. Matrix matched standards gave higher % recoveries for spiked samples and more repeatable results, and consequently resulted in reporting of higher liver SGAR concentrations in birds with detectable residues. It was concluded that matrix-matched standards provided a better analysis and would be used in this and future years analysis, but that a predicted solvent-matched equivalent SGAR residue would be calculated for use in time and spatial trend analysis that involved comparisons with data from previous years. This would eliminate biases that could otherwise be introduced into the analysis due to changes in analytical methodology. In birds that died in 2011, SGARs were detected in 84% of 58 barn owls analysed and the most prevalent compounds were difenacoum and bromadiolone. The majority of the residues were low and not diagnosed as directly causing mortality. The livers from 18 red kites were analysed in 2011. Most (94%) had detectable liver SGAR concentrations, again mainly difenacoum and bromadiolone, although brodifacoum was also detected in 78% of the birds. Six of the red kites analysed showed signs of haemorrhaging thought possibly to be associated with rodenticide poisoning. However, only two of these birds had relatively high sum SGAR liver concentrations (> 0.4 µg/g wet weight) and the contribution of SGARs, if any, to the death of the other four birds is uncertain. SGARs were detected in all 20 kestrel analysed. The most prevalent rodenticides detected in kestrel livers were difenacoum and bromadiolone. The co-occurrence of multiple residues was also prevalent with 19 out of 20 kestrels having more than one SGAR present in their liver. Due to a new collaborative arrangement with the Hawk Conservancy Trust the PBMS received a higher proportion of its barn owls and kestrels from the counties of Berkshire, Hampshire, Oxfordshire & Wilstshire. These counties are within a focus of rodenticide resistance in the Norway rat (Rattus norvegicus) and we tested whether there were significant differences for the prevalence and magnitude of SGAR residues in barn owls and kestrels between this area and other counties. There were no significant differences between either barn owls or kestrels from resistance and non-resistance counties in either the proportion of birds with detectable liver SGAR residues or the magnitude of liver SGAR concentrations in those birds with detected residues. However, the sample size examined was relatively small and it would be valuable in the future to conduct an analysis of the potential impact of resistance on residue prevalence and magnitude for birds collected over a longer time-scale and incorporating all counties where resistance to SGARs in rats has been documented. SGARs have been monitored in barn owls since 1983. Data on long-term trends have been adjusted to account for changes over time in sensitivity of analytical methods. This has meant that very low residues (<0.025 µg/g wet weight), which are now detectable, are not included in the time trend analysis. Overall, the proportion of barn owls with detectable liver concentrations of one or more SGAR has increased significantly over the course of monitoring. The highest value was recorded in 2008 while the value for 2011 was 25.9%. The proportion of barn owls with detectable SGAR residues over the period 1990-2011 was two-fold higher in England than in Scotland and Wales and also varied significantly between different regions of England. Between 1997 and 2011 there has not been any significant progressive increase or decrease in detectable SGAR residues in kestrels

Anticoagulant rodenticides in predatory birds 2009: a Predatory Bird Monitoring Scheme (PBMS) report.

The Predatory Bird Monitoring Scheme (PBMS; http://pbms.ceh.ac.uk/) is the umbrella project that encompasses the Centre for Ecology & Hydrology’s National Capability contaminant monitoring and surveillance work on avian predators. By monitoring sentinel vertebrate species, the PBMS aims to detect and quantify current and emerging chemical threats to the environment and in particular to vertebrate wildlife. Anticoagulant rodenticides, and in particular second generation anticoagulant rodenticides (SGARs), can be toxic to all mammals and birds. Predators that feed upon rodents are particularly likely to be exposed to these compounds. The PBMS, together with other studies, have shown that there is widespread exposure to SGARs of a diverse range of predators in Britain and that some mortalities occur as a result. This report summarises the PBMS monitoring for anticoagulant rodenticides in barn owls (Tyto alba), and red kites (Milvus milvus) that were found dead in 2009 and presents long term trend analysis for barn owls. Since 2006, anticoagulant rodenticide concentrations have been quantified using the more sensitive Liquid Chromatography – Mass Spectrometry (LC-MS) method. This has resulted in lower concentrations of these compounds being detected than was previously possible. Consequently, for samples from 2006 onwards, the proportion of birds in which anticoagulant rodenticides have been detected has increased compared to previous years. SGARs were detected in 89% of barn owls and the most prevalent compounds were difenacoum and bromadiolone. The majority of the residues were low and not diagnosed as directly causing mortality. Only five red kites were received by the scheme in 2009. Most of the red kites (4 o/5 birds) had detectable liver SGAR concentrations, again mainly difenacoum and bromadiolone although brodifacoum was also detected in over half the birds. Two of the five red kites analysed showed signs of haemorrhaging thought possibly to be associated with rodenticide poisoning. SGARs have been monitored in barn owls since 1983. Data on long-term trends have been adjusted to account for changes over time in sensitivity of analytical methods. This has meant that very low residues (<0.025µg/g wet weight), which are now easily detectable, are not included in the time trend analysis. The proportion of owls with detectable SGAR residues was found to be two-fold higher in England than in either Scotland or Wales. Overall, the proportion of barn owls with detectable liver concentrations of one or more SGAR has increased significantly over the course of monitoring. The highest value was recorded in 2008 but this was approximately twice that for the previous three years. The value for 2009 was lower than 2008 but remains one of the highest recorded since monitoring began. Continued monitoring is required to determine whether the high detection rate for SGARs in barn owls in 2008 and 2009 will change. Although our data for red kites in 2009 is limited, it is consistent with a high proportion of red kites being exposed to SGARs and some dying as a result. This species remains at particular risk from anticoagulant rodenticides