Modeling responses of Daphnia magna to pesticide pulse exposure under varying food conditions: intrinsic versus apparent sensitivity

Recent studies showed that limiting food conditions resulted in either increased or decreased sensitivity of Daphnia magna to toxicants. It remained unclear whether these contrasting food-dependent alterations in toxicity resulted from differences in intrinsic sensitivity of the daphnids or from changes in toxicokinetics and resource allocation. It is hypothesized here that, if food level only affects accumulation kinetics and resource allocation, then the intrinsic sensitivity to this toxicant should be the same for all food regimes. This hypothesis was investigated using the DEBtox model, which is based on the theory of Dynamic Energy Budgets. We examined results of two recently conducted life-cycle studies on the combined effects of food level and a pulsed exposure to the pyrethroid insecticide fenvalerate (FV) on D. magna. The model described the effects of the time-varying exposure well, and indicated that when the animals did not die from exposure to FV, full reversibility of toxic effects was possible, allowing a complete recovery. Results revealed furthermore that the data from both studies could be described by the same NECs for survival and assimilation, killing rate and tolerance concentration (132 (49.2-228) x 10(-6) microg/L, 0 (0-1.18 x 10(-5)) microg/L, 74.4 (55.6-96.4) L (microg d)(-1) and 5.39 (2.72-18.5) x 10(-3) microg/L, respectively). It is therefore concluded that food-dependent FV toxicity can be explained by altered toxicokinetics and resource allocation, but not by changes in the intrinsic sensitivity of the daphnids. This study implies that the effect of pesticide application in the field depends on the trophic state of the receiving water body, but also that full recovery of survivors is possible after FV application.     

重复性测定结果可接受性检查方法在滴定液标定中的应用

目的 总结分析我国国家标准和中国药典滴定液标定结果可接受性检查方法,对食品药品检验系统滴定液标定的管理提供建议。方法 查阅国内相关标准、规定以及文献,总结现状、分析问题、提出相应对策。结果与结论 应建立食品、保健食品和化妆品滴定液标定方法的技术指导文件,积累标定数据计算检验机构内部的标定重复性标准差,建立动态更新的数据库,进一步加强应用于国标方法滴定液的管理办法。     

Safety assessment of EPA-rich oil produced from yeast: Results of a 90-day subchronic toxicity study

The safety of eicosapentaenoic acid (EPA) oil produced from genetically modified Yarrowia lipolytica yeast was evaluated following 90 days of exposure. Groups of rats received 0 (olive oil), 98, 488, or 976 mg EPA/kg/day, or GRAS fish oil or deionized water by oral gavage. Rats were evaluated for in-life, neurobehavioral, anatomic and clinical pathology parameters. Lower serum cholesterol (total and non-HDL) was observed in Medium and High EPA and fish oil groups. Lower HDL was observed in High EPA and fish oil males, only at early time points. Liver weights were increased in High EPA and Medium EPA (female only) groups with no associated clinical or microscopic pathology findings. Nasal lesions, attributed to oil in the nasal cavity, were observed in High and Medium EPA and fish oil groups. No other effects were attributed to test oil exposure. Exposure to EPA oil for 90 days produced no effects at 98 mg EPA/kg/day and no adverse effects at doses up to 976 mg EPA/kg/day. The safety profile of EPA oil was comparable to that of GRAS fish oil. These results support the use of EPA oil produced from yeast as a safe source for use in dietary supplements.     

Alternative methods to safety studies in experimental animals: role in the risk assessment of chemicals under the new European Chemicals Legislation (REACH)

During the last two decades, substantial efforts have been made towards the development and international acceptance of alternative methods to safety studies using laboratory animals. In the EU, challenging timelines for phasing out of many standard tests using laboratory animals were established in the seventh Amending Directive 2003/15/EC to Cosmetics Directive 76/768/EEC. In continuation of this policy, the new European Chemicals Legislation (REACH) favours alternative methods to conventional in vivo testing, if validated and appropriate. Even alternative methods in the status of prevalidation or validation, but without scientific or regulatory acceptance may be used under certain conditions. Considerable progress in the establishment of alternative methods has been made in some fields, in particular with respect to methods predicting local toxic effects and genotoxicity. In more complex important fields of safety and risk assessment such as systemic single and repeated dose toxicity, toxicokinetics, sensitisation, reproductive toxicity and carcinogenicity, it is expected that the development and validation of in silico methods, testing batteries (in vitro and in silico) and tiered testing systems will have to overcome many scientific and regulatory obstacles which makes it extremely difficult to predict the outcome and the time needed. The main reasons are the complexity and limited knowledge of the biological processes involved on one hand and the long time frame until validation and regulatory acceptance of an alternative method on the other. New approaches in safety testing and evaluation using "Integrated Testing Strategies" (ITS) (including combinations of existing data, the use of chemical categories/grouping, in vitro tests and QSAR) that have not been validated or not gained wide acceptance in the scientific community and by regulatory authorities will need a thorough justification of their appropriateness for a given purpose. This requires the availability of knowledge and experience of experts in toxicology. The challenging deadlines for phasing out of in vivo tests in the Cosmetics Amending Directive 2003/15/EC appear unrealistic. Likewise, we expect that the application of validated alternative methods will only have a small or moderate impact on the reduction of in vivo tests under the regimen of REACH, provided that at least the same level of protection of human health as in the past is envisaged. As a consequence, under safety aspects, it appears wise to consider established in vivo tests to be indispensable as basic tools for hazard and risk assessment with respect to systemic single and repeated dose toxicity, sensitisation, carcinogenicity and reproductive toxicity, especially regarding quantitative aspects of risk assessment such as NOAELs, LOAELs and health-related limit values derived from them. Based on the overall evaluation in this review, the authors are of the opinion that in the short- and mid-term, the strategy of the development of alternative methods should be more directed towards the refinement or reduction of in vivo tests. The lessons learnt during these efforts will provide a substantial contribution towards the replacement initiatives in the long-term.     

Application of Resampling Techniques to Estimate Exact Significance Levels for Covariate Selection During Nonlinear Mixed Effects Model Building: Some Inferences

Purpose. One of the main objectives of the nonlinear mixed effects modeling is to provide rational individualized dosing strategies by explaining the interindividual variability using intrinsic and/or extrinsic factors (covariates). The aim of the current study was to evaluate, using computer simulations and real data, methods for estimating the exact significance level for including or excluding a covariate during model building. Methods. Original data were simulated using a simple one-compartment pharmacokinetic model with (full model) or without (null model) covariates (one or two). The covariate values in the original data were resampled (using either permutations or parametric bootstrap methods) to generate data under the null hypothesis that there is no covariate effect. The original and permuted data were fitted to null and full models, using first-order and first-order condition estimation (with or without interaction) methods in NONMEM, to compare the asymptotic and conditional p-value. Target log-likelihood ratio cutoffs for assessing covariate effects were derived. Results. The simulations showed that for sparse as well as dense data, the first-order condition estimation methods yielded the best results while the first-order method performs somewhat better for sparse data. Depending on the modeling objective, the appropriate asymptotic p-value can be substituted for the conditional significance level. Target log-likelihood ratio cutoffs should be determined separately for each covariate when exact p-values are important. Conclusions. Resampling methods can be employed to estimate the exact significance level for including a covariate during nonlinear mixed effects model building. Some reasonable inferences can be drawn for potential application to design future population analyses.     

Collapsing Mechanistic Models: An Application to Dose Selection for Proof of Concept of a Selective Irreversible Antagonist

When data fail to support fully mechanistic models, alternative modeling strategies must be pursued. Simpler, more empirical models or the fixing of various rate constants are necessary to avoid over-parameterization. Fitting empirical models can dilute information, limit interpretation, and cloud inference. Fixing rate constants requires external, relevant, and reliable information on the mechanism and can introduce subjectivity as well as complicate determining the validity of model extrapolation. Furthermore, both these methods ignore the possibility that failure of the data to support the mechanistic model could contain information about the pharmacodynamic process. If the pathway has processes with “fast” dynamics, these steps could collapse yielding parametrically simpler classes of models. The collapsed models would retain the mechanistic interpretation of the full model, which is crucial for performing substantive inference, while reducing the number of parameters to be estimated. These concepts are illustrated through their manifestations on the dose–effect relationship and ensuing dose selection for a proof of concept study. Specifically, a mechanistic model for a selective irreversible antagonist was posited and candidate classes of models were derived utilizing “fast dynamics” assumptions. Model assessment determined the rate-limiting step facilitating pertinent inference with respect to the mechanism. For comparison, inference using a more empirical modeling strategy is also presented. A general solution for the collapse of the typical PK–PD model differential equations is provided in Appendix A     

The intra- and inter-laboratory reproducibility and predictivity of the KeratinoSens assay to predict skin sensitizers in vitro: Results of a ring-study in five laboratories

Due to regulatory constraints and ethical considerations, research on alternatives to animal testing to predict the skin sensitization potential of novel chemicals has gained a high priority. Accordingly, different in vitro, in silico and in chemico approaches have been described in the scientific literature to achieve this goal. To replace regulatory approved animal tests, these alternatives need to be transferable to other labs, their within and between laboratory reproducibility must be assured, and their predictivity should be high. The KeratinoSens assay is a cell-based reporter gene assay to screen substances with a full dose-response assessment. It is based on a stable transgenic keratinocyte cell line. The induction of a luciferase gene under the control of the antioxidant response element (ARE) derived from the human AKR1C2 gene is determined. Here we report on the results of a ring-study with five laboratories performing the KeratinoSens assay on a set of 28 test substances. The assay was found to be easily transferable to all laboratories. Overall both the qualitative (sensitizer/non-sensitizer categorization) and the quantitative (concentration for significant gene induction) results were reproducible between laboratories. A detailed analysis of the transferability, the within- and between laboratory reproducibility and the predictivity is presented.     

Exposure-driven risk assessment: Applying exposure-based waiving of toxicity tests under REACH

The REACH Regulation 1907/2006/EC aims to improve knowledge of the potential risks to humans and the environment of the large number of chemicals produced and used in the EU. The testing requirements are likely to trigger numerous toxicological studies, potentially involving millions of experimental animals, despite the professed goal of REACH to reduce vertebrate testing. It may be necessary therefore to shift emphasis away from animal studies towards more pragmatic strategies, reserving animal tests for the substances of greatest concern. One approach is to waive certain tests based on levels of exposure to the substance. This review explores application of ‘Exposure-Based Waiving’ (EBW) of toxicity studies, with a particular focus on inhalation where possible, considering the potential qualitative and quantitative supporting arguments that might be made, including the use of thresholds of toxicological concern. Incorporating EBW into intelligent testing strategies for substance registration could advance the goals of REACH and the 3Rs (reduction, replacement and refinement of animals in research) by reducing the usage of animals in toxicity tests, whilst maintaining appropriate protection of human health and the environment. However greater regulatory evaluation, acceptance and guidance are required for EBW to achieve its full impact.