The utility of QSARs in predicting acute fish toxicity of pesticide metabolites: A retrospective validation approach

The European Plant Protection Products Regulation 1107/2009 requires that registrants establish whether pesticide metabolites pose a risk to the environment. Fish acute toxicity assessments may be carried out to this end. Considering the total number of pesticide (re-) registrations, the number of metabolites can be considerable, and therefore this testing could use many vertebrates. EFSA's recent “Guidance on tiered risk assessment for plant protection products for aquatic organisms in edge-of-field surface waters” outlines opportunities to apply non-testing methods, such as Quantitative Structure Activity Relationship (QSAR) models. However, a scientific evidence base is necessary to support the use of QSARs in predicting acute fish toxicity of pesticide metabolites. Widespread application and subsequent regulatory acceptance of such an approach would reduce the numbers of animals used.The work presented here intends to provide this evidence base, by means of retrospective data analysis. Experimental fish LC₅₀ values for 150 metabolites were extracted from the Pesticide Properties Database (http://sitem.herts.ac.uk/aeru/ppdb/en/atoz.htm). QSAR calculations were performed to predict fish acute toxicity values for these metabolites using the US EPA's ECOSAR software. The most conservative predicted LC₅₀ values generated by ECOSAR were compared with experimental LC₅₀ values.There was a significant correlation between predicted and experimental fish LC₅₀ values (Spearman r_s = 0.6304, p < 0.0001). For 62% of metabolites assessed, the QSAR predicted values are equal to or lower than their respective experimental values. Refined analysis, taking into account data quality and experimental variation considerations increases the proportion of sufficiently predictive estimates to 91%. For eight of the nine outliers, there are plausible explanation(s) for the disparity between measured and predicted LC₅₀ values.Following detailed consideration of the robustness of this non-testing approach, it can be concluded there is a strong data driven rationale for the applicability of QSAR models in the metabolite assessment scheme recommended by EFSA. As such there is value in further refining this approach, to improve the method and enable its future incorporation into regulatory guidance and practice.     

The usefulness of the bioconcentration factor as a tool for priority setting in chemicals control

The implementation of the REACH system will lead to the creation of a single, uniform legislation for industrial chemicals in Europe. An important aim of this legislation is to generate toxicity data for previously untested chemicals. Testing tens of thousands of chemicals can however not be done in one step, and criteria for priority setting is therefore an essential part of the proposed REACH system. In this study we investigate potential consequences of using bioaccumulation (B) data as a tool for priority setting in chemicals control. The results of this investigation suggests that the use of data for the bioconcentration factor (BCF, as an estimation of B) at first tier will not introduce bias towards a particular type of toxicity (i.e. carcinogenicity, reproductive toxicity or mutagenicity) in the priority setting process.     

Computational analysis of the ToxCast estrogen receptor agonist assays to predict vitellogenin induction by chemicals in male fish

Endocrine disruptors, especially estrogen receptor (ER) agonists, have received considerable research attention. While there are several mechanistic endpoints for ER agonism in the Endocrine Disruptor Screening Program, there have been growing efforts to develop high-throughput screening assays and computational models to reduce testing cost, time, and animal use. For example, there are 16 ER agonist assays and an integrated computational model in ToxCast. In the present study, we examined the relationship between ToxCast ER agonist assays and model activity to male vitellogenin induction in the Fish-Short Term Reproduction Assay. It was found 15/16 of the assays significantly predicted potency ranks for 10 common ER agonists, and 7/16 of the assays had a significant linear correlation. The integrated model also provided comparable performance to most assays. Thus, the ToxCast ER agonist assays and model may be useful to identify endocrine disruptors and predict reproductive outcomes in fish.     

鱼蛋白胨和鱼油对替考拉宁产量和组分含量的影响

研究鱼蛋白胨和鱼油对替考拉宁产量和各组分含量的影响。通过在发酵培养基中选用不同原材料，比较结果发现，鱼蛋白胨2″和鱼油能分别提高替考拉宁的产量15．8％和21．0％。进而采用正交试验设计，确定了最佳发酵培养基配方。与对照组相比，在优化配方中添加0．5％鱼蛋白胨2″和0．8％鱼油可增加产量达35％，效价高达3000u／ml以上，且产品中各组分的相对百分含量不发生明显改变。为探求其原理，分别对不同原材料进行氨基酸组分分析和碘值测定，分析结果推测鱼蛋白胨的作用与原料中含有较高量的替考拉宁生物合成氨基酸前体有关，而鱼油则通过提供饱和脂肪酸，参与替考拉宁起始氨基酸的生物合成。同时也证明了通过筛选含不同化学结构成份的发酵碳氮原材料．可定向控制替考拉宁的生物合成。     

鱼油降低尿钙作用的研究

目的探讨鱼油对尿石形成的作用。方法 20例含钙结石高尿钙患者,应用鱼油进行治疗。结果服用鱼油患者24h尿钙排泄从(461．37±80．67)mg/d下降到(312．63±40．57)mg／d,尿镁从(136．41 ±56．12)mg/d下降到(108．43±18．96)mg/d,尿镁/钙比值从(0．29±0．08)上升到(0．36±0．11),尿酸无明显变化。结论鱼油能降低高尿钙结石患者尿钙、尿镁的排出,提高尿镁/钙的比值,可能预防特发性高尿钙结石患者的复发。     

Enhancing and improving the extraction of omega-3 from fish oil

Omega-3 fatty acids DHA and EPA derived from fish oil are widely marketed across the world as valued dietary supplements offering numerous health benefits to children and adults alike. Traditional extraction processes are energy intensive and use organic solvents. Green and sustainable alternatives are needed with the aim to significantly expand and improve the production of omega-3 extracts, especially with the aim to obtain these essential polyunsaturated fatty acids from fish processing waste available in >20 million tonnes/year amount.     

Estimation of acute oral toxicity using the No Observed Adverse Effect Level (NOAEL) from the 28 day repeated dose toxicity studies in rats

Acute systemic toxicity is one of the areas of particular concern due to the 2009 deadline set by the 7th Amendment of the Cosmetics Directive (76/768/EEC), which introduces a testing and marketing ban of cosmetic products with ingredients tested on animals. The scientific community is putting considerable effort into developing and validating non-animal alternatives in this area. However, it is unlikely that validated and regulatory accepted alternative methods and/or strategies will be available in March 2009. Following the initiatives undertaken in the pharmaceutical industry to waive the acute oral toxicity testing before going to clinical studies by using information from other in vivo studies, we proposed an approach to identify non-toxic compounds (LD50>2000mg/kg) using information from 28 days repeated dose toxicity studies. Taking into account the high prevalence of non-toxic substances (87%) in the New Chemicals Database, it was possible to set a NOAEL threshold of 200mg/kg that allowed the correct identification of 63% of non-toxic compounds, while <1% of harmful compounds were misclassified as non-toxic. Since repeated dose toxicity studies can be performed in vivo until 2013, the proposed approach could have an immediate impact for the testing of cosmetic ingredients.     

Impact of bioavailability on the correlation between in vitro cytotoxic and in vivo acute fish toxic concentrations of chemicals

The lower sensitivity of in vitro cytotoxicity assays currently restricts their use as alternative to the fish acute toxicity assays for hazard assessment of chemicals in the aquatic environment. In vitro cytotoxic potencies mostly refer to nominal concentrations. The main objective of the present study was to investigate, whether a reduced availability of chemicals in vitro can account for the lower sensitivity of in vitro toxicity test systems. For this purpose, the bioavailable free fractions of the nominal cytotoxic concentrations (EC50) of chemicals determined with a cytotoxicity test system using Balb/c 3T3 cells and the corresponding free cytotoxic concentrations (ECu50) were calculated. The algorithm applied is based on a previously developed simple equilibrium distribution model for chemicals in cell cultures with serum-supplemented culture media. This model considers the distribution of chemicals between water, lipids and serum albumin. The algorithm requires the relative lipid volume of the test system, the octanol-water partition coefficient (K(ow)) and the in vitro albumin-bound fraction of the chemicals. The latter was determined from EC50-measurements in the presence of different albumin concentrations with the Balb/c 3T3 test system. Organic chemicals covering a wide range of cytotoxic potency (EC50: 0.16-527000 microM) and lipophilicity (logK(ow): -5.0-6.96) were selected, for which fish acute toxicity data (LC50-values) from at least one of the three fish species, medaka, rainbow trout and fathead minnow, respectively, were available. The availability of several chemicals was shown to be extensively reduced either by partitioning into lipids or by serum albumin binding, or due to both mechanisms. Reduction of bioavailability became more important with increasing cytotoxic potency. The sensitivity of the Balb/c 3T3 cytotoxicity assay and the correspondence between in vivo and in vitro toxic potencies were increased when the free cytotoxic concentrations instead of the nominal cytotoxic concentrations were used as measure of cytotoxic potency. The few remaining prominent differences between cytotoxic and acute toxic concentrations can be explained by a more specific mechanism of acute toxic action than basal cytotoxicity. It is concluded that the frequently observed low sensitivity of in vitro cytotoxicity test systems, compared to fish acute toxicity assays, at least in part, can be explained by differences in the availability of chemicals in vitro and in vivo. Moreover, neglecting these differences systematically causes a bias of the correlation between in vivo and in vitro toxic potencies of chemicals. Taking them into account, however, increases the predictivity of the in vitro assays.     

Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio)

The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH – Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24 h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.     

分子蒸馏分离深海鱼油药用组分的研究

深海鱼油富舍ω3型功能性脂肪酸（EPA、DHA和DPA）。采用刮膜式分子蒸馏小试装置对深海鱼油粗产品中的药用组分进行分离,馏余液中药用组分的含量（26．5％以上）达到了某药企对药品中ω-3型脂肪酸总含量的要求（大于25％）。通过研究蒸馏温度、进料速率和刮膜器转速对馏余液中药用组分总含量和收率的影响,得到分子蒸馏分离深海鱼油粗产品中药用组分的最佳工艺条件：蒸馏温度300C,进料速率0．7L·h-1。和刮膜转速300r·min-1。     

Updating the use of biochemical biomarkers in fish for the evaluation of alterations produced by pharmaceutical products

The evaluation of toxic effects in stressful environmental conditions can be determined through the imbalance between exogenous factors (environmental contaminants) and enzymatic and non-enzymatic defenses in biological systems. The use of fish for the identification of alterations in biochemical biomarkers provides a comprehensive vision of the effects that pharmaceutical products cause in the aquatic ecosystem, as they are organisms with high sensitivity to contaminants, filtering capacity, and potential for environmental toxicology studies. A wide range of pharmaceuticals can stimulate or alter a variety of biochemical mechanisms, such as oxidative damage to membrane lipids, proteins, and changes in antioxidant enzymes. This review includes a summary of knowledge of the last 20 years, in the understanding of the different biochemical biomarkers generated by exposure to pharmaceuticals in fish, which include different categories of pharmaceutical products: NSAIDs, analgesics, antibiotics, anticonvulsants, antidepressants, hormones, lipid regulators and mixtures. This review serves as a tool in the design of studies for the evaluation of the effects of pharmaceutical products, taking into account the most useful biomarkers, type of matrix, enzyme alterations, all taking the pharmaceutical group of interest.     

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.     

Interval estimators of relative potency in toxicology and radiation countermeasure studies: comparing methods and experimental designs

The relative potency of one agent to another is commonly represented by the ratio of two quantal response parameters; for example, the LD₅₀ of animals receiving a treatment to the LD₅₀ of control animals, where LD₅₀ is the dose of toxin that is lethal to 50% of animals. Though others have considered interval estimators of LD₅₀, here, we extend Bayesian, bootstrap, likelihood ratio, Fieller’s and Wald’s methods to estimate intervals for relative potency in a parallel-line assay context. In addition to comparing their coverage probabilities, we also consider their power in two types of dose designs: one assigning treatment and control the same doses vs. one choosing doses for treatment and control to achieve same lethality targets. We explore these methods in realistic contexts of relative potency of radiation countermeasures. For larger experiments (e.g., ≥100 animals), the methods return similar results regardless of the interval estimation method or experiment design. For smaller experiments (e.g., < 60 animals), Wald’s method stands out among the others, producing intervals that hold closely to nominal levels and providing more power than the other methods in statistically efficient designs. Using this simple statistical method within a statistically efficient design, researchers can reduce animal numbers.     

The regulatory use of the Local Lymph Node Assay for the notification of new chemicals in Europe

The regulatory use of the Local Lymph Node Assay (LLNA) for new chemicals registration was monitored by screening the New Chemicals Database (NCD), which was managed by the former European Chemicals Bureau (ECB) at the European Commission Joint Research Centre (JRC). The NCD centralised information for chemicals notified after 1981, where toxicological information has been generated predominantly according to approved test methods. The database was searched to extract notifications for which the information for skin sensitisation labelling was based on results derived with the LLNA. The details of these records were extracted and pooled, and evaluated with regard to the extent of use of the LLNA over time, as well as for analysing the information retrieved on critical aspects of the procedure e.g. strain and amount of animals used, lymph node processing, solvent and doses selected, stimulation indices, and for assessing their level of compliance to the OECD Test Guideline 429. In addition the accuracy of the reduced LLNA when applied to new chemicals was investigated.     

Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish

Cultures of vertebrate cells are widely applied in mechanistic studies in human toxicology as well as in toxicity identification in ecotoxicology. As in vitro models, they display many advantages over whole animal experimentation, pertaining to such characteristics as availability, reproducibility and costs. As well, they satisfy the societal desire to reduce the number of animals in toxicology. For these reasons vertebrate cell models also appear to be a desirable replacement for animals in regulatory tests. Several vertebrate cell models are now accepted for regulatory purposes in human health sciences, with the test for photocytotoxicity using the 3T3 mouse cell line being one example. However, an in vitro alternative to whole animal tests has not yet been established for regulatory risk assessment in ecotoxicology. This review sets out to outline why such a replacement has not yet been possible and explores avenues to improve vertebrate cell cultures so that a replacement of whole animal tests could more likely be achieved. Inasmuch as fish is the most widely used non-mammalian vertebrate in risk assessment and regulation, focus will be on the replacement, by in vitro vertebrate models, of fish.     

Opercular epithelial cells: a simple approach for in vitro studies of cellular responses in fish

This study evaluated the efficacy of fish opercular external (skin) and inner (opercular membrane) epithelium as an in vitro model for toxic and other substances studies. The rainbow trout (Oncorhynchus mykiss) operculum was cultured in 12-well dishes containing sterile Leibovitz 15 (L-15) supplemented with glutamine medium during 24h at 9 degrees C, and the effect of copper, a toxic agent, and/or cortisol, an endogenous agent, on the epithelial cells was analyzed using light microscopy techniques. The opercula were submitted to four treatments: (i) control (Cont), L-15 medium only, (ii) 0.28 microM cortisol (Cort), (iii) 100 microM CuSO(4) (Cu), and (iv) 0.28 microM cortisol+100 microM CuSO(4) (Cort-Cu). The tissue condition after 24h incubation was analyzed by staining the mucous cells for neutral and acid mucosubstances. Cellular necrosis was evaluated by measuring the lactate dehydrogenase (LDH) leakage at 12 and 24h incubation. Cellular proliferation, apoptosis, metallothionein (MT) and glucocorticoid receptor (GR) expression were evaluated by immunohistochemistry. The LDH leakage was higher and the proliferating cell nuclear antigen (PCNA) positive-stained cells were lower in Cu treatment in both, epidermis and opercular membrane. Apoptotic cells in the opercular membrane were higher in the Cort and Cort-Cu treatments while, in the epidermis, they were higher in Cu and Cort-Cu treatments. GR-positive stained cells decreased significantly in all treatments in both epithelia and the MT-positive cells increased in the Cu and Cort-Cu treated groups. Copper showed to be a potent toxic stressor killing the cells via necrosis, decreasing the number of PCNA-positive cells and inducing MT synthesis while cortisol did not affect the MT synthesis, although it might stimulate apoptosis. The results are evidence that the opercular epithelia serve as a suitable model for studying in vitro effects of toxic agents, as well as endogenous factors on the cellular responses without interference of the physiological state of fish being useful to predict in vivo toxicity.     

In vivo genotoxicity evaluation of atrazine and atrazine-based herbicide on fish Carassius auratus using the micronucleus test and the comet assay

Atrazine is a selective triazine herbicide used to control broadleaf and grassy weeds mainly in corn, sorghum, sugarcane, pineapple, and other crops, and in conifer reforestation planting fields. It has been showed that atrazine is one of the most frequently detected pesticides in agricultural streams and rivers, over the past two decades. Although the toxic properties of atrazine are well known, the data on the genotoxic effects of atrazine on aquatic organisms are rather scarce. Thus, in the present study we aimed to evaluate the genotoxic effects of atrazine and an atrazine-based herbicide (Gesaprim®) on a model fish species Carassius auratus L., 1758, (Pisces: Cyprinidae) using the micronucleus test and the comet assay in peripheral blood erythrocytes. Fish were exposed to 5, 10 and 15 μg/L atrazine and to its commercial formulation for 2, 4 and 6 days. Ethyl methane sulfonate (EMS) at a single dose of 5 mg/L was used as positive control. Our results revealed significant increases in the frequencies of micronuclei and DNA strand breaks in erythrocytes of C. auratus, following exposure to commercial formulation of atrazine and thus demonstrated the genotoxic potential of this pesticide on fish.     

微波消解-ICP-MS法同时测定鱼油类保健食品中6种微量元素

目的：建立微波消解电感耦合等离子体质谱法同时测定鱼油类保健食品中铝、镉、总铬、铜、锌、镍的含量。方法采用微波消解法处理样品,电感耦合等离子体质谱法测定。结果6种元素中在0~100 mg·L-1范围内,线性关系良好。平均加样回收率在98.8%~105.2%之间,精密度RSD在1.6%~2.8%,重复性RSD在0.66%~4.91%。结论本方法简便快速准确,可用于鱼油中6种微量元素的测定,为国家保健食品监督抽验和风险监测工作提供技术支持。     

毛细管气相色谱法测定鱼油脂肪乳注射液中DHA和EPA的含量

目的采用毛细管气相色谱法对鱼油脂肪注射液的DHA和EPA含量进行测量的效果进行研究分析。方法抽取同一批次的鱼油脂肪注射液,采用毛细管气象色谱法对DHA和EPA的含量进行测定,载气选择氦气,流速控制在0.5mL/min,柱温进行程序升温,采用FID检测器,分流比为5：1,进样口温度控制在300℃,进样量为1μL。结果经过仔细研究后笔者发现,被检测的鱼油脂肪注射液中DHA甲酯的浓度在0.216～0.712g/L,EPA甲酯的浓度在0.224～0.768g/L的现象关系非常明显,两种成分的回收率均在99%以上,RSD水平均在0.50%以上。结论采用毛细管气象色谱法对鱼油脂肪注射液的DHA和EPA含量进行测量主要具有操作简单、方便快捷、准确度高、重现性好等特点,可作为今后对该药物的成分含量进行测定的常规方法。