Assessing the combination effects of environmental estrogens in fish

The method on combined effects of environmental estrogens and mixture environmental risk assessment was discussed. Batch tests were conducted to assess the in vivo potency of mixtures of estrogens using plasma vitellogenin concentrations in male crucian carp (Carassius carassius) as the endpoint. A nonlinear regression was determined on the concentration response relationship for the single chemical of 17β-estradiol (E₂), 17α-ethynylestradiol (EE₂), 4-tert-octylphenol (OP), bisphenol A (BPA), and that of the mixed compounds at equipotent concentrations (E₂-EE₂, E₂-EE₂-OP-BPA), the mixture was tested using a fixed-ratio design. On the basis of statistical selection criteria, the best-fit model is chosen individually for each set of data. Furthermore, the bootstrap methodology is applied for constructing confidence intervals for the estimated effect concentrations. The combined effects of the mixture can be predicted using biomathematical models based on the concentration and potency of the individual mixture components. The finding of non-monotonic dose–response relationship and the combined effects can be accurately predicted in whole range of exposure concentration by the reference models, whereas the outcome of simple effect summation with a great deal of indetermination. Results suggested that there can be a risk of mixture effects. The potential impact of components on mixture would depend predominantly on its concentration, the mixture ratio, and its relative potency. Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures, therefore further improvement is needed.     

Toxicity classification and evaluation of four pharmaceuticals classes: antibiotics, antineoplastics, cardiovascular, and sex hormones

Four different classes of environmental concern are quantitatively and qualitatively assessed for environmental hazards; antibiotics (n = 226), antineoplastics (n = 81), cardiovascular (n = 272), and sex hormones (n = 92). These along with an ECOSAR scan of all pharmaceuticals (n = 2848) were then classified according to the OECD aquatic toxicity classification system. The predicted species susceptibility is: daphnid > fish > algae, and the predicted rank order of relative toxicity: sex hormones > cardiovascular = antibiotics > antineoplastics (Table 1). Generally, a relatively large proportion (1/3) of all pharmaceuticals are potentially very toxic to aquatic organisms (Table 2). The qualitative risk assessment ranking relative to probability and potential severity for human and environmental health effects is: antibiotics > sex hormones > cardiovascular > antineoplastics. (Q)SARs and pharmacodynamic information should be used to prioritize and steer experimental risk assessments of pharmaceuticals, and potentially, also be used in new drug discovery optimizing efficacy and in minimising environmental hazards of new products. Nuclear receptors are relatively well conserved in evolution. Currently, antibacterial resistance represents the most significant human health hazard, and potentially the largest non-target organism hazard is sex hormones acting as endocrine modulators in wildlife. Data for the individual compounds are accessible via.     

Vitellogenin induction by a mixture of steroidal estrogens in freshwater fishes and relevant risk assessment

The study method on combined effects of environmental contaminant mixture and ecological risk assessment was discussed. Batch tests were conducted to assess the in vivo potency of binary mixtures of estrogens using plasma vitellogenin concentrations in male crucian carp as the endpoint. The estrogenic potencies of 17β‐estradiol (E₂) and 17α‐ethynylestradiol (EE₂) were determined following 14 day exposure to the individual chemicals and equipotent binary mixtures. A Nonlinear regression was obtained and 95% confidence limits of effect concentration were achieved using the bootstrap method. Concentration‐response curve for fixed ratio binary mixtures of E₂ and EE₂ was compared with those for individual chemicals, using the biomathematical models of concentration addition (CA) and independent action (IA). A complete overlap was found for the CA predictions with the 95% confidence interval of the best‐fit regression line of the observed responses, and the IA predictions was shown lower than the observations. The observed mixture effects were considerably higher than those of the hormone alone and far exceeded the 95% confidence interval of the estrogen regression lines. The predicted effects of binary mixtures at different mixture ratios indicated that the potential impact of components on mixture would depend predominantly on its concentration, the mixture ratio and its relative potency. Results suggested that E₂ and EE₂ acted together in an additive manner and the combined effects can be accurately predicted in whole range of exposure concentration by the models of CA and IA, the model of CA might be realistic, but more useful for ecological risk assessment. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Suggesting a testing strategy for possible endocrine effects of drug metabolites

Most pharmaceuticals are extensively metabolized by organisms, which results in internal exposure to mixtures of parent compounds and various metabolites. Many of these metabolites are considered non-toxic, but some metabolites retain toxic properties of the parent compound or elicit other undesirable outcomes. Unfortunately, the effects of metabolites are often not considered when endocrine activities of chemicals are evaluated in vitro. In this study two approaches, an “effect-based” and a “compound-by-compound” testing design, were used to determine the effects of metabolites of the antidepressant sertraline on aromatase enzyme activity. In the “effect-based” approach, a mixture of sertraline metabolites, produced by liver microsomes, inhibited aromatase, but was less potent than sertraline. In the “compound-by-compound” testing design, three specific metabolites were evaluated individually and in mixtures. Though two N-desmethylated metabolites were more potent aromatase inhibitors than sertraline, hydroxyl ketone sertraline did not inhibit the enzyme and mixtures of these metabolites and sertraline were less potent than predicted from a concentration addition model. Our findings highlight the importance of considering aromatase inhibition, and potentially other biological activities, of pharmaceutical metabolites produced by liver microsome preparations and then comparing such observations to studies of specific metabolites available for testing in pure form. Subsequently, a five step integrated strategy for screening of the potential endocrine effects of drugs and their metabolites are proposed.     

The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal): evaluation of impacts on fish liver, by histopathology, stereology, vitellogenin and CYP1A immunohistochemistry, after sub-acute exposures of the zebrafish model

Qualitative and quantitative approaches were tested to assess zebrafish liver effects after sub-acute exposures of certain pharmaceuticals. Carbamazepine, fenofibric acid, propranolol, sulfamethoxazole and trimethoprim were tested individually and in mixtures, including low environmental levels. Overall, data showed sex specific reactions in liver, with the major alterations being observed in males. Males treated with propranolol, fenofibric acid and with mixtures, showed an increase of vitellogenin immunostaining, compared with the control. Males also evidenced a tendency for an increased hepatic mass, after individual and mixture exposures. The volume-weighted nuclear volume of hepatocytes was high in males after exposures to either mixture, which together with the greater cytoplasmic eosinophilia and changes in cytochrome P450 1A immunoreactivity, point to an increase in metabolic/detoxification activity. These investigations revealed distinct impacts depending on the exposure type, and strengthened the importance of studying non-steroidal compounds in mixtures, including environmental levels and both sexes.     

Environmental risk and toxicology of human and veterinary waste pharmaceutical exposure to wild aquatic host-parasite relationships

Pollution of the aquatic environment by human and veterinary waste pharmaceuticals is an increasing area of concern but little is known about their ecotoxicological effects on wildlife. In particular the interactions between pharmaceuticals and natural stressors of aquatic communities remains to be elucidated. A common natural stressor of freshwater and marine organisms are protozoan and metazoan parasites, which can have significant effects on host physiology and population structure, especially under the influence of many traditional kinds of toxic pollutants. However, little is known about the effects of waste pharmaceuticals to host-parasite dynamics. In order to assess the risk waste pharmaceuticals pose to aquatic wildlife it has been suggested the use of toxicological data derived from mammals during the product development of pharmaceuticals may be useful for predicting toxic effects. An additional similar source of information is the extensive clinical studies undertaken with numerous classes of drugs against parasites of human and veterinary importance. These studies may form the basis of preliminary risk assessments to aquatic populations and their interactions with parasitic diseases in pharmaceutical-exposed habitats. The present article reviews the effects of the most common classes of pharmaceutical medicines to host-parasite relationships and assesses the risk they may pose to wild aquatic organisms. In addition the effects of pharmaceutical mixtures, the importance of sewage treatment, and the risk of developing resistant strains of parasites are also assessed.     

Estrogenic activity of pharmaceuticals and pharmaceutical mixtures in a yeast reporter gene system

Pharmaceuticals enter aquatic environments in unchanged form or as metabolites. Little is known about their potential hormonal activity, which is of particular interest due to potential long-term effects on fertility and reproduction in aquatic organisms. Moreover, there is a need to assess the combined activity of pharmaceutical mixtures. In this study, 37 pharmaceuticals have been analysed in vitro for estrogenic activity using a recombinant yeast system expressing the human estrogen receptor alpha. Six pharmaceuticals belonging to different therapeutic classes, cimetidine, fenofibrate, furosemide, paracetamol, phenazone and tamoxifen, exhibited weak estrogenic activity. Furosemide showed an almost full concentration-response curve, whereas the other compounds showed low efficacy. The half-maximal activities of the pharmaceuticals were in the range of 0.66-25.53 mM. Furthermore, binary mixtures of furosemide and 17beta-estradiol (E2), and furosemide and phenazone, and mixtures of up to five active pharmaceuticals were assessed for their combinatory activity at different equipotent concentrations. The estrogenic activity of binary mixtures of furosemide with E2 and phenazone, respectively, followed the model of concentration addition (CA). Mixtures of other pharmaceuticals often deviated from the CA model, because extrapolations become inaccurate with only partial and non-parallel concentration-response curves having low efficacy. This demonstrates that full and parallel concentration-response curves are a prerequisite for accurate predictions of mixture activity. Our study demonstrates for the first time weak estrogenic activity in vitro of some common pharmaceuticals and their mixtures.     

Joint algal toxicity of 16 dissimilarly acting chemicals is predictable by the concept of independent action

For a predictive assessment of the aquatic toxicity of chemical mixtures, two competing concepts are available: concentration addition and independent action. Concentration addition is generally regarded as a reasonable expectation for the joint toxicity of similarly acting substances. In the opposite case of dissimilarly acting toxicants the choice of the most appropriate concept is a controversial issue. In tests with freshwater algae we therefore studied the extreme situation of multiple exposure to chemicals with strictly different specific mechanisms of action. Concentration response analyses were performed for 16 different biocides, and for mixtures containing all 16 substances in two different concentration ratios. Observed mixture toxicity was compared with predictions, calculated from the concentration response functions of individual toxicants by alternatively applying both concepts. The assumption of independent action yielded accurate predictions, irrespective of the mixture ratio or the effect level under consideration. Moreover, results even demonstrate that dissimilarly acting chemicals can show significant joint effects, predictable by independent action, when combined in concentrations below individual NOEC values, statistically estimated to elicit insignificant individual effects of only 1%. The alternative hypothesis of concentration addition resulted in overestimation of mixture toxicity, but differences between observed and predicted effect concentrations did not exceed a factor of 3.2. This finding complies with previous studies, which indicated near concentration-additive action of mixtures of dissimilarly acting substances. Nevertheless, with the scientific objective to predict multi-component mixture toxicity with the highest possible accuracy, concentration addition obviously is no universal solution. Independent action proves to be superior where components are well known to interact specifically with different molecular target sites, and provided that reliable statistical estimates of low toxic effects of individual mixture constituents can be given. With a regulatory perspective, however, fulfilment of both conditions may be regarded as an extraordinary situation, and hence concentration addition may be defendable as a pragmatic and precautionary default assumption.     

Regulatory assessment and risk management of chemical mixtures: challenges and ways forward

Abstract

This paper summarizes current challenges, the potential use of novel scientific methodologies, and ways forward in the risk assessment and risk management of mixtures. Generally, methodologies to address mixtures have been agreed; however, there are still several data and methodological gaps to be addressed. New approach methodologies can support the filling of knowledge gaps on the toxicity and mode(s) of action of individual chemicals. (Bio)Monitoring, modeling, and better data sharing will support the derivation of more realistic co-exposure scenarios. As knowledge and data gaps often hamper an in-depth assessment of specific chemical mixtures, the option of taking account of possible mixture effects in single substance risk assessments is briefly discussed. To allow risk managers to take informed decisions, transparent documentation of assumptions and related uncertainties is recommended indicating the potential impact on the assessment. Considering the large number of possible combinations of chemicals in mixtures, prioritization is needed, so that actions first address mixtures of highest concern and chemicals that drive the mixture risk. As chemicals with different applications and regulated separately might lead to similar toxicological effects, it is important to consider chemical mixtures across legislative sectors.     

Human pharmaceuticals in surface waters. Implementation of a prioritization methodology and application to the French situation

Human pharmaceuticals are widely used and can reach surface waters, where they have the potential to exert biological effects on aquatic non-target organisms. Due to the high number of pharmaceutical drugs used in human medicine throughout the world, it is necessary to select the pharmaceuticals to search for, prior to implementing any environmental measurements and any extensive environmental risk assessment (ERA). This paper describes a methodology developed in order to define this selection. The prioritization scheme consists in three tiers. First, a preliminary classification based on the assessment of exposure is implemented. This exposure assessment is determined by calculating predicted environmental concentrations (PECs) for each pharmaceutical according to the European Medicine Evaluation Agency's (EMEA's) environmental risk assessment guidelines [EMEA, 2006. European Medicine Agency Guideline on the Environmental Risk Assessment of Medicinal Products for Human Use. EMEA/CHMP/SWP/4447/00.]. In the second step, the preliminary classification is reviewed on a case-by-case hypothesis basis using all the biological data available: ecotoxicological, pharmacological (mechanism of action (MoA), enzyme modulation, adverse effects) and physicochemical data (log K(ow)). Finally, an additional step is used to select priority compounds among molecules showing the same chemical structure and the same mechanism of action. We applied this methodology to the French situation and prioritized 120 parent molecules and 30 metabolites. The final prioritization list gathers 40 parent compounds and 14 metabolites. Among the 40 parent molecules, 21 have already been found in the aquatic environment, indicating a good agreement between the theoretical approach and the environmental measurements. Parameters used to construct the effect criteria are discussed for their relevance.     

Risk assessment of human antibiotics in Korean aquatic environment

In Korea, antibiotic usage has received a lot of attention from the public due to the increasing number of bacteria resistant to the currently used antibiotics. In this study based on FDA regulation and EU draft guideline, the most concerned antibiotics regarding their environmental risk in Korea were assessed and the refined predicted environmental concentration in surface water (PEC(surface water)) was obtained by applying a GIS-based KORea ECOlogical Risk assessment (KOREOCORisk) model. Thirteen antibiotics, which expected introductory concentration (EIC) greater than the trigger value (1.0μg/L), were chosen to assess ecological risk and the PEC/PNEC ratio exceeded 1.0 for amoxicillin, erythromycin and roxithromycin. The results in this study using conservative assumptions did not represent that there is a risk for acute toxic effects in the environment based on today's use of pharmaceuticals in Korea. However, the results do not exclude the potential for chronic environmental effects.     

Outline and criteria for evaluating the safety of new chemicals

Risk assessments for chemicals manufactured in large quantities, used by many people, and disposed of broadly into the environment require a systematic process with emphasis on scientific fudgments, specific criteria for decision making, and an overall process for ensuring that human and material resources are used effectively and efficiently. A risk assessment process incorporating these features consists of careful prework in which the physical, chemical, and biological characteristics of the material are organized from known information or predicted from structural considerations as well as information related to the intended manufacture, distribution, use, and disposal. Interrelated programs involving a number of scientific disciplines are then developed for human and for environmental risk assessments. Both of these programs rely on the development of increasingly refined relationships between predicted doses or exposure and biological effects in animal models. Predictive tests for environmental fate and concentrations and for biological effects are selected and conducted progressively. Specific decision criteria based on comparisons of predicted exposures with biological effects data are used to decide when enough testing has been conducted to make a risk assessment. The application of these guidelines to three example chemicals is provided to illustrate the decision-making process.     

Ecotoxicology of human pharmaceuticals

Low levels of human medicines (pharmaceuticals) have been detected in many countries in sewage treatment plant (STP) effluents, surface waters, seawaters, groundwater and some drinking waters. For some pharmaceuticals effects on aquatic organisms have been investigated in acute toxicity assays. The chronic toxicity and potential subtle effects are only marginally known, however. Here, we critically review the current knowledge about human pharmaceuticals in the environment and address several key questions. What kind of pharmaceuticals and what concentrations occur in the aquatic environment? What is the fate in surface water and in STP? What are the modes of action of these compounds in humans and are there similar targets in lower animals? What acute and chronic ecotoxicological effects may be elicited by pharmaceuticals and by mixtures? What are the effect concentrations and how do they relate to environmental levels? Our review shows that only very little is known about long-term effects of pharmaceuticals to aquatic organisms, in particular with respect to biological targets. For most human medicines analyzed, acute effects to aquatic organisms are unlikely, except for spills. For investigated pharmaceuticals chronic lowest observed effect concentrations (LOEC) in standard laboratory organisms are about two orders of magnitude higher than maximal concentrations in STP effluents. For diclofenac, the LOEC for fish toxicity was in the range of wastewater concentrations, whereas the LOEC of propranolol and fluoxetine for zooplankton and benthic organisms were near to maximal measured STP effluent concentrations. In surface water, concentrations are lower and so are the environmental risks. However, targeted ecotoxicological studies are lacking almost entirely and such investigations are needed focusing on subtle environmental effects. This will allow better and comprehensive risk assessments of pharmaceuticals in the future.     

Predicted critical environmental concentrations for 500 pharmaceuticals

A growing number of pharmaceuticals are found in surface waters worldwide, raising concerns about their effects on aquatic organisms and it is a major challenge to develop a rational strategy for prioritizing drugs on which to focus the most extensive environmental research efforts. However, in contrast to most other chemicals, very good understanding of the human potency of pharmaceuticals has been obtained through efficacy and safety testing. Assuming that a drug acts primarily through the same target(s) also in a non-target species, it would be possible to predict the likelihood for pharmacological interactions in wildlife. Among aquatic organisms, fish most often share drug targets with humans. In this study, we have calculated the predicted critical environmental concentration (CECs), i.e. the surface water concentration expected to cause a pharmacological effect in fish, for 500 pharmaceuticals, assuming equivalent pharmacological activity. The CECs are based on literature data on human potencies together with a predicted bioconcentration factor in fish for each drug based on lipophilicity. We propose that CECs could be used as preliminary indicators of specific drugs’ potential to cause adverse pharmacological effects at specific water concentrations, used when selecting pharmaceuticals to include in screening campaigns and for assessing relevant detection limits.     

Assessment of the environmental fate and effects of ivermectin in aquatic mesocosms

Pharmaceuticals in the environment have been subject to increasing public concern and scientific investigation over the past years. More than 100 active pharmaceutical ingredients have been detected in surface waters worldwide at the ng to microg L(-1) range. At these low levels it is commonly assumed that only chronic and/or mixture toxic effects will be discernible in aquatic ecosystems and that there are orders of magnitude between exposure and effect concentrations. Assessment of potential ecosystem risk of pharmaceuticals are recommended but rarely performed in mesocosms, so for most risk assessments the final tier to reduce extrapolation uncertainty is missing. This paper describes the fate and effects of the anthelmintic drug ivermectin for a 265-day period following treatment (nominal concentration levels of 0, 30, 100, 1000 ng L(-1) (or parts per trillion (ppt)) in fifteen 12,000 L outdoor aquatic mesocosms. Although it is established that ivermectin is highly toxic towards invertebrates, it has been believed that ivermectin does not present notable risks to aquatic systems due to the rapid dissipation of the compound and binding to the sediment. Hence, fate and exchange of ivermectin between water and sediment were evaluated in this study. The ivermectin DT(50aqueous) in water was found to be 3-5 days, but concentrations increased and appeared to be stabile in the sediment at 20-30 ng kg(-1) with no assessable DT(50sed). Acute effects (first week) following ivermectin exposure were identified and cladocerans were particularly sensitive (nom. 100 ppt). Chronic responses (229 days) were identified for more sediment-active organisms (e.g. Chydoriae and Ephemeroptera) (nom. 1000 ppt). This is the first study to demonstrate the potential environmental risk of ivermectin at or below the predicted environmental concentration using a standardized test methodology (mesocosm) with minimal extrapolation uncertainty.     

Effects and interactions in an environmentally relevant mixture of pharmaceuticals.

With the goal of assessing the environmental risk of pharmaceuticals, we have previously observed that a mixture of 13 different drugs at environmentally relevant concentrations had adverse consequences on human and zebra fish cells in vitro. Here we aimed to identify both main and interaction effects within the same environmentally relevant mixture of pharmaceuticals. We studied in vitro cytotoxicity in Escherichia coli, human embryonic HEK293, and estrogen-responsive OVCAR3 tumor cells using fractional-factorial experimental design. Our approach identified a subset of compounds of primary environmental concern, namely atenolol, bezafibrate, ciprofloxacin, and lincomycin, that had statistically significant effects on prokaryotic and eukaryotic cells at environmentally relevant exposure levels (ng/l). Drugs could interact and behave as chemosensitizers, with joint effects representing a statistically significant element of mixture toxicity. Effects and interactions were concentration dependent, confirming the difficulty of dose extrapolation in mixture toxicity data. This study suggests that a thorough investigation of mixture effects can direct environmental concerns toward a handful of pharmaceuticals, which may represent an actual risk at environmental concentrations. We indicate that risk identification may strongly depend on the use of environmentally relevant exposure scenarios. Antagonistic-synergistic interactions and dose dependency of effects may hamper the modeling and prediction of mixture toxicity with pharmaceuticals. Hazard identification for micropollutants depends heavily on appropriate study designs, and we indicate the use of in vitro cytotoxicity threshold and statistical design of experiments (DOEs) as a valid approach.     

Approaches for applications of physiologically based pharmacokinetic models in risk assessment

Physiologically based pharmacokinetic (PBPK) models are particularly useful for simulating exposures to environmental toxicants for which, unlike pharmaceuticals, there is often little or no human data available to estimate the internal dose of a putative toxic moiety in a target tissue or an appropriate surrogate. This article reviews the current state of knowledge and approaches for application of PBPK models in the process of deriving reference dose, reference concentration, and cancer risk estimates. Examples drawn from previous U.S. Environmental Protection Agency (EPA) risk assessments and human health risk assessments in peer-reviewed literature illustrate the ways and means of using PBPK models to quantify the pharmacokinetic component of the interspecies and intraspecies uncertainty factors as well as to conduct route to route, high dose to low dose and duration extrapolations. The choice of the appropriate dose metric is key to the use of the PBPK models for the various applications in risk assessment. Issues related to whether uncertainty factors are most appropriately applied before or after derivation of human equivalent dose (or concentration) continue to be explored. Scientific progress in the understanding of life stage and genetic differences in dosimetry and their impacts on variability in susceptibility, as well as ongoing development of analytical methods to characterize uncertainty in PBPK models, will make their use in risk assessment increasingly likely. As such, it is anticipated that when PBPK models are used to express adverse tissue responses in terms of the internal target tissue dose of the toxic moiety rather than the external concentration, the scientific basis of, and confidence in, risk assessments will be enhanced.     

Approaches for Applications of Physiologically Based Pharmacokinetic Models in Risk Assessment

Physiologically based pharmacokinetic (PBPK) models are particularly useful for simulating exposures to environmental toxicants for which, unlike pharmaceuticals, there is often little or no human data available to estimate the internal dose of a putative toxic moiety in a target tissue or an appropriate surrogate. This article reviews the current state of knowledge and approaches for application of PBPK models in the process of deriving reference dose, reference concentration, and cancer risk estimates. Examples drawn from previous U.S. Environmental Protection Agency (EPA) risk assessments and human health risk assessments in peer-reviewed literature illustrate the ways and means of using PBPK models to quantify the pharmacokinetic component of the interspecies and intraspecies uncertainty factors as well as to conduct route to route, high dose to low dose and duration extrapolations. The choice of the appropriate dose metric is key to the use of the PBPK models for the various applications in risk assessment. Issues related to whether uncertainty factors are most appropriately applied before or after derivation of human equivalent dose (or concentration) continue to be explored. Scientific progress in the understanding of life stage and genetic differences in dosimetry and their impacts on variability in susceptibility, as well as ongoing development of analytical methods to characterize uncertainty in PBPK models, will make their use in risk assessment increasingly likely. As such, it is anticipated that when PBPK models are used to express adverse tissue responses in terms of the internal target tissue dose of the toxic moiety rather than the external concentration, the scientific basis of, and confidence in, risk assessments will be enhanced.