Developmental Toxicity Assay Research Articles

The ecotoxicological evaluation of Cylindrospermopsis raciborskii from Lake Balaton (Hungary) employing a battery of bioassays and chemical screening

Ecotoxicity of four Cylindrospermopsis raciborskii strains (ACT 9502, ACT 9503, ACT 9504, ACT 9505) isolated from Lake Balaton (Hungary) was evaluated in four aquatic bioassays including the Thamnocephalus platyurus acute lethality test; Daphnia magna acute immobilization assay; D. magna feeding inhibition assay and Danio rerio embryo developmental toxicity assay, assisted by chemical screening for known toxins by HPLC-MS. For reference, we analyzed in parallel the toxin content and toxic effects of two previously characterized toxin-producing strains: the Australian cylindrospermopsin producer AQS C. raciborskii and the anatoxins producer Oscillatoria sp. PCC 6506. Bioassays were used to evaluate the overall toxicity of the hydrophilic bioactive metabolites pool synthesized by the selected cyanobacteria.Chemical screening has proven that the ACT C. raciborskii extracts investigated did not contained cylindrospermopsins and anatoxins. The relative toxicity of the ACT C. raciborskii aqueous extracts observed in each bioassay was comparable to the effects recorded for the anatoxins producer PCC 6506 strain while toxicity values (EC50/LC50) calculated for the AQS extract were in general one order of magnitude lower.Concerning sublethal effects of ACT C. raciborskii extracts to the D. rerio embryogenesis, the general morphological abnormality observed was a significant retardation of development.Overall, our results suggest that C. raciborskii populating Lake Balaton produce metabolites with significant bioactive potencies. Therefore, continued investigation of these unknown compounds is required.

Establishment of a Molecular Embryonic Stem Cell Developmental Toxicity Assay

The mouse embryonic stem cell test (EST) is a 10-day screen for teratogenic potential developed to reduce animal use for embryotoxicity testing of chemicals (Spielmann, 2005; Spielmann et al., 1997). In this study, we used the cytotoxicity IC(50) values and transcriptional expression changes as primary endpoints in a shorter 4-day version of the EST, the molecular embryonic stem cell assay. Mouse D3 embryonic stem cells were used for cytotoxicity assessment (monolayers) or grown as embryoid bodies in low attachment plates for transcriptional profiling. Sixty-five compounds with known in vivo teratogenicity (33 teratogens and 32 nonteratogens) were evaluated to develop a model for classifying compounds with teratogenic potential. The expression of 12 developmentally regulated gene targets (nanog, fgf5, gsc, cd34, axin2, apln, chst7, lhx1, fgf8, sox17, foxa2, and cxcr4) was measured following exposure of embryoid bodies to a single compound concentration (0.1 × the cytotoxicity IC(20)) for 4 days. In the decision-tree model, compounds with IC(50) values < 22 µM were categorized as teratogens, whereas compounds in the two groups with IC(50) values between 22-200 µM and > 200 µM were categorized as teratogens if ≥ 8 and 12 genes, respectively, were deregulated by at least 10%. Forty-seven of 65 compounds of the training set were correctly identified (72% total concordance). In a test set of 12 additional compounds (5 teratogens, 7 nonteratogens), 10 were correctly classified by this approach (83% concordance). The false positive rate in the training and test sets was 24 and 0%, respectively, indicating that this assay has potential to identify teratogens.

Early Zebrafish Embryogenesis Is Susceptible to Developmental TDCPP Exposure

Background: Chlorinated phosphate esters (CPEs) are widely used as additive flame retardants for low-density polyurethane foams and have frequently been detected at elevated concentrations within indoor environmental media.Objectives: To begin characterizing the potential toxicity of CPEs on early vertebrate development, we examined the developmental toxicity of four CPEs used in polyurethane foam: tris(1,3-dichloro-2-propyl) phosphate (TDCPP), tris(2-chloroethyl) phosphate (TCEP), tris(1-chloro-2-propyl) phosphate (TCPP), and 2,2-bis(chloromethyl)propane-1,3-diyl tetrakis(2-chlorethyl) bis(phosphate) (V6).Methods: Using zebrafish as a model for vertebrate embryogenesis, we first screened the potential teratogenic effects of TDCPP, TCEP, TCPP, and V6 using a developmental toxicity assay. Based on these results, we focused on identification of susceptible windows of developmental TDCPP exposure as well as evaluation of uptake and elimination of TDCPP and bis(1,3-dichloro-2-propyl)phosphate (BDCPP, the primary metabolite) within whole embryos. Finally, because TDCPP-specific genotoxicity assays have, for the most part, been negative in vivo and because zygotic genome remethylation is a key biological event during cleavage, we investigated whether TDCPP altered the status of zygotic genome methylation during early zebrafish embryogenesis.Results: Overall, our findings suggest that the cleavage period during zebrafish embryogenesis is susceptible to TDCPP-induced delays in remethylation of the zygotic genome, a mechanism that may be associated with enhanced developmental toxicity following initiation of TDCPP exposure at the start of cleavage.Conclusions: Our results suggest that further research is needed to better understand the effects of a widely used and detected CPE within susceptible windows of early vertebrate development.

Introducing Environmental Toxicology in Instructional Labs: The Use of a Modified Amphibian Developmental Toxicity Assay to Support Inquiry-Based Student Projects

Introducing students to the process of scientific inquiry is a major goal of high school and college labs. Environmental toxins are of great concern and public interest. Modifications of a vertebrate developmental toxicity assay using the frog Xenopus laevis can support student-initiated toxicology experiments that are relevant to humans. Teams of students formulate hypotheses, perform experiments, analyze data, and present their results. By performing experiments to investigate the toxicity of household chemicals, pharmaceuticals, or agricultural chemicals, students will gain an appreciation of the environmental effects of improper disposal of common chemicals and industrial or agricultural run-off.

Cell differentiation markers as endpoints for in vitro developmental toxicity assays

Cell differentiation markers as endpoints for in vitro developmental toxicity assays

Inter-laboratory assessment of a harmonized zebrafish developmental toxicology assay – Progress report on phase I

This report provides a progress update of a consortium effort to develop a harmonized zebrafish developmental toxicity assay. Twenty non-proprietary compounds (10 animal teratogens and 10 animal non-teratogens) were evaluated blinded in 4 laboratories. Zebrafish embryos from pond-derived and cultivated strain wild types were exposed to the test compounds for 5 days and subsequently evaluated for lethality and morphological changes. Each of the testing laboratories achieved similar overall concordance to the animal data (60–70%). Subsequent optimization procedures to improve the overall concordance focused on compound formulation and test concentration adjustments, chorion permeation and number of replicates. These optimized procedures were integrated into a revised protocol and all compounds were retested in one lab using embryos from pond-derived zebrafish and achieved 85% total concordance. To further assess assay performance, a study of additional compounds is currently in progress at two laboratories using embryos from pond-derived and cultivated-strain wild type zebrafish.

Developmental toxicity and endocrine disrupting potency of 4-azapyrene, benzo[b]fluorene and retene in the zebrafish Danio rerio

This study examined the developmental toxicity of the polycyclic aromatic hydrocarbons (PAHs) 11H-benzo(b)fluorene (BBF) and 4-azapyrene (AP) in comparison to the known teratogen retene. Developmental toxicity assays were performed in zebrafish embryos exposed for 120h. BBF and retene induced a similar dioxin-like phenotype, whereas AP showed distinct effects, particularly craniofacial malformations. Microarray analysis revealed that for BBF and retene, drug metabolism pathways were induced, which were confirmed by subsequent studies of cyp1a gene expression. For AP, microarray analysis revealed the regulation of genes involved in retinoid metabolism and hematological functions. Studies with a panel of CALUX® bioassays to screen for endocrine disrupting activity of the compounds also revealed novel antagonistic effects of BBF and retene on androgen and progesterone receptors. Classification analysis revealed distinct gene expression profiles for both individual and combined PAH exposure. This study highlights the potential health risk of non priority PAHs.

Transcriptomic Concentration-Response Evaluation of Valproic Acid, Cyproconazole, and Hexaconazole in the Neural Embryonic Stem Cell Test (ESTn)

Alternative developmental toxicity assays are urgently needed to reduce animal use in regulatory developmental toxicology. We previously designed an in vitro murine neural embryonic stem cell test (ESTn) as a model for neurodevelopmental toxicity testing (Theunissen et al., 2010). Toxicogenomic approaches have been suggested for incorporation into the ESTn to further increase predictivity and to provide mechanistic insights. Therefore, in this study, using a transcriptomic approach, we investigated the concentration-dependent effects of three known (neuro) developmental toxicants, two triazoles, cyproconazole (CYP) and hexaconazole (HEX), and the anticonvulsant valproic acid (VPA). Compound effects on gene expression during neural differentiation and corresponding regulated gene ontology (GO) terms were identified after 24 h of exposure in relation to morphological changes on day 11 of culture. Concentration-dependent responses on individual gene expression and on biological processes were determined for each compound, providing information on mechanism and concentration-response characteristics. All compounds caused enrichment of the embryonic development process. CYP and VPA but not HEX significantly enriched the neuron development process. Furthermore, specific responses for triazole compounds and VPA were observed within the GO-term sterol metabolic process. The incorporation of transcriptomics in the ESTn was shown to enable detection of effects, which precede morphological changes and provide a more sensitive measure of concentration-dependent effects as compared with classical morphological assessments. Furthermore, mechanistic insight can be instrumental in the extrapolation of effects in the ESTn to human hazard assessment.

The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles

Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The most pronounced effect of silver nanoparticles and the role of particle size in determining these effects, also in comparison to silver ions, are largely unknown. Effects of silver nanoparticles of different sizes (20, 80, 113 nm) were compared in in vitro assays for cytotoxicity, inflammation, genotoxicity and developmental toxicity. Silver nanoparticles induced effects in all endpoints studied, but effects on cellular metabolic activity and membrane damage were most pronounced. In all toxicity endpoints studied, silver nanoparticles of 20 nm were more toxic than the larger nanoparticles. In L929 fibroblasts, but not in RAW 264.7 macrophages, 20 nm silver nanoparticles were more cytotoxic than silver ions. Collectively, these results indicate that effects of silver nanoparticles on different toxic endpoints may be the consequence of their ability to inflict cell damage. In addition, the potency of silver in the form of nanoparticles to induce cell damage compared to silver ions is cell type and size-dependent.

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

Zebrafish developmental toxicity assay: A fishy solution to reproductive toxicity screening, or just a red herring?

The zebrafish embryotoxicity/teratogenicity assay is described as a useful alternative screening model to evaluate the effect of drugs on embryofoetal development. Fertilized eggs were exposed to different concentrations of 15 compounds with teratogenic (8) and non-teratogenic (7) potential until 96 h post-fertilization when 28 morphological endpoints and the level of compound uptake was assessed. The majority of drugs testing positive in mammals was also positive in zebrafish (75% sensitivity), while a relative high number of false positives were noted (43% specificity). Compound uptake determination appears useful for clarifying classifications as teratogenic or potential overdose although assay sensitivity could be improved to 71% if the exposure threshold, previously suggested as ∼50 ng/larvae, is reconsidered. The zebrafish assay shows some potential, though limited in its current form, as a screening tool for developmental toxicity within Janssen drug development. Further assay refinement with respect to endpoints and body burden threshold is required.

Time-Response Evaluation by Transcriptomics of Methylmercury Effects on Neural Differentiation of Murine Embryonic Stem Cells

Current globally harmonized Organisation for Economic Co-operation and Development (OECD) animal test guidelines for developmental toxicity require high numbers of experimental animals. To reduce animal use in this field, alternative developmental toxicity assays are highly desirable. We previously developed a dynamic in vitro model for screening effects of possible neurodevelopmental toxicants, using neural cell differentiation of pluripotent murine embryonic stem cells. To further mechanistically characterize the mouse neural embryonic stem cell test (ESTn) and to improve detection of possible neurodevelopmental toxicants, gene expression patterns were studied describing neural cell differentiation over time, as well as the impact on gene expression of exposure to the well-known neurotoxicant methylmercury (MeHg). A transcriptomics study was performed to examine whole-genome expression changes during the first 7 days of the cell differentiation protocol. Specific gene clusters were identified and enrichment analysis of Gene Ontology (GO) terms and gene sets derived from literature was performed using DAVID and T-profiler. Over time, a decrease of blastocyst and trophectoderm GO terms was observed, which included well-characterized pluripotency genes. Furthermore, an increase in the range of neural development-related GO terms, such as neuron differentiation and the wnt pathway, was observed. Analysis of gene expression using principle component analysis showed a time-dependent track in untreated cells, describing the process of neural differentiation. Furthermore, MeHg was shown to induce deviation from the predefined differentiation track. The compound inhibited general development GO terms and induced neural GO terms over time. This system appears promising for studying compound effects on neural differentiation in a mechanistic approach.

Innovation and Creation

Innovation and Creation

Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity

The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental toxicity. The results reveal that the potency ranking obtained in the ES-D3 cell differentiation assay is similar to the reported potency rankings in the two other in vitro assays for developmental toxicity. TTNPB (( E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) was the most potent retinoid, whereas etretinate and retinol had the lowest potency. All- trans-retinoic acid, 13- cis-retinoic acid, 9- cis-retinoic acid and acitretin showed an intermediate potency. In vivo potency rankings of the developmental toxicity of retinoids appear to be dependent on the species and/or exposure regimens used. The obtained in vitro potency ranking does not completely correspond with the in vivo potency rankings, although TTNPB is correctly predicted to be the most potent and retinol the least potent congener. The lack of in vivo kinetic processes in the ES-D3 cell differentiation assay might explain the deviating potency predictions of some retinoids. Therefore, knowledge on the species-dependent in vivo kinetics is essential when using in vitro toxicity data for the estimation of in vivo developmental toxicity potencies within series of related compounds.

A different approach to validating screening assays for developmental toxicity

There continue to be many efforts around the world to develop assays that are shorter than the traditional embryofetal developmental toxicity assay, or use fewer or no mammals, or use less compound, or have all three attributes. Each assay developer needs to test the putative assay against a set of performance standards, which traditionally has involved testing the assays against a list of compounds that are generally recognized as "positive" or "negative" in vivo. However, developmental toxicity is highly conditional, being particularly dependent on magnitude (i.e. dose) and timing of exposure, which makes it difficult to develop lists of compounds neatly assigned as developmental toxicants or not. Here we offer an alternative approach for the evaluation of developmental toxicity assays based on exposures. Exposures are classified as "positive" or "negative" in a system, depending on the compound and the internal concentration. Although this linkage to "internal dose" departs from the recent approaches to validation, it fits well with widely accepted principles of developmental toxicology. This paper introduces this concept, discusses some of the benefits and drawbacks of such an approach, and lays out the steps we propose to implement it for the evaluation of developmental toxicity assays.

Research Article: Evaluation of developmental toxicity of interaction between caffeine and pseudoephedrine using frog embryo teratogenesis assay-Xenopus (Fetax)

Pseudoephedrine and caffeine are found in many over-the-counter drugs including decongestants and weight loss chemicals. Both are proven central nervous system stimulants, and they may potentially interact on human health. Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was used to determine the developmental toxicity of pseudoephedrine and caffeine mixtures and to determine if synergism or antagonism occurs between the two on developmental toxicity. FETAX is a 96-hour developmental toxicity assay that screens for direct acting teratogens. Both have both been evaluated for developmental toxicity in FETAX but not as mixtures. The 96-hr LC50, 96-hr EC50, MCIG, and TI were determined for pseudoephedrine and caffeine in various mixtures. We tested five binary mixturesofthe two chemicals. The mixtures were basedonthe toxic unitsofeach chemical; where one toxic unit was equal to the 96-hr LC50. The toxic unit mixtures were pseudoephedrine to caffeine at toxic unit ratios of 0:1, 1:0, 3:1, 1:1, and 1:3 based on published literature. Toxic units were plotted on an isobole graph to determine if synergism, concentration response or antagonism occurred. Mixtures acted in a concentration additive interaction on the developmental toxicity. Malformations in mixtures were similar to those seen in caffeine with the exception of the mixture containing the most pseudoephedrine.

Validation of alternative methods for developmental toxicity testing

Developmental toxicity testing according to current international guidelines involves exposure of pregnant animals, mostly rats and rabbits, and subsequent assessment of toxic effects in their fetuses. Alternative methods have been developed since the early 1980s. They include cell differentiation assays using either primary cell cultures or immortalized cell lines. At a higher integration level the development of organ anlagen in vitro has been employed in assays for developmental toxicity. The most complex assays in this area make use of isolated postimplantation rodent embryos which are cultured in vitro during the phase of major organogenesis. The possibilities for their application as toxicity screens have been investigated in various validation studies. The most elaborate validation study of embryotoxicity assays carried out to date included the embryonic stem cell test EST, the limb bud micromass MM, and whole embryo culture WEC. In each test 20 chemical compounds were tested in four independent laboratories. The results have shown a marked success in the reproducibility of results. The extrapolation of these results of a limited set of chemicals to a generalized judgment of the tests for the world of chemicals remains a matter for further discussion.

Embryonic development assay with Daphnia magna: application to toxicity of chlorophenols

Cladoceran embryos, derived from Daphnia magna, were used for the developmental toxicity assay system of chlorophenols (CPs) and the quantitative structure-activity relationships (QSAR) studies. As the toxicity end point, the median effective concentration (EC50s) to reduce hatchability was estimated. The EC50s for embryo hatchabilities were found to be significantly correlated to acute immobilization toxicity (24-h EC50s) with juveniles, and the embryo assay appeared to be more sensitive to CPs with higher water solubility than the juveniles assay. The EC50s for embryos were correlated with six physicochemical parameters, and the Pow (n-octanol/water partition coefficient) gave a good correlation in simple linear regression analysis, as is frequently stated in toxicity studies with aquatic organisms. These results suggest that in vitro hatchability assay using D. magna embryos are useful for ecotoxicity screening of aquatic pollutants.

Exposure of Drosophila melanogaster embryonic cell cultures to 60-Hz sinusoidal magnetic fields: assessment of potential teratogenic effects.

There is considerable concern about potential detrimental health effects associated with exposure to environmentally relevant magnetic fields. One specific concern relates to potential effects of magnetic field (MF) exposure on reproduction and development. Consequently, an in vitro teratogenesis (developmental toxicity) assay employing embryonic Drosophila cells has been used to determine whether exposure to a 60-Hz MF of 100 microT for 16-18 hr is itself teratogenic and whether such an exposure could potentiate the teratogenic response induced by a chemical teratogen (developmental toxicant). The results demonstrated that (1) MF exposure alone did not induce a teratogenic response, whether the MF was oriented parallel or perpendicular to the plane of the culture dishes; and (2) MF exposure did not alter the teratogenic response induced by optimal or suboptimal concentrations of three chemical teratogens (retinoic acid, hydroxyurea, and cadmium). Furthermore, in additional studies, Drosophila embryos were exposed to 60-Hz MFs of 10 and 100 microT for 24 hr or for their entire development time (i.e., until adult ecolsion, about 10 days). Results demonstrated that MF exposure did not produce an increase in developmental abnormalities over those observed in unexposed controls.

Evaluation of Subchronic (13 Week), Reproductive, and in Vitro Genetic Toxicity Potential of 2-Ethylhexyl-2-cyano-3,3-diphenyl Acrylate (Octocrylene)

Evaluation of Subchronic (13 Week), Reproductive, and in Vitro Genetic Toxicity Potential of 2-Ethylhexyl-2-cyano-3,3-diphenyl Acrylate (Octocrylene). Odio, M. R., Azri-Meehan, S., Robison, S. H., and Kraus, A. L. (1994). Fundam. Appl. Toxicol. 22, 355-368.Use of 2-ethylhexyl-2-cyano-3,3-diphenyl acrylate (Octocrylene) in commercial sunscreen products has increased considerably in recent years. To support larger scale human exposure to this compound, additional toxicological information was needed in several key areas. The present studies evaluated subchronic toxicity, developmental toxicity, and in vitro genotoxic potential of Octocrylene. In the subchronic study, male and female New Zealand white (NZW) rabbits treated topically with concentrations of octocrylene up to 534 mg/kg/day for 13 weeks showed slight to moderate dose-dependent skin irritation that correlated positively with a mild depression in body weight gain. Lack of associated histopathologic or clinical hematology abnormalities suggested that the body weight effect probably reflected a nonspecific response to topical irritation. In percutaneous developmental toxicity studies, NZW does were treated topically with Octocrylene at levels up to 267 mg/kg/day on Days 6 through 18 of gestation. Body weight gain, food consumption, and all maternal, reproductive, and offspring parameters evaluated were comparable between Octocrylene-treated and control animals. In the oral developmental toxicity assay, female CD-1 mice received oral doses of Octocrylene up to 1000 mg/kg/day on Days 8-12 of gestation. No evidence of maternal or developmental toxicity was seen at any dose tested. Genotoxicity was evaluated in vitro using the Chinese hamster ovary cell assay to assess clastogenicity and the mouse lymphoma cell assay to assess forward gene mutations. Octocrylene did not induce any significant increase in genotoxicity. This evaluation of toxicological potential supports the use of Octocrylene as a human photoprotectant.