Maximum Test Concentration Research Articles

Structure-based design, synthesis, and biological characterization of indolylarylsulfone derivatives as novel human immunodeficiency virus type 1 inhibitors with potent antiviral activities and favorable drug-like profiles.

In the current antiretroviral landscape, continuous efforts are still needed to search for novel chemotypes of human immunodeficiency virus type 1(HIV-1) inhibitors with improved drug resistance profiles and favorable drug-like properties. Herein, we report the design, synthesis, biological characterization, and druggability evaluation of a class of non-nucleoside reverse transcriptase inhibitors. Guided by the available crystallographic information, a series of novel indolylarylsulfone derivatives were rationally discovered via the substituent decorating strategy to fully explore the chemical space of the entrance channel. Among them, compound 11h bearing the cyano-substituted benzyl moiety proved to be the most effective inhibitor against HIV-1 wild-type and mutant strains (EC50 = 0.0039-0.338 μM), being far more potent than or comparable to etravirine and doravirine. Besides, 11h did not exhibit cytotoxicity at the maximum test concentration. Meanwhile, the binding target of 11h was further confirmed to be reverse transcriptase (IC50 = 0.055 μM). Preliminary structure-activity relationship were discussed to guide further optimization work. Molecular docking and dynamics simulation studies were investigated in detail to rationalize the biological evaluation results. Further drug-likeness assessment indicated that 11h possessed excellent physicochemical properties. Moreover, no apparent hERG blockade liability and cytochrome P450 inhibition were observed for 11h. Notably, 11h was characterized by favorable in vitro metabolic stability with moderate clearance rates and long half-lives in human plasma and liver microsomes. Overall, 11h holds great promise as an ideal Anti-HIV-1 lead compound due to its potent antiviral efficacy, low toxicity, and favorable drug-like profiles.

Sodium borohydride mediated synthesis of nano-sized silver particles: Their characterization, anti-microbial and cytotoxicity studies

This work reports the one-step, fast, easy, simple and economical process for the synthesis of nano-sized and stable silver particles. AgNPs were synthesized at six (0.02 M, 0.04 M, 0.06 M, 0.08 M, 0.10 M and 1.00 M) different concentrations of metal precursor and reducing agent. Sodium borohydride was used as both reducing and stabilizing agent. Characterization studies revealed that all the silver nanoparticles (AgNPs) synthesized were in the range of 10 nm–50 nm, spherical shaped, crystalline nature and with high negative surface charge (−30 mV to −47 mV). Among the AgNPs evaluated for their anti-microbial activity through well diffusion method against Escherichia coli (Gram negative bacteria), Bacillus subtilis (Gram positive bacteria) and Candida albicans (fungi), AgNPs-1.00 showed maximum zone of inhibition with 11.5 mm, 18.5 mm and 10 mm at highest test concentration (100 mg/L), respectively. Cytotoxicity of the AgNPs was determined against MCF-7, PVA1 and Caco-2 cancer cell lines through standard MTT assay. AgNPs-0.08 showed highest mortality% with 90% and 80% against MCF-7 and PVA1 at maximum test concentration (100 mg/L), respectively. However, none of the AgNPs showed toxicity towards Caco-2 cancer cell line. Therefore, AgNPs can be used as anti-microbial agents in the biomedical applications.

High throughput embryonic zebrafish test with automated dechorionation to evaluate nanomaterial toxicity

Engineered nanomaterials pose occupational health and environmental concerns as they possess unique physical and chemical properties that can contribute to toxicity. High throughput toxicity screening methods are needed to address the increasing number of nanomaterials in production. Here we used a zebrafish photomotor response (PMR) test to evaluate a set of fifteen nanomaterials with military relevance. Automated dechorionation of zebrafish embryos was used to enhance nanomaterials bioavailability. Optimal PMR activity in zebrafish embryos was found at 30–31 hours post-fertilization (hpf). Behavioral and toxicological responses were measured at 30 and 120 hpf; behavioral responses were found for thirteen of the fifteen nanomaterials and acute toxicity (LC50) levels for nine of the fifteen nanomaterials below the maximum test concentration of 500 μg/ml. Physico-chemical characterization of the nanomaterials detected endotoxin and bacterial contamination in two of the tested samples, which may have contributed to observed toxicity and reinforces the need for physical and chemical characterization of nanomaterials use in toxicity testing. The zebrafish PMR test, together with automated dechorionation, provides an initial rapid assessment of the behavioral effects and toxicity of engineered nanomaterials that can be followed up by physico-chemical characterization if toxicity is detected, reducing the amount of time and monetary constraints of physico-chemical testing.

Rational Framework for the Design of Trp- and Arg-Rich Peptide Antibiotics Against Multidrug-Resistant Bacteria.

The threat of antibiotic resistance warrants the discovery of agents with novel antimicrobial mechanisms. Antimicrobial peptides (AMPs) directly disrupting bacterial membranes may overcome resistance to traditional antibiotics. AMP development for clinical use has been mostly limited to topical application to date. We developed a rational framework for systematically addressing this challenge using libraries composed of 86 novel Trp- and Arg-rich engineered peptides tested against clinical strains of the most common multidrug-resistant bacteria known as ESKAPE pathogens. Structure-function correlations revealed minimum lengths (as low as 16 residues) and Trp positioning for maximum antibacterial potency with mean minimum inhibitory concentration (MIC) of 2–4 μM and corresponding negligible toxicity to mammalian cells. Twelve peptides were selected based on broad-spectrum activity against both gram-negative and -positive bacteria and <25% toxicity to mammalian cells at maximum test concentrations. Most of the selected PAX remained active against the colistin-resistant clinical strains. Of the selected peptides, the shortest (the 16-residue E35) was further investigated for antibacterial mechanism and proof-of-concept in vivo efficacy. E35 killed an extensively-resistant isolate of Pseudomonas aeruginosa (PA239 from the CDC, also resistant to colistin) by irreversibly disrupting the cell membranes as shown by propidium iodide incorporation, using flow cytometry and live cell imaging. As proof of concept, in vivo toxicity studies showed that mice tolerated a systemic dose of up to 30 mg/kg peptide and were protected with a single 5 mg/kg intravenous (IV) dose against an otherwise lethal intraperitoneal injection of PA239. Efficacy was also demonstrated in an immune-compromised Klebsiella pneumoniae infection model using a daily dose of 4mg/kg E35 systemically for 2 days. This framework defines the determinants of efficacy of helical AMPs composed of only cationic and hydrophobic amino acids and provides a path for a potential departure from the restriction to topical use of AMPs toward systemic application.

Synthesis of Hydroxypropyltrimethyl Ammonium Chitosan Derivatives Bearing Thioctate and the Potential for Antioxidant Application

Hydroxypropyltrimethyl ammonium chloride chitosan (HACC) is one of the most important water-soluble chitosan derivatives; its derivatives have gained growing attention due to their potential biomedical applications. Here, hydroxypropyltrimethyl ammonium chitosan derivatives bearing thioctate (HACTs), with different degrees of substitution of thioctate, were prepared using HACC and α-lipoic acid as the reaction precursors, using an ion exchange method. The structural characteristics of the synthesized derivatives were confirmed by FTIR, 1H NMR, and 13C NMR spectroscopy. In addition, their antioxidant behaviors were also investigated in vitro by the assays of reducing power, and scavenging activities against hydroxyl radicals and DPPH radicals. The antioxidant assay indicated that HACTs displayed strong antioxidant activity compared with HACC, especially in terms of reducing power. Besides, the antioxidant activities of the prepared products were further enhanced with the increase in the test concentration and the degrees of substitution of thioctate. At the maximum test concentration of 1.60 mg/mL, the absorbance value at 700 nm of HACTs, under the test conditions, was 4.346 ± 0.296, while the absorbance value of HACC was 0.041 ± 0.007. The aforementioned results support the use of HACTs as antioxidant biomaterials in food and the biomedical field.

Chronic toxicity of 50 metals to Ceriodaphnia dubia

Metals are essential elements for human life but may cause disorders when exposure is excessive. Previously, we reported on the acute toxicity of 50 metals; however, the chronic toxicity data of some metals are not available. Therefore, we conducted chronic toxicity tests to determine the effects of 50 metals on the water flea, Ceriodaphnia dubia. The IC20 of 20 metals (Be, Sc, Cr, Co, Ni, Cu, Zn, Y, Ru, Ag, Cd, In, Te, W, Os, Pt, Au, Hg, Tl and Pb) were <100 μg/L; nine metals (Al, V, As, Se, Zr, Nb, Rh, Sb and Bi) were 100 ≤ IC20 < 1000 μg/L; 16 metals (Li, Mg, K, Ti, Mn, Fe, Ga, Ge, Rb, Sr, Mo, Sn, Cs, Ba, Re and Ir) were 1000 ≤ IC20 ≤ 100 000 μg/L; and two metals (Na and Ca) were >100 000 μg/L. Three metals (Pd, Hf and Ta) did not show IC20 at the upper limit of respective aqueous solubility, and IC20 s were not obtained. The maximum test concentrations (almost aqueous solubility) of Pd, Hf and Ta were 83, 2400 and 5.3 μg/L, respectively. These data show the high correlation between our IC50 s for C. dubia and those for Dahpnia magna published previously. The IC50 s of 47 metals were not correlated with electronegativity, first ionization energy, atomic weight, atomic number, covalent radius, atomic radius or ionic radius.

Effect of cosmetic chemical preservatives on resident flora isolated from healthy facial skin.

Healthy skin harbors numerous microbes known to maintain its health and prevent attacks from external pathogens. The influence of chemical preservatives commonly used in cosmetic products on facial resident flora remains poorly characterized. In this study, we aimed to investigate the antibacterial activity of five such preservatives on in vitro cultivated skin-resident bacteria. Both Gram-positive and Gram-negative bacteria were isolated on blood agar, tryptic soy agar, and nutrient agar; Gram-negative bacteria were then selected on Hank's balanced salt solution containing antibiotics and Reasoner's 2A. The minimum inhibitory concentrations (MICs) of methylisothiazolinone (MTI), iodopropynyl butylcarbamate (IPBC), ethylhexylglycerin (EHG), methylparaben (MP), and phenoxyethanol (PE) were estimated for nine facial resident bacteria, Escherichia coli, and Staphylococcus aureus using serial broth dilution in vitro. The maximum test concentrations coincided with the upper limits set by the "Cosmetic Safety and Technical Specification" (2015 edition, China). Nine facial resident bacteria were isolated from 14 healthy adults: Staphylococcus epidermidis, Staphylococcus capitis, Kocuria, Micrococcus luteus, Bacillus, Acinetobacter, Pseudomonas parafulva, Pseudomonas oleovorans, and Roseomonas cervicalis. MTI and IPBC displayed the strongest effect on all tested strains (MICs ≤0.01%), followed by EHG and MP (MICs ≤0.3%), and finally PE with the weakest effect (MIC ≤1%). The five chemical preservatives assayed inhibited survival of the nine facial resident bacteria isolates, when tested at the maximum allowed limit. The corresponding MICs will provide a reference for the effective utilization of these compounds in product formulations.

Antioxidant Effects of Four Heartwood Extractives on Midgut Enzyme Activity in Heterotermes indicola (Blattodea: Rhinotermitidae).

Heterotermes indicola (Wasmann) (Blattodea: Rhinotermitidae) is a species of subterranean termite that is a destructive pest of wood and wood products in Pakistan. This study evaluated the antioxidant and antienzyme potential of heartwood extractives against H. indicola. Heartwood extractives of four durable wood species, Tectona grandis (L.f), Dalbergia sissoo (Roxb.), Cedrus deodara (Roxb.), and Pinus roxburghii (Sarg.) were removed from wood shavings via soxhlet extraction with an ethanol:toluene solvent system. The antioxidant potential of the extractive compounds was determined using the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging test. Results showed maximum antioxidant activity for extractives of D. sissoo. D. sissoo had the lowest IC50 (the concentration where 50% inhibition of the DPPH radical is obtained) at 28.83 µg/ml among the heartwood extractives evaluated. This antioxidant activity, however, was not concentration dependent as was observed in the other heartwood extractives tested. At the maximum test concentration, T. grandis showed the highest percent inhibition at 89.7%, but this inhibition was lower compared to the positive control antioxidant compounds butylated hydroxytoluene and quercetin. When termites were fed filter paper treated with IC50s of the extractives and control compounds, glutathione S-transferase activity in the guts of H. indicola workers was significantly reduced by T. grandis and D. sissoo extractives. Similarly, esterase activity was reduced more by P. roxburghii extractives compared to control antioxidant treatments and other tested extractives. However, none of the extractives examined significantly reduced the activity of catalase enzymes in H. indicola compared to treatments with the antioxidant control compounds.

In search of a comprehensible set of endpoints for the routine monitoring of neurotoxicity in vertebrates: sensory perception and nerve transmission in zebrafish (Danio rerio) embryos.

In order to develop a test battery based on a variety of neurological systems in fish, three sensory systems (vision, olfaction, and lateral line) as well as nerve transmission (acetylcholine esterase) were analyzed in zebrafish (Danio rerio) embryos with respect to their suitability as a model for the screening of neurotoxic trace substances in aquatic ecosystems. As a selection of known or putative neurotoxic compounds, amidotrizoic acid, caffeine, cypermethrin, dichlorvos, 2,4-dinitrotoluene, 2,4-dichlorophenol, 4-nonylphenol, perfluorooctanoic acid, and perfluorooctane sulfonic acid were tested in the fish embryo test (OECD test guideline 236) to determine EC10 values, which were then used as maximum test concentration in subsequent neurotoxicity tests. Whereas inhibition of acetylcholinesterase was investigated biochemically both in vivo and in vitro (ex vivo), the sensory organs were studied in vivo by means of fluorescence microscopy and histopathology in 72- or 96-h-old zebrafish embryos, which are not regarded as protected developmental stages in Europe and thus - at least de jure- represent alternative test methods. Various steps of optimization allowed this neurotoxicity battery to identify neurotoxic potentials for five out of the nine compounds: Cypermethrin and dichlorvos could be shown to specifically modulate acetylcholinesterase activity; dichlorvos, 2,4-dichlorophenol, 4-nonylphenol, and perfluorooctane sulfonic acid led to a degeneration of neuromasts, whereas both vision and olfaction proved quite resistant to concentrations ≤ EC10 of all of the model neurotoxicants tested. Comparison of neurotoxic effects on acetylcholinesterase activity following in vivo and in vitro (ex vivo) exposure to cypermethrin provided hints to a specific enzyme-modulating activity of pyrethroid compounds. Enhancement of the neuromast assay by applying a simultaneous double-staining procedure and implementing a 4-scale scoring system (Stengel et al. 2017) led to reduced variability of results and better statistical resolution and allowed to differentiate location-dependent effects in single neuromasts. Since acetylcholinesterase inhibition and neuromast degeneration can be analyzed in 72- and 96-h-old zebrafish embryos exposed to neurotoxicants according to the standard protocol of the fish embryo toxicity test (OECD TG 236), the fish embryo toxicity test can be enhanced to serve as a sensitive neurotoxicity screening test in non-protected stages of vertebrates.

P53 induction and cell viability modulation by genotoxic individual chemicals and mixtures.

The binding of the p53 tumor suppression protein to DNA response elements after genotoxic stress can be quantified by cell-based reporter gene assays as a DNA damage endpoint. Currently, bioassay evaluation of environmental samples requires further knowledge on p53 induction by chemical mixtures and on cytotoxicity interference with p53 induction analysis for proper interpretation of results. We investigated the effects of genotoxic pharmaceuticals (actinomycin D, cyclophosphamide) and nitroaromatic compounds (4-nitroquinoline 1-oxide, 3-nitrobenzanthrone) on p53 induction and cell viability using a reporter gene and a colorimetric assay, respectively. Individual exposures were conducted in the absence or presence of metabolic activation system, while binary and tertiary mixtures were tested in its absence only. Cell viability reduction tended to present direct correlation with p53 induction, and induction peaks occurred mainly at chemical concentrations causing cell viability below 80%. Mixtures presented in general good agreement between predicted and measured p53 induction factors at lower concentrations, while higher chemical concentrations gave lower values than expected. Cytotoxicity evaluation supported the selection of concentration ranges for the p53 assay and the interpretation of its results. The often used 80% viability threshold as a basis to select the maximum test concentration for cell-based assays was not adequate for p53 induction assessment. Instead, concentrations causing up to 50% cell viability reduction should be evaluated in order to identify the lowest observed effect concentration and peak values following meaningful p53 induction.

Physicochemical properties and antimicrobial activity of new spirocyclic thieno[2,3-d]pyrimidin-4(3H)-one derivatives

A number of 6'-methylidene-2,3-dihydro-1H-spiro[pyridine-4,5'-thieno[2,3-d]pyrimidin]-4'(3'H)-one derivatives, obtained as major products of a domino reaction between 5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3-d]pyrimidines and electron-deficient alkynes, were characterized for their acid-base properties and lipophilicity and evaluated for the antimicrobial activity against a number of clinical isolates of bacterial and fungal strains. The cytotoxicity against four tumor cell lines was also screened for some compounds. The dissociation constants (pK a) and partition coefficients (log P) in 1-octanol–water system were determined using a potentiometric technique. The negative difference between the observed and calculated log P values could be explained in the light of the conformational rigidity. Most of the studied compounds showed a moderate and selective activity against Gram-positive bacteria strains (S. agalactiae, E. faecalis, S. epidermidis), whereas did not exhibit any effect against Gram-negative bacteria and fungi at the maximum test concentration (500 μM). Implications of the physicochemical properties in modulating the antimicrobial activity and cytotoxicity of the examined spirocyclic thieno[2,3-d]pyrimidin-4(3H)-one derivatives are also discussed.

Dose rates of antimicrobial substances in boar semen preservation-time to establish new protocols.

To achieve a standardized number of spermatozoa in the final AI dose, varying amounts of extender fluid with a fixed concentration of antimicrobial substances are currently added to boar ejaculates. This practice ignores the different degrees of dilution of the antimicrobials in the end product. In calculating the final concentration of gentamicin in AI doses from 27,538 processed boar ejaculates, we demonstrated varying gentamicin concentrations in the resultant extended boar semen samples. The median concentration was 220.37mg/L. In 25 of the samples (0.09%), the gentamicin concentration fell below 5mg/L, which is close to or below the epidemiological cut-off value for many bacteria. We calculated the minimum inhibitory concentration of gentamicin for bacteria isolated from raw and extended ejaculates. Five of the isolates from extended ejaculates exceeded the maximum test concentration of 512mg/L. As a result, we are presenting an alternative method of boar semen preservation whereby a particular combination of gentamicin concentrate and antibiotic-free extender is incorporated that standardizes the antibiotic concentration in the diluted semen. The addition of standardized antibiotic concentrations did not negatively affect sperm quality when compared to the use of ready-to-use extenders. In conclusion, an end volume-based and standardized addition of gentamicin to boar ejaculates can be a helpful alternative to prevent insufficient dosage of antibiotics in liquid preserved boar semen without affecting semen quality.

Identification of the AntiListerial Constituents in Partially Purified Column Chromatography Fractions of Garcinia kola Seeds and Their Interactions with Standard Antibiotics.

Partially purified fractions of the n-hexane extract of Garcinia kola seeds were obtained through column chromatography and their constituents were identified through the use of gas chromatography coupled to mass spectrometry (GC-MS). Three fractions were obtained by elution with benzene as the mobile phase and silica gel 60 as the stationery phase and these were named Benz1, Benz2, and Benz3 in the order of their elution. The antiListerial activities of these fractions were assessed through MIC determination and only Benz2 and Benz3 were found to be active with MIC's ranging from 0.625 to 2.5 mg/mL. The results of the GC-MS analysis showed Benz2 to have 9 compounds whilst Benz3 had 7 compounds, with the major compounds in both fractions being 9,19-Cyclolanost-24-en-3-ol, (3.β.) and 9,19-Cyclolanostan-3-ol,24-methylene-, (3.β.). The Benz2 fraction was found to have mainly indifferent interactions with ampicillin and penicillin G whilst mainly additive interactions were observed with ciprofloxacin. The Benz3 fraction's interactions were found to be 50% synergistic with penicillin G and 25% synergistic with ciprofloxacin and ampicillin. A commercially available 9,19-Cyclolanost-24-en-3-ol, (3.β.) was found not to exhibit any antiListerial activities at maximum test concentrations of 5 mg/mL, suggesting that the compound could be acting in synergy with the other compounds in the eluted fractions of Garcinia kola seeds.

Development of biodegradable methylprednisolone microparticles for treatment of articular pathology using a spray-drying technique

In this work, microparticles were prepared by spray-drying using albumin, chondroitin sulfate, and hyaluronic acid as excipients to create a controlled-release methylprednisolone system for use in inflammatory disorders such as arthritis. Scanning electron microscopy demonstrated that these microparticles were almost spherical, with development of surface wrinkling as the methylprednisolone load in the formulation was increased. The methylprednisolone load also had a direct influence on the mean diameter and zeta potential of the microparticles. Interactions between formulation excipients and the active drug were evaluated by x-ray diffraction, differential scanning calorimetry, and thermal gravimetric analysis, showing limited amounts of methylprednisolone in a crystalline state in the loaded microparticles. The encapsulation efficiency of methylprednisolone was approximately 89% in all formulations. The rate of methylprednisolone release from the microparticles depended on the initial drug load in the formulation. In vitro cytotoxic evaluation using THP-1 cells showed that none of the formulations prepared triggered an inflammatory response on release of interleukin-1β, nor did they affect cellular viability, except for the 9.1% methylprednisolone formulation, which was the maximum test concentration used. The microparticles developed in this study have characteristics amenable to a therapeutic role in inflammatory pathology, such as arthritis.

Improved Soil Mixing and Delivery System for a Storm Runoff Simulator

An earlier version of a storm runoff simulator to test conservation buffers reproduced target hydrographs and sedigraphs using uniform, fine sand; however, it was unable to uniformly mix and deliver native sediment. The objectives of this work reported were to create a method to process native agricultural sediment, mix a uniform sediment slurry at a target concentration, and create a control system that will deliver the slurry in varying flow rates corresponding to a target sedigraph. Eroded silty clay (14% sand) was scraped, dried, and processed with a hammer mill. A sand (93% sand) and loam (44% sand) were dried and screened for organic debris and large clods. Each soil type was mixed by an axial flow impeller in an 1890-L cone-bottom tank. Recirculation through a trash pump was used to further break down aggregates and maintain a uniform sediment concentration in the tank. A V-Port ball valve under pressurized flow was used to achieve outflow control. The sediment mixing system was capable of producing concentrations within 3.3% of the target concentration with a maximum test concentration of 0.294 kg L -1 with the silty clay soil. Simulated hydrographs had a Nash Sutcliffe Efficiency of 0.998, a Root Mean Square Error of 0.06 L s -1 , and a peak flow rate within 1% of the target flow. Simulated sedigraphs with silty clay had similar performance. Neither the sandy soil, nor the loam, were successfully delivered through the system to match target sedigraphs. The sand could not be uniformly mixed in the tank, but the loam was uniformly mixed to the target concentration.

Experimental and theoretical studies on the photoinduced acute toxicity of a series of anthraquinone derivatives towards the water flea ( Daphnia magna)

The photoinduced acute toxicity of fourteen anthraquinone derivatives to Daphnia magna was investigated. Whilst in the dark, all of dyes exhibited no observable toxicity at the maximum test concentration used, in the presence of visible light, all of the colorants, with the exception of three nitroanthraquinones, were acutely toxic towards the water flea over a wide range of medium effective concentrations (4.3–4186.7 nmol L −1). Generally, the acute toxicity of the dyes was higher in the presence of full-spectrum simulated solar radiation compared with visible light; in addition, the photoinduced toxicity of the dyes increased with increase in their hydrophobicity. The energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital was found to indicate the relative photoinduced toxicity of the dyes. Time-dependent density functional theory calculations revealed that singlet oxygen and the superoxide anion could be generated through direct energy transfer or autoionization of the excited state of the dyes.

Evaluation of the genotoxic potential of three phenyltetrahydropyridinyl butylazole-derived sigma-receptor ligand drug candidates

Three structurally related phenyltetrahydropyridinyl butylazole (PTHPB)-derived drug candidates with sigma receptor-binding properties were evaluated for genotoxic potential in the ICH standard battery of genetic toxicology assays. These comprised an Ames test, a mouse-lymphoma assay, and a mouse bone-marrow micronucleus test. The maximum test concentrations in the in vitro assays were determined by the solubility and/or the cytotoxicity of the compounds. In the mouse micronucleus assay, the compounds were administered orally at three levels up to the maximum tolerated dose (MTD). Negative results were obtained for all three drug candidates in the Ames test and in the mouse-lymphoma assay, both in the absence or presence of metabolic activation. In the mouse micronucleus test, there was no effect on the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after oral administration of any of the three test compounds, at any dose level or sampling time (24 and 48 h). Administration of all three compounds at the MTD induced a clear decrease in mouse body-temperature of 3.1–4.8 °C below normal; the temperature returned to normal within 8 h of dose administration. The produced mild hypothermia and absence of micronucleus induction was in contrast to the induction of MNPCE secondary to marked hypothermia reported for a structurally similar PTHPB-derived sigma-receptor ligand, the antipsychotic compound E-5842. The results obtained in the current series of studies suggest that exposure to the three tested PTHPB-derived drug candidates would not pose a genotoxic risk under clinical conditions.

Effects on litter‐dwelling earthworms and microbial decomposition of soil‐applied imidacloprid for control of wood‐boring insects

Imidacloprid is an effective, systemic insecticide for the control of wood-boring insect pests in trees. Systemic applications to trees are often made by soil injections or drenches, and the resulting imidacloprid concentrations in soil or litter may pose a risk of harm to natural decomposer organisms. The authors tested effects of imidacloprid on survival and weight gain or loss of the earthworms Eisenia fetida (Savigny) and Dendrobaena octaedra (Savigny), on leaf consumption rates and cocoon production by D. octaedra and on microbial decomposition activity in laboratory microcosms containing natural forest litter. Dendrobaena octaedra was the most sensitive of the two earthworm species, with an LC(50) of 5.7 mg kg(-1), an LC(10) of about 2 mg kg(-1) and significant weight losses among survivors at 3 mg kg(-1). Weight losses resulted from a physiological effect rather than from feeding inhibition. There were no effects on cocoon production among survivors at 3 mg kg(-1). The LC(50) for E. fetida was 25 mg kg(-1), with significant weight losses at 14 mg kg(-1). There were no significant effects on microbial decomposition of leaf material at the maximum test concentration of 1400 mg kg(-1). The results indicate that, when imidacloprid is applied as a systemic insecticide to the soil around trees, it is likely to cause adverse effects on litter-dwelling earthworms if concentrations in the litter reach or exceed about 3 mg kg(-1).

Evaluation of the genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) in a battery of in vitro and in vivo genotoxicity assays

The genotoxic potential of the natural neurotoxin Tetrodotoxin (TTX) was evaluated in a battery of in vitro and in vivo genotoxicity assays. These comprised a bacterial reverse-mutation assay (Ames test), an in vitro human lymphocyte chromosome-aberration assay, an in vivo mouse bone-marrow micronucleus assay and an in vivo rat-liver UDS assay. Maximum test concentrations in in vitro assays were determined by the TTX limit of solubility in the formulation vehicle (0.02% acetic acid solution). In the Ames test, TTX was tested at concentrations of up to 200 μg/plate. In the chromosome-aberration assay human lymphocytes were exposed to TTX at concentrations of up to 50 μg/ml for 3 and 20 h in the absence of S9, and for 3 h in the presence of S9. For the in vivo assays, maximum tested dose levels were determined by the acute lethal toxicity of TTX after subcutaneous administration. In the mouse micronucleus assay TTX dose levels of 2, 4 and 8 μg/kg were administered to male and female animals, and bone-marrow samples taken 24 and 48 h (high-dose animals only) after administration. In the UDS assay, male rats were given TTX on two occasions with a 14-h interval at dose levels of 2.4 and 8 μg/kg, the last dose being administered 2 h before liver perfusion and hepatocyte culturing. Relevant vehicle and positive control cultures and animals were included in all assays. TTX was clearly shown to lack in vitro or in vivo genotoxic activity in the assays conducted in this study. The results suggest that administration of TTX as a therapeutic analgesic agent would not pose a genotoxic risk to patients.

The Comparative Testing of Eight Coded Chemicals in the Rat Limb Bud Micromass and Rat Embryo Culture Systems

When cultured at high density, mesenchymal cells from rat limb buds proliferate and differentiate into chondrocytes. Inhibition of this in vitro chondrogenic process has been used for the preliminary evaluation of teratogenic potential. Alternatively, intact post-implantation rat embryos, maintained in short-term culture, provide a system for the in vitro study of abnormal development not limited to the skeletal system. Both systems isolate the test agent from maternal metabolism and pharmacokinetic restraints. In this study, drug-associated selective inhibition of alcian blue uptake by cartilage proteoglycans, in micromass cultures of limb bud cells prepared from 13-day-old rat embryos, was used to assess teratogenic potential in vitro following exposure for 48 hours to eight coded compounds (acetylsalicylic acid, isoniazid. penicillin G, saccharine, vincristine sulphate, 6-aminonicotinamide, retinoic acid, and amaranth). Following drug exposure, cultures were incubated for another 96 hours, and the cells were then fixed and stained with 0.5% alcian blue. Bound dye was then extracted and quantitated. In parallel cultures, cell viability was measured by neutral red uptake, and protein content was assayed by using the bicinchoninic acid method. Except for retinoic acid and vincristine sulphate, the maximum test concentration was 1000μg/ml. Inhibition of alcian blue uptake (&gt; 50%) was noted at 0.001μg/ml vincristine sulphate, 0.5/μg/ml retinoic acid and 5μg/ml 6-aminonicotinamide, demonstrating that strong teratogens inhibit differentiation in micromass cultures at lower concentrations than those which affect limb cell viability. When the same eight compounds were tested in a 24-hour embryo culture model, dysmorphogenesis was evident at 0.005μg/ml vincristine sulphate, 0.1μg/ml retinoic acid and 0.3μg/ml 6-aminonicotinamide. For the other five chemicals, little or no toxicity was noted up to the maximum test concentration in either model. We conclude that the two test systems, both based on the developing rat embryo, are consistent with each other, and that either of them would be useful for the preliminary screening of potential teratogens.