Józef Sawicki

Evaluation of toxicity of medical devices using Spirotox and Microtox tests : I. Toxicity of selected toxicants in various diluents

Significant effort has been directed toward developing in vitro alternatives, which can be the first step of toxicity analysis. Tissue culture assays are currently the most popular in vitro tests for evaluating acute toxicity. The possibility of applying two bioassays using microorganisms in assessing the toxicity of extracts of medical devices was investigated. The Microtox test system--a luminescent bacteria toxicity test--assesses changes in light output from a luminescent bacteria, Vibrio fischeri. Spirotox used a large ciliate protozoan: Spirostomum ambiguum. The most widely used extraction solvent, 0.9% NaCl, must be concentrated up to 2% for Microtox and diluted nine times for the Spirotox test. The organic solvents ethanol, DMSO, and polyethylene glycol 400 were not toxic in either test in concentrations of 1-2%. The toxicity of reference compounds Hg, Cd, Zn, Pb, and SDS was examined in various diluents. The sequence of toxicity of the tested compounds in Spirotox and Microtox was: Hg > Cd > Zn > Pb > SDS, and Hg > Pb = Zn > SDS > Cd, respectively. Addition of organic solvents changed the toxicity of compounds tested in 60% of Spirotox tests and only in 25% of Microtox tests. Changes were low, not exceeding 100% in almost all cases. No correlation was observed between diluent and toxicant in either bioassay.

Evaluation of in vitro biotransformation of propranolol with HPLC, MS/MS, and two bioassays.

The majority of human drugs enter aquatic systems after ingestion and subsequent excretion in the form of parent compounds and metabolites. Environmental exposure to drug metabolites has not been reported so far. The goal of the present study was to apply the in vitro method of biotransformation of compounds with S9 fraction to the ecotoxicological analysis. β‐adrenoceptor antagonist propranolol was metabolized with S9 rat liver fraction. The parent compound was quantified with HPLC, and the metabolites were identified with QToF MS. Propranolol was metabolized rapidly, during the first hour its level decreased by 80 and 50% of the initial 20 and 100 mg L−1, respectively. Ten peaks were observed on the HPLC‐RF chromatogram. Four peaks were identified with QToF MS/MS propranolol (m/z = 260), N‐desisopropylpropranolol (m/z = 218), hydroxypropranolol (m/z = 276), and hydroxy N‐desisopropranolol glycol (m/z = 235). Then the ecotoxicity of the reaction mixture was studied with two bioassays Spirotox with the protozoan Spirostomum ambiguum and Thamnotoxkit F™ with the anostracean crustacean Thamnocephalus platyurus. Propranolol is twofolds more toxic to Spirotox than to Thamnotoxkit F with 24 h‐EC50 = 1.77 mg L−1 and 24 h‐LC50 = 3.86 mg L−1, respectively. No statistically significant differences were found between the toxicity of the reaction mixtures after S9 biotransformation and the propranolol solution. These results indicate that the biological activity of the metabolites is similar to that of the parent drug. The presented method of in vitro biotransformation of drugs with S9 fraction followed by HPLC and ecotoxicity tests, may be used as screening method for evaluation of the toxicity of drug metabolites.

Application of microbial assay for risk assessment biotest in evaluation of toxicity of human and veterinary antibiotics

The microbial assay for risk assessment (MARA) is a multispecies, growth inhibition microplate toxicity test with 11 microorganisms individually lyophilized in microplate wells. The microbial species representing wide diversity, generated 11 microbial toxic concentration (MTC) values were obtained giving a unique “toxic fingerprint” profile of the test sample. The toxicity of 14 antibiotics was evaluated with the MARA test. The fingerprints for each group of antibiotics differ, indicating a particular toxic profile. Tetracyclines were the most toxic antibacterials with the minimum MTC value of 3.6 μg L−1. In the group of tetracyclines the order of the three most sensitive microbial strains was the same 2 > 6 > 7. Quinolones affected the most sensitive strain(s) at concentrations of 12–75 μg L−1. The MARA bioassay exhibited different spectra of toxic responses to microbial strains for the first and second generation quinolones. However, for first generation quinolones strain 6 was substantially more sensitive than the other microorganisms, while second generation quinolones were most toxic to strain 3, followed by 6 then 4. In this instance, the three strains belong to two different phylogenetic groups–strain 3 is γ‐proteotype and strains 4 and 6 are β‐proteotype.

Influence of pH on the toxicity of nitrophenols to Microtox and Spirotox tests

The toxicity of mononitrophenols and dinitrophenols (DNP) to luminescent bacteria Vibrio fischeri (Microtox test) and ciliated protozoan Spirostomum ambiguum (Spirotox test) was evaluated. Spirotox was more sensitive to the tested nitrophenols (NPs) than the Microtox test. 2,5-DNP was the most toxic and 2-NP was the least toxic to the both bioindicators. The toxicity depended greatly on the pH of the medium. The highest changes were observed for DNPs, where the toxicity decreased more than 20-times when the pH increased from 6 to 8. No significant decrease of the toxicity was found for NPs, when the pH increased from 6 to 7. Greater increase of the pH to 8 caused from 1.5 to 4-fold decrease of the toxicity.

The toxicity of tri-substituted benzenes to the protozoan ciliate Spirostomum ambiguum

The Spirotox test utilises a large ciliate protozoan Spirostomum ambiguum as a test organism. The goal of the present study was to evaluate the toxicity of tri-substituted benzenes in the Spirotox test. Twenty-six organic compounds were tested in this study and included: dimethylphenols (DMPs), dichlorophenols (DCMs), trichlorobenzenes (TCBs), dichloroanilines (DCAs), dinitrophenols (DNPs), dinitroaniline (DNA), dinitrochlorobenzene (DNCB) and dinitrofluorobenzene (DNFB). The toxicity of the compounds tested varied almost four orders of a magnitude. DMPs and DCAs were the least toxic, whereas dinitro derivatives were the most toxic to S. ambiguum. When chlorine or fluorine atoms were replaced by amino or hydroxy substituents, the toxicity increased dramatically. The results of the Spirotox test were compared with three other bioassays that are widely used around the world: Microtox, Tetrahymena pyriformis and Daphnia magna. The Spirotox was less sensitive than these other bioassays for the majority of these compounds, with an exception found for the dinitro derivatives.