Growing worldwide efforts to replace (reduce) animal testing and to improve alternative in vitro tests which may be more efficient in terms of both time, cost and scientific validity include also genotoxicity/mutagenicity endpoints. The aim of the review article was to summarize currently available in vitro testing approaches in this field, their regulatory acceptance and recommended combinations for classification of chemicals. A study using the combination of Comet Assay performed on two cell lines and the Chromosomal Aberration test on human peripheral lymphocytes was performed with the aim to predict the genotoxic potential of selected paraben esters, serving as a model chemical group. Parabens are widely used in consumer products as preservatives and have been reported to exhibit inconclusive results in numerous genotoxicity studies. The Comet Assay identified Ethylparaben and Benzylparaben as potentially genotoxic. The Chromosomal Aberration test revealed weak genotoxic potential in case of Ethylparaben and positive genotoxicity in case of Butylparaben, Propylparaben and Isopropylparaben. The main reasons for variability seem to be limited water solubility of parabens, determining their bioavailability at the cellular level, and absence of metabolic activation in the Comet Assay. The results confirmed that the Comet Assay should serve as a screening test and should not be used as a stand-alone method for classification of genotoxicity. The weight of evidence approach in risk assessment should be supported with data generated with the use of human relevant in vitro methods based on cells / tissues of human origin.
The Fish Embryo Acute Toxicity (FET) Test was adopted by the Organisation for Economic Co-operation and Development as OECD TG 236 in 2013. The test has been designed to determine acute toxicity of chemicals on embryonic stages of fish and proposed as an alternative method to the Fish Acute Toxicity Test performed according to OECD TG 203. In recent years fish embryos were used not only in the assessment of toxicity of chemicals but also for environmental and wastewater samples. In our study we investigated the acute toxicity of treated wastewater from seven hospitals in the Czech Republic. Our main purpose was to compare the suitability and sensitivity of zebrafish embryos with the sensitivity of two other aquatic organisms commonly used for wastewater testing – Daphnia magna and Aliivibrio fischeri. For the aim of this study, in addition to the lethal endpoints of the FET test, sublethal effects such as delayed heartbeat, lack of blood circulation, pericardial and yolk sac edema, spinal curvature and pigmentation failures were evaluated. The comparison of three species demonstrated that the sensitivity of zebrafish embryos is comparable or in some cases higher than the sensitivity of D. magna and A. fischeri. The inclusion of sublethal endpoints caused statistically significant increase of the FET test efficiency in the range of 1-12 %. Based on our results, the FET test, especially with the addition of sublethal effects evaluation, can be considered as a sufficiently sensitive and useful additional tool for ecotoxicity testing of the acute toxicity potential of hospital effluents.