Methodological approaches for potential in vitro toxicity of manufactured nanomaterials relevant for chemicals and cosmetics

Andrea ZIJNO, Istituto Superiore di Sanità

Due to their unique physico-chemical properties, nanomaterials (NMs) are commonly used in various applications in industrial, electrical, pharmaceutical and biomedical fields and are included in several consumer products, such as cosmetics and food. The same physico-chemical properties of NMs may be responsible of interaction with living system that are unpredictable solely on  the basis of their chemical composition. Hence, their possible adverse effects must be thoroughly evaluated. With this aim, in vitro tests are especially relevant in an early phase of NMs evaluation for screening purposes and for identification of potential toxicity endpoints. In vitro approaches, commonly applied for testing traditional chemicals, are suitable also for testing NMs providing some adaptation are included in order to take into account the NMs specificity. Indeed, though there is increasing amount of information of the hazard potential on several NMs, there is a dramatic lack of systematic information on the potential hazards associated with them. As first step, towards definition of reliable strategy for NMs evaluation, developing preparation and characterization procedures based on standardized protocols are mandatory. Furthermore, in the last years scientific community is dealing with the identification of limits of available in vitro tests and setting new methods to overcome such limits.

At ISS several research groups are participating to national and international projects aimed to evaluate the performance of available tests and to establish the correct procedures to test NMs. As far as the methods for NMs preparation and characterization are concerned, ISS is involved in the NANoREG project (EC FP7 project), where several NMs are tested after set up of specific procedures for sample preparation, based on sonication performed according to selected parameters, in order to obtain comparable suspension among participating groups. Further, characterization of NMs batch dispersion and NMs dispersion in cell culture media were also performed, according to a common protocol.

Concerning cytotoxicity evaluation we reported, in a study funded by the Health Ministry, that in Neutral Red Uptake, a conventional cytotoxicity assay, NMs interacted with the colorimetric detection of viable cells hampering the results of the assay. Conversely, Colony Forming Efficiency and the MTS assays, whose performances have been evaluated in a OECD round robin study and in NANoREG project, respectively, showed better performance and reproducibility among different laboratories. Screening of genotoxic properties of NMs is part of risk assessment evaluation  of NMs. Among the genotoxic assays not all tests can be applied to NMs (for instance the Ames test) or, alternatively, need some modifications due to the possible interference of NMs with assay procedures (for instance Comet or Micronucleus assays). In order to optimize micronucleus assay protocol, in the above mentioned projects we tested titanium dioxide and silicas NMs, modifying the time of treatment. However, in spite of a positive genotoxic effects observed after evaluation of oxidative DNA damage, no micronucleus induction was observed suggesting the repair of induced damage not converted in frank chromosome damage.Finally, immunotoxic effects of NMs have to be carefully investigated. So far, procedures for evaluating these effects are lacking and cannot be identified a single in vitro test to detect potential immunotoxicological effects of NMs. A panel of tests have to be applied covering different effects on immunological system. In the framework of NANoREG project several tests have been developed to cover different immunotoxic effects.

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