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Antimicrobial resistance is a very worrying global health problem caused among others, by the overuse of antibiotics in livestock production. A silver-kaolin formulation with bactericidal activity has been developed to be used as an alternative to antimicrobials in animal feed. This formulation contains silver nanoparticles (AgNPs) embedded on the surface of a kaolin matrix. The aim of the thesis was to carry out the genotoxicity characterization of the mentioned silver-kaolin formulation, following the strategy suggested by the EFSA “Guidance on the risk assessment for the application of nanoscience and nanotechnologies in the food and feed chain”. The specific objectives were: to perform a bibliographic revision of the genotoxicity of AgNPs and to carry out the in vitro and in vivo genotoxicity evaluation of the silver-kaolin formulation. The bibliographic revision was conducted by compiling the genotoxicity studies carried out with AgNPs since 2013. The articles that used well-established standard assays (i.e., in vitro mouse lymphoma assays, in vitro micronucleus tests, in vitro comet assays, in vivo micronucleus tests, in vivo chromosome aberration tests and in vivo comet assays), were selected. The results showed that AgNPs produce genotoxic effects at all DNA damage levels evaluated, in both in vitro and in vivo assays. However, a higher proportion of positive results was obtained in the in vitro studies. None of the studies included a complete battery of assays, as recommended by ICH and EFSA guidelines, and few of the authors followed OECD guidelines when performing assays. Previous to the in vitro evaluation, the washing methods after treatment were adapted to the material. Then, preliminary cytotoxicity and proliferation assays were carried out to establish the concentrations tested in the genotoxicity assays. The in vitro genotoxicity evaluation included the mouse lymphoma assay in L5178Y TK+/− cells and the micronucleus (MN) test in TK6 cells, following the principles of the OECD guidelines 490 and 487, respectively. As a complement, the standard and Fpg-modified comet assays for the evaluation of strand breaks, alkali labile sites and oxidative DNA damage were also performed in TK6 cells. The formulation was tested without metabolic activation after an exposure of 3 h and 24 h. Its corresponding release in medium, after the continuous agitation of the silver-kaolin for 24 h, was also evaluated. Under the conditions tested, the test compound did not produce gene mutations, chromosomal aberrations or DNA damage (i.e., strand breaks, alkali labile sites or oxidized bases). Due to the complexity of the nanomaterials (NMs), the lack of guidelines and methods adaptation for testing them, and the evidence of secondary genotoxicity mechanisms caused by NMs, the genotoxicity evaluation of the silver-kaolin formulation was continued by using in vivo models. As a previous step, a dose finding study was conducted to establish the maximum dose to be administered in a repeated-dose study. Then, a combined genotoxicity study including the MN test and the standard and Fpg-modified comet assays integrated in a repeated dose 28-day oral toxicity study were carried out. Three doses of the formulation were administered; the high dose was established in the dose finding study, the low dose corresponded to the effective dose and the medium dose was the intermediate. A reversion study was included for the negative control and high dose group. The studies were conducted following the OECD guidelines 487, 489 and 407. Under the conditions tested, a NOAEL of 2000 mg/kg b.w. was determined. The silver-kaolin formulation did not induce chromosomal aberrations in bone marrow or DNA damage (i.e., strand breaks, alkali labile sites or oxidized bases) in liver, kidney, spleen or duodenum. |
