Brazil's Regulatory Context for Using New Approach Methodologies (NAMs) on the Registration of Products.

New Approach Methodologies (NAMs) are any non-animal-based approaches that can provide information in the context of chemical hazard and safety assessment. The goal is to develop information with equivalent or better scientific quality and relevance than that provided by traditional animal models. Starting with ethical issues, these approaches are gaining regulatory relevance in different global agencies. Since 2008, with the enactment of the Arouca Law-the first Brazilian legislation dedicated to laboratory animals, NAMs are gathering pace in Brazil's regulations. Specific regulations from different sectors include the acceptance of these new methods. However, some regulation is controversial about what is needed to address specific toxicological endpoints. The resulting regulatory uncertainty induces companies to keep on adopting the traditional methods, slowing NAM's development in the country. This work brings a perspective on the regulatory acceptance of NAMs in Brazilian Legislation for the registration of pharmaceuticals, medical devices, food/supplements, and agrochemical products. This text discusses the main issues of NAM adoption for each specific regulation. Therefore, legal acceptance of NAMs results in Brazil is still a process in progress. A collective effort including regulators, industry, contract research organizations (CROs), and the academic environment is needed to build regulatory confidence in the use of NAMs.

ZnO nanoparticles alter redox metabolism of Limnoperna fortunei.

Nanoparticles such as zinc oxide nanoparticles (ZnO-NP) that are incorporated in consumer and industrial products have caused concern about their potential ecotoxicological impact when released into the environment. Bivalve mollusks are susceptible targets for nanoparticle toxicity since nanomaterials can enter the cells by endocytosis mechanisms. The aim of this study was to evaluate the influence of ZnO-NP on the redox metabolism in Limnoperna fortunei and the DNA damage after exposure to ZnO-NP. Adult bivalves were incubated with 1-, 10-, and 50-µg mL-1 ZnO-NP for 2, 4, and 24 h. Ionic Zn release, enzymatic and non-enzymatic antioxidant activity, oxidative damage, and DNA damage were evaluated. Oxidative damage to proteins and lipids were observed after 4-h exposure and returned to baseline levels after 24 h. Superoxide dismutase levels decreased after 4-h exposure and increased after 24 h. No significant alteration was observed in the catalase activity or even DNA double-strand cleavage. The dissociation of ZnO may occur after 24 h, releasing ionic zinc (Zn2+) by hydrolysis, which was confirmed by the increase in the ionic Zn concentration following 24-h exposure. In conclusion, ZnO-NP were able to induce oxidative stress in exposed golden mussels. The golden mussel can modulate its own antioxidant defenses in response to oxidative stress and seems to be able to hydrolyze the nanoparticles and consequently, release Zn2+ into the cellular compartment.

Cytotoxicity and genotoxicity induced by coal and coal fly ash particles samples in V79 cells.

Exposure to coal and coal ashes can cause harmful effects in in vitro and in vivo systems, mainly by the induction of oxidative damage. The aim of this work was to assess cytotoxic and genotoxic effects using the V79 cell line treated with coal and coal fly ash particles derived from a coal power plant located in Santa Catarina, Brazil. Two coal samples (COAL11 and COAL16) and two coal fly ash samples (CFA11 and CFA16) were included in this study. COAL16 was co-firing with a mixture of fuel oil and diesel oil. The comet assay data showed that exposure of V79 cells to coal and coal fly ash particles induced primary DNA lesions. Application of lesion-specific endonucleases (FPG and ENDO III) demonstrated increased DNA effects indicating the presence of high amounts of oxidative DNA lesions. The cytokinesis-block micronucleus cytome assay analysis showed that exposure of V79 cells to high concentrations of coal and coal fly ash particles induced cytotoxic effects (apoptosis and necrosis) and chromosomal instability (nucleoplasmic bridges, nuclear buds, and micronucleus (MN) formation). These results may be associated with compounds contained in the surface of the particles as hazardous elements, ultrafine/nanoparticles, and polycyclic aromatic hydrocarbons (PAHs) which were detected in the samples. Graphical abstract ᅟ.

Evaluation of the cytotoxicity, genotoxicity and mutagenicity of diphenyl ditelluride in several biological models.

Diphenyl ditelluride (DPDT) is a potential prototype for the development of novel biologically active molecules. Thus, it is important to evaluate the toxic effects of this compound. In the present study, we evaluated the cytotoxic, genotoxic and mutagenic properties of DPDT in Chinese hamster fibroblast (V79) cells, in strains of the yeast Saccharomyces cerevisiae both proficient and deficient in several DNA repair pathways and in Salmonella typhimurium. DPDT induced frameshift mutations in both S.typhimurium and a haploid wild-type strain of S.cerevisiae. Mutants of S.cerevisiae defective in base excision repair and recombinational repair were more sensitive to DPDT. The results of a lactate dehydrogenase leakage assay suggest that DPDT is cytotoxic to V79 cells. At cytotoxic concentrations, this compound increased thiobarbituric reactive species levels and decreased the glutathione:GSSH ratio in yeast and V79 cells. DPDT generated single- and double-strand DNA breaks in V79 cells, both with and without metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, an induction of oxidative DNA base damage was indicated by a modified comet assay using formamidopyrimidine DNA glycosylase and endonuclease III. Treatment with DPDT also induced micronucleus formation in V79 cells. Pre-incubation with N-acetylcysteine reduced DPDT's oxidative, genotoxic and mutagenic effects in yeast and V79 cells. Our results suggest that the toxic and mutagenic properties of DPDT may stem from its ability to disturb the redox balance of the cell, which leads to oxidative stress and the induction of DNA damage.

3'3-ditrifluoromethyldiphenyl diselenide: a new organoselenium compound with interesting antigenotoxic and antimutagenic activities.

The trace element selenium (Se), once known only for its potential toxicity, is now a well-established essential micronutrient for mammals. The organoselenium compound diphenyl diselenide (DPDS) has shown interesting antioxidant and neuroprotective activities. On the other hand, this compound has also presented pro-oxidant and mutagenic effects. The compound 3'3-ditrifluoromethyldiphenyl diselenide (DFDD), a structural analog of diphenyl diselenide, has proven antipsychotic activity in mice. Nevertheless, as opposed to DPDS, little is known on the biological and toxicological properties of DFDD. In the present study, we report the genotoxic effects of the organoselenium compound DFDD on Salmonella typhimurium, Saccharomyces cerevisiae and Chinese hamster lung fibroblasts (V79 cells). DFDD protective effects against hydrogen peroxide (H(2)O(2))-induced DNA damage in vitro are demonstrated. DFDD did not cause mutagenic effects on S. typhimurium or S. cerevisiae strains; however, it induced DNA damage in V79 cells at doses higher than 25 microM, as detected by comet assay. DFDD protected S. typhimurium and S. cerevisiae against H(2)O(2)-induced mutagenicity, and, at doses lower than 12.5 microM, prevented H(2)O(2)-induced genotoxicity in V79 cells. The in vitro assays demonstrated that DFDD mimics catalase activity better than DPDS, but neither presents superoxide dismutase action. The products of the reactions of DFDD or DPDS with H(2)O(2) were different, as determined by electrospray mass spectrometry analysis (ESI-MS). These results suggest that DFDD is not mutagenic for bacteria or yeast; however, it may induce weak genotoxic effects on mammalian cells. In addition, DFDD has a protective effect against H(2)O(2)-induced damage probably by mimicking catalase activity, and the distinct products of the reaction DFDD with H(2)O(2) probably have a fundamental role in the protective effects of DFDD.

Assessment of environmental stress by the micronucleus and comet assays on Limnoperna fortunei exposed to Guaíba hydrographic region samples (Brazil) under laboratory conditions.

The Guaíba Basin is a source of drinking water for Porto Alegre (RS, Brazil). The water from this basin receives industrial, urban, and rural waste from many sources. The mussel species Limnoperna fortunei was chosen based on population data, distribution, and sensitivity. Previous tests with comet assay and micronuclei frequency in this freshwater mussel have shown to be successful in biomonitoring studies. The aim of this study was to evaluate the genotoxic contamination of the Guaíba Lake Hydrographic Region, through the determination of damage by the micronuclei and comet assays in L. fortunei (golden mussel). Nine sampling sites were evaluated in three different seasons: five sites in the mouths of the main rivers that flow into Guaíba lake; one site at the mouth of a stream; one major site of sewage discharge; two sites at Guaíba lake, near a sewage discharge; and the control site in a preservation area. DNA damage was detected by the single cell gel assay, as well as the frequency of micronuclei in hemocytes of mussels exposed under laboratory conditions for 7 days to water and sediment samples. Significant results were found in different seasons in almost all sampling sites (P<0.05, ANOVA Dunnet's test). Most of the positive results were found in samples affected mainly by urban effluents. It was possible to observe that there was a weak relation between mutagenic and genotoxic responses and mussels inorganic elements contents. Seasonal variation was observed at different sampling sites, but always indicating a huge contamination near urban sewage discharge. These results are consistent with previous studies, allowing us to infer that urban contamination is the biggest problem in this region. It is also possible to infer that L. fortunei is a good sentinel organism for the Guaíba Basin.

DNA damage and repair in haemolymph cells of golden mussel (Limnoperna fortunei) exposed to environmental contaminants.

The development of methodologies for biomonitoring freshwater ecosystems is of particular relevance in view of the serious problem of aquatic environmental pollution. The mussel species Limnoperna fortunei (golden mussel) was chosen to be tested as a biomonitor organism based on its population data and distribution. L. fortunei individuals were exposed to UV radiation in vitro, and in vivo to pentachlorophenol (PCP) and copper sulphate (CuSO(4)), with the aim of standardizing comet assay and micronucleus test methodologies and evaluating the potential of this organism as a biomonitor. Haemolymph cells immobilized in agarose on slides exposed to UV radiation showed a dose-response relationship with maximum damage at 4.2 J/m(2). For the chemical tests, individuals were exposed for 2h for the comet assay and 24 and 48 h for the micronucleus test. A dose-response relationship was observed for both chemicals. 3x10(-5) M CuSO(4) induced high genotoxicity, also producing some toxicity after 48 h of exposure. PCP induced maximum damage in both assays at 150 µg/L. Individuals exposed to PCP showed 100% repair 2 h after the exposure period, as assessed by the comet assay. Exposure to an environmental sample over 7 days confirmed the mussel sensitivity to water contaminants, detected both by the comet assay and the micronucleus test. The results allow us to suggest the golden mussel as a potential biomonitor organism.