Brazilian National Network of Alternative Methods (RENAMA) 10th Anniversary: Meeting of the Associated Laboratories, May 2022.

The Brazilian National Network of Alternative Methods (RENAMA), which is linked to the Ministry of Science, Technology and Innovation, is currently comprised of 51 laboratories from CROs, academia, industry and government. RENAMA's aim is to develop and validate new approach methodologies (NAMs), as well as train researchers and disseminate information on their use - thus reducing Brazilian, and consequently Latin American, dependence on external technology. Moreover, it promotes the adoption of NAMs by educators and trained researchers, as well as the implementation of good laboratory practice (GLP) and the use of certified products. The RENAMA network started its activities in 2012, and was originally comprised of three central laboratories - the National Institute of Metrology, Quality and Technology (INMETRO); the National Institute of Quality Control in Health (INCQS); and the National Brazilian Biosciences Laboratory (LNBio) - and ten associated laboratories. In 2022, RENAMA celebrated its 10th anniversary, a milestone commemorated by the organisation of a meeting attended by different stakeholders, including the RENAMA-associated laboratories, academia, non-governmental organisations and industry. Ninety-six participants attended the meeting, held on 26 May 2022 in Balneário Camboriú, SC, Brazil, as part of the programme of the XXIII Brazilian Congress of Toxicology 2022. Significant moments of the RENAMA were remembered, and new goals and discussion themes were established. The lectures highlighted recent innovations in the toxicological sciences that have translated into the assessment of consumer product safety through the use of human-relevant NAMs instead of the use of existing animal-based approaches. The challenges and opportunities in accepting such practices for regulatory purposes were also presented and discussed.

Assessment of surface water quality of the bois river (Goiás, Brazil) using an integrated physicochemical, microbiological and ecotoxicological approach.

The data on water pollution is scarce in developing countries, including Brazil. The water quality assessment is important implementing the monitoring and remediation programs to minimize the risk of hazardous substances in freshwaters. Thus, this study evaluated the surface water quality of a stretch of the Bois River (Brazil), based on the physicochemical, microbiological and ecotoxicological analyses conducted in 2017, using Standard Methods and fish embryo acute toxicity (FET) test with zebrafish (Danio rerio). The results indicated that the quality of water samples located close to the discharge of tannery effluents was most impaired. Total phosphorus, BOD, DO, ammoniacal nitrogen, and thermotolerant coliforms parameters in P4 were not in accordance with the standards of current Brazilian legislation. Iron, lead, and copper levels were higher than environmental standards. The physicochemical quality of water samples was lower in the dry season than the rainy season. All samples (P1, P3, and P5) in rainy and dry seasons did not induce significant acute toxicity for zebrafish early-life stage; however other trophic levels (algae and microcrustacean) should be investigated to gain a better understanding of the toxicity during water quality analysis. In conclusion, the physicochemical and microbiological changes in the water of the Bois River can affect aquatic organisms as well as humans when it is used for drinking or in agriculture.

Ecotoxicological assessment and electrochemical remediation of doxorubicin.

Doxorubicin (DOX) is an anthracycline widely used in treatments of several cancers, so it has found in hospital effluents with a significant concentration (above 1 µg L-1). Electrochemical remediation is an alternative to promote its degradation. The aim of this work was to evaluate the ability of nanostructured graphite electrodes with metallic oxides to degrade DOX by electro-oxidation (EO). Graphite, TiO2@graphite and AuO-TiO2@graphite electrodes were used in medium with tap water or 10 mmol L-1 NaCl. DOX treatments at concentrations of 1.25-5 mg L-1 were carried out in a voltage source with 1.5-5 V. The cathode used was the platinum electrode. The treatment of DOX 1.25 mg L-1 with 10 mmol L-1 NaCl electrolyte using the AuO-TiO2@graphite electrode at 5 V and 1 mA was the best methodology to promote its degradation. Also, the modified electrode was efficient to DOX degradation after 17 cycles of reuse. An energy expenditure of 1.11 and 0.2 kWh m-3 were obtained for 3 and 50 mL of treatment, respectively. Fish embryo acute toxicity test with zebrafish (Danio rerio) were performed before and after treatment by EO using NaCl. This treatment caused no effect on embryo-larval development, however it induced significant damage in the DNA of the zebrafish larvae after 96 h of exposure, which emphasizes the importance of a depth ecotoxicological evaluation during the development of EO methodologies.

Eco- and genotoxicological assessments of two reactive textile dyes.

Contamination of natural waters has been one of the major problems of modern society and the textile industry is rated as an important polluting source, due to the generation of large amounts of wastewaters. The aim of this study was to assess textile dyes Reactive Blue 19 (RB19, anthraquinone dye) and Reactive Red 120 (RR120, azo dye) in terms of the potential to induce adverse effects on aquatic organisms and humans. Thus, these dyes were tested using the following assays: Microtox assay (Vibrio fischeri); brine shrimp (Artemia salina); Daphnia similis; and Comet with normal human dermal fibroblasts as well as Ames test (TA98, TA100, YG1041, YG1042--with and without S9). RB19 was relatively nontoxic to all aquatic bioindicators analyzed with an EC50 of more than 100 mg/L, whereas RR120 was only moderately toxic to A. salina with a EC50-48h of 81.89 mg/L. Mutagenicity through base pair substitution was observed with RB19 in the presence of S9 (Ames-positive). The comet assay did not demonstrate any apparent genotoxic effects for any tested dye. Although mutagenicity was detected with RB19, the mutagenic effect observed may be considered weak compared to the ability to induce DNA damage by other classes of dyes such as disperse dyes. Therefore, these dyes may be classified as nonmutagens (RR120) or weak mutagens (RB19) and relatively nontoxic for aquatic organisms. However, it is noteworthy that the weak acute toxicity to A. salina induced by RR120 is sufficient to suggest potential damage to the aquatic ecosystem and emphasizes the need for biomonitoring dye levels in wastewater systems.

Sens-ocular model: Cell-based assay to evaluate eye stinging potential of chemicals and baby cosmetic formulations.

The TRPV1 receptor, which is known to contribute significantly to pain perception, has recently been identified as a useful tool for predicting eye stinging potential in cosmetics. In this study, HEK-293 cells with high TRPV1 expression were utilized to evaluate calcium influx related to receptor activation triggered by chemicals and cosmetic formulations. The cells were exposed to increasing concentrations of substances to cause or not some aggression to the eye, and TRPV1 activity was assessed by measuring intracellular FURA-2 AM fluorescence signal. To confirm TRPV1 channel activation, capsazepine, a capsaicin antagonist, was employed in addition to using capsaicin as a positive control. The study's results indicate that this novel model can identify compounds known to cause some aggression to the eye, such as stinging, considering a cut-off value of 60% of Ca2+ influx exposed to the lowest evaluated concentration (0.00032%). When applied to the cosmetic baby formulation, although the presented model exhibited higher sensitivity by classifying as stinging formulations that had previously undergone clinical testing and were deemed non-stinging, the assay could serve as a valuable in vitro tool for predicting human eye stinging sensation and can be used as a tier 1 in an integrated testing strategy.

Evaluation of the acute toxicity, phototoxicity and embryotoxicity of a residual aqueous fraction from extract of the Antarctic moss Sanionia uncinata.

BACKGROUND: Ultraviolet (UV) radiation is the main exogenous inductor of skin damage and so photoprotection is important to control skin disorders. The Antarctic moss Sanionia uncinata is an important source of antioxidants and the photoprotective activity of its organic extracts has been investigated. This study aimed to evaluate the potential photoprotection, cytotoxicity and embryotoxicity of residual aqueous fraction (AF) from the moss S. uncinata. METHODS: UV-visible spectrum and SPF (sun protection factor) were determined by spectrophotometry. Embryotoxicity potential was evaluated by Fish embryo-larval toxicity test using zebrafish (Danio rerio) as organism model. Cell death assays by water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) were investigated using HaCaT keratinocyte cell line cultured in monolayers and three dimensions (3D). Phototoxicity and association with UV-filters were performed by 3T3 neutral red uptake test. RESULTS: The AF showed sharp absorption bands in the UV region and less pronounced in the visible region. The SPF was low (2.5 ± 0.3), but the SPF values of benzophenone-3 and octyl-methoxycinnamate increased ~ 3 and 4 times more, respectively, in association with AF. The AF did not induce significant lethal and sublethal effects on zebrafish early-life stages. In monolayers, the HaCaT cell viability, evaluated by WST-1, was above 70% by ≤0.4 mg AF/mL after 48 and 72-h exposure, whereas ≤1 mg AF/mL after 24-h exposure. The LDH assay showed that the cell viability was above 70% by ≤0.4 mg AF/mL even after 72-h exposure, but ≤1 mg/mL after 24 and 48-h exposure. In 3D cell culture, an increased cell resistance to toxicity was observed, because cell viability of HaCaT cell by WST-1 and LDH was above ~ 90% when using ≤1 and 4 mg AF/mL, respectively. The AF demonstrated values of photo irritation factor < 2 and of photo effect < 0.1, even though in association with UV-filters. CONCLUSIONS: The residual AF absorbs UV-vis spectrum, increased SPF values of BP-3 and OMC and does not induce embryotoxicity to zebrafish early life-stage. The cell death assays allowed establishing non-toxic doses of AF and phototoxicity was not detected. AF of S. uncinata presents a good potential for skin photoprotection against UV-radiation.

Toxicological evaluation of nail polish waste discarded in the environment.

Nail polish has been widely used around the world. However, the hazards of nail polishes discarded in the environment are still poorly investigated. Thus, the toxicogenetic effects of solubilized (SE) and leached (LE) extracts from nail polishes were investigated, simulating their disposal on water and landfill, respectively, and identifying their physicochemical properties and chemical constituents. Organic compounds and metals were detected in both extracts. SE and LE only induced mutagenic effects in TA98 Salmonella strain in the presence and absence of exogenous metabolic activation. Although both extracts did not significantly increase the frequency of micronucleated HepG2 cells, the cell viability was affected by 24-h exposure. No DNA damage was observed in gonad fish cells (RTG-2) exposed to both extracts; however, the highest SE and LE concentrations induced significant lethal and sublethal effects on zebrafish early-life stages during 96-h exposure. Based on our findings, it can be concluded that if nail polishes enter aquatic systems, it may cause negative impacts to the environment.

Removal of azo dye using Fenton and Fenton-like processes: Evaluation of process factors by Box-Behnken design and ecotoxicity tests.

The conventional treatment of textile effluents is usually inefficient in removing azo dyes and can even generate more toxic products than the original dyes. The aim of the present study was to optimize the process factors in the degradation of Disperse Red 343 by Fenton and Fenton-like processes, as well as to investigate the ecotoxicity of the samples treated under optimized conditions. A Box-Behnken design integrated with the desirability function was used to optimize dye degradation, the amount of residual H2O2 [H2O2residual], and the ecotoxicity of the treated samples (lettuce seed, Artemia salina, and zebrafish in their early-life stages). Dye degradation was affected only by catalyst concentration [Fe] in both the Fenton and Fenton-like processes. In the Fenton reaction, [H2O2residual] was significantly affected by initial [H2O2] and its interaction with [Fe]; however, in the Fenton-like reaction, it was affected by initial [H2O2] only. A. salina mortality was affected by different process factors in both processes, which suggests the formation of different toxic products in each process. The desirability function was applied to determine the best process parameters and predict the responses, which were confirmed experimentally. Optimal conditions facilitated the complete degradation of the dye without [H2O2residual] or toxicity for samples treated with the Fenton-like process, whereas the Fenton process induced significant mortality for A. salina. Results indicate that the Fenton-like process is superior to the Fenton reaction to degrade Disperse Red 343.

A test battery for assessing the ecotoxic effects of textile dyes.

The textile dyeing industry is one of the main sectors contributing to environmental pollution, due to the generation of large amounts of wastewater loaded with dyes (ca. 2-50% of the initial amount of dyes used in the dye baths is lost), causing severe impacts on human health and the environment. In this context, an ecotoxicity testing battery was used to assess the acute toxicity and genotoxicity of the textile dyes Direct Black 38 (DB38; azo dye) and Reactive Blue 15 (RB15; copper phthalocyanine dye) on different trophic levels. Thus these dyes were tested using the following assays: Filter paper contact test with earthworms (Eisenia foetida); seed germination and root elongation toxicity test (Cucumis sativus, Lactuca sativa and Lycopersicon esculentum); acute immobilization test (Daphnia magna and Artemia salina); and the Comet assay with the rainbow trout gonad-2 cell fish line (RTG-2) and D. magna. Neither phytotoxicity nor significant effects on the survival of E. foetida were observed after exposure to DB38 and RB15. Both dyes were classified as relatively non-toxic to D. magna (LC50 > 100 mg/L), but DB38 was moderately toxic to A. salina with a LC50 of 20.7 mg/L. DB38 and RB15 induced significant effects on the DNA of D. magna but only DB38 caused direct (alkaline comet assay) and oxidative (hOGG1-modified alkaline comet assay) damage to RTG-2 cells in hormetic responses. Therefore, the present results emphasize that a test battery approach of bioassays representing multiple trophic levels is fundamental in predicting the toxicity of textile dyes, aside from providing the information required to define their safe levels for living organisms in the environment.

Electrochemical remediation of amoxicillin: detoxification and reduction of antimicrobial activity.

Amoxicillin (AMX) is one of the most commonly prescribed antibiotics around the world to treat and prevent several diseases in both human and veterinary medicine. Incomplete removal of AMX during wastewater treatment contributes to its presence in water bodies and drinking water. AMX is an emerging contaminant since its impact on the environment and human health remains uncertain. This contribution was aimed to evaluate the electrochemical oxidation (EO) of AMX using different anodes in tap water, NaCl or Na2SO4 solutions and to evaluate the potential toxicity of remaining AMX and its by-products on zebrafish early-life stages. Chemical intermediates generated after EO were determined by mass spectrometry and their resulting antimicrobial activity was evaluated. AMX did not induce significant mortality in zebrafish during extended exposure but affected zebrafish development (increased body length) from 6.25 mg/L to 25 mg/L and inhibited enzymatic biomarkers. Carbon modified with titanium oxide (TiO2@C) anode achieved complete AMX removal in just a few minutes and efficiency of the supported electrolytes occurred in the following order: 0.1 M NaCl > 0.1 M Na2SO4 > 0.01 M NaCl > tap water. The order of potential toxicity to zebrafish early life-stages related to lethal and sublethal effects was as follows: 0.1 M Na2SO4 > 0.1 M NaCl >0.01 M NaCl = tap water. Additionally, the EO of AMX using TiO2@C electrode with 0.01 M NaCl was able to inhibit the antimicrobial activity of AMX, reducing the possibility of developing bacterial resistance.

In vitro genotoxicity and in vivo subchronic evaluation of the anti-inflammatory pyrazole compound LQFM021.

Scientific evidences have highlighted 5-(1-(3-fluorophenyl)-1H-pyrazol-4-yl)-2H-tetrazole (LQFM021) as a promising anti-inflammatory, analgesic and antinociceptive agent due to its effects on peripheral opioid receptors associated with activation of the nitric oxide/cGMP/KATP pathway. Despite these important pharmacological findings, toxicity data of LQFM021 are scarce. Thus, this study investigated the in vitro genotoxicity of LQFM021 through cytokinesis-block micronucleus assay (OECD Nº 487/2014). Moreover, zebrafish model was used to assess the embryotoxicity potential of LQFM021 using fish embryo toxicity test (OECD Nº 236/2013) with extended exposure to evaluate subchronic larval development. In vivo subchronic toxicity of LQFM021 in rats (OECD Nº 407/2008) was also conducted. This compound at the lower concentrations tested (3.1 and 31 µg/mL) did not promote changes in micronuclei frequency in HepG2 cells. However, in the higher concentrations of LQFM021 (310 and 620 µg/mL) triggered a significant increase of micronucleated HepG2 cells, showing an alert signal of potential genotoxicity. Regarding the oral treatment of rats with LQFM021 (62.5, 125 or 250 mg/kg) for 28 days, the main findings showed that LQFM021 promoted renal and liver changes in a dose-dependent manner, being irreversible damage for kidneys while liver tissue showed a recovery after 14 days post treatment. Regarding embryotoxicity, although the lower concentrations used did not show toxicity, the concentration of LQFM021 (39.8 and 100 mg/L) promoted malformations in zebrafish embryo-larvae stage, in especial cardiac tissue changes. In conclusion, anti-inflammatory compound LQFM021 seems to have some limiting factors as a new therapeutic option to be used orally and in high repeated doses, related to those found in the non-steroidal anti-inflammatory drugs (NSAIDs).

Topotecan-loaded lipid nanoparticles as a viable tool for the topical treatment of skin cancers.

OBJECTIVES: This work aimed to evaluate semisolid formulations containing topotecan (TPT) loaded nanostructured lipid carriers (NLC) for topical treatment of skin cancers, as TPT is effective against a variety of tumours. A formulation which increases TPT skin permeation would be extremely desirable. METHODS: TPT-NLC were prepared and incorporated in hydrogels with hydroxyethyl cellulose and chitosan (TPT-NLC-HEC and TPT-NLC-Ch, respectively). Control formulations were obtained by dispersing TPT in HEC and Ch hydrogels (TPT-HEC and TPT-Ch). KEY FINDINGS: TPT-NLC-HEC and TPT-NLC-Ch showed to maintain the drug and nanoparticle dispersions stable for up to 30 days. When nanoparticles were incorporated into gels, TPT release was significantly decreased (P < 0.05). Still, TPT-NLC-HEC increased 2.37 times permeation compared with TPT-HEC (11.9 and 5.0 µg/cm2 , respectively). Cell culture experiments with B16F10 melanoma demonstrated that nanoencapsulation significantly increased TPT cytotoxicity (P < 0.05). TPT-NLC was more toxic than free TPT, with IC50 value of 5.74 µg/ml, whereas free TPT had an IC50 > 20 µg/ml. As skin permeated values of TPT from developed formulation (TPT-NLC) were superior to melanoma IC50, it can be extrapolated that chemotherapeutic permeated amounts may be sufficient for a therapeutic effect. CONCLUSIONS: TPT-NLC-HEC may be a valuable tool for the topical treatment of skin cancers.

Ecotoxicological assessment of glyphosate-based herbicides: Effects on different organisms.

Glyphosate-based herbicides are the most commonly used worldwide because they are effective and relatively nontoxic to nontarget species. Unlimited and uncontrolled use of such pesticides can have serious consequences for human health and ecological balance. The present study evaluated the acute toxicity and genotoxicity of 2 glyphosate-based formulations, Roundup Original (Roundup) and Glyphosate AKB 480 (AKB), on different organisms: cucumber (Cucumis sativus), lettuce (Lactuca sativa), and tomato (Lycopersicon esculentum) seeds, and microcrustacean Artemia salina and zebrafish (Danio rerio) early life stages. For the germination endpoint, only L. esculentum presented significant sensitivity to AKB and L. sativa to Roundup, whereas both formulations significantly inhibited the root growth of all species tested. Both AKB and Roundup induced significant toxicity to A. salina; both are classified as category 3, which indicates a hazard for the aquatic environment, according to criteria of the Globally Harmonized Classification System. However, Roundup was more toxic than AKB, with 48-h median lethal concentration (LC50) values of 14.19 mg/L and 37.53 mg/L, respectively. For the embryo-larval toxicity test, Roundup proved more toxic than AKB for the mortality endpoint (96-h LC50 values of 10.17 mg/L and 27.13 mg/L, respectively), whereas for the hatching parameter, AKB was more toxic than Roundup. No significant genotoxicity to zebrafish larvae was found. We concluded that AKB and Roundup glyphosate-based formulations are phytotoxic and induce toxic effects in nontarget organisms such as A. salina and zebrafish early life stages. Environ Toxicol Chem 2017;36:1755-1763. © 2016 SETAC.