The alamar blue assay in the context of safety testing of nanomaterials

Longhin, Eleonora; Yamani, Naouale El; Rundén-Pran, Elise; Dušinská, Maria

doi:10.3389/ftox.2022.981701

Cited by 47 publications

(31 citation statements)

References 29 publications

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“…MTT is used in several ISO standards (ISO10993-5:2009) and OECD test guidelines (OECD 431, 439, 492). The inclusion of the different assays as output-related features is also relevant since NFs could interfere with the tests and the final outcome. , Intestinal cells are exposed with either digested or non-digested AgNFs at concentrations ranging from 1.25 to 100 ppm with the WST-1 assay. All experiments refer to a 24 h duration of exposure.…”

Section: Methodsmentioning

confidence: 99%

Data-Driven Quantitative Intrinsic Hazard Criteria for Nanoproduct Development in a Safe-by-Design Paradigm: A Case Study of Silver Nanoforms

Furxhi

Bengalli

Motta

et al. 2023

ACS Appl. Nano Mater.

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The current European (EU) policies, that is, the Green Deal, envisage safe and sustainable practices for chemicals, which include nanoforms (NFs), at the earliest stages of innovation. A theoretically safe and sustainable by design (SSbD) framework has been established from EU collaborative efforts toward the definition of quantitative criteria in each SSbD dimension, namely, the human and environmental safety dimension and the environmental, social, and economic sustainability dimensions. In this study, we target the safety dimension, and we demonstrate the journey toward quantitative intrinsic hazard criteria derived from findable, accessible, interoperable, and reusable data. Data were curated and merged for the development of new approach methodologies, that is, quantitative structure−activity relationship models based on regression and classification machine learning algorithms, with the intent to predict a hazard class. The models utilize system (i.e., hydrodynamic size and polydispersity index) and non-system (i.e., elemental composition and core size)-dependent nanoscale features in combination with biological in vitro attributes and experimental conditions for various silver NFs, functional antimicrobial textiles, and cosmetics applications. In a second step, interpretable rules (criteria) followed by a certainty factor were obtained by exploiting a Bayesian network structure crafted by expert reasoning. The probabilistic model shows a predictive capability of ≈78% (average accuracy across all hazard classes). In this work, we show how we shifted from the conceptualization of the SSbD framework toward the realistic implementation with pragmatic instances. This study reveals (i) quantitative intrinsic hazard criteria to be considered in the safety aspects during synthesis stage, (ii) the challenges within, and (iii) the future directions for the generation and distillation of such criteria that can feed SSbD paradigms. Specifically, the criteria can guide material engineers to synthesize NFs that are inherently safer from alternative nanoformulations, at the earliest stages of innovation, while the models enable a fast and cost-efficient in silico toxicological screening of previously synthesized and hypothetical scenarios of yet-to-be synthesized NFs.

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Section: Methodsmentioning

confidence: 99%

Data-Driven Quantitative Intrinsic Hazard Criteria for Nanoproduct Development in a Safe-by-Design Paradigm: A Case Study of Silver Nanoforms

Furxhi

Bengalli

Motta

et al. 2023

ACS Appl. Nano Mater.

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“…Quantitative measurement of cell death used the alamarBlue assay. , After 48 h incubation, the extraction medium in the 24-well plates was replaced by 10% (v/v) alamarBlue reagent (alamarBlue dye, Thermo Scientific) mixed with a fresh cell growth medium, and the samples were incubated for 2 h at 37 °C with 5% CO 2 . After 2 h incubation, the medium containing alamarBlue reagent was transferred to a 96-well plate (100 μL/well) to obtain fluorescence intensity (excitation/emission: 530/590 nm, triplicate measurements).…”

Section: Methodsmentioning

confidence: 99%

Facile Modification of Medical-Grade Silicone for Antimicrobial Effectiveness and Biocompatibility: A Potential Therapeutic Strategy against Bacterial Biofilms

Wynne,

Zolotarskaya,

Jarrell

et al. 2023

ACS Appl. Mater. Interfaces

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A one-step modification of biomedical silicone tubing with N,N-dimethyltetradecylamine, C14, results in a composition designated WinGard-1 (WG-1, 1.1 wt % C14). A surface-active silicon-amine phase (SAP) is proposed to account for increased wettability and increased surface charge. To understand the mechanism of antimicrobial effectiveness, several procedures were employed to detect whether C14 leaching occurred. An immersion-growth (IG) test was developed that required knowing the bacterial Minimum Inhibitory Concentrations (MICs) and Minimum Biocidal Concentrations (MBCs). The C14 MIC and MBC for Gm– uropathogenic E. coli (UPEC), commonly associated with catheter-associated urinary tract infections (CAUTI), were 10 and 20 μg/mL, respectively. After prior immersion of WG-1 silicone segments in a growth medium from 1 to 28 d, the IG test for the medium showed normal growth for UPEC over 24 h, indicating that the concentration of C14 must be less than the MIC, 10 μg/mL. GC-MS and studies of the medium inside and outside a dialysis bag containing WG-1 silicone segments supported de minimis leaching. Consequently, a 5 log UPEC reduction (99.999% kill) in 24 h using the shake flask test (ASTM E2149) cannot be due to leaching and is ascribed to contact kill. Interestingly, although the MBC was greater than 100 μg/mL for Pseudomonas aeruginosa, WG-1 silicone affected an 80% reduction via a 24 h shake flask test. For other bacteria and Candida albicans , greater than 99.9% shake flask kill may be understood by proposing increased wettability and concentration of charge illustrated in the TOC. De minimis leaching places WG-1 silicone at an advantage over conventional anti-infectives that rely on leaching of an antibiotic or heavy metals such as silver. The facile process for preparation of WG-1 silicone combined with biocidal effectiveness comprises progress toward the goals of device designation from the FDA for WG-1 and clearance.

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“…The most used ones are based on colorimetric methods such as AlamarBlue, MTS, MTT, WST-1. However, potential interference of NMs with these methods needs to be tested (see Longhin et al, 2022 this collection of manuscripts). Cytotoxicity should always be tested with the same cells and the same set-up as for the comet assay—plate layout and amount of medium, NMs dispersion etc.—and ideally performed in the same experiment as the comet assay ( El Yamani et al, 2017 ).…”

Section: Methodsmentioning

confidence: 99%

The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials

Yamani

Rundén-Pran²,

Collins³

et al. 2022

Front. Toxicol.

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The in vitro comet assay is a widely applied method for investigating genotoxicity of chemicals including engineered nanomaterials (NMs). A big challenge in hazard assessment of NMs is possible interference between the NMs and reagents or read-out of the test assay, leading to a risk of biased results. Here, we describe both the standard alkaline version of the in vitro comet assay with 12 mini-gels per slide for detection of DNA strand breaks and the enzyme-modified version that allows detection of oxidized DNA bases by applying lesion-specific endonucleases (e.g., formamidopyrimidine DNA glycosylase or endonuclease III). We highlight critical points that need to be taken into consideration when assessing the genotoxicity of NMs, as well as basic methodological considerations, such as the importance of carrying out physicochemical characterization of the NMs and investigating uptake and cytotoxicity. Also, experimental design—including treatment conditions, cell number, cell culture, format and volume of medium on the plate—is crucial and can have an impact on the results, especially when testing NMs. Toxicity of NMs depends upon physicochemical properties that change depending on the environment. To facilitate testing of numerous NMs with distinct modifications, the higher throughput miniaturized version of the comet assay is essential.

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The alamar blue assay in the context of safety testing of nanomaterials

Cited by 47 publications

References 29 publications

Data-Driven Quantitative Intrinsic Hazard Criteria for Nanoproduct Development in a Safe-by-Design Paradigm: A Case Study of Silver Nanoforms

Data-Driven Quantitative Intrinsic Hazard Criteria for Nanoproduct Development in a Safe-by-Design Paradigm: A Case Study of Silver Nanoforms

Facile Modification of Medical-Grade Silicone for Antimicrobial Effectiveness and Biocompatibility: A Potential Therapeutic Strategy against Bacterial Biofilms

The miniaturized enzyme-modified comet assay for genotoxicity testing of nanomaterials

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