A Human Cell-based Assay to Assess the Induction of Vasculature Formation for Non-genotoxic Carcinogenicity Testing Purposes: A Pilot Study.

The induction of vasculature formation is proposed to be a significant mechanism behind the non-genotoxic carcinogenicity of a chemical. The vasculature formation model used in this study is based on the coculture of human primary HUVECs and hASCs. This model was used to develop an assay to assess the induction of vasculature formation. Three assay protocols, based on different conditions, were developed and compared in order to identify the optimal conditions required. Some serum supplements and growth factors were observed to be essential for initiating vasculature formation. Of the studied putative positive reference chemicals, aspartame, sodium nitrite, bisphenol A and nicotine treatment led to a clear induction of vasculature formation, but arsenic and cadmium treatment only led to a slight increase. This human cell-based assay has the potential to be used as one test within a next generation testing battery, to assess the non-genotoxic carcinogenicity of a chemical through the mechanism of vasculature formation induction.

Angiogenic Effect of Pravastatin Alone and with Sera from Healthy and Complicated Pregnancies Studied by in vitro Vasculogenesis/Angiogenesis Assay.

OBJECTIVE: To determine the direct effect of pravastatin on angiogenesis and to study the interaction between pravastatin and maternal sera from women with early- or late-onset pre-eclampsia (PE), intrauterine growth restriction, or healthy pregnancy. METHODS: We collected 5 maternal serum samples from each group. The effect of pravastatin on angiogenesis was assessed with and without maternal sera by quantifying tubule formation in a human-based in vitro assay. Pravastatin was added at 20, 1,000, and 8,000 ng/mL concentrations. Concentrations of angiogenic and inflammatory biomarkers in serum and in test medium after supplementation of serum alone and with pravastatin (1,000 ng/mL) were measured. RESULTS: Therapeutic concentration of pravastatin (20 ng/mL) did not have significant direct effect on angiogenesis, but the highest concentrations inhibited angiogenesis. Pravastatin did not change the levels of biomarkers in the test media. There were no changes in angiogenesis when therapeutic dose of pravastatin was added with maternal sera, but there was a trend to wide individual variation towards enhanced angiogenesis, particularly in the early-onset PE group. CONCLUSIONS: At therapeutic concentration, pravastatin alone or with maternal sera has no significant effect on angiogenesis, but at high concentrations the effect seems to be anti-angiogenic estimated by in vitro assay.

Association between [68Ga]NODAGA-RGDyK uptake and dynamics of angiogenesis in a human cell-based 3D model.

Radiolabeled RGD peptides targeting expression of αvß3 integrin have been applied to in vivo imaging of angiogenesis. However, there is a need for more information on the quantitative relationships between RGD peptide uptake and the dynamics of angiogenesis. In this study, we sought to measure the binding of [68Ga]NODAGA-RGDyK to αvß3 integrin in a human cell-based three-dimensional (3D) in vitro model of angiogenesis, and to compare the level of binding with the amount of angiogenesis. Experiments were conducted using a human cell-based 3D model of angiogenesis consisting of co-culture of human adipose stem cells (hASCs) and of human umbilical vein endothelial cells (HUVECs). Angiogenesis was induced with four concentrations (25%, 50%, 75%, and 100%) of growth factor cocktail resulting in a gradual increase in the density of the tubule network. Cultures were incubated with [68Ga]NODAGA-RGDyK for 90 min at 37 °C, and binding of radioactivity was measured by gamma counting and digital autoradiography. The results revealed that tracer binding increased gradually with neovasculature density. In comparison with vessels induced with a growth factor concentration of 25%, the uptake of [68Ga]NODAGA-RGDyK was higher at concentrations of 75% and 100%, and correlated with the amount of neovasculature, as determined by visual evaluation of histological staining. Uptake of [68Ga]NODAGA-RGDyK closely reflected the amount of angiogenesis in an in vitro 3D model of angiogenesis. These results support further evaluation of RGD-based approaches for targeted imaging of angiogenesis.

Qualitative and Quantitative Analysis of Certain Aspects of the Cytotoxic and Genotoxic Hazard of Hospital Wastewaters by Using a Range of In Vitro Assays.

Health care facilities and hospitals generate significant amounts of wastewater which are released into the sewage system, either after a preliminary treatment or without any further treatment. Hospital wastewater may contain large amounts of hazardous chemicals and pharmaceuticals, some of which cannot be eliminated entirely by wastewater treatment plants. Moreover, hospital effluents may be loaded with a plethora of pathogenic microorganisms or other microbiota and microbiome residues. The need to monitor hospital effluents for their genotoxic hazard is of high importance, as detailed information is scarce. DNA-based information can be acquired directly from samples through the application of various molecular methods, while cell-based biomonitoring assays can provide important information about impaired cellular pathways or mechanisms of toxicity without prior knowledge of the identity of each toxicant. In our study, we evaluated samples of chlorinated hospital wastewater discharged into the sewage system after this disinfection process. The assessment of cytotoxicity, genotoxicity and mutagenicity of the hospital effluents was performed in vitro by using a broad battery of biomonitoring assays that are relevant for human health effects. All the tested hospital wastewater samples could be classified as potentially genotoxic, and it is concluded that the microbiota present in hospital wastewater might contribute to this genotoxic potential.

Toxicological testing of a photoactive phthalocyanine-based antimicrobial substance.

The aim of the study was toxicological testing of an innovative and efficient antimicrobial agent based on photoactive phthalocyanine (Pc) derivative. A promising Aluminium phthalocyanine (AlPc) with efficient and stable antimicrobial effects was subjected to a battery of toxicological tests to avoid local and systemic toxicity hazard. In compliance with the current European legislation restricting the use of experimental animals, the methods comprised exclusively in vitro procedures based on cellular and tissue models of human origin or mimicking human tissues. The battery of toxicological tests to identify local toxicity included skin corrosion/irritation, eye irritation, and phototoxicity. The basic systemic toxicity tests included acute toxicity, skin sensitization, genotoxicity, and endocrine disruption. The results showed that AlPc induced skin and eye irritation, exhibited borderline sensitization potential and mutagenic potential in one test strain of the Ames test, which was not confirmed in the chromosome aberration test. The AlPc was found to be phototoxic. The results from the cytotoxicity test designed for acute oral toxicity estimation were not conclusive, the acute toxicity potential has to be determined by conventional tests in vivo. Regarding endocrine disruption, no agonistic activity of the AlPc on human estrogen receptor α, nor human androgen receptor was observed. The skin penetration/absorption test revealed that the AlPc has not penetrated into the dermis and receptor fluid, confirming no risk of systemic exposure via the bloodstream.

In Vitro Vascular Network Modified to Function as Culture Platform and Angiogenic Induction Potential Test for Cancer Cells.

Drug treatments have been designed to inhibit tumor angiogenesis in hope of stopping tumor growth. However, not all tumor types respond to this type of treatment. A screening method which identifies angiogenesis inducing cancer types would help predict the efficacy of angiogenesis-inhibiting drugs for the patients. Our goal is to develop (1) a cell assay to assess the angiogenic induction potential of patient-derived tumor cells, and (2) a protocol for culturing cancer cells on a vascular platform. We optimized the media composition and seeding density of cells (hASC, HUVEC, and cancer cells) to 48-, 96-, and even 384-well plate sizes to allow vascular formation and cancer cell proliferation and subsequent analysis with high throughput. The angiogenic induction potential of patient-derived cancer cells was investigated by quantifying the formation of tubular structures and the drug response of cancer cells grown on a vascular platform was evaluated using gene expression and cell viability (WST-1) assay. Immunocytochemistry was performed with von Willebrand factor, collagen IV, CD44, cytokeratin 19 and ALDH1A1. The angiogenic induction potential test was shown to be responsive to the induction of angiogenesis by cancer cells. The responses of cancer cells were different when grown on a vascular platform or on plastic, seen in gene expression level and viability results. These two protocols are promising novel tools for aiding the selection of efficient cancer drugs for personalized medicine and as an alternative cancer cell culture platform.

Angiogenic capacity in pre-eclampsia and uncomplicated pregnancy estimated by assay of angiogenic proteins and an in vitro vasculogenesis/angiogenesis test.

OBJECTIVE: The purpose of the study was to determine the angiogenic capacity of sera in early and late pregnancy and in umbilical blood serum after childbirth, and to define how angiogenic properties assessed in a functional in vitro test are related to individual angiogenic proteins in six women with pre-eclampsia and in six healthy pregnant controls. METHODS: Maternal first and third trimester serum samples, and umbilical blood samples after childbirth, were tested in an in vitro human adipose stromal cell-human umbilical vein endothelial cell (hASC-HUVEC) vasculogenesis/angiogenesis assay. The angiogenic properties of the samples were measured by quantifying tubule formation. Concentrations of total placental growth factor (PlGF), total vascular endothelial growth factor (VEGF), soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sEng) were determined by immunoassay. RESULTS: First-trimester maternal sera of both groups had a stimulatory effect on angiogenesis in vitro and levels of angiogenic proteins did not differ between the groups. Third-trimester maternal sera in the pre-eclampsia group had an inhibitory effect on tubule formation, while those from normal pregnancies remained stimulatory. Compared with the first trimester there was a significant change in the concentrations of angiogenic proteins toward an anti-angiogenic state in pre-eclampsia. Umbilical blood serum exhibited strong anti-angiogenic effects without a significant difference between groups. CONCLUSIONS: Third-trimester serum of pre-eclamptic patients is anti-angiogenic. This phenomenon is not yet present in the first trimester. Umbilical blood serum shows inhibitory effects on angiogenesis after normal as well as pre-eclamptic pregnancy.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood.

BACKGROUND AIMS: Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. METHODS: CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. RESULTS: We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor-rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor-rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. CONCLUSIONS: Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Better science with human cell-based organ and tissue models.

At present, animal-based models are the major test systems for assessing the tolerability and safety of chemical substances for regulatory purposes, and also for pivotal efficacy testing in pharmaceutical development. In spite of the high genetic similarity between many laboratory animals and humans, animal models are very poor predictors of human health effects and pathophysiological processes. Thus, models and testing strategies that are more relevant to human biology, are needed for these purposes. The best predictability is achieved with human organotypic models that mimic the microenvironment of human tissues. During their development, such models have to be characterised at the structural, genetic and functional levels, and compared to the respective human tissues. Their predictivity should be confirmed by using known reference chemicals with corresponding human data. The use of these methods in safety assessment and biomedical research, combined with the knowledge gained of the underlying biological processes on gene and protein expression, as well as on cellular signalling, will ultimately lead to better human science and animal welfare.

Toxicity of silver nanoparticle in rat ear and BALB/c 3T3 cell line.

BACKGROUND: Silver nanoparticles (AgNPs) displayed strong activities in anti-bacterial, anti-viral, and anti-fungal studies and was reportedly efficient in treating otitis media .The potential impact of AgNPs on the inner ear was missing. OBJECTIVE: Attempted to evaluate the potential toxicity of AgNPs in the inner ear, middle ear, and external ear canal after transtympanic injection in rats. RESULTS: In in vitro studies, the IC50 for AgNPs in neutral red uptake assay was lower than that in NAD(P)H-dependent cellular oxidoreductase enzyme assay (WST-1) and higher than that in total cellular ATP and nuclear membrane integrity (propidium iodide) assessments. In in vivo experiments, magnetic resonance imaging (MRI) showed that significant changes in the permeability of biological barriers occurred in the middle ear mucosa, the skin of the external ear canal, and the inner ear at 5 h post-transtympanic injection of AgNPs at concentrations ranging from 20 µg/ml to 4000 µg/ml. The alterations in permeability showed a dosage-response relationship, and were reversible. The auditory brainstem response showed that 4000 µg/ml AgNPs induced hearing loss with partial recovery at 7 d, whereas 20 µg/ml caused reversible hearing loss. The functional change in auditory system was in line with the histology results. In general, the BALB/c 3T3 cell line is more than 1000 times more sensitive than the in vivo studies. Impairment of the mitochondrial function was indicated to be the mechanism of toxicity of AgNPs. CONCLUSION: These results suggest that AgNPs caused significant, dose-dependent changes in the permeability of biological barriers in the middle ear mucosa, the skin of the external ear canal, and the inner ear. In general, the BALB/c 3T3 cell line is more than 1000 times more sensitive than the in vivo studies. The rat ear model might be expended to other engineered nanomaterials in nanotoxicology study.

LASU: An efficient and stable phthalocyanine dye with tolerable safety profile for self-disinfecting anti-COVID textiles activated by ambient light.

BACKGROUND: Recent COVID crisis has demonstrated that modern society urgently needs an accessible protection against mass infections, especially viruses, as the new strains are appearing at an ever-increasing pace and cause severe harm to the population and the world economy. METHODS: We have developed an efficient phthalocyanine photosensitizer LASU, that is suitable for dyeing textiles and allows to prepare reusable self-disinfecting fabrics with strong antiviral properties. The safety profile of LASU was evaluated in accredited laboratories by several in vitro assays according to the OECD-guidelines. RESULTS: The textiles impregnated with LASU phthalocyanine showed a significant antiviral photodynamic effect even under moderate indoor and outdoor light. The dye did not show any genotoxic potential in human lymphocyte micronucleus assay. It showed a possible indication for eye irritation in human EpiOcular™ model and was phototoxic when tested in mouse BALB/c 3T3 cell test in the presence and absence of UVA-irradiation. CONCLUSION: Novel phthalocyanine-dyed textiles are suitable for general use as self-disinfecting antiviral barriers and materials in hospitals, households, and public places. The safety profile of LASU is the phototoxic effect which is related to LASU´s mode of action.

[Non-animal toxicology in the safety testing of chemicals]. / Eläinkokeeton toksikologia kemikaalien turvallisuustestauksessa.

There is an urgent need to develop predictive test methods better than animal experiments for assessing the safety of chemical substances to man. According to today's vision this is achieved by using human cell based tissue and organ models. In the new testing strategy the toxic effects are assessed by the changes in the critical parameters of the cellular biochemical routes (AOP, adverse toxic outcome pathway-principle) in the target tissues. In vitro-tests are rapid and effective, and with them automation can be applied. The change in the testing paradigm is supported by all stakeholders: scientists, regulators and people concerned on animal welfare.

In Search of Clinical Markers: Indicators of Exposure in Dampness and Mold Hypersensitivity Syndrome (DMHS).

Potential markers were sought to diagnose mold hypersensitivity. Indoor air condensed water and human macrophage THP-1 test were applied to evaluate the buildings. Basophil activation tests (BAT) were conducted and mold-specific immunoglobulins (IgE, IgG, IgA, and IgD) were measured in study subjects' serum and feces. Exposed subjects reported markedly more symptoms from occupational air than controls. Basophils from exposed subjects died/lost activity at 225 times lower concentrations of toxic extracts from the target building than recommended in the common BAT protocol. Fecal IgG and IgD levels against Acrostalagmus luteoalbus and Aspergillus versicolor produced receiver operating curves (ROC) of 0.928 and 0.916, respectively, when plotted against the inflammation marker MRP8/14. Assaying serum immunoglobulin concentrations against the toxic Chaetomium globosum (MTAV35) from another building, a test control, did not differentiate study individuals. However, if liver metabolism produced the same core molecule from other Chaetomium globosum strains, this would explain the increased response in fecal immunoglobulins in the exposed. The altered immunoglobulin values in the samples of exposed when compared to controls revealed the route of mold exposure. The toxicity of indoor air condensed water samples, BAT and serology confirmed the severity of symptoms in the target building's employees, supporting earlier findings of toxicity in this building.

Adipose stromal cell tubule network model provides a versatile tool for vascular research and tissue engineering.

The current limitation in designing three-dimensional tissue models is the lack of adequate vascularization with mature and stable vessels. Adipose tissue is known to secrete several angiogenic factors, and human adipose stromal cells (hASC) are known to promote vessel growth, maturation and stabilization. In this study, hASC were induced to angiogenesis with growth factor-enriched medium either in monoculture or in coculture with human umbilical vein endothelial cells (HUVEC) and analyzed for vascular, pericytic and smooth muscle cell markers. hASC and HUVEC cocultures showed an accelerated proliferation rate and the cells self-assembled, independent of the cell passage number, into multilayered three-dimensional tubular networks. The networks of hASC and HUVEC expressed endothelial markers, a complete basement membrane and vessel-supporting cells with contractile properties. A hASC and green fluorescence protein-HUVEC-infection model revealed that cocultures consisted of a mosaic of von Willebrand factor-positive cells derived from both cell populations - hASC and HUVEC. hASC monoculture had passage- and donor-dependent ability to form tubular networks, with half of the cultures presenting tubule structures and basement membrane formation. Pericytic and smooth muscle cell markers were expressed in hASC monoculture even when tubules were absent. By combining the potential properties of hASC and features from the present angiogenesis assays, we generated a natural-like, xeno-free, prevascular-like network in vitro model with excellent reproducibility and minimal limitations in technical performance. This tubular network model is an excellent tool for studying cell interactions during vascular development, for chemical and drug testing and for developing natural-like, multilayered, vascularized, scaffold-free tissue models.

Direct Contraction Force Measurements of Engineered Cardiac Tissue Constructs With Inotropic Drug Exposure.

Contractility is one of the most crucial functions of the heart because it is directly related to the maintenance of blood perfusion throughout the body. Both increase and decrease in contractility may cause fatal consequences. Therefore, drug discovery would benefit greatly from reliable testing of candidate molecule effects on contractility capacity. In this study, we further developed a dual-axis piezoelectric force sensor together with our human cell-based vascularized cardiac tissue constructs for cardiac contraction force measurements. The capability to detect drug-induced inotropic effects was tested with a set of known positive and negative inotropic compounds of isoprenaline, milrinone, omecamtiv mecarbil, propranolol, or verapamil in different concentrations. Both positive and negative inotropic effects were measurable, showing that our cardiac contraction force measurement system including a piezoelectric cantilever sensor and a human cell-based cardiac tissue constructs has the potential to be used for testing of inotropic drug effects.

Toxic Indoor Air Is a Potential Risk of Causing Immuno Suppression and Morbidity-A Pilot Study.

We aimed to establish an etiology-based connection between the symptoms experienced by the occupants of a workplace and the presence in the building of toxic dampness microbiota. The occupants (5/6) underwent a medical examination and urine samples (2/6) were analyzed by LC-MS/MS for mycotoxins at two time-points. The magnitude of inhaled water was estimated. Building-derived bacteria and fungi were identified and assessed for toxicity. Separate cytotoxicity tests using human THP-1 macrophages were performed from the office's indoor air water condensates. Office-derived indoor water samples (n = 4/4) were toxic to human THP-1 macrophages. Penicillium, Acremonium sensu lato, Aspergillus ochraceus group and Aspergillus section Aspergillus grew from the building material samples. These colonies were toxic in boar sperm tests (n = 11/32); four were toxic to BHK-21 cells. Mycophenolic acid, which is a potential immunosuppressant, was detected in the initial and follow-up urine samples of (2/2) office workers who did not take immunosuppressive drugs. Their urinary mycotoxin profiles differed from household and unrelated controls. Our study suggests that the presence of mycotoxins in indoor air is linked to the morbidity of the occupants. The cytotoxicity test of the indoor air condensate is a promising tool for risk assessment in moisture-damaged buildings.

Functional human cell-based vascularised cardiac tissue model for biomedical research and testing.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) are widely used in in vitro biomedical research and testing. However, fully matured, adult cardiomyocyte characteristics have not been achieved. To improve the maturity and physiological relevance of hiPSC-derived cardiomyocytes, we co-cultured them with preconstructed vascular-like networks to form a functional, human cell-based cardiac tissue model. The morphology and gene expression profiles indicated advanced maturation in the cardiac tissue model compared to those of a cardiomyocyte monoculture. The cardiac tissue model's functionality was confirmed by measuring the effects of 32 compounds with multielectrode array and comparing results to human data. Our model predicted the cardiac effects with a predictive accuracy of 91%, sensitivity of 90% and specificity of 100%. The correlation between the effective concentration (EC50) and the reported clinical plasma concentrations was 0.952 (R2 = 0.905). The developed advanced human cell-based cardiac tissue model showed characteristics and functionality of human cardiac tissue enabling accurate transferability of gained in vitro data to human settings. The model is standardized and thus, it would be highly useful in biomedical research and cardiotoxicity testing.

New approach methods for assessing indoor air toxicity.

Indoor air is typically a mixture of many chemicals at low concentrations without any adverse health effects alone, but in mixtures they may cause toxicity and risks to human health. The aim of this study was by using new approach methods to assess the potential toxicity of indoor air condensates. In specific, different in vitro test methods including cyto-and immunotoxicity, skin sensitization and endocrine disruption were applied. In addition to biological effects, the indoor air samples were subjected to targeted analysis of 25 volatile organic compounds (VOCs) and Genapol X-80 (a nonionic emulsifier) suspected to be present in the samples, and to a non-targeted "total chemical scan" to find out whether the chemical composition of the samples is associated with the biological effects. The results confirm that assessing health risks of indoor air by analysing individual chemicals is not an adequate approach: We were not able to detect the VOCs and Genapol X-80 in the indoor air samples, yet, several types of toxicity, namely, cytotoxicity, immunotoxicity, skin sensitization and endocrine disruption were detected. In the non-targeted total chemical scan of the indoor air samples, a larger number of compounds were found in the cytotoxic samples than in the non-cytotoxic samples supporting the biological findings. If only one biological method would be selected for the screening of indoor air quality, THP-1 macrophage/WST-1 assay would best fit for the purpose as it is sensitive and serves as a good representative for different sub-toxic end points, including immunotoxicity, (skin) sensitization and endocrine disruption.