GHS Category Research Articles

Evaluating the QileX-RhE skin corrosion test for chemical subcategorization in accordance with OECD TG 431

Skin corrosion testing is integral to evaluating the potential harm posed by chemicals, impacting regulatory decisions on safety, transportation, and labeling. Traditional animal testing methods are giving way to in vitro alternatives, such as reconstructed human epidermis (RhE) models, aligning with evolving ethical standards. This study evaluates the QileX-RhE test system's performance for chemical subcategorization within the OECD TG 431 framework. Results demonstrate its ability to differentiate subcategories, accurately predicting 83% of UN GHS Category 1A and 73% of UN GHS Category 1B/1C chemicals with 100% sensitivity in corrosive prediction. Additionally, this study provides a comprehensive assessment of the test method's performance by employing nuanced parameters such as positive predictive value (PPV), negative predictive value (NPV), post-test odds and likelihood rations, offering valuable insights into the applicability and effectiveness of the QileX-RhE test method.

Ocular irritation reversibility assessment of a laundry detergent using the Porcine Corneal Opacity Reversibility Assay (PorCORA): a focused study

Consumer product manufacturers utilise a spectrum of alternative ocular irritation assays, as these tests do not require the use of live animals. Despite their usefulness, no regulatory-accepted assay assesses the reversibility of ocular damage, a key criterion of GHS ocular classification, like the rabbit eye test (i.e., Draize Rabbit Eye Test [DRET]) . The Porcine Corneal Opacity Reversibility Assay (PorCORA), an ex vivo intact corneal tissue culture model, predicts the reversibility of damage by ocular irritants. Inclusion of the damage reversibility endpoint in the PorCORA supplements other alternative test methods for ocular irritation, by assessing induced eye damage and the ability of this damage to reverse (heal) without the use of live animals to distinguish between Globally Harmonised System of Classification and Labelling of Chemicals (GHS) ocular classifications. In this focused study, results of a Bovine Corneal Opacity and Permeability (BCOP) test of a laundry detergent, neat and 10% dilution, (product mixture from S.C. Johnson & Son, Inc. [SCJ]) classified the product into GHS Category 1; however, the BCOP test cannot assess the reversibility of ocular damage. The laundry detergent was evaluated using the PorCORA, where ocular damage induced by the detergent was fully reversed within seven days. Evaluation of the reversibility of ocular damage using the PorCORA in this focused study can add strength to the weight-of-evidence (WoE) analysis approach in ocular hazard assessment. This WoE approach strengthens the argument that the PorCORA can be used to supplement BCOP data, and that this laundry detergent is not an irreversible eye irritant.

Assessment of Biological Activities, Acute and Sub-chronic Toxicity of Liang (Gnetum gnemon var. tenerum) Leaves Powder, a Natural Product.

Gnetum gnemon var. tenerum (Gnetaceae) is a shrub plant native to South-East Asia. In Thailand, Liang leaves are commonly consumed in South of Thailand as vegetable. According to literature, they have an antihyperglycemic capacity because of their rich chlorophyll, fiber, and protein. However, there is need to assess the safety since natural food products are not completely devoid of toxicity. This study aimed to assess the biological activities as well as the acute and sub-chronic oral toxicity of Liang leaves powder (LLP). The evaluation of LLP for acute oral toxicity was performed at dose level 2000 mg/kg body weight in Wistar rats while the sub-chronic oral toxicity of LLP was performed at the effective dose (1.47 g/kg) for antihyperglycemic property according to Organisation for Economic Co-operation and Development (OECD)-425. The results showed that LLP demonstrated anti-inflammatory activities. It also showed no clinical signs of toxic effects and mortality in rats throughout 90 d. Thus, LLP could be classified in GHS category 5 which are of relatively low acute toxicity and then the lethal dose, 50% (LD50) cut off at 5000 mg/kg body weight to infinity (∞). Administration of LLP to the experimental rats significantly increased (p < 0.05) the concentration of triglyceride and increased concentration of creatinine as a result of kidney malfunction was also noticed in the experimental rats. Hematological alteration was not noticed in the treated female rats, but red blood cell, hemoglobin and hematocrit concentrations significantly increased in the treated male rats. The study concludes that sub-chronic administration of 1.47 g/kg LLP is relatively safe.

Further optimisation of a macromolecular ocular irritation test (OptiSafeTM)

Purpose OptiSafeTM (OS) is a shelf stable, nonanimal test for ocular irritation. A recent database search found that half of the OS false positive (FP) materials were associated with reactive oxygen chemistries but were not eye irritants in vivo (based on historical rabbit studies by other groups). We hypothesized that naturally occurring tear antioxidants protect the eye from reactive chemistries in vivo and that specific tear chemistries might help explain why some materials are FP for nonanimal tests but are reported as nonirritants in the live animal. To test this hypothesis, a prior study evaluated tear antioxidants and found that the tear antioxidant ascorbic acid, added at human physiological tear levels to the OS test, specifically reduced the measured values for these FPs but did not reduce the true-positive rate. Based on these findings, the OS test method was further optimized. The purpose of the current study was to comprehensively evaluate the performance of the further optimized test method for detection of ocular irritants. Materials and methods The OS test measures chemically induced damage to macromolecules and relates these measured values to ocular irritancy. To improve the performance of OS, we made updates to the material to be tested physiochemical handling procedures, prediction model, and test method to include tear-level concentrations of ascorbic acid. We then retested the 78 chemicals from the prior OS-coded validation study in triplicate and compared the accuracy of the ‘nonirritant versus irritant’ prediction for the further optimized method with the prior results. Results We report that for the detection of ‘nonirritant’ versus ‘irritant’ (GHS NC versus categories 2B/A and 1) test substances, the further optimized OS test with ascorbic acid compared with the original version has a FP rate that is reduced from 40.0 to 22.2%, the false-negative (FN) rate remains at 0.0%, and the accuracy improved from 80.3% to 89.2%. Conclusion A comparison to OECD-adopted tests demonstrates that the further optimized OS test, like the original method, has a higher accuracy and lower FN rate for the detection of ‘nonirritants’ versus ‘irritants’ (GHS Category NC versus 2B/A and 1) than the other eye irritation tests (BCOP, EpiOcularTM Eye Irritation Test, ICE, Ocular Irritection®, and STE).

Expansion of the application domain of a macromolecular ocular irritation test (OptiSafe™)

The OptiSafe (OS) test is shelf-stable, macromolecular eye irritation test that does not include any animal ingredient or component (“vegan”). The purpose of this study was to evaluate the test's accuracy for an expanded application domain for both the original and recently updated OS method. This study involved the testing of additional ocular corrosives and previously excluded foaming agents (“surfactants”) using both the original and updated OS methods and then combining these data with prior validation data for a total of 147 chemicals. Predictivity was evaluated by a statistical comparison of the OptiSafe predictions with historical in vivo “Draize” rabbit eye data for the same chemicals (from public databases). We report that for the detection of chemicals not requiring classification for eye irritation [Globally Harmonized System of Classification and Labeling of Chemicals (GHS) No Category], the accuracy, specificity, and sensitivity were 92.8%, 79.6%, and 100.0%, respectively, for the updated method; for the detection of chemicals inducing extreme eye damage/corrosion (GHS Category 1), the accuracy, specificity, and sensitivity were 79.4%, 71.8%, and 91.7%, respectively, for the updated method. Results indicate that both the original and updated methods have a high accuracy for the expanded application domain that included ocular corrosives and surfactants.

Acceptable surface limits (ASLs) of skin sensitizers derived from the local lymph node assay (LLNA): BrdU-ELISA EC1.6 values and their relationships to known sensitization potency information.

Skin sensitization is an extremely important risk factor for occupational health and safety, and it would be desirable to set health-based exposure limits (HBELs) for the quantitative risk assessment (QRA) based on the skin sensitizing potencies of chemical. We attempted to set acceptable surface limits (ASLs) as HBELs for skin sensitizers in the workplace based on the local lymph node assay (LLNA): BrdU-ELISA EC1.6 values. To calculate the ASLs, a safety assessment factor (SAF)interspecies value of 6, based on the EC1.6 values/human repeat insult patch test (HRIPT) NOEL ratios, a SAFinterindividual value of 10, and a SAFfrequency/duration value of 3 were applied, referring to previous literatures on SAFs for skin sensitization QRA, and the composite SAF was calculated as 180. The ASLs (mg/100 cm2 ) derived thus for 33 chemicals ranged from 0.001 to 10.417. Comparison of the ranges with known human sensitization potency classes and GHS subcategories revealed that use of GHS Category 1A chemicals needs to be controlled to ensure surface residue levels of less than 1mg/100 cm2 . To minimize sensitization risks, a quantitative sensitization risk assessment method for chemicals and appropriate risk management are necessary. This report provides a potentially useful ASL-based method of managing sensitization risk derived from LLNA: BrdU-ELISA EC1.6 values, comparison of the ASLs and known human sensitization potency data showed that GHS subcategorization results would be a primary information notifying ASL ranges to be required for minimizing the sensitization risk.

ISO 10993-23 In vitro irritation testing for medical devices: Substantiating applicability to mild irritants and non-extractables

Irritation testing is an integral part of the biocompatibility assessment of medical devices and has historically been conducted on animals, either by direct contact or with polar and non-polar solvent extracts. In 2018 an ISO-sponsored interlaboratory validation study demonstrated that two reconstituted human epidermis (RhE) based assays, which were adapted from validated methods used for industrial chemicals, produced results essentially equivalent to those obtained with in vivo tests. This led to the publication of the ISO 10993-23:2021 standard on irritation testing, which states that RhE-based assays are now the preferred method. The 2018 validation study evaluated strong irritants, so we tested nine mild irritants (GHS Category 3), neat and spiked at different concentrations into medical device extracts, per ISO 10993-23:2021. The results substantiated the applicability of RhE-based assays for evaluating mild irritants in medical device extracts. Moreover, the 2018 validation study tested solid extractable medical device materials but did not consider non-extractable medical device materials (e.g., creams, gels, or sprays). By testing nine marketed non-extractable materials, either neat or spiked with irritants, we also confirmed that RhE-based assays are readily applicable to such medical device materials.

Evaluation of potential eye or skin irritation/corrosion in rabbit exposed to TiO2 photocatalyst (GST).

TiO2 NPs photocatalyst is widely used in a variety of applications and products in the environmental and energy fields, including self-cleaning surfaces, air and water purification systems, sterilization, hydrogen evolution, and photoelectrochemical conversion. The possible biological and safety effects of TiO2 dermal exposure and absorption have not been well studied and more investigations on the potential health hazards of the TiO2 are needed. This study aimed to investigate potential effect of local lesions (eye and skin irritation/corrosion) for new TiO2 material powder, GST produced through sludge recycling of the sewage treatment plant in according to the OECD test guideline (TG 404, 405) and imaging evaluation (micro-computed tomography analysis), histopathology examination. Also, P-25, commercial photocatalyst was used to compare with GST. For the eye or skin irritation/corrosion test, the test substances (GST, P-25) showed no irritation/corrosion for local lesions and the GHS category was identified as a “No hazard class”. The imaging analysis indicated that GST did not penetrate or distribute in the local lesions (eye, skin) and the treatment-related effect was not observed in histopathology. Therefore, the present study revealed that new TiO2 powder, GST was considered to be no potential effects (irritation/corrosion), penetration or distribution in the local lesions (eye, skin).

In vitro acute inhalation toxicity for TiO2 (GST) using 3D human tissue model (EpiAirwayTM).

The present study was performed to screen in vitro potential acute inhalation toxicity using an EpiAirway™ tissue model (human tracheal/bronchial tissue) for the nano-sized titanium dioxide, GST manufactured as a photocatalyst through of sludge recycling and to compare with P-25 a commercialized photocatalytic material. According to the protocol provided by in vitro tissue manufacturer, the GST was exposure to the tissue for 3 hours in 450, 500, 650, 850 mg/mL concentration after preliminary dose range finding study and then tissue viability (%, IC75) was calculated using the MTT assay. Besides, the histopathological observation was performed to compare to the MTT assay. As a result of study, IC75 could not be confirmed at 850 mg/mL in both GST and P-25 and the grade was confirmed to be IC75> 600 mg/mL in vitro model tissue category. Therefore, it was considered that the GHS category could be classified as ‘No classification’ in screening method for potential acute inhalation toxicity. Also, not the morphological effects of epithelial cells in tissue model were observed compared with the vehicle control and histological findings were similar to the results of MTT Viability assay. Based on these results, the potential acute inhalation toxicity for GST produced through sludge recycling using in vitro tissue model inhalation toxicity showed that it could be non-hazardous substance. However, further study (in vivo study, etc.) is thought to be needed to ascertain whether GST is a toxic effect or safe.

Validation study of a new reconstructed human epidermis model EPiTRI for in vitro skin irritation test according to OECD guidelines

Following the global trend of reducing animal testing, various reconstructed human epidermis (RHE) models for skin irritation test (SIT) have been developed, verified, validated and included in OECD TG 439. We developed a new RHE called EPiTRI and a SIT method using EPiTRI (EPiTRI-SIT model) following the OECD guidelines. EPiTRI possesses morphological, biochemical and physiological properties similar to human epidermis with well-differentiated multilayered viable cells with barrier function. The EPiTRI-SIT model was tested for 20 reference chemicals in Performance Standard of OECD TG 439 (GD 220), showing good predictive capacity with 100% sensitivity, 70% specificity and 85% accuracy. EPiTRI had sensitivity in detecting di-n-propyl disulphate, as an irritant chemical (UN GHS Category 2), whereas most validated reference methods detected it as a non-irritant. An international validation study of EPiTRI-SIT was conducted in four laboratories to confirm the within- and between-laboratory reproducibility, as well as predictive capacity. The phase I/II within-laboratory and between-laboratory reproducibility was 100%/95% and 95%, respectively. The overall sensitivity, specificity and accuracy of EPiTRI-SIT was 96%, 70% and 83%, respectively, which fulfilled the OECD criteria. Thus, EPiTRI, meets the criteria of Performance Standards of OECD TG 439 (GD 220) and is suitable for screening irritating chemicals in vitro.

Aqueous extract from Chrysophyllum cainito bark exhibits embryonic toxicity in Danio rerio and negligible acute toxicity in adult wistar rats

Chrysophyllum cainito has been used as a traditional medicine to treat a wide range of diseases, but the toxicity profile of this plant remains unknown. This study aimed to evaluate the acute toxicity of the aqueous extract of C. cainito (CE) bark based on OECD guidelines in two different in vivo experimental models: acute single-dose oral toxicity in adult Wistar rats and the zebrafish embryo acute toxicity test. All concentrations of CE (500-4000 mg/kg) tested during a 14-day period in both male and female rats showed no effect on behavior, body weight, organ weights, biochemical and hematological parameters. In contrast, CE significantly delayed zebrafish embryo hatching and decreased embryo survival rates in a dose-dependent manner. Hatched larvae were notably sensitive to CE-induced toxicity compared to unhatched fish embryos. Acridine orange staining showed that CE induced apoptosis in the yolk sac region that is responsible for supplying nutrients to support larval growth and development. According to OECD guidelines, CE was identified as GHS category 5, a substance with low to no acute toxicity. However, as embryotoxicity was observed in zebrafish, CE use during pregnancy should be exercised with caution until further examination of its safety.

In Silico Assessment of Acute Oral Toxicity for Mixtures

While exposure of humans to environmental hazards often occurs with complex chemical mixtures, the majority of existing toxicity data are for single compounds. The Globally Harmonized System of chemical classification (GHS) developed by the Organization for Economic Cooperation and Development uses the additivity formula for acute oral toxicity classification of mixtures, which is based on the acute toxicity estimate of individual ingredients. We evaluated the prediction of GHS category classifications for mixtures using toxicological data collected in the Integrated Chemical Environment (ICE) developed by the National Toxicology Program (United States Department of Health and Human Services). The ICE database contains in vivo acute oral toxicity data for ∼10,000 chemicals and for 582 mixtures with one or multiple active ingredients. By using the available experimental data for individual ingredients, we were able to calculate a GHS category for only half of the mixtures. To expand a set of components with acute oral toxicity data, we used the Collaborative Acute Toxicity Modeling Suite (CATMoS) implemented in the Open Structure-Activity/Property Relationship App to make predictions for active ingredients without available experimental data. As a result, we were able to make predictions for 503 mixtures/formulations with 72% accuracy for the GHS classification. For 186 mixtures with two or more active ingredients, the accuracy rate was 76%. The structure-based analysis of the misclassified mixtures did not reveal any specific structural features associated with the mispredictions. Our results demonstrate that CATMoS together with an additivity formula can be used to predict the GHS category for chemical mixtures.

Anti-inflammatory and antinociceptive activity profile of a new lead compound – LQFM219

LQFM219 is a molecule designed from celecoxibe (COX-2 inhibitor) and darbufelone (inhibitor of COX-2 and 5-LOX) lead compounds through a molecular hybridisation strategy. Therefore, this work aimed to investigate the antinociceptive and anti-inflammatory activities of this new hybrid compound. The acute oral systemic toxicity of LQFM219 was evaluated via the neutral red uptake assay. Acetic acid-induced abdominal writhing and CFA-induced mechanical hyperalgesia were performed to evaluate the antinociceptive activity, and the anti-oedematogenic activity was studied by CFA-induced paw oedema and croton oil-induced ear oedema. Moreover, the acute anti-inflammatory activity was determined by carrageenan-induced pleurisy. In addition, cell migration, myeloperoxidase enzyme activity, and TNF-α and IL-1β levels were determined in pleural exudate. Moreover, a redox assay was conducted using electroanalytical and DPPH methods. The results demonstrated that LQFM219 was classified as GHS category 4, and it showed better free radical scavenger activity compared to BHT. Besides, LQFM219 decreased the number of writhings induced by acetic acid and the response to the mechanical stimulus in the CFA-induced mechanical hyperalgesia test. Furthermore, LQFM219 reduced oedema formation, cell migration, and IL-1β and TNF-α levels in the pleural cavity and inhibited myeloperoxidase enzyme activity. Thus, our study provides that the new pyrazole derivative, LQFM219, demonstrated low toxicity, antinociceptive and anti-inflammatory potential in vitro and in vivo.

Antiangiogenic and antitumoral activity of LQFM126 prototype against B16F10 melanoma cells

Inhibition of mouse double minute 2 homolog (MDM2)-p53 interaction and reactivation of p53 signaling have been explored as effective anticancer therapeutic strategy. The potent and specific antitumor activity shown by Nutlins, first class of MDM2-p53 inhibitors discovered, has made these compounds potential antitumor candidates. To this end, we synthesized Nutlin-1 and Nutlin-2 analogs through molecular simplification and selected the compound with the most efficient antitumoral activity. Cytotoxicity of Nutlin-2 analog LQFM126 on B16F10 melanoma cells induced intense cytoplasmic vacuolization, reduction of cell size, chromatin condensation, cytoplasmic degeneration and nuclear fragmentation. LQFM126 antiproliferative effects mediated cell cycle retention in G0/G1 phase and increased the levels of cell cycle regulatory proteins p21 and p27. This Nutlin analog increased mitochondrial membrane potential, activated caspase-8, -9 and -3/7 and reduced VEGF levels in B16F10 cells. Therefore, LQFM126 promoted alterations suggestive of apoptosis, G0/G1 cell cycle arrest and suppression of angiogenesis through modulation of VEGF expression in B16F10 cells. Additionally, LQFM126 was classified as UN GHS category 4 (LD50 > 300–2000 mg/kg), suggesting it has low acute systemic toxicity. LQFM126 can be a promising prototype for anticancer therapy.

Toxic assessment, growth disrupting and neurotoxic effects of red seaweeds' botanicals against the dengue vector mosquito Aedes aegypti L.

Application of synthetic pesticides over decades to control insects, pests, and disease vectors has resulted in negative impacts on environment and health. The current study assessed the toxicological effects of 12 botanicals obtained from 4 different red seaweeds against the dengue vector mosquito Aedes aegypti L. (Diptera: Culicidae). Four species of red seaweeds, namely Laurencia karachiana, Gracilaria foliifera, Jania rubens, Asparagopsis taxiformis, were collected from Karachi coast and extracted with hexane, dichloromethane and methanol. The efficiency of these extracts was determined by using a dose-response bioassay method against 4th instar larvae of Ae. aegypti. Separate investigations on the toxicity and IGI effects were done. Comparative studies showed that the hexane extracts induced more toxic effects. Based on the LC50 values, obtained after 24h of treatments, hexane extract of J. rubens (HJ) exhibited toxic effects with LC50 32μg/mL, (equivalent to GHS category 3), followed by G. foliifera (HG) (LC50 76.8μg/mL). HJ also showed prominent neurotoxic effects within 1-6h. Comparatively, higher morphological abnormalities and growth inhibiting (IGI) effects were obtained in the dichloromethane and methanol extracts treated larvae, after 48-96h, resulting in the formation of immature life forms such as larvi-pupae and pupi-adult. Presumptive growth inhibiting effects were also noted. These included formation of albino and black pupae, deformities in the internal structure of the treated larvae and the chitin synthesis related effects such as 'inhibiting effect on adult emergence'. Finding revealed that red seaweeds, harvested from the Arabian Sea, have potentials to affect Ae. aegypti survival and thus can be utilized as green pesticides.

Laser Light-Based Opacitometer ‘Peira LLBO 180’: A new and validated opacitometer for use in the Bovine Corneal Opacity and Permeability (BCOP) eye irritation test method

The “Peira LLBO 180” is a Laser Light-Based Opacitometer that can be used as an alternative for the standard OP-KIT device in the Bovine Corneal Opacity and Permeability (BCOP) test Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 437 to identify chemicals inducing serious eye damage as defined by United Nations Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), i.e. chemicals to be classified as UN GHS Category 1 and chemicals not requiring classification for eye irritation or serious eye damage under the UN GHS classification system (No Category).• The Peira LLBO 180 offers the advantage of analysing the complete corneal surface and is therefore able to detect more efficiently opaque spots located around the periphery of the excised corneas.• This new device will allow not only a more accurate definition of the eye irritating potential of compounds, but also a more precise ranking of moderate to mild and non-irritating compounds.• The value of Peira LLBO 180 is confirmed during in-house and multi-laboratory evaluation studies and is now included in the updated OECD TG 437, dated 26th of June 2020.The results demonstrate that the presented methodology is an improved new approach methodology (NAM) for ocular irritation testing of liquids and solids.

Dose-dependent cell necrosis induced by silica nanoparticles

In recent years, much attention has been given to nanoparticles (NPs) due to their many possible applications, and as research has progressed, these NPs have become valuable tools for medical purposes. Among many different types of NPs, silica nanoparticles (SiO2NPs) have been specifically evaluated for medical purposes and have also been used in many different types of products. Although SiO2NPs have already been applied and are believed to be nontoxic, there is still a concern regarding possible adverse effects that may be triggered after SiO2NP exposure. Therefore, in the present study, we employed a recommended cell line (BALB/c 3T3) for the toxicity evaluation to investigate the cytotoxic effects of SiO2NPs produced by chemical synthesis at a laboratory scale. First, we employed OECD guideline 129 in order to evaluate cytotoxicity effects and also estimate the starting doses for acute oral systemic toxicity tests. We evaluated the cytotoxic effects of two types of SiO2NPs (nonfluorescent and fluorescent) and found that they were not significantly different (IC50 = 1986.39 ± 237 μg/mL and IC50 = 1861.13 ± 186.72 μg/mL, respectively). Then, we used the predicted LD50 of both types of SiO2NPs to suggest that they could be categorized as GHS category 4 substances. By ultrastructural evaluation, we found that SiO2NPs are internalized by 3 T3 cells and are located in vacuole-like structures with no other significant changes in cell structure. We also found that SiO2NPs lead to cell necrosis in a dose-dependent manner.

Mechanistic-based non-animal assessment of eye toxicity: Inflammatory profile of human keratinocytes cells after exposure to eye damage/irritant agents

Eye toxicity is a mandatory parameter in human risk and safety evaluation for products including chemicals, pesticides, medicines and cosmetics. Historically, this endpoint has been evaluated using the Draize rabbit eye test, an in vivo model that was never formally validated. Due to advances in scientific knowledge, economic and ethical issues, non-animal methods based on mechanisms of toxicity are being developed and validated for increasing the capability of these models to predict eye toxicity. In this study, the Cytometric Bead Array (CBA) and ELISA assays were used to evaluate the inflammatory cytokine profile produced by HaCaT human keratinocytes after exposure to chemicals with different UN GHS eye toxicity classifications, aiming to stablish a correlation between inflammatory endpoints and eye toxicity (damage/irritation) potential. As a first step, cytotoxic profile of the chemicals, including 3 non-irritants and 10 eye toxicants (GHS Category 1, 2A and 2B), was evaluated after 24 h exposure using MTT assay and Inhibitory Concentration of 20% of cell viability (IC20) was calculated for each chemical. Then, the cells were exposed to these chemicals at IC20 for 24 h and supernatants and cell lysates were analyzed by CBA assay for quantification of the following cytokines: IL-6, IL-8, IL-10, IL-1β, TNF and IL-12p70. Regarding cytotoxicity evaluation, chemicals showed different cytotoxicity profiles and data demonstrated no correlation with their UN GHS classification. Among the cytokines evaluated, IL-1β production has changed after exposure and such alterations were confirmed by quantification employing ELISA method. The higher intracellular levels of IL-1β were found in GHS Category 1 chemicals, followed by Category 2A and 2B, while non irritants did not induce such increase. Thus, these findings show that IL-1β measurement, using HaCaT model, can be a considerable biomarker to identify chemicals according to their potential in promote eye toxicity, differentiating damage from irritation potential.

4-Fluorobenzaldehyde limonene-based thiosemicarbazone induces apoptosis in PC-3 human prostate cancer cells

AimsThis study evaluated parameters of toxicity and antiproliferative effects of (+)-N(1)-4-fluorobenzaldehyde-N(4)-{1-methyl-1-[(1R)-4-methylcyclohexene-3-il]-ethyl}-thiossemicarbazone (4-FTSC) in PC-3 adenocarcinoma prostate cells. Main methodsCytotoxicity of 4-FTSC in PC-3 cells was evaluated using MTT assay. Morphology examination of PC-3 cells treated with 4-FTSC was also performed as well as the cell death mechanisms induced were investigated using flow cytometry. Parameters of toxicity of 4-FTSC was conducted by the investigation of its potential myelotoxicity and lymphotoxicity, hemolytic activity and acute oral toxicity profile. Key findings4-FTSC showed promising cytotoxic effects against PC-3 cells (IC50 = 18.46 μM). It also triggered apoptotic morphological changes, phosphatidylserine externalization and a significant increase of DNA fragmentation in PC-3 cells. Moreover, 4-FTSC did not show changes in the PC-3 cell cycle with levels of p21, p27, NFĸB and cyclin D1 similar to those found in both control and treated cells. 4-FTSC also promoted an increase of p53 levels associated with mitochondrial impairment through loss of ∆Ψm and ROS overproduction. 4-FTSC-induced cell death mechanism in PC-3 cells involved activation of caspase-3/-7 through apoptosis intrinsic pathway via caspase-9. Regarding toxicological profile, 4-FTSC showed in vitro lymphotoxicity, although with low cytotoxicity for bone marrow progenitors and no hemolytic potential. Moreover, it was classified as GHS category 5 (LD50 > 2000–5000 mg/Kg), suggesting it has low acute oral systemic toxicity. Significance4-FTSC seems to be a promising candidate to be used as a clinical tool in prostate cancer treatment. Further studies are required to better clarify its toxicopharmacological effects found in this compound.

The novel piperazine-containing compound LQFM018: Necroptosis cell death mechanisms, dopamine D4 receptor binding and toxicological assessment

Piperazine is a promising scaffold for drug development due to its broad spectrum of biological activities. Based on this, the new piperazine-containing compound LQFM018 (2) [ethyl 4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate] was synthetized and some biological activities investigated. In this work, we described its ability to bind aminergic receptors, antiproliferative effects as well as the LQFM018 (2)-triggered cell death mechanisms, in K562 leukemic cells, by flow cytometric analyses. Furthermore, acute oral systemic toxicity and potential myelotoxicity assessments of LQFM018 (2) were carried out. LQFM018 (2) was originally obtained by molecular simplification from LASSBio579 (1), an analogue compound of clozapine, with 33% of global yield. Binding profile assay to aminergic receptors showed that LQFM018 (2) has affinity for the dopamine D4 receptor (Ki = 0.26 μM). Moreover, it showed cytotoxicity in K562 cells, in a concentration and time-dependent manner; IC50 values obtained were 399, 242 and 119 μM for trypan blue assay and 427, 259 and 50 μM for MTT method at 24, 48 or 72 h, respectively. This compound (427 μM) also promoted increase in LDH release and cell cycle arrest in G2/M phase. Furthermore, it triggered necrotic morphologies in K562 cells associated with intense cell membrane rupture as confirmed by Annexin V/propidium iodide double-staining. LQFM018 (2) also triggered mitochondrial disturb through loss of ΔΨm associated with increase of ROS production. No significant accumulation of cytosolic cytochrome c was verified in treated cells. Furthermore, it was verified an increase of expression of TNF-R1 and mRNA levels of CYLD with no involviment in caspase-3 and -8 activation and NF-κB in K562 cells. LQFM018 (2) showed in vitro myelotoxicity potential, but it was orally well tolerated and classified as UN GHS category 5 (LD50 > 2000–5000 mg/Kg). Thus, LQFM018 (2) seems to have a non-selective action considering hematopoietic cells. In conclusion, it is suggested LQFM018 (2) promotes cell death in K562 cells via necroptotic signaling, probably with involvement of dopamine D4 receptor. These findings open new perspectives in cancer therapy by use of necroptosis inducing agents as a strategy of reverse cancer cell chemoresistance.