Acute Systemic Toxicity Test Research Articles

A biocompatible silicon nitride dental implant material prepared by digital light processing technology

For decades, titanium has been the preferred material for dental implant fabrication. However, metallic ions and particles can cause hypersensitivity and aseptic loosening. The growing demand for metal-free dental restorations has also promoted the development of ceramic-based dental implants, such as silicon nitride. In this study, silicon nitride (Si3N4) dental implants were fabricated for biological engineering by photosensitive resin based digital light processing (DLP) technology, comparable to conventionally produced Si3N4 ceramics. The flexural strength was (770 ± 35) MPa by the three-point bending method, and the fracture toughness was (13.3 ± 1.1) MPa · m1/2 by the unilateral pre-cracked beam method. The elastic modulus measured by the bending method was (236 ± 10) GPa. To confirm whether the prepared Si3N4 ceramics possessed good biocompatibility, in vitro biological experiments were performed with the fibroblast cell line L-929, and preferable cell proliferation and apoptosis were observed at the initial stages. Hemolysis test, oral mucous membrane irritation test, and acute systemic toxicity test (oral route) further confirmed that the Si3N4 ceramics did not exhibit hemolysis reaction, oral mucosal stimulation, or systemic toxicity. The findings indicate that Si3N4 dental implant restorations with personalized structures prepared by DLP technology have good mechanical properties and biocompatibility, which has great application potential in the future.

Biosafety evaluation of BaSi2O2N2:Eu2+/PDMS composite elastomers.

In recent years, mechanoluminescent (ML) materials have shown great potential in stress sensing, mechanical energy collection and conversion, so they have attracted wide attention in the field of stomatology. In the early stage of this study, BaSi2O2N2:Eu2+ ML phosphors were synthesized by two-step high temperature solid state method, and then mixed with Polydimethylsiloxane (PDMS) in different proportions to obtain BaSi2O2N2:Eu2+/PDMS ML composites with different mass fractions (10%,20%,30%,40%,50%). Then its biosafety was evaluated by Cell Counting Kit-8 (CCK-8), Calcein-AM/PI fluorescence staining, hemolysis, oral mucosal irritation, acute and subacute systemic toxicity tests. The experimental results show that the biosafety of BaSi2O2N2:Eu2+/PDMS ML composite elastomers with different mass fraction is in line with the existing standards, and other related properties can be further studied.

First Food and Drug Administration Cleared Thin-Film Electrode for Intracranial Stimulation, Recording, and Monitoring of Brain Activity-Part 1: Biocompatibility Testing.

Subdural strip and grid invasive electroencephalography electrodes are routinely used for surgical evaluation of patients with drug-resistant epilepsy (DRE). Although these electrodes have been in the United States market for decades (first FDA clearance 1985), their fabrication, materials, and properties have hardly changed. Existing commercially available electrodes are made of silicone, are thick (>0.5 mm), and do not optimally conform to brain convolutions. New thin-film polyimide electrodes (0.08 mm) have been manufactured to address these issues. While different thin-film electrodes are available for research use, to date, only one electrode is cleared by Food and Drug Administration (FDA) for use in clinical practice. This study describes the biocompatibility tests that led to this clearance. Biocompatibility was tested using standard methods according to International Organization for Standardization (ISO) 10993. Electrodes and appropriate control materials were bent, folded, and placed in the appropriate extraction vehicles, or implanted. The extracts were used for in vitro and in vivo tests, to assess the effects of any potential extractable and leachable materials that may be toxic to the body. In vitro studies included cytotoxicity tested in L929 cell line, genotoxicity tested using mouse lymphoma assay (MLA) and Ames assay, and hemolysis tested in rabbit whole blood samples. The results indicated that the electrodes were non-cytotoxic, non-mutagenic, non-clastogenic, and non-hemolytic. In vivo studies included sensitization tested in guinea pigs, irritation tested in rabbits, acute systemic toxicity testing in mice, pyrogenicity tested in rabbits, and a prolonged 28-day subdural implant in sheep. The results indicated that the electrodes induced no sensitization and irritation, no weight loss, and no temperature increase. Histological examination of the sheep brain tissue showed no or minimal immune cell accumulation, necrosis, neovascularization, fibrosis, and astrocyte infiltration, with no differences from the control material. In summary, biocompatibility studies indicated that these new thin-film electrodes are appropriate for human use. As a result, the electrodes were cleared by the FDA for use in clinical practice [510(k) K192764], making it the first thin-film subdural electrode to progress from research to clinic. Its readiness as a commercial product ensures availability to all patients undergoing surgical evaluation for DRE.

Nutritional and biological value of natural-bio shoots mung bean “Mungoltin”. Food and biological values

Based on the results of our own research, examination of scientific dossier materials and reference literature data, it was established that dry powder Mungoltin made from the shoots of mung beans produced by Oriona-Scorpion LLC (Uzbekistan) contains a sufficient amount of protein, minerals, vitamins and dietary fiber, does not have a negative impact on the health status of experimental animals and does not result in functional and material cumulation. Acute systemic toxicity testing with intragastric administration of Mungoltin was carried out in 18 adult white male rats. Animals were divided into 4 groups. The animals of the first group received a nutrition dose of 5000 mg/ kg; the white rats of the second group were administered with a dose of 7500 mg/kg, and the rodents in the third group were given a dose of 10000 mg/kg, respectively. The animals in group 4 (the reference group) received distilled water. Upon a prolonged intragastric exposure to Mungoltin, no changes in biochemical parameters were detected. The activity indicators of alkaline phosphatase, trans-aminase enzymes and total protein in the blood serum did not differ significantly from those found in the reference group. Therefore, using Mungoltin will not cause a cytotoxic effect in relation to normal highly proliferating cells in an organism. The results of histomorphological studies of tissues of internal organs upon intragastric administration of Mungoltin within 30 days confirm the absence of toxic effects. According to toxicity parameters under the conditions of the above acute experiments, Mungoltin can be attributed to class 5 practically as a non-toxic substance.

Biosafety evaluation of Li2Si2O5 whisker-reinforced glass-ceramics

Lithium disilicate (Li2Si2O5) glass-ceramic is a commonly used dental ceramic material. In this study, Li2Si2O5 whiskers were prepared by the hydrothermal method, mixed with Li2Si2O5 glass powders, and Li2Si2O5 whisker-reinforced glass-ceramics were prepared by reaction sintering. The biosafety of the new Li2Si2O5 glass-ceramics were evaluated by in vitro cytotoxicity, hemolysis, oral mucosal irritation, acute systemic toxicity, and subacute systemic toxicity (oral route) tests according to ISO 7405/ISO 10993 standards. The cytotoxicity test results showed that the cell growth of the experimental group was good, and the cell number and morphology were not significantly different from those of the blank group (P> 0.05). The toxicity grading for both experimental and blank control groups were 0. The hemolysis rate of the material was 1.25%, which indicated that it did not cause hemolytic reaction. The material was non-irritating to the oral mucosa. In acute systemic toxicity test, animals in the experimental group showed increased body weight, moved freely, with no signs of poisoning. The food utilization rate and relative growth rate (change of the weight) of rats in the subacute systemic toxicity test were not statistically different from those of the control group (P > 0.05). Preliminary evaluation of the biosafety of the Li2Si2O5 whisker-reinforced glass-ceramics showed that it met the existing regulatory standards, and further biosafety experiments can be conducted, following which the material may be expected to be applied in clinical practice.

In vivo biological safety study of porous zinc oxide/hydroxyapatite composite materials

To evaluate the in vivo biological safety of porous zinc oxide (ZnO)/hydroxyapatite (HA) composite materials. The porous ZnO/HA composite materials and porous HA materials were prepared by the spark plasma sintering technology. First, the materials were characterized, including scanning electron microscopy to observe the material structure, in vitro degradation experiments to detect the degradation rate of the materials, and inductively coupled plasma emission spectrometer to detect the concentration of Zn 2+ dissolved out of the composite material degradation. Then the two kinds of material extracts were prepared for acute systemic toxicity test. Fifteen male Kunming mice were randomly divided into groups A, B, and C ( n=5) and injected intraperitoneally with normal saline, HA extracts, and ZnO/HA extracts, respectively. The body mass of the mice was recorded before injection and at 24, 48, and 72 hours after injection. The liver and kidney tissues were taken at 72 hours for HE staining to evaluate the safety of the composite material. Finally, the biological safety of the material in vivo was evaluated by implantation experiment. The eighteen male New Zealand white rabbits were randomly divided into HA group and ZnO/HA group ( n=9); a bilateral radius defect model (1 cm) was established, and the right forelimbs of the two groups were implanted with porous HA materials and porous ZnO/HA composite materials, respectively; the left untreated as a blank control. The general condition of the animals were observed after operation. The rabbit blood was collected at 1 day before operation and at 1 day, 1 week, 4 weeks, and 8 weeks after operation for routine blood test (inflammation-related indicators) and blood biochemistry (liver and kidney function-related indicators). X-ray films were taken at 4, 8, and 12 weeks after operation to observe the repair of bone defects. Material characterization showed that porous ZnO/HA composite materials had interconnected large and small pore structures with a pore size between 50 and 500 μm, which degraded faster than porous HA materials, and continuously and slowly dissolved Zn 2+. The acute systemic toxicity test showed that the mice in each group had no abnormal performance after injection, and the body mass increased ( P<0.05). HE staining showed that the cells shape and structure of liver and kidney tissue were normal. Animal implantation experiments showed that all rabbits survived until the experiment was completed; routine blood tests showed inflammation in each group (neutrophils, monocytes, and lymphocytes increased) at 1 day after operation, and all returned to normal at 8 weeks ( P>0.05); compared with 1 day before operation, the content of inflammatory cells in the HA group increased at 1 day, 1 week, and 4 weeks after operation ( P<0.05), and the ZnO/HA group increased at 1 day after operation ( P<0.05); blood biochemistry showed that the liver and kidney function indexes were in the normal range; X-ray films showed that the ZnO/HA group had better osseointegration than the HA group at 4 weeks after operation. The porous ZnO/HA composite material has good in vivo biological safety and good bone repair ability, which is a potential bone repair material.

Manufacture and preliminary evaluation of acellular tooth roots as allografts for alveolar ridge augmentation.

Alveolar ridge augmentation can be used to obtain appropriate alveolar ridge for dental implantation. A variety of bone graft materials including autogenous bone, allograft, xenograft, and alloplastic material are used in alveolar ridge augmentation. Autogenous tooth-derived bone graft material has received much attention for the past few years, because the structure and physicochemical characteristics of tooth are similar to those of bones. Compared to autogenous tooth, allogenic tooth has the advantage of extensive resources. However, the problem of cell-derived immunological rejection of allogenic tooth remains unresolved. In the present study, acellular tooth root (ATR) is obtained by an innovative combination procedure. The biocompatibility of ATR is assessed using cytotoxicity test, hemolysis test, intracutaneous reactivity test, and acute systemic toxicity test. Osseointegration is evaluated in vivo by implanting ATR into the rat tibia defect as an allograft material. The results show that the ATR has fine biocompatibility, and there is an osseointegration between ATR and bone bed at 8 weeks post operation. This study demonstrates that the ATR could be used in alveolar ridge augmentation as a kind of new tooth-derived bone graft material.

CATMoS: Collaborative Acute Toxicity Modeling Suite.

Background:Humans are exposed to tens of thousands of chemical substances that need to be assessed for their potential toxicity. Acute systemic toxicity testing serves as the basis for regulatory hazard classification, labeling, and risk management. However, it is cost- and time-prohibitive to evaluate all new and existing chemicals using traditional rodent acute toxicity tests. In silico models built using existing data facilitate rapid acute toxicity predictions without using animals.Objectives:The U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Acute Toxicity Workgroup organized an international collaboration to develop in silico models for predicting acute oral toxicity based on five different end points: Lethal Dose 50 ( value, U.S. Environmental Protection Agency hazard (four) categories, Globally Harmonized System for Classification and Labeling hazard (five) categories, very toxic chemicals [ ()], and nontoxic chemicals ().Methods:An acute oral toxicity data inventory for 11,992 chemicals was compiled, split into training and evaluation sets, and made available to 35 participating international research groups that submitted a total of 139 predictive models. Predictions that fell within the applicability domains of the submitted models were evaluated using external validation sets. These were then combined into consensus models to leverage strengths of individual approaches.Results:The resulting consensus predictions, which leverage the collective strengths of each individual model, form the Collaborative Acute Toxicity Modeling Suite (CATMoS). CATMoS demonstrated high performance in terms of accuracy and robustness when compared with in vivo results.Discussion:CATMoS is being evaluated by regulatory agencies for its utility and applicability as a potential replacement for in vivo rat acute oral toxicity studies. CATMoS predictions for more than 800,000 chemicals have been made available via the National Toxicology Program’s Integrated Chemical Environment tools and data sets (ice.ntp.niehs.nih.gov). The models are also implemented in a free, standalone, open-source tool, OPERA, which allows predictions of new and untested chemicals to be made. https://doi.org/10.1289/EHP8495

Efficacy, safety, and physicochemical properties of a flowable hemostatic agent made from absorbable gelatin sponge via vacuum pressure steam sterilization.

An effective and viable hemostatic agent is important for stopping bleeding during surgery. However, it is difficult to achieve hemostasis at uneven or deep bleeding sites using a gelatin sponge. A flowable hemostatic agent has therefore been developed by processing and improving gelatin sponge, to address bleeding under these conditions. In this study, we evaluated the efficacy, safety, and physical and chemical properties of this flowable hemostatic agent in various experiments. We examined its efficacy for stopping bleeding in a rabbit model of liver abrasion in vivo, and compared its efficacy in dynamic coagulation and erythrocyte aggregation tests with gelatin sponge in vitro. We also investigated its safety in rat histocompatibility and acute systemic toxicity tests in mice in vivo, and in hemolysis tests in vitro, to determine if the flowable hemostatic agent induced any pathological reactions or adverse events. In terms of its physical and chemical properties, we analyzed the morphology and chemical bonds of the flowable hemostatic agent by optical and electron microscopy and infrared spectroscopy, and its absorbency and density. The flowable hemostatic agent resulted in a shorter mean bleeding time, less bleeding, greater likelihood of successful hemostasis, and reduced clotting time compared with gelatin sponge. The flowable agent produced some changes in physical morphology, but no pathological changes or undesirable outcomes were detected. This flowable topical hemostatic agent thus provides a safe and more effective hemostatic method than gelatin sponge, and more promising results for intraoperative hemostasis, especially on uneven or deep bleeding surfaces.

Papain grafted into the silica coated iron-based magnetic nanoparticles ‘IONPs@SiO2-PPN’ as a new delivery vehicle to the HeLa cells

The present study aims at engineering, fabrication, characterization, and qualifications of papain (PPN) conjugated SiO2-coated iron oxide nanoparticles ‘IONPs@SiO2-PPN’. Initially fabricated iron oxide nanoparticles (IONPs) were coated with silica (SiO2) using sol–gel method to hinder the aggregation and to enhance biocompatibility. Next, PPN was loaded as an anticancer agent into the silica coated IONPs (IONPs@SiO2) for the delivery of papain to the HeLa cancer cells. This fabricated silica-coated based magnetic nanoparticle is introduced as a new physiologically-compatible and stable drug delivery vehicle for delivering of PPN to the HeLa cancer cell line. The IONPs@SiO2-PPN were characterized using FT-IR, AAS, FESEM, XRD, DLS, and VSM equipment. Silica was amended on the surface of iron oxide nanoparticles (IONPs, γ-Fe2O3) to modify its biocompatibility and stability. The solvent evaporation method was used to activate PPN vectorization. The following tests were performed to highlight the compatibility of our proposed delivery vehicle: in vitro toxicity assay, in vivo acute systemic toxicity test, and the histology examination. The results demonstrated that IONPs@SiO2-PPN successfully reduced the IC50 values compared with the native PPN. Also, the structural alternations of HeLa cells exposed to IONPs@SiO2-PPN exhibited higher typical hallmarks of apoptosis compared to the cells treated with the native PPN. The in vivo acute toxicity test indicated no clinical signs of distress/discomfort or weight loss in Balb/C mice a week after the intravenous injection of IONPs@SiO2 (10 mg kg−1). Besides, the tissues architectures were not affected and the pathological inflammatory alternations detection failed. In conclusion, IONPs@SiO2-PPN can be chosen as a potent candidate for further medical applications in the future, for instance as a drug delivery vehicle or hyperthermia agent.

Use of LUCS (Light-Up Cell System) as an alternative live cell method to predict human acute oral toxicity

LUCS (Light-Up Cell System) is a new live cell test that allows assessment of a cell’s homeostasis and its alteration by a toxic agent. To evaluate the effectiveness of LUCS as an alternative test method for acute oral toxicity, we compared EC50s determined in HepG2 cells treated with 53 chemicals selected from the ACuteTox EU database with corresponding human blood LC50s derived from human acute poisoning cases. Linear regression analysis showed that LUCS results predict human data to 69 %. Rodent oral LD50s and LUCS EC50s were then correlated to human LC50s using shared data sets. Linear regression analyses comparing LUCS and animal data clearly showed that LUCS always predicts human toxicity better than animal data do.These successful prediction values prompted us to simplify the LUCS test, adapting it to regulatory and high throughput applications, resulting in a new protocol with consistent dose-response profiles and EC50s.This study demonstrates that the LUCS test method could be relevant for assessing human acute oral toxicity with a simplified protocol adapted to commercially available fluorescence readers. We suggest that this new alternative method can be used for acute systemic toxicity testing in combination with other tests under European REACH and other regulations, wherever pertinent alternative methods are still lacking.

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.

Qualification of a non-animal vaginal irritation method admitted as nonclinical assessment model (NAM) in the Incubator Phase of the United States Food and Drug Administration (US FDA) Medical Devices Development Tool (MDDT)

The U.S. Food and Drug Administration (FDA) Center for Devices and Radiological Health (CDRH) classifies personal lubricants as Class II medical devices. Because of this status and the nature of body contact common to personal lubricants, CDRH reviewers routinely recommend a standard biocompatibility testing battery that includes: an in vivo rabbit vaginal irritation (RVI) test; an in vivo skin sensitization test, such as the guinea pig maximization test (GPMT); and an in vivo acute systemic toxicity test using mice or rabbits. These tests are conducted using live animals, despite the availability of in vitro and other non-animal test methods that may be suitable replacements. The only test included in the biocompatibility battery currently conducted using in vitro assay(s) is cytotoxicity. FDA's recently launched Predictive Toxicology Roadmap calls for the optimization of non-animal methods for the safety evaluation of drugs, consumer products and medical devices. In line with these goals, a Consortium comprising the Institute for In Vitro Sciences, Inc. (IIVS), industry, the Consumer Healthcare Products Association (CHPA), and the PETA International Science Consortium (PETA-ISC) is qualifying the use of an in vitro testing method as replacement for the RVI test. Participating companies include manufacturers of personal lubricants and those interested in the advancement of non-animal approaches working collaboratively with the FDA CDRH to develop an in vitro testing approach that could be used in place of the RVI in pre-market submissions. Personal lubricants and vaginal moisturizers with diverse chemical and physical properties (e.g., formulation, viscosity, pH, and osmolality) in their final undiluted form will be the focus of the program. In vitro vaginal irritation data generated using commercially available human reconstructed vaginal tissue model(s) will be paired with existing in vivo RVI data and analyzed to develop a Prediction Model for the safety assessment of these products. This research plan has been accepted into the FDA CDRH Medical Device Development Tools (MDDT) program as a potential non-clinical assessment model (NAM). The proposed NAM aligns with the goals of the recently launched FDA Roadmap to integrate predictive toxicology methods into safety and risk assessment with the potential to replace or reduce the use of animal testing.

Comparative Safety Testing (acute systemic toxicity in mice) of Two Materials Prepared from Polypropylene-Polyester (Codubix S) or Acrylate Resin (Mendec Cranio) Used for the Manufacturing of a Calvaria Prosthesis

The aim of the study was a comparison of the acute toxicity of two popular prostheses used in the reconstruction of the bones of the skull. For the tests, the following materials were used: a polypropylene-polyester knitted Codubix S cranial bone prosthesis, made by TRICOMED SA, and polymethyl methacrylate Mednec Cranio resin. The tests were carried out in accordance with the following standards – PN-EN ISO 10993-11:2009 Biological evaluation of medical devices - Part 11: Tests for systemic toxicity, and PN-EN ISO 10993-12:2012 Biological evaluation of medical devices – Part 12: Sample preparation and reference materials. During the evaluation, adult male and female Balb/c mice were used. The animals were injected intravenously using extracts of both materials in 0.9% NaCl and intraperitoneally with the same extracts in sesame oil. The tests lasted 7 days, during which the health of the animals and their behavior were assessed. Both in the control and test groups, there was no mortality of the animals, and the health and behaviour of mice were unchanged when compared with the normal. After 7 days the internal organs of the chest and abdominal cavity of the animals were subjected to macroscopic pathomorphological examination, during which no changes indicating the toxic action of Codubix S and Mednec Cranio resin were found. Before the acute systemic toxicity tests, the chemical purity of both implants was assessed. The chemical purity of a product is one of the factors determining its biological properties. A product which is characterised by a higher degree of chemical purity contains fewer substances which may have a negative impact on biological reactions. Both prostheses meet the requirements of purity for medical devices.

Physicochemical and Biocompatibility Properties of Type I Collagen from the Skin of Nile Tilapia (Oreochromis niloticus) for Biomedical Applications.

The aim of this study is to investigate the physicochemical properties, biosafety, and biocompatibility of the collagen extract from the skin of Nile tilapia, and evaluate its use as a potential material for biomedical applications. Two extraction methods were used to obtain acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) from tilapia skin. Amino acid composition, FTIR, and SDS-PAGE results showed that ASC and PSC were type I collagen. The molecular form of ASC and PSC is (α1)2α2. The FTIR spectra of ASC and PSC were similar, and the characteristic peaks corresponding to amide A, amide B, amide I, amide II, and amide III were 3323 cm−1, 2931 cm−1, 1677 cm−1, 1546 cm−1, and 1242 cm−1, respectively. Denaturation temperatures (Td) were 36.1 °C and 34.4 °C, respectively. SEM images showed the loose and porous structure of collagen, indicting its physical foundation for use in applications of biomedical materials. Negative results were obtained in an endotoxin test. Proliferation rates of osteoblastic (MC3T3E1) cells and fibroblast (L929) cells from mouse and human umbilical vein endothelial cells (HUVEC) were increased in the collagen-treated group compared with the controls. Furthermore, the acute systemic toxicity test showed no acute systemic toxicity of the ASC and PSC collagen sponges. These findings indicated that the collagen from Nile tilapia skin is highly biocompatible in nature and could be used as a suitable biomedical material.

Investigating cell type specific mechanisms contributing to acute oral toxicity_suppl2

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of integrated approaches to testing and assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evalu­ation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system, and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritize the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Investigating cell type specific mechanisms contributing to acute oral toxicity_suppl1

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of integrated approaches to testing and assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evalu­ation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system, and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain in vitro misclassifications only on the basis of mechanistic information. This analysis will help to prioritize the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Predictive models for acute oral systemic toxicity: A workshop to bridge the gap from research to regulation

In early 2018, the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM) published the “Strategic Roadmap for Establishing New Approaches to Evaluate the Safety of Chemicals and Medical Products in the United States” [1]. Cross-agency federal workgroups have been established to implement this roadmap for various toxicological testing endpoints, with an initial focus on acute toxicity testing. The ICCVAM acute toxicity workgroup (ATWG) helped organize a global collaboration to build predictive in silico models for acute oral systemic toxicity, based on a large dataset of rodent studies and targeted towards regulatory needs identified across federal agencies. Thirty-two international groups across government, industry, and academia participated in the project, culminating in a workshop in April 2018 held at the National Institutes of Health (NIH). At the workshop, computational modelers and regulatory decision makers met to discuss the feasibility of using predictive model outputs for regulatory use in lieu of acute oral systemic toxicity testing. The models were combined to yield consensus predictions which demonstrated excellent performance when compared to the animal data, and workshop outcomes and follow-up activities to make these tools available and put them into practice are discussed here.

Investigating cell type specific mechanisms contributing to acute oral toxicity.

The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of Integrated Approaches to Testing and Assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evaluation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral in vivo and in vitro data, 98 were identified with target organ specific effects, of which 93% were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% as non-toxic. This analysis will help to prioritise the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.

Toxico-pharmacological evaluations of the small-molecule LQFM166: Inducer of apoptosis and MDM2 antagonist

Inhibition of p53-MDM2 complex has been emerging as a strategy for antitumoral drug development considering the pro-apoptotic role of functional p53 in tumor cells. In our study, the prototype LQFM166 (2), designed through molecular simplification strategy inspired in the Nutlins compounds, was synthetized, characterized and the mechanisms of cell death were investigated. In addition, we estimated the starting doses for acute oral systemic toxicity tests according to the OECD Guidance Document No.129 - 3T3 NRU. The cytotoxic profile of LQFM166 (2) was determined in K-562 cells, a p53-null cell line, since previous studies also showed activity of LQFM166 (2) on this cells. After 24, 48 or 72 h of compound treatment, using MTT reduction assay, the IC50 values found were 100.1 μM, 56.76 μM and 45.11 μM, respectively. LQFM166 (2) was cytotoxic for leukemia cells in a concentration-time-dependent manner. Cell death mechanisms studies of LQFM166 on K-562 cells, revealed that the compound induced cell cycle arrest, increased the expression of caspase 3/7, 8 and 9, cytochrome c, Bax, p21 and p27. Additionally, a decrease in the expression of the Bcl-2 and cyclin-B1 was observed. The apoptotic inducer profile of the compound was confirmed by phosphatidylserine externalization. Investigation of complexation of p53/MDM2 was carried out by ELISA assay using 3T3 cell, showing a decrease in the p53-MDM2 complex induced by the compound. Furthermore, the cytotoxicity in basal fibroblasts 3T3 was determined to estimate LD50. LQFM166 (2) reduced 3T3 cells viability with the IC50 of 185.3 μM and estimated LD50 of 706.7 mg/kg (category 4 of GHS). The rationally designed of the prototype LQFM166 (2) induced cell death by apoptotic mechanisms in leukemic cells and showed MDM2 complexation antagonism in 3T3 cells.