Tolerance to the vascular effect of a novel nitric oxide-donating vasodilator, FK409

We investigated whether tolerance develops to the vasorelaxant effects of a new vasodilator, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409), in isolated canine coronary artery strips and to its hypotensive effect in rats, and whether FK409 activates soluble guanylate cyclase isolated from vascular tissues in the absence of -cysteine. No tolerance to FK409 (0.46 nM to 0.46 μM or 1–1000 μg/kg, i.v.) or cross-tolerance between FK409 and glyceryl trinitrate was demonstrated in vitro and in vivo experiments, whereas the tolerance to glyceryl trinitrate (0.44 nM to 4.4 μM or 1–1000 μg/kg, i.v.) was marked in both conditions. In addition, FK409 (0.1–10 μM) activated soluble guanylate cyclase without -cysteine, but glyceryl trinitrate (1–100 μM) required the addition of -cysteine (5 mM) for the activation of the enzyme. The results suggest that FK409 may be advantageous compared to tolerance-producing nitrates currently in clinical use, and that this property of FK409 is probably due to its independence of a sulfhyhydryl donor.     

Beneficial effects of GW274150, a novel,potent and selective inhibitor of iNOS activity,in a rodent model of collagen-induced arthritis

The aim of this study was to investigate the role of inducible nitric oxide synthase (iNOS) on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis. Collagen-induced arthritis was induced in wild-type mice (iNOS-WT) treated with GW274150, a novel, potent and selective inhibitor of iNOS activity, and in mice lacking the gene for iNOS (iNOS 'knock-out', iNOS-KO), by an intradermal injection of 100 microl of emulsion containing 100 microg of bovine type II collagen and complete Freund's adjuvant at the base of the tail. After 21 days, a second injection of type II collagen in complete Freund's adjuvant was administered. iNOS-WT mice developed erosive hind paw arthritis when immunised with type II collagen in complete Freund's adjuvant. Over a 35-day period, macroscopic clinical evidence of collagen-induced arthritis first appeared as periarticular erythema and oedema in the hind paws. By day 28, the incidence of collagen-induced arthritis was 100% in type II collagen-challenged iNOS-WT mice and the severity of collagen-induced arthritis progressed with radiographic evaluation revealing resorption of bone. Histopathology of collagen-induced arthritis mice demonstrated erosion of the cartilage at the joint margins. iNOS-WT mice treated with GW274150 (5 mg/kg, i.p. daily) starting at the onset of arthritis (day 23), and iNOS-KO mice showed a delay of the development of the clinical signs at days 24-35 and an improvement of the histological status in the knee and paw. Immunohistochemical analysis for nitrotyrosine and for poly(ADP-ribose) polymerase revealed positive staining in inflamed joints from type II collagen-treated iNOS-WT mice. The degree of staining for nitrotyrosine and poly(ADP-ribose) polymerase were markedly reduced in tissue sections obtained from type II collagen-treated iNOS-WT mice, who had received GW274150 and from iNOS-KO mice. Furthermore, radiographic signs of protection against bone resorption were present in the joints of iNOS-WT mice treated with GW274150 as well as in the joint from iNOS-KO mice. This study provides the first evidence that GW274150, a novel, potent and selective inhibitor of iNOS activity, attenuates the degree of chronic inflammation and tissue damage associated with collagen-induced arthritis in mice. Furthermore, these results suggest that the induction of iNOS and NO production are essential for the up-regulation of the inflammatory response during experimental collagen-induced arthritis.     

NCX 6560, a nitric oxide-releasing derivative of atorvastatin, inhibits cholesterol biosynthesis and shows anti-inflammatory and anti-thrombotic properties

We compared the lipid-lowering, vasodilating, anti-thrombotic and anti-inflammatory properties of NCX 6560, a novel NO-releasing derivative of atorvastatin, with those of atorvastatin. NCX 6560 and atorvastatin induced similar inhibition of cholesterol biosynthesis in rat smooth muscle cells (IC₅₀ = 1.9 ± 0.4 and 3.9 ± 1.0 μM, respectively). However, in hyperlipidemic mice, a 5-week oral treatment with NCX 6560 (46.8 mg/kg/day, p.o.) was more effective than equivalent atorvastatin (40 mg/kg/day, p.o.) at lowering serum cholesterol (NCX 6560: − 21% vs controls, P < 0.05; atorvastatin: − 14% vs control, P = NS). In norepinephrine-precontracted rabbit aortic rings, NCX 6560-induced vasodilation (EC₅₀ = 53.5 ± 8.3 μM) and in PC12 cells it stimulated cGMP formation (EC₅₀ = 1.8 ± 0.7 μM), while atorvastatin was inactive. In lipopolysaccharide from Escherichia coli (LPS)-treated RAW 264.7 macrophages, NCX 6560 reduced iNOS expression and dimer assembly more efficiently than atorvastatin and inhibited nitrite accumulation (IC₅₀ = 6.7 ± 1.6 μM) and TNF release. U46619- or collagen plus epinephrine-induced platelet pulmonary thromboembolism in mice was reduced by NCX 6560 at 46.8 mg/kg p.o. (mortality: − 44% and − 56% vs vehicle, respectively; P < 0.05), but not by atorvastatin 40 mg/kg, p.o. In the U46619-induced mortality model, isosorbide mononitrate (ISMN) (20 mg/kg, p.o.), a pure NO-donor, was also active (mortality: − 40%, P < 0.05). NCX 6560 significantly reduced ex vivo platelet adhesion to collagen at high shear (− 31 ± 1.3% vs vehicle), and so did ISMN (− 33.3 ± 1.7% vs vehicle). Atorvastatin was ineffective. NCX 6560, but not atorvastatin, reduced blood pressure in eNOS knockout mice (− 16%, P < 0.001 vs vehicle), an effect not observed in wild type mice. On the contrary, ISMN provoked a significant drop of blood pressure both in wild type (− 20%, P < 0.05 vs vehicle) and in eNOS−/− mice (− 21%, P < 0.05 vs vehicle). In conclusion, NCX 6560 exerts greater lipid-lowering, anti-thrombotic and anti-inflammatory effects than atorvastatin, due to a large extent to NO release.     

Toxic effects of carvacrol, caryophyllene oxide, and ascaridole from essential oil of Chenopodium ambrosioides on mitochondria

Chenopodium ambrosioides have been used for centuries in the Americas as a popular remedy for parasitic diseases. The essential oil of this plant possesses anthelmintic activity and is still used in some regions to treat parasitosis and leishmaniasis. However, the Chenopodium oil caused also some fatalities, leading to its commercial disuse. In this work, we studied the mechanism of toxicity of the essential oil and its major pure ingredients (carvacrol, caryophyllene oxide, and ascaridole, which was synthesized from α-terpinene) with respect to mammalian cells and mitochondria. We observed that all products, but especially caryophyllene oxide, inhibited the mitochondrial electron transport chain. This effect for carvacrol and caryophyllene oxide was mediated via direct complex I inhibition. Without Fe²⁺, ascaridole was less toxic to mammalian mitochondria than other major ingredients. However, evidence on the formation of carbon-centered radicals in the presence of Fe²⁺ was obtained by ESR spin-trapping. Furthermore, it was shown that Fe²⁺ potentiated the toxicity of ascaridole on oxidative phosphorylation of rat liver mitochondria. The increase of the α-tocopherol quinone/α-tocopherol ratio under these conditions indicated the initiation of lipid peroxidation by Fe²⁺-mediated ascaridole cleavage. Further ESR spin-trapping experiments demonstrated that in addition to Fe²⁺, reduced hemin, but not mitochondrial cytochrome c can activate ascaridole, explaining why ascaridole in peritoneal macrophages from BALB/c mice exhibited a higher toxicity than in isolated mitochondria.     

葡萄籽原花青素对2型糖尿病大鼠血清中SOD GSH MDA及NO表达的影响

目的观察葡萄籽原花青素(GSPE)对2型糖尿病大鼠血清中超氧化物歧化酶(SOD)、还原型谷胱甘肽(GSH)、丙二醛(MDA)及一氧化氮(NO)表达的影响。方法选取SPF级雄性SD大鼠30只,出生180d,体质量180～220g,高脂饲料喂养4周后再小剂量腹腔注射链脲佐菌素(STZ)诱导2型糖尿病模型,随机分为糖尿病对照组,GSPE250mg·kg-1·d-1、50mg·kg-1·d-1剂量干预组;另设6只为普食对照组。检测各组喂养第6周、10周后的血糖水平,并检测第10周后大鼠血清中SOD、MDA、GSH及NO的变化。结果GSPE能降低血糖,但效果不明显。在抗氧化指标的检测中,与糖尿病对照组相比,GSPE250mg·kg-1·d-1剂量干预组SOD、GSH明显升高(P<0.05),MDA、NO明显降低(P<0.05);50mg·kg-1·d-1剂量干预组SOD、GSH有所升高,MDA、NO有所降低,但差异无统计学意义(P>0.05)。结论较高剂量的GSPE能有效改善2型糖尿病大鼠的抗氧化能力,对其作用机制的进一步研究,可为GSPE对糖尿病及其并发症的营养干预治疗提供新的思路和依据。     

CL 284,846, a novel sedative-hypnotic: Evaluation of its metabolites for pharmacological activity in vitro and in vivo

CL 284,846, N-[3-3-cyanopyrazolo[1,5-a]pyrimidin-7-yl(phenyl)]-N-ethylacetamide, is a novel non-benzodiazepine sedative-hypnotic with benzodiazepine-like sedative effects, but with less apparent liability for accompanying undesired side effects. Three metabolites of the parent sedative-hypnotic have been isolated and identified, a desethyl metabolite, CL 284,859, a desethyl-5-keto metabolite, CL 345,644, and a 5-keto metabolite, CL 345,905. In experiments to determine the ability of these compounds to displace [³H]-flunitrazepam from rat cortical benzodiazepine receptors, the IC₅₀ values were 205 nM for CL 284,846 compared to 4.5 nM for diazepam as a positive control, and 11 μM for CL 284,859. The other two metabolite, CL 345,644 and CL 345,905, failed to displace [³H]-flunitrazepam. In a second experiment designed to evaluate in vivo receptor-mediated activity, CL 284,846 (3.0 mg/kg; 9.836 μmol/kg) was established as a discriminative stimulus (DS) in rats. While CL 284,846 (0.03ndash;3.0 mg/kg; 0.098–9.836 μmol/kg) showed a doserelated increase in drug-appropriate responding and one dose of triazolam (0.3 mg/kg; 0.875 μmol/kg) substituted as a positive control, all three metabolites (3.0–100.0 mg/kg; 10.3–341.3 μmol/kg for CL 284,859; 6.0–201.2 μmol/kg for CL 345,644; 9.3–311.5 μmol/kg for CL 345,905) failed to substitute for the DS effects of CL 284,846. These results suggest that CL 284,859, CL 345,644, and CL 345,905, the metabolites of the sedative-hypnotic CL 284,846, have no significant neuropharmacological effects at central benzodiazepine receptors and, thus, do not contribute to the activity of the parent compound. © 1994 Wiley-Liss, Inc.     

Neuroprotective effects of PMC, a potent alpha-tocopherol derivative, in brain ischemia-reperfusion: reduced neutrophil activation and anti-oxidant actions

2,2,5,7,8-Pentamethyl-6-hydroxychromane (PMC) is the most potent analogue of alpha-tocopherol for anti-oxidation. It is more hydrophilic than other alpha-tocopherol derivatives and has potent free radical-scavenging activity. In the present study, PMC significantly attenuated middle cerebral artery occlusion (MCAO)-induced focal cerebral ischemia in rats. Administration of PMC at 20mg/kg, showed marked reductions in infarct size compared with that of control rats. MCAO-induced focal cerebral ischemia was associated with increases in HIF-1alpha, active caspase-3, iNOS, and nitrotyrosine expressions in ischemic regions. These expressions were markedly inhibited by treatment with PMC (20mg/kg). In addition, PMC (4-12 microM) inhibited respiratory bursts in human neutrophils stimulated by fMLP (800 nM) and PMA (320 nM). Furthermore, PMC (6, 12, and 60 microM) also significantly inhibited neutrophil migration stimulated by leukotriene B(4) (160 nM). An electron spin resonance (ESR) method was conducted on the scavenging activity of PMC on the free radicals formed. PMC (12 microM) greatly reduced the ESR signal intensities of superoxide anion, hydroxyl radical, and methyl radical formation. In conclusion, we demonstrate a potent neuroprotective effect of PMC on MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by inhibition of free radical formation, followed by inhibition of HIF-1alpha activation, apoptosis formation (active caspase-3), neutrophil activation, and inflammatory responses (i.e., iNOS and nitrotyrosine expressions), resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. Thus, PMC treatment may represent a novel approach to lowering the risk or improving function in ischemia-reperfusion brain injury-related disorders.     

钙拮抗剂TMB-8对培养牛大脑中动脉内皮细胞[Ca2+]i和一氧化氮释放的影响

目的旨在观察TMB-8对血管内皮细胞[Ca²⁺]i水平和NO释放的影响,探讨扩张脑血管的机制。用AR-CM-MIC阳离子测定系统,测量单个细胞内游离钙浓度([Ca²⁺]i),用血红蛋白法测量一氧化氮(NO)的释放。结果表明,在细胞外钙浓度为1.3mmol·L^-1时,TMB-8 12.5及25.0μmol·L^-1对静息[Ca²⁺]i和甲基血红蛋白ΔE无明显影响,而50及100μmol·L^-1时可升高静息[Ca²⁺]i和甲基血红蛋白ΔE。表明TMB-850及100μmol·L^-1升高脑血管内皮[Ca²⁺]i,激活NO合酶,促进NO合成和释放,这可能是其扩张脑血管的重要机制之一。     

BE-I-PLA2, a novel acidic phospholipase A2 from Bothrops erythromelas venom: isolation, cloning and characterization as potent anti-platelet and inductor of prostaglandin I2 release by endothelial cells

A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.     

Ramipril prevents the detrimental sequels of chronic NO synthase inhibition in rats: hypertension,cardiac hypertrophy and renal insufficiency

Inhibition of the angiotensin converting enzyme (ACE) with ramipril was studied in male Wistar rats during long-term inhibition of nitric oxide (NO) synthase by N^G-nitro-l-arginine methyl ester (l-NAME). Chronic treatment with l-NAME in a dose of 25 mg/kg per day over 6 weeks caused myocardial hypertrophy and a significant increase in systolic blood pressure (245 ± 16 mmHg) as compared to controls (155+4 mmHg). Animals receiving simultaneously l-NAME and ramipril were protected against blood pressure increase and partially against myocardial hypertrophy. L-NAME caused a significant reduction in glomerular filtration rate (GFR: 2.56+0.73 ml·kg^–1·min^–1) and renal plasma flow (RPF: 6.93±1.70ml·kg^–1·min^–1) as compared to control (GFR: 7.29±0.69, RPF: 21.36±2.33ml·kg^–1·min^–1). Addition of ramipril prevented l-NAME-induced reduction in GFR and renal plasma flow. l-NAME produced an elevation in urinary protein excretion and serum creatinine and a decrease in potassium excretion which was antagonised by ramipril. L-NAME-induced increase in plasma renin activity (PRA) was further elevated with ramipril treatment. Isolated hearts from rats treated with l-NAME showed increased post-ischaemic reperfusion injuries. Compared to controls duration of ventricular fibrillation was increased and coronary flow reduced. During ischaemia the cytosolic enzymes lactate dehydrogenase and creatine kinase, as well as lactate in the venous effluent were increased. Myocardial tissue values of glycogen, ATP, and creatine phosphate were decreased, whereas lactate was increased. Coadministration of ramipril reversed these effects. l-NAME treatment reduced the cyclic GMP content in urine and renal arteries, and was not changed by additional ramipril-treatment. In the kidney hyalinosis of arterioles and of glomerular capillaries, as well as mesangial expansion and tubular atrophies seen after long-term inhibition of NO synthase were reduced by coadministration of ramipril. In conclusion, long-term ACE inhibition by ramipril prevented l-NAME-induced hypertension and cardiac hypertrophy, and attenuated functional and morphological changes in the kidneys. In addition, cardiac-dynamic and -metabolic deterioration induced by L-NAME was normalised by co-treatment with ramipril. Correspondence to: Max Hropot at the above address     

Development and validation of an HPLC assay for the quantitation of mefunidone,a novel derivative of pirfenidone,and an initial pharmacokinetics study in liver microsomes

本研究开发了一种简单可靠的HPLC-UV方法用于美氟尼酮的测定。生物分析步骤包括从500 μL肝微粒体系统中通过甲醇沉淀蛋白质提取美氟尼酮。色谱条件:色谱柱为Agilent TC-C₁₈柱(4.6 mm×250 mm, 5 μm), 流动相为10 mM甲酸铵(用10%的甲酸调PH至2.9)–乙腈(70:30, v/v),流速为1.0 mL/min, UV检测波长设定在245 nm。美氟尼酮和吡非尼酮(内标物)分别在6.0和9.7分钟洗脱,总运行时间为12分钟。根据美国食品和药物管理局生物分析指南,进行了方法验证,结果符合验收标准。美氟尼酮在肝微粒体中的标准曲线在0.5–16 μg/mL范围内呈线性关系。美氟尼酮内和外间精确度低于9.0％,偏差在±10.0％以内。美氟尼酮在肝微粒体中孵育后,该方法成功应用于药代动力学研究。     

Anti-proliferative and anti-leukemic activity of DDE46 (compound WHI-07), a novel bromomethoxylated arylphosphate derivative of zidovudine, and related compounds. Studies using human acute lymphoblastic leukemia cells and the zebrafish model

The anti-proliferative effects of a novel bromomethoxylated arylphosphate derivative of zidovudine (compound DDE46, CAS 213982-96-8) were first examined in a zebra fish embryo model. DDE46 blocked the cell division at the 2-cell stage of the embryonic development followed by total cell fusion. DDE46 also inhibited the proliferation of the leukemic cell lines NALM-6 and MOLT-3. DDE46 enhanced the activity of the pro-apoptotic enzymes Caspase-3, Caspase-6, Caspase-8, and Caspase-9 leading to the apoptotic death of the leukemic cell line Jurkat. These results justify the further development of this agent as a new anti-leukemic drug candidate.     

Development,optimization, and validation of a novel extraction procedure for the removal of opiates from human hair's surface

Room temperature ionic liquids (ILs) have proved to be efficient extraction media for several systems, and their ability to capture volatile compounds from the atmosphere is well established. We report herein a contactless extraction procedure for the removal of opiate drugs from the surface of human hair. The compounds were chosen as a model drug, particularly due to their low volatility. Equal amounts of IL and hair (about 100 mg) were introduced in a customized Y‐shaped vial, and the process occurred simply by heating. After testing several ILs, some of them (e.g. 1‐methyl‐3‐ethanol‐imidazolium tetrafluoroborate, phenyl‐trimethyl‐ammonium triflate or bis(dimethyl) diheptylguanidinium iodide) showed extraction efficiencies higher than 80% for the two studied compounds, morphine and 6‐monoacetylmorphine. Using the design of experiments (DOE) approach as an optimization tool, and bearing in mind the hygroscopic properties of the ILs (in particular, 1‐methyl‐3‐ethanol‐imidazolium tetrafluoroborate), the process was optimized concerning the following variables: temperature (50–120 ºC), extraction time (8–24 h), IL amount (50–200 mg) and water content of the IL (0.01–60%). This study not only provided the optimum conditions for the process (120 ºC, 16 h, 100 mg of IL containing 40% of water), but has also showed that the water content of the IL represents the variable with the most significant effect on the extraction efficiency. Finally, we validated our method through the comparison of the results obtained by treating hair samples with the described procedure to those obtained using a standard washing method and criteria for positivity. Copyright © 2014 John Wiley & Sons, Ltd.     

Diaminomethane dihydrochloride,a novel reagent for the synthesis of primary amides of amino acids and peptides from active esters

Amide formation from acids, N-protected amino acids and peptides was achieved in an easy and convenient way by treating “active esters” such as succinimidyl or 4-nitrophenyl esters or acyl chlorides with diami–nomethane dihydrochloride in dioxane in the presence of EtsN. Diaminomethane dihydrochloride behaves as a slow ammonia-releasing agent. The method is a good alternative to the use of concentrated aqueous ammonia; it avoids solubility problems and allows better control of the stoichiometry of the reaction and of the pH. It gives good yields and does not induce racemization. The mechanism of the reaction is discussed.     

Alterations on the morphology, nitric oxide synthesis and activity of platelets reproduced in rats as possible biomarkers for depression are reversed by fluoxetine

Biochemical markers associated with the prognosis of depression in humans are being described in the literature, whereas experimental studies in animal models in search for antidepressant strategies are lacking. The aim of this study was to evaluate platelet morphology, platelet activity and nitric oxide (NO) synthesis as possible biomarkers of depressive-like behavior by using FST alone and in the presence of fluoxetine. Naïve rats were compared to those receiving vehicle or fluoxetine at 10 mg/kg i.p. in acute, subchronic and chronic administration in the FST. After behavioral assessment, platelets were isolated from blood samples and analyzed by flow cytometry to determine the platelet mitochondrial membrane potential and NO synthesis. In addition, HPLC and electron microscopy were used to examine 5-HT and tryptophan levels and morphology of platelets, respectively. Rats receiving vehicle and exposed to FST showed depressive-like behavior at all the times tested; after chronic FST rats showed a similar pattern of alteration in platelet morphology and in the studied as possible biochemical markers as those previously recognized in depressive humans. Depressive-like behavior in rats exposed to FST was prevented in the presence of fluoxetine administration at all the times tested and associated with the prevention of alterations in platelet morphology, platelet activity and NO synthesis, and/or in 5-HT concentrations. The results of the present study suggest that platelet function and morphology might be relevant markers for the prognosis of depression and the search for functional treatments. Besides, the relevance of FST as model to study this psychiatric illness is reinforced.     

Efficient synthesis and formulation of (R)‐(−)‐[11C]Deprenyl,a selective radioligand for the quantification of MAO‐B activity using PET

Carbon‐11 labeled (R)‐(?)‐Deprenyl is the tracer of reference for the quantification of monoamine oxidase (MAO)‐B activity with PET. In this paper, its radiosynthesis is re‐investigated and oriented towards the preparation of multi‐milliCuries of radiotracer. Typically, using no‐carrier‐added [¹¹C]methyl triflate as the alkylating agent, 140–190 mCi (5.1–7.0 GBq) of (R)‐(?)‐[¹¹C]Deprenyl was obtained within 30 min of radiosynthesis (including HPLC purification and formulation) with specific radioactivities ranging from 0.8 to 1.2 Ci/μmol (29.6–44.4 GBq/μmol). The high efficiency of these radiosyntheses allows for multi‐injection protocols and kinetic approaches for absolute quantification of the tracer. Copyright © 2002 John Wiley & Sons, Ltd.     

Case studies putting the decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping) into practice

Case studies covering carbonaceous nanomaterials, metal oxide and metal sulphate nanomaterials, amorphous silica and organic pigments were performed to assess the Decision-making framework for the grouping and testing of nanomaterials (DF4nanoGrouping). The usefulness of the DF4nanoGrouping for nanomaterial hazard assessment was confirmed. In two tiers that rely exclusively on non-animal test methods followed by a third tier, if necessary, in which data from rat short-term inhalation studies are evaluated, nanomaterials are assigned to one of four main groups (MGs). The DF4nanoGrouping proved efficient in sorting out nanomaterials that could undergo hazard assessment without further testing. These are soluble nanomaterials (MG1) whose further hazard assessment should rely on read-across to the dissolved materials, high aspect-ratio nanomaterials (MG2) which could be assessed according to their potential fibre toxicity and passive nanomaterials (MG3) that only elicit effects under pulmonary overload conditions. Thereby, the DF4nanoGrouping allows identifying active nanomaterials (MG4) that merit in-depth investigations, and it provides a solid rationale for their sub-grouping to specify the further information needs. Finally, the evaluated case study materials may be used as source nanomaterials in future read-across applications. Overall, the DF4nanoGrouping is a hazard assessment strategy that strictly uses animals as a last resort.