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Vegetable oils are frequently used as a vehicle in the administration of lipophilic drugs in animal tests. However, the composition of these oils may interfere with the results. One alternative to reduce this potential bias is the use of mineral oil, which is not supposed to interfere in the physiology of experimental models, since this oil is considered to be innocous. The present study shows for the first time the effects of the oral administration of corn and mineral on the prostate, demonstrating their interference in homeostasis and revealing their potential to act as endocrine disruptors. Mineral oil treatment increased the expression of AR and ERα and serum estradiol concentrations, while corn oil increased the expression of positive cells for both types of estrogen receptors. The variation in the expression of these hormone receptors resulted in morphological changes in the prostate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The use of silver nanoparticles (AgNP) raises safety concerns during susceptible life stages such as pregnancy. We hypothesized that acute intravenous exposure to AgNP during late stages of pregnancy will increase vascular tissue contractility, potentially contributing to alterations in fetal growth. Sprague Dawley rats were exposed to a single dose of PVP or Citrate stabilized 20 or 110nm AgNP (700μg/kg). Differential vascular responses and EC 50 values were observed in myographic studies in uterine, mesenteric arteries and thoracic aortic segments, 24h post-exposure. Reciprocal responses were observed in aortic and uterine vessels following PVP stabilized AgNP with an increased force of contraction in uterine artery and increased relaxation responses in aorta. Citrate stabilized AgNP exposure increased contractile force in both uterine and aortic vessels. Intravenous AgNP exposure during pregnancy displayed particle size and vehicle dependent moderate changes in vascular tissue contractility, potentially influencing fetal blood supply., (Copyright © 2017 Elsevier Inc. All rights reserved.)

An intravenously injectable liquid formulation of the poorly water-soluble isosteviol sodium (ISVNa) that has a great clinical potential for cardiovascular diseases was developed using the co-solvent technology. The pH and composition of the co-solvent were optimized to obtain a stable liquid formulation (termed as STVNa) based on saline at pH 10.0 containing 25% (v/v) of ethanol and 20% (v/v) of propylene glycol. STVNa was physicochemically stable upon storage for more than 3 months under various conditions. In vitro studies showed that STVNa did not induce hemolytic effects up to 9.1% (v/v) after 3 h of incubation and it was cytocompatible up to 50 μg/mL in H2C9 cells. Furthermore, STVNa showed acceptable safety and pharmacokinetic parameters comparable with those of ISVNa in saline (dissolved at 60 °C) upon i.v. injection in Wistar rats. Overall, the results demonstrated that STVNa is a promising formulation of ISVNa for clinical translation.

The selection of a vehicle for oral formulations of compounds to be used in non-clinical safety studies is a challenge for poorly soluble compounds. Typically a compromise between solubility and tolerability has to be reached. Vehicle tolerability data are not readily available for a number of vehicles, and a series of oral tolerability studies were, therefore, conducted with Gelucire and Gelucire:PEG400 formulations in rats, dogs and minipigs in order to determine tolerable daily dose volumes in these species. Gelucire and Gelucire:PEG400 formulations were assessed in studies for up to 5 days in minipigs, 7 days in rats and up to 39 weeks in dogs. Gastrointestinal side effects in terms of soft and/or liquid faeces were noted in all species, but the sensitivity to these effects differed between species with the dog being the most sensitive. It was concluded that Gelucire:PEG400 (90:10) was tolerated in Beagle dogs when administered at 1 ml kg(-1) once daily for 39 weeks, and 100% Gelucire was tolerated in the rat and the minipig when administered once daily at 5 ml kg(-1) for 5 days. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

This study was conducted to investigate the use of a nanosuspension for intravenous injection into dogs to increase exposure without toxic additives for preclinical studies in the discovery stage. Nanosuspensions were prepared with a mixer mill and zirconia beads with a vehicle of 2% (w/v) poloxamer 338, which was confirmed to lead to no histamine release in dogs. Sterilized nanosuspensions of poorly water-soluble compounds, cilostazol (Cil), spironolactone (Spi) and probucol (Pro), at 10 mg ml(-1) were obtained by milling for 30 min, followed by autoclaving for 20 min at 121 °C and milling for 30 min (mill-autoclave-mill method). The particle sizes (d50) of Cil, Spi and Pro were 0.554, 0.484 and 0.377 µm, respectively, and the percentages of the nominal concentration were 79.1%, 99.6% and 75.4%, respectively. In chromatographic data, no extra peaks were observed. The particle size of Cil was 0.564 µm after storage for 16 days at 2-8 °C. Cil in nanosuspension, but not in microsuspension, rapidly dissolved in dog plasma. Cil nanosuspension at 0.4 mg kg(-1) and Cil saline solution at 0.03 mg kg(-1) , around the saturation solubility, were intravenously administered to dogs. Nanosuspension increased exposure. The versatility of the mill-autoclave-mill method was checked for 15 compounds, and the particle size of 12 compounds was in the nano range. The nanosuspension optimized in this study may be useful for intravenous toxicological and pharmacological studies in the early stage of drug development. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Potential new drugs are assessed in pre-clinical in vivo studies to determine their safety profiles. The drugs are formulated in vehicles suitable for the route of administration and the physicochemical properties of the drug, aiming to achieve optimal exposure in the test species. The availability of safety data on vehicles is often limited (incomplete data, access restricted/private databases). Nineteen potentially useful vehicles that contained new and/or increased concentrations of excipients and for which little safety data have been published were tested. Vehicles were dosed orally once daily to HanWistar rats for a minimum of 28 days and a wide range of toxicological parameters were assessed. Only 30% (w/v) hydroxypropyl-ß-cyclodextrin was found unsuitable owing to effects on liver enzymes (AST, ALT and GLDH), urinary volume and the kidneys (tubular vacuolation and tubular pigment). 20% (v/v) oleic acid caused increased salivation and hence this vehicle should be used with caution. As 40% (v/v) tetraethylene glycol affected urinary parameters, its use should be carefully considered, particularly for compounds suspected to impact the renal system and studies longer than 1 month. There were no toxicologically significant findings with 10% (v/v) dimethyl sulphoxide, 20% (v/v) propylene glycol, 33% (v/v) Miglyol®812, 20% (w/v) Kolliphor®RH40, 10% (w/v) Poloxamer 407, 5% (w/v) polyvinylpyrrolidone K30 or 10% (v/v) Labrafil®M1944. All other vehicles tested caused isolated or low magnitude effects which would not prevent their use. The aim of sharing these data, including adverse findings, is to provide meaningful information for vehicle selection, thereby avoiding repetition of animal experimentation., (Copyright © 2015 John Wiley & Sons, Ltd.)

The aim of the present paper was to investigate the promoting activity of borneol on the transdermal permeation of drugs with differing lipophilicity, and probe its alterations in molecular organization of stratum corneum (SC) lipids. The toxicity of borneol was evaluated in epidermal keratinocyte HaCaT and dermal fibroblast CCC-HSF-1 cell cultures and compared to known enhancers, and its irritant profile was also assessed by transepidermal water loss (TEWL) evaluation. The promoting effect of borneol on the transdermal permeation of five model drugs, namely 5-fluorouracil, antipyrine, aspirin, salicylic acid and ibuprofen, which were selected based on their lipophilicity denoted by logp value, were performed using in vitro skin permeation studies. Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) was employed to monitor the borneol-induced alteration in molecular organization of SC lipids. The enhancer borneol displayed lower cytotoxicity or irritation in comparison to the well-established and standard enhancer Azone. Borneol could effectively promote the transdermal permeation of five model drugs, and its enhancement ratios were found to be parabolic curve with the logp values of drugs, which exhibited the optimum permeation activity for relatively hydrophilic drugs (an estimated logp value of -0.5 ∼0.5). The molecular mechanism studies suggested that borneol could perturb the structure of SC lipid alkyl chains, and extract part of SC lipids, resulting in the alteration in the skin permeability barrier.

Miglyol 812(®), a mixture of medium-chain triglycerides, has been identified as an oral vehicle that could improve the solubility and possibly the bioavailability of orally administered drugs during the non-clinical safety assessment. The toxicity of Miglyol was assessed in Göttingen(®) minipigs upon daily oral administration (gavage) for six weeks, at dosing-volumes of 0.5 and 2 mL/kg/day, compared to controls receiving 0.5% CarboxyMethylCellulose/0.1% Tween(®) 80 in water at 2 mL/kg/day. The control vehicle did not induce any findings. Miglyol at 0.5 and 2 mL/kg/day induced transient tremors, abnormal color of feces and increase in triglycerides. Miglyol at 2 ml/kg/day also induced reduced motor activity, decreased food intake, respiratory signs (2/6 animals) and increased total and LDL-cholesterol. At necropsy, the lung of 3/6 animals treated at 2 mL/kg/day presented abnormal color and/or irregular surface correlated with a chronic bronchiolo-alveolar inflammation. This finding is probably due to aspiration pneumonia in relation to the administration method and the high viscosity of Miglyol. Overall, the oral administration of pure Miglyol 812(®) for six weeks up to 2 mL/kg was less tolerated than that of the control vehicle. Miglyol as vehicle for sub-chronic oral toxicity studies in minipigs should be used with a limited dosing-volume., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Delivery of therapeutic macromolecules is limited by the physiological limitations of the gastrointestinal tract including poor intestinal permeability, low pH and enzymatic activity. Several permeation enhancers have been proposed to enhance intestinal permeability of macromolecules; however their utility is often hindered by toxicity and limited potency. Here, we report on a novel permeation enhancer, Dimethyl palmitoyl ammonio propanesulfonate (PPS), with excellent enhancement potential and minimal toxicity. PPS was tested for dose- and time-dependent cytotoxicity, delivery of two model fluorescent molecules, sulforhodamine-B and FITC-insulin in vitro, and absorption enhancement of salmon calcitonin (sCT) in vivo. Caco-2 studies revealed that PPS is an effective enhancer of macromolecular transport while being minimally toxic. TEER measurements in Caco-2 monolayers confirmed the reversibility of the effect of PPS. Confocal microscopy studies revealed that molecules permeate via both paracellular and transcellular pathways in the presence of PPS. In vivo studies in rats showed that PPS enhanced relative bioavailability of sCT by 45-fold after intestinal administration. Histological studies showed that PPS does not induce damage to the intestine. PPS is an excellent permeation enhancer which provides new opportunities for developing efficacious oral/intestinal delivery systems for therapeutic macromolecules., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Three multiple water-in-oil-in-water (W/O/W) nanoemulsions have been designed for potential inclusion of either lipophilic or hydrophilic drugs using a two-step emulsification process exclusively based on low-energy self-emulsification. The W/O primary emulsion was constituted by a blend of oil (medium chain triglyceride), a mixture (7:3) of two surfactants, and a 10% water phase. The surfactants were a mixture of Polysorbate-85/Labrasol(®), Polysorbate-85/Cremophor(®) EL or glycerol/Polysorbate-85. The final W/O/W nanoemulsions were obtained by the addition of water, with a weight ratio nanoemulsion/water of 1:2. The multiple emulsion stability was found to increase from 24 hours to 2 and 6 months with Labrasol, glycerol, and Cremophor, respectively. Cytotoxicity was found for formulations including Labrasol and Cremophor EL. The concentration of emulsion inhibiting 50% cell viability (IC(50)) was determined using the alamarBlue(®) test, giving after 24 hours of incubation, IC(50) = 10.2 mg/mL for the Labrasol formulation and IC(50) = 11.8 mg/mL for the Cremophor EL formulation. Corresponding calculated IC(50) values for surfactants were 0.51 mg/mL for Labrasol and 0.59 mg/mL for Cremophor EL. In both cases, cytotoxicity was due to an apoptotic mechanism, evidenced by chromatin condensation and P2X7 cell death receptor activation. The formulation including glycerol, investigated between 1 and 100 mg/mL concentration of nanoemulsion, did not affect cell viability. Moreover, neither chromatin condensation nor P2X7 activation was found between the 10 and 30 mg/mL final concentration of the emulsion. This last formulation would therefore be of major interest for further developments.

Background: The present study investigated the enhancement efficiency between liposomes and microbubbles for insulin pulmonary absorption., Methods: Two types of phospholipid-based vesicle-liposomes and microbubbles-were prepared, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) cytotoxicity test was used to evaluate their in vitro toxicity in A549 cells. Cellular uptake of insulin combined with liposomes or microbubbles was determined using A549 cells. With intratracheal insufflation of Sprague-Dawley rats, an insulin mixture with liposomes or microbubbles was administered to assess its potential for promoting drug pulmonary absorption., Results: Both liposomes and microbubbles had a narrow and monodispersed size distribution with average diameter of 3.1 μm and 1.0 μm, respectively. From the MTT cytotoxicity test, a phospholipid-based vesicle concentration of <25% (vol/vol) in the final volume was the safe dosage range that could avoid severe cytotoxic effects. The intracellular uptake amount of insulin in the insulin-microbubble mixture was significantly higher than that in the insulin-liposome mixture. The minimum reductions of the blood glucose concentration produced by insulin-microbubble and insulin-liposome mixtures were 60.8% and 35.0% of the initial glucose levels, respectively, and their bioavailabilities relative to subcutaneous injection were 48.6% and 30.8%, respectively., Conclusions: Microbubbles have much better efficiency than liposomes in the rate and extent of insulin pulmonary absorption. Microbubbles might be recommended as a potential agent for enhancing protein intrapulmonary absorption.

Diethylene glycol (DEG), a chemical that has been implicated in multiple medication-associated mass poisonings, can result in renal and neurological toxicity if ingested. Three previous such mass poisonings implicated Chinese manufacturers as the origin of contaminated ingredients. No literature exists on potential DEG or triethylene glycol (TEG), a related compound, contamination of health products imported from Asian countries to the USA. Our primary objective was to quantitatively assess the amount of DEG present in a convenience sampling of these health products. The study's secondary objectives were to: (1) evaluate for, and quantify TEG levels in these samples; (2) compare DEG and TEG levels in these products directly to levels in medications implicated in previous similar mass poisonings; and (3) to estimate DEG dose (in mg/kg) based on the manufacturer's instructions and compare these values to toxic doses from past mass poisonings and the literature. A quantitative assessment of DEG and TEG was performed in a convenience sampling of over-the-counter health products imported from Asian countries. Results were converted to volume to volume (v/v) % and compared with DEG levels in medications implicated in previous mass poisonings. Estimated doses (based on the manufacturer's instructions) of each product with detectable levels of DEG for a 70 kg adult were compared to toxic doses of DEG reported in the literature. Seventeen of 85 (20%) samples were not able to be analyzed for DEG or TEG due to technical reasons. Fifteen of 68 (22%) samples successfully tested had detectable levels of DEG (mean, 18.8 μg/ml; range, 0.791-110.1 μg/ml; and volume to volume (v/v) range, 0.00007-0.01%). Two of 68 (3%) samples had TEG levels of 12.8 and 20.2 μg/ml or 0.0012% and 0.0018% TEG v/v. The product with the highest DEG% by v/v was 810 times less than the product involved in the Panama DEG mass poisoning (8.1%). The lowest reported toxic dose from a past DEG mass poisoning (14 mg/kg) was more than 150 times higher than the highest daily dose estimated in our study (0.09 mg/kg). Sixty-eight of 85 (80%) samples were able to be successfully analyzed for DEG and TEG. DEG and TEG were detectable in 15/68 (22%) and 2/68 (3%) samples, respectively. Based on current standards, these levels probably do not represent an acute public health threat. Additional research focusing on why DEG is found in these products and on the minimum amount of DEG needed to result in toxicity is needed., (© American College of Medical Toxicology 2010)

Background: A series of studies were conducted to assess Polysorbate 80 (PS80), Propylene Glycol (PG), and Hydroxypropyl-β-Cyclodextrin (HPβCD), when compared with Hydroxypropyl Methylcellulose (MC) in developmental and reproductive toxicology (DART) studies., Methods: In the rat fertility study, 20 mg/kg MC, 10 mg/kg PS80, 1,000 mg/kg PG, 500 mg/kg HPβCD or 1,000 mg/kg HPβCD were administered orally before/during mating, and on gestation Day (GD) 0-7, followed by an assessment of embryonic development on GD 14. In the rat and rabbit teratology studies, the doses of MC, PS80, PG, and HPβCD were the same as those in the fertility study. In these teratology studies, pregnant females were dosed during the period of organogenesis, followed by an assessment of fetal external, visceral, and skeletal development., Results: In the rat fertility and rat teratology studies, PS80, PG, and HPβCD did not exhibit toxicity, when compared with MC. Similarly, in the rabbit teratology study, there was no PS80 or PG-related toxicity, when compared with MC. However, individual rabbits in the 500 and 1,000 mg/kg HPβCD groups exhibited maternal toxicity, which included stool findings, decreased food consumption, and body weight gain. Furthermore, one rabbit each in the 500 and 1,000 mg/kg HPβCD groups exhibited evidence of abortion, which was considered secondary to maternal toxicity., Conclusions: Although HPβCD was not well tolerated in rabbits at doses of 500 and 1,000 mg/kg, PS80 and PG were comparable to MC and should be considered for use in developmental and reproductive toxicology studies., (© 2010 Wiley-Liss, Inc.)

This study was conducted to assess the safety and tolerability of the alternative formulation vehicles polysorbate 80 (PS80), propylene glycol (PG), and hydroxypropyl-beta-cyclodextrin (HPβCD) in general toxicology studies in the mouse, rat, dog, and monkey. Twenty (20) mg/kg of hydroxypropyl methylcellulose (MC, control), 10 mg/kg PS80, 1000 mg/kg PG, 500 mg/kg HPβCD, or 1000 mg/kg HPβCD were administered by oral gavage to mice, rats, dogs, and cynomolgus monkeys for approximately 90 days. The effects of these formulations on clinical observations, body weight and food consumption parameters, clinical pathology, and histopathology were evaluated across all species. The suitability of formulations containing up to 20 mg/kg MC, 10 mg/kg PS80, and 1000 mg/kg PG for use in preclinical safety studies was confirmed by a lack of effects on all parameters examined. However, formulations containing HPβCD produced elevated transaminase (aspartate and alanine aminotransferase) levels in rats and mice and fecal changes (loose and soft stool) in large animals. Although the etiology and toxicological significance of the transaminase elevations in rats and mice is uncertain, this finding could represent a significant liability for a preclinical formulation because of the critical importance of these biomarkers in the risk assessment of novel therapeutic agents. Based on these data, PS80 and PG are considered to be practical alternatives to MC in preclinical toxicology studies. However, formulations containing HPβCD should be used with caution because of the elevations in rodent transaminase levels.

We report a facile silica nanovehicle preparation procedure for hydrophobic drug delivery, which is carried out in water without adding any surfactant and additional catalyst. This strategy includes hydrophobic drug nanopaticle preparation by reprecipitation method and in situ hydrolyzation and polymerization to encapsulate this naoparticle using only N-(beta-amimoethyl)-gamma-aminopropyltriethoxysilane (AETPS). To demonstrate this technique hypocrellin A (HA), a hydrophobic photosensitizing anticancer drug, is embedded into silica nanovehicle using this simple method. The resulting HA encapsulated nanovehicles (HANV) are monodisperse and stable in aqueous solution. Comparative studies with free HA and entrapped HA have demonstrated that the encapsulation effect on the embedded photosensitizer nanoparticle significantly enhances the efficacy of singlet oxygen generation and, thereby, the in vitro photodynamic efficacy., (Copyright 2009 Elsevier B.V. All rights reserved.)

Following initial signs of dural infection, or as prophylactic postoperative therapy, three cynomologus macaques were given an intramuscular or a subcutaneous injection of ceftriaxone dissolved in a 1% lidocaine diluent (CL). Two to 15 min later, all three monkeys experienced a long-lasting generalized apnoeic clonic convulsive seizure. The injected doses of lidocaine (0.7-2 mg/kg) were as low as 7-20-fold less than the experimental intravenous dosage (14.2 +/- 3.2 mg/kg) previously reported to induce seizure in healthy rhesus monkeys. Under different clinical conditions, the same three animals were either once (1 animal) or repeatedly given CL without any resultant neurological alterations. The monkeys had a cranial device implanted and two of them were craniotomized. This first report of accidental lidocaine-induced seizure in laboratory non-human primates following CL injection strongly suggests increased susceptibility when lidocaine administration is associated with central nervous system alteration. A novel hypothesis, the possible role of cytokine in lowering the lidocaine seizure threshold, is suggested.

The parasites Tritrichomonas foetus and Trichomonas gallinae present veterinary and economic importance since they cause bovine and avian trichomonosis, respectively. The absence of a specific treatment and the necessity of effective and safe drugs against these etiologic agents have stimulated the search for new antiprotozoal drugs with high activity, low toxicity to the animal, and low cost. Screening of potential antiprotozoal molecules is currently a common practice and different kinds of organic solvents and surfactant vehicles are used, since many bioactive compounds have low water solubility. Thus, it is important to determine the toxicity and to provide the minimal inhibitory concentration (MIC) values of the most common solubilization vehicles used in biological activity in vitro evaluation: ethanol, methanol, isopropanol, acetone, DMSO, tween 20 and tween 80. The assays were conducted employing the resazurin microtiter assay, which demonstrated a rapid, safe, and quantitative method for the in vitro determination of T. foetus and T. gallinae trophozoites viability. In summary, all solvents and surfactants, except ethanol, could be used in cytotoxicity assays against T. foetus, and acetone, tween 20 and tween 80 are the preferential vehicles for in vitro analysis of potential bioactive compounds against T. gallinae, though these must be used with caution.

Purpose: To investigate the potential toxicity of the vehicle used for triamcinolone acetonide (TA) to the cornea, lens, ciliary body, and retina of pigmented rabbits., Methods: Forty chinchilla rabbits (40 eyes) were divided into four groups: group A (control group) eyes received an intravitreal injection of 0.1 ml sterile saline solution; group B eyes received 0.1 ml (1.3 mg) TA plus vehicle; group C eyes received 0.1 ml (1.3 mg) TA alone (vehicle eliminated); group D eyes received 0.1 ml vehicle only. Intraocular pressure (IOP) was measured pre-injection and at 1, 7, 14, 30, and 90 days post-injection. Scotopic and photopic electroretinograms (ERG) were examined pre-injection and 7, 30, and 90 days post-injection. Animals were sacrificed 7, 30, and 90 days post-injection. Eyes were enucleated and examined by light microscopy (LM) and electron microscopy (EM)., Results: The IOP of groups B and C was higher than that of other groups on days 1, 7, and 14 post-injection (p < 0.05). ERG amplitudes of groups B and D were lower than those of other groups on days 7 and 30 post-injection (p < 0.05). Histopathological sections indicated morphologic changes in the ciliary body, lens, and retina of eyes in groups B and D., Conclusions: Vehicle used for TA is toxic to the lens, ciliary body, and retina of pigmented rabbit eyes after injection of intravitreal TA.

The superporous hydrogel containing poly(acrylic acid-co-acrylamide)/O-carboxymethyl chitosan (O-CMC) interpenetrating polymer networks (SPH-IPN) that had been developed as an oral delivery vehicle for protein drugs was subject to cytotoxicity and genotoxicity testing, thus evaluating its biological safety in use. In a battery of cytotoxicity assays on RBL-2H3 and Caco-2 cells, the SPH-IPN caused minimal damage towards cell viability, lysosomal activity, and metabolic activity following both direct and indirect treatment. The SPH-IPN did not induce cell apoptosis or DNA breakage in the above cell lines; it did not increase micronucleus (MN) incidence in mouse bone marrow, either. Therefore, the SPH-IPN was preliminarily considered to be biocompatible and might be a safe carrier for protein drugs. In addition, using the HPLC method, residual acrylic acid, acrylamide, and glutaraldehyde in the SPH-IPN were quantified to be 1.4, 2.0, and below 0.2 ppm, respectively. Lack of these low molecular monomers and crosslinker that were mainly responsible for the toxicity provided evidence for the good biocompatibility of the SPH-IPN.

Tetrodotoxin (TTX) injection is currently being studied in clinical trials for potential antinociceptive applications. This work tries to increase the knowledge of its biological tolerability by using a behavioral procedure that can detect aversive effects of drug treatments, as well as in vitro cytotoxicity studies in non-excitable cell systems. Place conditioning studies with Sprague-Dawley male rats showed that pharmacologically active TTX injections (2.5 microg/kg, subcutaneous) were devoid of negative reinforcing properties, the drug being able to prevent the aversive effect of the vehicle. Similarly, TTX was not cytotoxic by itself as evaluated with the neutral red test and the MTT assay in HepG2 cells incubated for 24h with TTX concentrations as high as 400 microM. The results support the idea that low doses of TTX can be well tolerated.

Purpose: To evaluate the toxic effects of two triamcinolone acetonide (TA) vehicles on rabbit retina at different volumes., Methods: Vehicle A and B were prepared from two TA injections by centrifugation. Thirty-six New Zealand white rabbits were intravitreally injected with vehicle A, B, or balanced saline solution at 0.1 mL or 0.2 mL respectively. The eyes were examined by indirect ophthalmoscope, light microscope, and transmission electron microscope up to week 8 postinjection., Results: Eyes with vehicle A appeared normal under the ophthalmoscope, but showed disorganization in retinal inner nuclear layer and photoreceptor layer in pathologic analyses. Eyes with vehicle B displayed more significant retinal changes including retinal hemorrhage, vascular narrowing, myelinated fiber edema, retinal necrosis and atrophy, and photoreceptor apoptosis. There was an increase in degree of the above damages as the volume of either vehicle increased., Conclusion: Both vehicle A and B caused volume-related toxicity in rabbit retina. The intensity of the toxic effects of different vehicles may differ. Reducing or decanting the vehicles should be considered before intravitreal use of TA injections.

Purpose: This work was intended to develop and evaluate a new polymeric system based on amphiphilic carboxymethylpullulans (CMP(49)C(8) and CMP(12)C(8)) that can spontaneously self-assemble in aqueous solutions and efficiently solubilize hydrophobic drugs., Methods: The self-assembling properties of CMP(49)C(8) and CMP(12)C(8) were characterized by fluorescence spectroscopy and surface tension measurements. The solubilization of benzophenone and docetaxel was assessed from surface tension measurements, UV spectrometry and HPLC assays. The in vitro cytoxicity of CMP(49)C(8) solutions and the docetaxel commercial vehicle (Tween 80/Ethanol-water) were evaluated in the absence and in the presence of docetaxel., Results: Compared to CMP(12)C(8), CMP(49)C(8) in aqueous solutions appeared to self-organize into monomolecular aggregates containing hydrophobic nanodomains, and to significantly increase the apparent solubility of benzophenone. Docetaxel solubility could also be improved in the presence of CMP(49)C(8) but to a lower extent due to the surface properties of the drug. Nevertheless, in vitro, the cytotoxicity studies revealed that against cancer cells, the CMP(49)C(8)-docetaxel formulation was equipotent to the commercial docetaxel one. Furthermore, in the absence of the drug, CMP(49)C(8) appeared less cytotoxic against macrophages than the Tween 80/Ethanol-water., Conclusions: CMP(49)C(8) is a good candidate for solubilizing hydrophobic drugs and could be applied to docetaxel formulations.

Purpose: Ocular side effects in patients using eye drops may be due to intolerance to the vector used in eye drops. Castor oil is the commonly used lipophilic vector but has been shown to be cytotoxic. Effects on cells of four oils (olive, camelina, Aleurites moluccana, maize) were compared with those of castor oil in human conjunctival cells., Methods: Human conjunctival cells were incubated with the oils for 15 minutes. After a 24-hour recovery period, cells were tested for viability, proliferation, apoptosis (P2X7 cell death receptor and caspase 3 activation), intracellular redox potential, and reactive oxygen species production. Fatty acid incorporation in cell membranes was also analyzed. In vivo ocular irritation was assessed using the Draize test., Results: Compared to the four other oils, castor oil was shown to induce significant necrosis and P2X7 cell death receptor and caspase 3 activation and to enhance intracellular reactive oxygen species production. Aleurites moluccana and camelina oils were not cytotoxic and increased cell membrane omega-3 fatty acid content. None of the five tested oils showed any in vivo ocular irritation., Conclusions: The results demonstrated that castor oil exerts cytotoxic effects on conjunctival cells. This cytotoxicity could explain the side effects observed in some patients using eye drops containing castor oil as a vehicle. The lack of cytotoxic effects observed with the four other oils, Aleurites, camelina, maize, and olive, suggest that they could be chosen to replace castor oil in ophthalmic formulations.

Purpose: To investigate the toxic effects of triamcinolone acetonide (TA) suspensions on human retinal pigment epithelial (RPE) cells., Methods: Cultured human RPE cells were exposed for up to 2 hours to one of seven solutions: control (balanced salt solution, BSS; Alcon Laboratories, Ft. Worth TX), commercial TA suspension (cTA), cTA from which the vehicle (which contains the preservative benzyl alcohol) had been removed (vehicle-removed TA, -vTA), vehicle of the cTA (V), or a 1:10 dilution (in BSS; Alcon) of cTA, -vTA or V. Solution effects were evaluated by phase-contrast microscopy of cells stained in situ with trypan blue and in vitro by trypan blue exclusion assay. RPE cell function was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The mechanism of TA toxicity was studied by acridine orange-ethidium bromide staining and epifluorescence microscopy, and ultrastructural changes were examined by transmission electron microscopy (TEM)., Results: The effects of vehicle-removed solutions (-vTA and 1:10 -vTA) were similar to those of the control solution. Exposure for 1 hour or longer to a vehicle-containing solution (cTA and V) resulted in similar and significant degrees of cell damage that were dose and time dependent. The major mechanism of cell death was necrosis, and the early ultrastructural change was swelling of organelles in the cytoplasm., Conclusions: Preserved commercial TA suspensions damaged human RPE cells, but vehicle-free solutions did not. The authors suggest removing the vehicle as completely as possible from TA solutions before they are administered intravitreally. Furthermore, they recommend that a commercial formulation of preservative-free TA suspension be made available for intraocular use.

Purpose: To assess retinal toxicity of the vehicle of triamcinolone, benzyl alcohol (BA), when injected into the vitreous cavity of rabbits., Methods: This prospective comparative experimental study included 24 pigmented rabbits assigned into two groups: group 1 (experimental, n = 12) received intravitreal 0.1 ml of BA, and group 2 (control, n = 12) received intravitreal 0.1 ml of balanced salt solution (BSS); all injections were done in the right eyes. Clinical examinations [slit lamp biomicroscopy, indirect ophthalmoloscopy, and three intraocular pressure (IOP) measurements] were done on both eyes before injection, at 1 and 3 h post injection, together with electroretinograms (ERGs) at 3 days, 1, 2, 4, and 6 weeks following injections. Three rabbits from each group were euthanased at 1, 2, 4, or 6 weeks and eyes were sent for light and electron microscopic examination for quantitative morphometric measurements., Results: The mean amplitudes of the a and b waves of the BA-injected eyes were 6.42 +/- 9.02 microv and 11.18 +/- 15.18 microv at 3 days, respectively, which were significantly reduced compared with the BSS-injected eyes (30.87 +/- 8.22 microv and 57.90 +/- 13.38 microv, respectively; P < 0.01 t-test) and the non-injected contralateral eyes (36.20 +/- 7.85 microv and 64.10 +/- 9.36 microv, respectively; P < 0.01 t-test). These ERG responses continued to be significantly reduced in the BA-injected eyes (P < 0.01 t-test) throughout the study period. The mean ganglion cell count was significantly reduced (P < 0.005 t-test) in the BA-injected eyes (8.42 +/- 2.4) compared with the BSS- and non-injected eyes (16.42 +/- 3.9 and 16.5 +/- 4.2, respectively). The mean thicknesses of the inner nuclear layer (INL) and outer nuclear layer (ONL) were significantly reduced (P < 0.005 t-test) in the BA-injected eyes (3.78 +/- 0.96 microm and 11.77 +/- 1.29 microm, respectively) compared with the BSS- (6.1 +/- 0.92 microm and 21.82 +/- 0.95 microm, respectively) and non-injected eyes (7.05 +/- 1.9 microm and 22.49 +/- 1.01 microm, respectively). Electron microscopy showed moderate to severe intracellular changes in the ganglion cell layer, INL, ONL, and photoreceptor layer at 6 weeks in BA-injected eyes, with no significant changes in BSS-injected eye. There was no significant rise in the IOP or clinical evidence of increased lens density during the study period in any of the eyes., Conclusions: Triamcinolone acetonide's vehicle, BA, produced severe ERG and structural damage to the retina when injected intravitreally.

The murine local lymph node assay (LLNA) is used for the prospective identification of contact allergens. This method is not only accepted as a stand-alone test for the identification of contact allergenic hazard but also used increasingly for the measurement of the relative potency of skin-sensitizing chemicals as an integral component of the risk assessment process. During the development and validation phases of the method, a list of standard vehicles for use in the LLNA was identified, among them being the vehicle most commonly used acetone/olive oil (4:1, AOO). We have now explored the performance in the LLNA of a non-standard vehicle, ethanol:diethyl phthalate (1:3, EtOH:DEP), that is used frequently to evaluate dermal effects of fragrance materials in both human and experimental studies. Current investigations have demonstrated that EtOH:DEP induces similar levels of background proliferative responses in lymph nodes comparable with the standard vehicle AOO. Moreover, expected levels of activity are observed when EtOH:DEP is used to deliver a known contact allergen in the LLNA. The conclusion drawn is that EtOH:DEP provides a suitable vehicle for use in the LLNA and that the approach described provides a basis for future evaluation of novel vehicles.

The laboratory toxicologist is frequently faced with the challenge of selecting appropriate vehicles or developing utilitarian formulations for use in in vivo nonclinical safety assessment studies. Although there are many vehicles available that may meet physical and chemical requirements for chemical or pharmaceutical formulation, there are wide differences in species and route of administration specific to tolerances to these vehicles. In current practice, these differences are largely approached on a basis of individual experience as there is only scattered literature on individual vehicles and no comprehensive treatment or information source. This approach leads to excessive animal use and unplanned delays in testing and development. To address this need, a consulting firm and three contract research organizations conducted a rigorous data mining operation of control (vehicle) data from studies dating from 1991 to present. The results identified 65 single component vehicles used in 368 studies across multiple species (dog, primate, rat, mouse, rabbit, guinea pig, minipig, chick embryo, and cat) by multiple routes. Reported here are the results of this effort, including maximum tolerated use levels by species, route, and duration of study, with accompanying dose limiting toxicity. Also included are basic chemical information and a review of available literature on each vehicle, as well as guidance on volume limits and pH by route and some basic guidance on nonclinical formulation development.

Background: Intravitreal triamcinolone acetonide (TA) has been widely used as a therapeutic method for many ocular diseases, but a consensus on an appropriate safe therapeutic window of dosage for intravitreal injection, and whether vehicle of TA should be reduced or eliminated, has not yet been reached. The aim of this article is to investigate these issues., Methods: Forty New Zealand white rabbits were divided into four experimental groups and one control group. Four or 25 mg TA, with vehicle either reduced or not, was injected into the vitreous cavity of rabbits in experimental groups. Rabbits in the control group received 0.2 ml intravitreal sterile saline solution. Intraocular pressures (IOP) were measured by a Tonopen tonometer. Values of lens density were measured by a Pentacam system. Soluble protein, total antioxidation capacity, reduced glutathione (GSH), glutathion peroxidase (GSH-px), and superoxide dismutase (SOD) in lens were measured by specific kits. ERG and pathological examinations, including light and electron microscopy of the retina, were also performed., Results: Elevation of IOP was noted in all experimental groups after intravitreal TA (P<0.01, paired t-test). Significant increase of lens density was noticed at 1 week after intravitreal TA in the 25 mg vehicle-containing group (P<0.0001, paired t-test). Significant loss of GSH-px activity was noticed at the end of the study (P<0.05, paired t-test), while SOD activity increased (P<0.05, paired t-test). Amplitudes of ERG waves declined significantly in vehicle-containing groups (P<0.01, paired t-test) at the end of the study. Pathological examination showed obvious retinal toxicity in vehicle-containing groups., Conclusions: Vehicle of TA should be eliminated or reduced before intravitreal injection to avoid potential retinal toxicity and transient increase in lens density.

Allyl esters, frequently used in the fragrance industry, often contain a certain percentage of free allyl alcohol. Allyl alcohol is known to have a potential for delayed skin irritation. Also present in the finished product are different solvent systems, or vehicles, which are used to deliver the fragrances based upon their intended application. This study was conducted to determine whether different vehicles affect the skin irritation potential of five different allyl esters. The allyl esters tested were allyl amyl glycolate, allyl caproate, allyl (cyclohexyloxy)acetate, allyl cyclohexylpropionate, and allyl phenoxyacetate in the vehicles diethyl phthalate, 3:1 diethyl phthalate:ethanol, and 1:3 diethyl phthalate:ethanol at concentrations of 0.1%, 0.5%, 1.0%, and 2.0% (w/w). A modified cumulative irritation test was conducted in 129 human subjects. Test materials (0.3 ml) were applied under occlusion to skin sites on the back for 1 day (24 h) using Hill Top chambers. Irritation was assessed at 1, 2, 4, and 5 days following application of test materials. Cumulative irritation scores varied considerably among test materials. There were no delayed irritation observations. The highest irritation scores were observed at the 2.0% concentration for all test materials. The irritation scores for allyl amyl glycolate, allyl (cyclohexyloxy)acetate, and allyl phenoxyacetate were highest in 1:3 diethyl phthalate:ethanol, thus the resulting calculated no-observed-effect levels, 0.12%, 0.03%, and 0%, respectively, were much lower for this vehicle compared to the diethyl phthalate vehicle, 0.33%, 0.26%, 0.25%, respectively. These data showed a trend for lower concentration thresholds to induce irritation when higher levels of ethanol were used in the vehicle.

The effects of polyoxyethylenglycerol triricinoleate 35 (Cremophor EL, CrEL) on markers of oxidative stress, nuclear factor kappa B (NF-kappaB) activation and inducible nitric oxide synthase (iNOS) expression were studied in the liver of male Wistar rats. Animals were randomly divided into three groups. Group Cr1 received, i.p., CrEL at 0.046ml/kg daily for 7 days, group Cr2 received CrEL at 0.33ml/kg and the controls were injected with CrEL vehicle (saline solution with 25% ethanol). Both alanine transaminase (ALT) and aspartate transaminase (AST) serum activities were significantly increased in the Cr2 group (+16% and +25%, respectively). AST activity was also higher in the Cr1 group when compared to control animals (+20%). The cytosolic concentration of thiobarbituric acid reactive substances (TBARS) increased in both groups of rats receiving CrEL (Cr1: +24%; Cr2: +33%). Reduced glutathione (GSH) concentration was not significantly modified at any of the CrEL doses, but both the hepatic concentration of oxidised glutathione (GSSG) (Cr1: +37%; Cr2: +84%) and the GSH/GSSG ratio (Cr1: -21%; Cr2: -45%) were significantly modified. CrEL induced no significant NF-kappaB activation, changes in p50 and p65 NF-kappaB subunits or induction of iNOS protein. Data obtained indicate that although high doses of CrEL cause oxidative stress, this is not enough to induce changes in NF-kappaB activation or iNOS expression.

Purpose: To test the toxicity of intravitreal injections of benzyl alcohol., Methods: Nine New Zealand rabbits were injected with either a control or a test article at elevating concentrations. The test article was benzyl alcohol calculated to give final injected concentrations of 0.0073%, 0.022%, 0.073%, 0.222%, and 0.733% benzyl alcohol. The 0.022% concentration corresponds to the concentration of benzyl alcohol in human eyes when 0.1 mL of commercial Kenalog (Bristol-Myers Squibb, Princeton, NJ) is used. Baseline examination of the rabbits was performed along with postinjection examinations on days 1, 3, 7, and 14. The eyes were enucleated and examined by light and electron microscopic examinations., Results: Eyes injected with benzyl alcohol concentrations of 0.073%, 0.222%, and 0.733% displayed changes in the outer retina including loss of, and shortening of, outer segments and photoreceptors., Conclusions: Benzyl alcohol at concentrations modestly higher than what is present in commercial Kenalog is toxic to the rabbit eye. This has been shown in other organ systems. If commercial preserved Kenalog is to be used clinically, decanting the supernatant or using other means to remove the benzyl alcohol may be considered, especially if a volume of >0.1 mL of solution is used. We hypothesize that the noninfectious inflammation seen clinically after Kenalog injection is due to the presence of a toxic preservative at unsafe concentrations.

Drug metabolism and pharmacokinetic (DMPK) studies are an important phase in drug discovery research. Compounds are administered via the intravascular or extravascular routes to animals to calculate various pharmacokinetic parameters. An important step in this process is dissolving the novel compound in a safe vehicle. This procedure is particularly challenging for compounds that must be administered intravenously, as the solution must be clear before injection. There are no published guidelines on which vehicles, or combination of vehicles, are acceptable in a particular species, nor are there published data on the effects these vehicles have on clinical chemistry or hematology parameters, particularly in dogs. In this study, 9 vehicles commonly used at sanofi-aventis USA (propylene glycol, polyethylene glycol 400, glycofurol, hydroxypropyl Beta-cyclodextrin, dimethyl sulfoxide, N-methyl-2-pyrrolidone, dimethylacetamide, ethyl alcohol, and saline) were tested for adverse clinical reactions (such as vomiting or diarrhea) and for their effect on hematology and clinical chemistry parameters. Each vehicle was administered to a group of 8 Beagles by slow intravenous infusion, and blood was collected prior to infusion and at 24 h and 7 d postinfusion. Of 8 dogs given propylene glycol, 2 developed mild gastrointestinal signs (vomitus, diarrhea) after their infusions. None of the vehicles tested induced significant hematology or serum clinical chemistry abnormalities, nor were significant clinical signs noted after administration. We conclude that at the dose, route, and manner described, all of the vehicles tested in this study are clinically safe to use and have no acute effects on hematology or serum chemistry parameters.

Objective: The aim of this study was to evaluate the biocompatibility of calcium hydroxide suspended in HTC20 or calcium hydroxide plus CMCP and their effects on the healing of periapical lesions in dogs., Study Design: Experimental apical periodontitis was induced by opening the pulp chamber of 36 mandibular premolars in 6 3-year old dogs. The teeth were left opened for 1 week and then closed with IRM for 60 days in order to induce periapical lesions. The teeth were divided into 1 control and 2 experimental groups. Both experimental groups received intracanal dressings with a calcium hydroxide slurry. In the first experimental group calcium hydroxide was mixed with a detergent vehicle and in the second group with camphorated parachlorophenol. The medications were kept in the root canal for 2 periods of 30 days each. Afterwards, the dogs were killed and the periapical areas were studied histologically., Results: Based on biocompatibility and tissue remodeling in the periapical area, the best results were observed when calcium hydroxide was mixed with the detergent. The difference was statistically significant (P < .05)., Conclusion: Calcium hydroxide plus CMCP intracanal dressing was the most irritating to the periapical region, and the group of calcium hydroxide plus HCT 20 showed the best results whenever biocompatibility and the capacity of new bone formation was considered.

The pharmaceutical uses of cremophor EL, a non-ionic surfactant, are similar to those of polysorbate 80. In our previous study, polysorbate 80 exerted some adverse actions on rat thymocytes under in vitro condition. Therefore, the effects of cremophor EL on thymic lymphocytes were examined using a flow cytometer with appropriate fluorescent dyes. Cremophor EL at 10 microg/ml or more (up to 300 microg/ml) concentration-dependently decreased cellular content of glutathione. The cell viability of thymocytes under control condition was 95.4 +/- 1.2% (n = 7, mean +/- S.D.). The incubation of thymocytes with 300 microg/ml cremophor EL or 3 mM hydrogen peroxide for 2 h, respectively, decreased the cell viability to 90.8 +/- 2.8% or 91.2 +/- 2.6%. However, the simultaneous incubation with cremophor EL and hydrogen peroxide decreased the cell viability to 28.7 +/- 8.2%. Cremophor EL at 100 microg/ml accelerated the process of cell death induced by hydrogen peroxide. Results suggest that cremophor EL increases the susceptibility to oxidative stress. Cremophor EL at clinically relevant concentrations may increase the therapeutic potential of some anticancer agents to produce oxidative stress.

Diethyl phthalate (DEP; CAS No. 84-66-2) has many industrial uses, as a solvent and vehicle for fragrance and cosmetic ingredients and subsequent skin contact. This review focuses on its safety in use as a solvent and vehicle for fragrance and cosmetic ingredients. Available data are reviewed for acute toxicity, eye irritation, dermal irritation, dermal sensitization, phototoxicity, photoallergenicity, percutaneous absorption, kinetics, metabolism, subchronic toxicity, teratogenicity, reproductive toxicity, estrogenic potential, genetic toxicity, chronic toxicity, carcinogenicity, in vitro toxicity, ecotoxicity, environmental fate and potential human exposure. No toxicological endpoints of concern have been identified. Comparison of estimated exposure (0.73 mg/kg/day) from dermal applications of fragrances and cosmetic products with other accepted industrial (5 mg/m(3) in air) and consumer exposures (350 mg/l in water; 0.75 mg/kg/day oral exposure) indicates no significant toxic liability for the use of DEP in fragrances and cosmetic products.

Phenylsilsesquioxane fluid (PSF) is used widely in the personal care industry and is a common component of skin and oral care products. The potential developmental toxicity of PSF was evaluated in rats and rabbits. Groups of 25 sperm-positive Sprague-Dawley rats (Taconic Farms) and 15 sperm-positive New Zealand White rabbits (HRP) were administered dose levels of 50, 500, or 1000 mg/kg PSF in corn oil. Vehicle control groups of equal size were administered corn oil alone. Rats were dosed daily (5 ml/kg) on gestation d 6-15 and sacrificed on gestation d 20, while rabbits were dosed daily (1.5 ml/kg) on gestation d 6-18 and sacrificed on gestation d 29. The fetuses were removed by cesarean section and examined for gross external, visceral, cephalic, and skeletal anomalies. No treatment-related clinical signs of toxicity were observed. No marked effects upon maternal food consumption, body weight, body weight gain, or uterus or liver weight were detected. Fetal viability and body weight, as well as developmental endpoints, were unaffected by treatment. Accordingly, exposure of pregnant rats or rabbits to 50, 500, or 1000 mg/kg of PSF during the period of major organogenesis did not result in any biologically significant adverse or teratogenic effects in the dams or fetuses.

In order to examine whether immunosuppressive treatment could be responsible for the reduced exercise capacity of heart transplant recipients (HTR), we studied the effects of long-term immunosuppressive treatment with cyclosporin A (CsA) and its vehicle (2/3 cremophor and 1/3 alcohol diluted in olive oil) on in situ mitochondrial respiration of different muscles. Rats were fed for 3 weeks with 10 or 25 mg/kg/day CsA in its vehicle (CsA10 and CsA25 groups), or vehicle or H(2)O. Oxygen consumption rate was measured in saponin skinned fibers without (V(0)) and with ADP until maximal respiration (V(max)) was reached and K(M)for ADP as well as V(max)were calculated using non-linear fit of the Michaelis-Menten equation. In the cardiac muscle of the CsA25 group, V(0)and V(max)were decreased by immunosuppressive treatment respectively from 6.33+/-0.51 to 3.18+/-0.3micromol O(2)/min/g dw (P<0.001) and from 29.0+/-1.5 to 18.1+/-1.6micromol O(2)/min/g dw (P<0.001), an effect which could be entirely attributed to the vehicle itself, with no difference between CsA10 and CsA25. Regulation of cardiac mitochondrial respiration by ADP was altered by vehicle with the K(M)for ADP decreasing from 371+/-37 to 180+/-21microm(P<0.001). A similar trend was observed in the diaphragm or soleus, although to a lesser extent. In contrast, V(0)and V(max)decreased in glycolytic gastrocnemius muscle respectively from 1.7+/-0.2 to 0.94+/-0.14 (P<0. 01) and from 6.8+/-0.3 to 5.1+/-0.4micromol O(2)/min/g dw (P<0.001) in the CsA25 group, but the main effects could be attributed to CsA itself. It was concluded that immunosuppressive treatment induces a deleterious effect on cardiac and skeletal muscle oxidative capacities, mainly due to cremophor, the main component of vehicle., (Copyright 2000 Academic Press.)

The development of contact hypersensitivity (CHS) greatly depends on the allergenicity of the inducing agent. However, various cofactors are known to influence the outcome of the response as well. From this perspective, we have compared the effects of five different vehicles: acetone, ethanol, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), and a 4 to 1 mixture of acetone and olive oil (AOO) on the cellular and humoral immune responses to epicutaneously applied oxazolone in female BALB/c mice. A single application of 0.2% oxazolone dissolved in acetone or ethanol induced stronger proliferative responses and higher lymph node cell numbers than the other three vehicles. Moreover, both vehicles led to higher numbers of oxazolone-specific Ab forming cells in the draining lymph nodes of sensitized animals. When the IgG2a/IgG1 ratios were determined to indicate the type of T helper cell involved, the highest values were obtained with AOO and lowest with DMF and DMSO, while acetone and ethanol were in between. Moreover, no correlation was found between oxazolone-specific antibody production and cellular responses, measured as [3H]thymidine incorporation of draining lymph node cells after sensitization and increased ear thickness after challenge. From this study it can be concluded that cellular and humoral responses in CHS to oxazolone are dissimilarly affected by the vehicles used.

The acute hemodynamic effects of ciclosporin A (Cs-A; 40 and 20 mg/kg), FK506 (1.5 and 0.4 mg/kg), and their vehicles were studied in an in situ autoperfused rat kidney model. Cs-A (60 +/- 7 and 66 +/- 5% of the initial value, respectively; p < 0.05 vs. control group) and FK506 (89 +/- 3 and 77 +/- 3% of the initial value respectively; p < 0.05 vs. control group) induced a significant fall in renal blood flow. Cs-A significantly increased the renal vascular resistance, whereas FK506 had no effect. The glomerular filtration rate (inulin clearance) declined significantly in all groups. Cremophore and FK506 vehicle had no hemodynamic effect on the glomerular filtration rate. In our model, FK506 had less vasoactive effects on the renal hemodynamics than Cs-A.

A rapid and reproducible model of fatty liver in rats was developed by injecting corn oil (s.c.). In preliminary experiments, the mortality due to acute ethanol intoxication was significantly higher in this model of acutely fattened animals. Lipid peroxidation is a process that involves free radicals and consumes as substrate unsaturated fatty acids, which are present in great amounts in corn oil. Thus, in this work we explored whether the acute loads of corn oil increased hepatic lipid peroxidation. The three markers of cellular oxidative stress measured in fatty livers from rats injected with corn oil were: the production of thiobarbituric acid-reactive substances (TBARS), liver content of triacylglycerides (TAG), and total glutathione (GSH-GSSG). All were significantly modified. We also studied the effect of butylated hydroxytoluene (BHT), a free radical scavenger frequently used in the food industry to prevent lipid oxidation, and found that it prevented the effect of corn oil on TBARS and TAG but enhanced the depletion of GSH-GSSG caused by the acute administration of large loads of corn oil.

The effect of a single oral dose of 73 or 294 mg propylene glycol/100 g bw, in Group I or II respectively, on hematological parameters, plasma osmolality and erythrocyte morphology was investigated in groups of 6 female rats each at different time intervals after dosing. A statistically significant and progressive decrease was observed in hemoglobin, packed cell volume and red cell counts for 2 d, which returned to basal values on the 8th day. Reticulocyte counts, plasma hemoglobin and osmolality increased after PG dosing in both the groups, and the changes were more pronounced after 2 d. The osmotic fragility of erythrocytes remained unaffected after PG dosing whereas electron microscope morphology revealed rough cell surface, ruptured membranes and increased cell adherence throughout the observation period, but these features were not marked on the 8th day. These modified red cell surface characteristics could promote removal by the reticulo-endothelial system resulting in the significant increases in spleen weight in both groups.

Cyclosporin A (CsA) is widely used as the immunosuppressant of choice for preventing graft rejection. However, its clinical use is hampered by certain side effects, especially its nephrotoxicity and other cardiovascular side effects. CsA for intravenous infusion contains cremophor (Cre) and this vehicle has significant adverse effects on endothelial function and vascular muscle. The present study was aimed at investigating the direct effects of CsA and Cre on isolated and perfused rat hearts in the dosage that closely approximates the peak level achieved for the prevention of graft rejection in the rat. Transplantation is a clinical setting in which the myocardium may be exposed to transient ischemia. In this study, we have shown that the vehicle of CsA, namely Cre, has significant adverse effects on cardiac function. We observed a reduction in coronary flow and aortic output. Addition of CsA appeared to induce a further reduction of aortic flow. We have also shown that a significant increase of thiobarbituric acid reactive substances, considered as an index of lipid peroxidation, occurred in the reperfused heart in the presence of Cre+CsA. Our experimental study shows that Cre turned out to be toxic to myocardium by itself. In the heart, potential Cre-CsA interactions possibly potentiating CsA toxicity could not be excluded. The increase of lipid peroxidation in the heart perfused with CsA suggests that reactive oxygen species may be involved in the detrimental effects of this substance on the heart.

Chloroanilines are widely used chemical intermediates for the manufacture of dyes, agricultural chemicals and industrial compounds. Nephrotoxicity occurs as one toxicity following intraperitoneal (i.p.) administration of chloroaniline hydrochlorides to rats. The purpose of this study was to examine the effect of chemical form, route of administration and vehicle on 3,5-dichloroaniline-induced nephrotoxicity. In one set of studies, male Fischer 344 rats (four to eight per group) were administered a single i.p. injection of 3,5-dichloroaniline free base or hydrochloride salt, cysteine hydrochloride or ornithine hydrochloride (0.8, 1.0 or 1.5 mmol kg-1) or an appropriate vehicle and renal function monitored for 48 h. Only 3,5-dichloroaniline hydrochloride induced nephrotoxicity that was characterized as acute renal failure. When 3,5-dichloroaniline free base (0.8 mmol kg-1) was administered in dimethyl sulfoxide (DMSO), all rats died within 24 h. In a second experiment, the free base or hydrochloride form of 3,5-dichloroaniline (1.5 mmol kg-1) or vehicle (0.9% saline or sesame oil, respectively) were administered orally and renal function monitored for 48 h. No evidence of nephrotoxicity was observed following either treatment. However, when the hydrochloride salt was given in 25% DMSO in 0.9% saline, all rats died within 24 h, with two rats demonstrating increased proteinuria, glucosuria and hematuria within the first 6 h after treatment. These results demonstrate that 3,5-dichloroaniline nephrotoxicity is potentiated by the administration of systemic acid, but that acid alone has no effect on renal function at the dose tested. Also, 3,5-dichloroaniline (hydrochloride or free base form) is less toxic orally than when administered i.p.(ABSTRACT TRUNCATED AT 250 WORDS)

Bromodichloromethane (BDCM) is a disinfection by-product of drinking water chlorination and is the second most common trihalomethane (THM) in finished drinking water. THMs have generally been administered to experimental animals in corn oil, rather than drinking water, which can influence the site and magnitude of toxicity. To examine the effects of gavage vehicle on the acute renal and hepatic toxicity of orally administered BDCM, 95-day-old male F344 rats were given single doses of 0, 200, or 400 mg BDCM/kg in corn oil or an aqueous 10% Emulphor solution. Activities of serum hepatoxicity indicators were significantly greater 48 hr after administration of 400 mg BDCM/kg in corn oil compared to the aqueous vehicle, but delivery of the low dose in either dosing vehicle had little effect on serum enzymes. In contrast, significant elevations in urinary renal toxicity indicators were noted at 200 and 400 mg BDCM/kg in both vehicles after 24 hr, indicating that the kidney is more sensitive to low doses of BDCM than the liver. Significantly greater increases were observed in urinary indicators after delivery of 200 mg BDCM/kg in 10% Emulphor compared to corn oil. However, administration of the high dose in corn oil resulted in greater nephrotoxicity than in the aqueous vehicle. Significant interactions between vehicle of administration and BDCM dose observed for both urinary and serum parameters further indicate that vehicle differences noted in BDCM acute toxicity are dose dependent. This observation may be due to pharmacokinetic differences in gastrointestinal rates of absorption of BDCM from corn oil as compared to an aqueous solution.

Nervous system complications resulting from i.v. administration of cyclosporine (CS) are especially frequent in liver transplant recipients. Because CS is insoluble in water, the i.v. preparation is formulated in a polyoxyethylated castor oil and ethyl alcohol. Rat dorsal root ganglion neurons exposed in vitro to the i.v. preparation exhibited axonal swelling and degeneration. No effect of CS (dissolved directly in serum) was seen on testing individual components of the i.v. solution. However, 0.1% polyoxyethylated castor oil (volume of solute/volume of solvent) produced axonal swelling and degeneration and 0.001% polyoxyethylated castor oil produced demyelination in vitro. Polyoxyethylated castor oil is manufactured by reacting castor oil with ethylene oxide, and we speculate that residual ethylene oxide or a polymerization product may be responsible for the in vitro neurotoxicity. Although little is known about the pharmacokinetics of polyoxyethylated castor oil, plasma levels of 0.001 to 0.01% polyoxyethylated castor oil (volume of solute/volume of solvent) are probably achieved with therapeutic doses of the i.v. CS preparation.

The predominant hypothesis for cyclosporine-induced acute renal failure is postulated to be prerenal vasoconstriction with concomitant hemodynamic changes; an alternate hypothesis, however, may be that cyclosporine (CsA) affects intrarenal processes, i.e., direct renal parenchymal cell injury. However, reports on this direct effect of CsA on renal parenchymal cells are contradictory. Therefore, the purpose of this study was to address whether CsA is directly toxic to renal parenchymal cells in a primary culture system of rat renal cortical epithelial cells. The cytotoxicity of Sandimmune, the commercial form of CsA in a polyoxyethylated castor oil vehicle (Cremophor), CsA without vehicle, and the Cremophor vehicle was assessed by plasma membrane integrity (lactate dehydrogenase leakage), mitochondrial metabolic activity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction], and gross morphology (phase-contrast microscopy). The cytotoxicity of Sandimmune was also assessed by lysosomal activity (neutral red uptake), by proximal tubular enzyme activity (alkaline phosphatase), and by three fluorescent probes using a multiwell scanner. The three fluorescent probes were propidium iodide which stains nuclei of nonviable cells; bis-carboxyethyl-carboxyfluorescein which is retained by viable cells; and rhodamine 123, which assesses mitochondrial membrane potential. The results of this study demonstrated that Sandimmune caused dose- (10, 25, and 50 microM) and time- (12, 24, and 48 hr) dependent cytotoxicity, while Cremophor caused cytotoxicity only at high concentrations and long incubations. We conclude that (1) CsA is directly toxic to renal parenchymal cells in vitro and this system potentially represents a sensitive model for further mechanistic studies; (2) CsA plus vehicle (Sandimmune) was more cytotoxic to renal cells than CsA alone (without the polyoxyethylated castor oil vehicle).

To explore the feasibility of using human recombinant tissue-type plasminogen activator in the management of subretinal hemorrhage, we examined the toxic effects of a commercial preparation of human recombinant tissue-type plasminogen activator in the subretinal space of the holangiotic retina of the cat. Various concentrations of human recombinant tissue-type plasminogen activator and controls were infused into the subretinal space with a glass micropipette (40- to 60-microns tip) to form a retinal bleb. Concentrations from 2.5 mg/L to 200 mg/L were well tolerated without ultrastructural toxic effects, while 1000 mg/L of human recombinant tissue-type plasminogen activator caused severe, irreversible toxic effects to the photoreceptor-retinal pigment epithelium complex. This toxic effect appeared to be due to the carrier vehicle rather than the human recombinant tissue-type plasminogen activator protein itself. Our results demonstrate that commercially prepared human recombinant tissue-type plasminogen activator can be safely used in the subretinal space of the cat with the range necessary for fibrinolysis.