Genotoxicity studies of a desealant solvent mixture, SR-51.

The Royal Australian Air Force (RAAF) has reported that personnel involved in F-111 fuel tank maintenance were concerned that exposure to a range of chemicals during the period 1977 to mid-1990s was the cause of health problems, including cancer. Particular concern was directed at SR-51, a desealant chemical mixture containing the following four solvents: aromatic 150 solvent (Aro150), dimethylacetamide, thiophenol (TP), and triethylphosphate. The present study examined the mutagenic potential of SR-51 using a range of well-known mutagen and genotoxin assays. The tests used were i) a modified version of the Ames test, ii) the mouse lymphoma assay, iii) the comet assay (a single-cell gel electrophoresis assay), and iv) a mouse micronucleus test. The modified Ames test used mixed bacterial strains in liquid suspension media. The Ames test results showed that SR-51 (tested up to the cytotoxic concentration of 36 microg/ml, 30 min incubation) in the presence and absence of S9 metabolic activation was not mutagenic. The mouse lymphoma assay used cultured mouse lymphoma cells in a microwell suspension method. The mouse lymphoma assay was also negative with SR-51 (tested up to the cytotoxic concentration of 22.5 microg/ml, 3 h incubation) in the presence and absence of S9 metabolic activation. The Comet assay, using cultured mouse lymphoma cells, showed no evidence of DNA damage in cells exposed up to the cytotoxic concentration of SR-51 at 11.25 microg/ml. The in-vivo mouse micronucleus test was undertaken in wild-type C57Bl6J male mice dosed orally with SR-51for 14 days with a single daily dose up to 360 mg/kg/day (the maximum-tolerated dose). No increases were observed in micronuclei (MN) frequency in bone marrow collected (24 h after final dose) from SR-51-treated mice compared to the number of MN observed in bone marrow collected from untreated mice. Tissues collected from treated mice at necropsy demonstrated a significant increase in spleen weights in the high dose mice. Gas chromatography analysis of SR-51 identified more than 40 individual components and an oxidation product, diphenyldisulfide derived from TP under conditions of mild heating. In conclusion, there was no evidence that SR-51 is mutagenic.

An evaluation of three-dimensional modeling of compaction cycles by analyzing the densification behavior of binary and ternary mixtures.

The aim of the study is to use the 3D modeling technique of compaction cycles for analysis of binary and ternary mixtures. Three materials with very different deformation and densification characteristics [cellulose acetate (CAC), dicalcium phosphate dihydrate (EM) and theophylline monohydrate (TM)] have been tableted at graded maximum relative densities (rhorel, max) on an eccentric tableting machine. Following that, graded binary mixtures from CAC and EM have been compacted. Finally, the same ratios of CAC and EM have been tableted in a ternary mixture with 20 vol% TM. All compaction cycles have been analyzed by using different data analysis methods. Three-dimensional modeling, conventional determination of the slope of the Heckel function, determination of the elastic recovery during decompression, and calculations according to the pressure-time function were the methods of choice. The results show that the 3D model technique is able to gain the information in one step instead of three different approaches, which is an advantage for formulation development. The results show that this model enables one to better distinguish the compaction properties of mixtures and the interaction of the components in the tablet than 2D models. Furthermore, the information by 3D modeling is more precise since in the slope K of the Heckel-plot (in die) elasticity is included, and in the parameters of the pressure-time function beta and gamma plastic deformation due to pressure is included. The influence of time and pressure on the displacement can now be differentiated.

Time dependence of elastic recovery for characterization of tableting materials.

The purpose of this study was to complete information on elastic recovery during a compression cycle by measuring the expansion of the tablet after ejection, and thus, to measure the whole recovery process, which means the time dependency of elastic recovery. Two methods were applied: manual measurement by a micrometer screw, and a continuous measurement by thermomechanical analysis, always at a constant temperature and humidity. Elastic recovery of the tablet continued after ejection from the die, and the amount of expansion was different for the tableting materials used. The results showed that expansion continued for most of the materials over several days, until a steady state of the tablet and its physical properties was reached. The extent and the profiles of the elastic recovery curves were different for the tableting materials dicalcium phosphate dihydrate, carrageenan, microcrystalline cellulose, cellulose acetate, hydroxypropyl methylcellulose and theophylline monohydrate. There were slight differences between both the methods used. The profiles of the materials could be related to some properties of the materials.

Three-dimensional modeling to determine properties of tableting materials on rotary machines using a rotary tableting machine simulator.

A new three-dimensional modeling technique of tableting data has been used for data measured with a rotary tableting machine simulator. The use of the tableting machine simulator is helpful in this case because a scale up or a change of equipment is easily possible. The model substances used were hydroxypropyl methylcellulose (HPMC 15.000), microcrystalline cellulose (Avicel PH 101), dicalcium phosphate dihydrate (Emcompress) and theophylline monohydrate, four very differently deforming substances. Tablets were produced by simulating a Manesty Betapress with 100 rev./min. The materials were tableted to five graded maximum relative densities (rho(rel, max)) of 0.75, 0.80, 0.85, 0.90 and 0.95. A twisted plane was fitted to the measured data and three parameters: d, the time-plasticity: e, the pressure plasticity and omega, the elastic decompression, resulted for each material at the given rho(rel, max) according to the three-dimensional tableting technique. The results show different parameter-plots for the tableting materials, allowing to differentiate between the tableting characteristics of various substances. Three-dimensional modeling of data from rotary machines is shown to be a valuable tool not only for material characterization on eccentric machines but on rotary tableting machines as well.

A new theoretical model to characterize the densification behavior of tableting materials.

The purpose of the study was to develop a new three-dimensional model using force, time and displacement to characterize the densification behavior of tableting materials. Normalized time (x), displacement converted to ln(1/1 - D(rel)) according to Heckel (y) and force presented as pressure (z) were used to plot a graph. A twisted plane was fitted to this three-dimensional plot. This plane was characterized by three parameters d, the slope over time called 'time plasticity', e, the slope over pressure called 'pressure plasticity' and omega, the angle of rotation called 'fast elastic decompression'. These parameters were used to characterize the densification behavior of the well-known materials microcrystalline cellulose, dicalcium phosphate dihydrate, theophylline monohydrate, cellulose acetate and hydroxypropyl methylcellulose at different rho(rel, max). It could be shown that brittle, elastic and plastic compression properties could be very well distinguished and differentiated. Further on, it could be shown whether these properties were due to pressure or time. Thus this model has the prevailing advantage to characterize tableting materials in one step according to time and pressure and it is a useful tool to develop tablet formulations or new excipients.

The automatic micrometer screw.

A new analytical method - the automatic micrometer screw - has been established to measure the edge height of tablets. The equipment offers many advantages compared with other methods. The precision is slightly increased compared to the traditional micrometer screw and the measurement with a small punch and a linear voltage transducer. No longer any touch of the tablet is necessary and influences results. The method works automatically and continuously, no manual measurement of the tablets is necessary. Up to ten tablets can be analyzed at the same time because of a rotary table on which they are positioned. Thus the method is not personal intensive. By combining the results from the measurement of punch displacement which means tablet height in the die and the results of the measurement with the automatic micrometer screw which means tablet height outside the die, a convenient measurement for the decompression process is possible.

The use of carrageenan in mixture with microcrystalline cellulose and its functionality for making tablets.

The modulation of tableting and release behavior of combinations of kappa-carrageenan Gelcarin((R)) GP 911 NF and microcrystalline cellulose (MCC) Avicel((R)) PH 101 has been evaluated. Graded binary mixtures were tableted to a maximum relative density of 0.850 at the maximum displacement of the upper punch. Additionally, ternary mixtures with the same ratios of kappa-carrageenan and MCC and a constant percentage of theophylline monohydrate (20% (v/v)) were tested for their release behavior. Tablets produced from pure kappa-carrageenan deformed more elastically than pure MCC, the tablets produced were stable but not at the same degree as those made from MCC. Scanning electron microscopy (SEM) pictures showed that for MCC a smooth surface of the tablets resulted, tablets made from kappa-carrageenan showed less 'fusion' and thus more mechanical interlocking is responsible for their stability. Binary mixtures showed a continuous change in compaction properties from plastic to elastic deformation. All ternary mixtures with theophylline deformed more plastically than the binary mixtures, the change in deformation properties remained the same. Theophylline reduced the crushing strength due to its different fracture properties. The ternary mixtures showed different release mechanisms: Fast release up to 20% (v/v) kappa-carrageenan, slower release starting from 30% (v/v). The kinetics of release tended at 70% (v/v) more clearly towards zero-order kinetics. This change in release is in accordance with a change in swelling of tablets made of the binary mixtures.

Matrix tablets of carrageenans. I. A compaction study.

Carrageenans can be used as excipients for controlled-release tablets. The aim of this study was to determine their compaction and consolidation behavior to prove their usefulness for tableting. The Carr indices of the three carrangeenans, two kappa-carrageenans(Gelcarin GP-812 NF and GP-911 NF) and one tau-carrageenan (Gelcarin GP-379 NF), indicate that the materials are free flowing. They are polymers in the rubbery state. Their glass transition-temperature is about 0 degree C analyzed by differential scanning calorimetry (DSC). The powders were analyzed regarding their compression behavior using an eccentric tableting machine. From data obtained during one compaction cycle, porosity-pressure and pressure-time plots were made. Compaction behavior is evaluated by fitting the pressure-time function to the pressure-time plot and by fitting the Heckel function to the porosity-pressure plot. The polymers show "viscoelastic" tableting behavior. Several additional tableting parameters were analyzed for strengthening the results obtained, namely, maximum work, maximum power, and the time between maximum upper punch force and maximum displacement of the upper punch. The crushing strength of the tablets is high; therefore, the carrageenans are able to form strong compacts. However, they remain in the rubbery state, as shown by thermomechanical analysis. In addition, elastic recovery is regarded at several times after ejection. Finally, after 10 days, it is about 30% as determined from the minimum of tablet height during the compression cycle. These results indicate that the carrageenans are suitable tableting excipients for controlled-release tablets. They show good compactibility and good consolidation behavior. Strong compacts with a high elastic recovery are formed; this means that the materials are able to embed drugs softly. Only a little stress and strain remains in the tablet. All three carrageenans show similar tableting behavior, and a flexible dosage form design is possible.

Matrix tablets of carrageenans. II. Release behavior and effect of added cations.

Carrageenans are hydrocolloids in the rubbery state at standard conditions. They are useful excipients for controlled-release tablets. Three carrageenans, two kappa-carrageenans (Gelcarin GP-812 NF and GP-911 NF) and one iota-carrageenan (Gelcarin GP-379 NF), are analyzed regarding their release behavior in combination with sorption, swelling, and rheology. The iota-carrageenan has a higher substitution by sulfate groups. The kappa-carrageenan Gelcarin GP-812 NF contains a small amount of potassium chloride left over from processing. Water sorption of the pure materials was studied gravimetrically, and the rheology of different solutions (2% and 5% w/w) was studied by cup-cylinder rotation viscosimetry. Swelling was determined as the vertical expansion of the tablets with a specially designed swelling apparatus. Drug release from the tablets was performed by the USP paddle method for 8 hr. The data indicate that drug release increases when water sorption and swelling extent decrease and as viscosity increases. The order of release is nearly zero-order kinetics for theophylline monohydrate, a nonionic drug. Diffusion of the anionic drug diclofenac sodium is anomalous. In addition, the influence of the added salts potassium and calcium chloride on swelling and release was studied. Before tableting, physical mixtures of these salts with and without theophylline monohydrate were prepared. Swelling and release change in the same order, but this is only valid when the ionic interactions responsible for this are strong enough. Besides this, physical mixing of salts with the carrageenans can result in an increased release of drug caused by decreased cohesion of the matrix during drug release, mainly for calcium chloride.

In vitro assessment of the effect of halogenated hydrocarbons: chloroform, dichloromethane, and dibromoethane on embryonic development of the rat.

Halogenated hydrocarbons are widely used in industry, the laboratory, and in the home. In the present study three of these solvents--chloroform, dichloromethane, and dibromoethane--were examined for embryotoxic/teratogenic potential using rat embryo culture. The results showed that each of the solvents had a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. The effect and no-effect concentrations (expressed in mumol/ml culture medium), respectively, for each of the halogenated hydrocarbons tested were: dibromoethane--0.33, < 0.18; chloroform--2.06, 1.05; dichloromethane--6.54, 3.46. The levels of chloroform and dichloromethane found to be embryotoxic in the present study were compared to reported blood levels attained following controlled human exposure. In the industrial situation, if the current exposure levels are adhered to, chloroform and dichloromethane appear to have little potential for reproductive toxicity in the human. Fatal or near fatal solvent levels would be required in the mother for the embryotoxic level to be reached. For dibromoethane, there are no reports following controlled human exposure presumably due to its carcinogenicity. In an attempt to elucidate the mechanism of embryotoxicity, histological studies were performed after exposure of rat embryos to an embryotoxic level of each of the halogenated hydrocarbons studied, for increasing time periods up to the standard 40-hour culture. Marked cell death in the neuroepithelium of the developing neural tube was a prominent feature in all embryos exposed to an embryotoxic level of these solvents for periods of 16 hours of longer.

Effect of relative humidity during tabletting on matrix formation of hydrocolloids: densification behavior of cellulose ethers.

The aim of this research was to investigate the elastic-plastic deformation behavior of the cellulose ethers hydroxypropylmethylcellulose (HPMC), hydroxyethylmethylcellulose (HEMC), and sodium carboxymethylcellulose (NaCMC) at relative humidities (RH) of 38, 57, and 75% and assess how the release of drugs embedded in such matrices is affected by the inner structure of the tablets formed during tabletting. Sorption and desorption isotherms and glass transition temperature were determined between 32 and 75% RH. The materials were equilibrated at 38, 57, and 75% RH and tabletted to a range of graded maximum relative densities. Pressure-time and displacement-time curves were analyzed by use of the Heckel function and a modified Weibull function (pressure-time only). After equilibration at the different RHs, all materials were in the glassy state. The respective degrees of polymerization had negligible effect on the absolute content of water, the sorption isotherms, and finally the densification behavior. At 38% RH, NaCMC contains the same amount of water as HPMC and HEMC, but deforms less plastically than the latter. This is attributed to tight binding of the water of hydration in the former. With increasing RH, NaCMC becomes only a little more plastic than both HPMC and HEMC, although it contains more than twice the amount of water. The binding strength of water and its molecular mobility, not the amount, seems to determine the readiness for volume reduction under load.

In vitro assessment of the effect of methanol and the metabolite, formic acid, on embryonic development of the rat.

Inhalation studies in rats have indicated that methanol is embryotoxic at levels that are only mildly maternally toxic. In the present study, the embryotoxicity of methanol and its metabolite, formic acid, was evaluated using rat embryo culture. The results showed that both methanol and formic acid have a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. The no-effect concentration of methanol was 211.7 mumol/ml culture medium, while embryotoxicity was observed at 286.5 mumol/ml. The no-effect concentration of formic acid was 3.74 mumol/ml, while a concentration of 18.66 mumol/ml was associated with severe embryotoxicity. When embryos were grown in sera containing 18.66 mumol sodium formate/ml or in sera adjusted with hydrochloric acid to pH values similar to those achieved with formic acid, the results indicated that both low pH and formate contributed to the observed embryotoxicity of formic acid. When the level of methanol found to be embryotoxic in the present study is compared to blood levels in the human following controlled industrial exposure there appears to be a large margin of safety. However, plasma methanol levels are only one aspect of methanol toxicity in the human. Of greater significance is the formate level and the associated acidosis. However, it appears that embryotoxicity due to low pH or high formate levels would only occur after very severe methanol intoxication. Based on these in vitro studies, current industrial safety limits would appear to provide protection for the developing embryo.

In vitro assessment of individual and interactive effects of aromatic hydrocarbons on embryonic development of the rat.

There have been reports of disruption of embryonic development following exposure of pregnant women to aromatic hydrocarbons. In the present study, the embryotoxicity of toluene, xylene, benzene, styrene, and its metabolite, styrene oxide, was evaluated using the in vitro culture of postimplantation rat embryos. Possible interactions between toluene, xylene, and benzene were also studied using mixtures of these solvents. The results of the study showed that toluene, xylene, benzene, and styrene all have a concentration-dependent embryotoxic effect on the developing rat embryo in vitro. Styrene was embryotoxic at a lower concentration (1.00 mumol/mL) than benzene (1.56 mumol/mL), toluene (2.25 mumol/mL), or xylene (1.89 mumol/mL). The metabolite of styrene, styrene oxide, was embryotoxic at a concentration (0.038 mumol/mL). more than 20 times less than the parent compound. There was no evidence of a synergistic interaction between toluene, xylene, and benzene in causing embryotoxicity; the solvents interacted in an additive manner. The embryos were exposed to the solvents for 40 h of the organogenic period. When the levels of solvents found to be embryotoxic in the present study are compared to blood levels in the human following industrial exposure or solvent abuse, it appears unlikely that the threshold blood levels for embryotoxicity would be exceeded in the workplace. However, the possibility that exposure to solvents earlier or later or throughout the entire organogenic period might result in a different conclusion cannot be excluded.

Triterpenoid glycosides from Schefflera lucantha.

Three new triterpenoid glycosides were isolated from the leaves of Schefflera lucantha Viguier. Based on the spectroscopic data, especially 2D NMR, the structures of glycosides have been established as 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->2)-beta-D-glucuronopyranosyl] betulinic acid, 3-O-[alpha-L-rhamnopyranosyl (1-->2)-beta-D-xylopyranosyl (1-->2)-beta-D-glucuronopyranosyl]betulinic acid and 3-O-[alpha-L-rhamnopyranosyl(1-->2)-beta-D-glucopyranosyl (1-->2)-beta-D-glucuronopyranosyl]oleanolic acid.

Embryotoxicity of xylene and toluene: an in vitro study.

Exposure to aromatic hydrocarbon solvents during pregnancy has been reported to adversely affect human embryonic development. This exposure may be due to deliberate abuse or may occur in the workplace. Xylene and toluene are the most common solvents encountered in the workplace and toluene is a constituent of commonly abused substances. This study was performed in an endeavour to fulfil two requirements for proof of teratogenicity of a substance, namely development of an animal model and demonstration of a dose-response relationship of teratogenicity. To fulfil these aims, the possible teratogenic and embryotoxic effects of xylene and toluene on rat embryos during the organogenic period was investigated in vitro. Rat embryos were explanted on day 9.5 of gestation and cultured in heat-inactivated rat serum to which xylene or toluene (0.1, 0.5 or 1.0 microL/mL) had been added, dispersed in 0.1% DMSO. The amount of solvent in the culture medium was quantitated using gas chromatography. Neither xylene nor toluene had any observable teratogenic effect on the embryos in terms of increased malformations. However, both solvents were embryotoxic and caused a dose-dependent retardation of growth and development. A no-effect level was not established for either xylene or toluene, however, the lowest levels used for each of these compounds caused only a slight retardation of growth. Although there was no indication that exposure to these solvents caused a teratogenic effect, there was clear evidence of embryotoxicity. The embryotoxic levels of these solvents needed in culture were higher than blood levels likely to occur in the human following industrial exposure or recreational abuse.