Potential impurities in drug substances: Compound-specific toxicology limits for 20 synthetic reagents and by-products, and a class-specific toxicology limit for alkyl bromides.

This paper provides compound-specific toxicology limits for 20 widely used synthetic reagents and common by-products that are potential impurities in drug substances. In addition, a 15 µg/day class-specific limit was developed for monofunctional alkyl bromides, aligning this with the class-specific limit previously defined for monofunctional alkyl chlorides. Both the compound- and class-specific toxicology limits assume a lifetime chronic exposure for the general population (including sensitive subpopulations) by all routes of exposure for pharmaceuticals. Inhalation-specific toxicology limits were also derived for acrolein, formaldehyde, and methyl bromide because of their localized toxicity via that route. Mode of action was an important consideration for a compound-specific toxicology limit. Acceptable intake (AI) calculations for certain mutagenic carcinogens assumed a linear dose-response for tumor induction, and permissible daily exposure (PDE) determination assumed a non-linear dose-response. Several compounds evaluated have been previously incorrectly assumed to be mutagenic, or to be mutagenic carcinogens, but the evidence reported here for such compounds indicates a lack of mutagenicity, and a non-mutagenic mode of action for tumor induction. For non-mutagens with insufficient data to develop a toxicology limit, the ICH Q3A qualification thresholds are recommended. The compound- and class-specific toxicology limits described here may be adjusted for an individual drug substance based on treatment duration, dosing schedule, severity of the disease and therapeutic indication.

Top-down design of magnonic crystals from bottom-up magnetic nanoparticles through protein arrays.

We show that chemical fixation enables top-down micro-machining of large periodic 3D arrays of protein-encapsulated magnetic nanoparticles (NPs) without loss of order. We machined 3D micro-cubes containing a superlattice of NPs by means of focused ion beam etching, integrated an individual micro-cube to a thin-film coplanar waveguide and measured the resonant microwave response. Our work represents a major step towards well-defined magnonic metamaterials created from the self-assembly of magnetic nanoparticles.

An investigation of the genotoxic potential of a well-characterized yerba mate extract.

Yerba Mate (Ilex paraguariensis) is historically used as a beverage and its extracts are considered traditional medicine in South America. Extract use has been expanding to North American and European markets and the currently available genetic toxicology literature indicate discrepancies in genotoxicity findings for yerba mate. As botanical extract use expands, assumption in safety should be made with caution assuring a good understanding of the test material characterization. Authoritative agencies suggest a two-step paradigm to investigate genotoxicity, and this was implemented to evaluate the safety of yerba mate hydroxycinnamic acid extract. Four OECD compliant assays were employed: bacterial reverse mutation, in vitro micronucleus and a parallel in vivo micronucleus, and comet assay. No evidence of mutagenicity was observed in the in vitro Ames assay, but the results of an in vitro micronucleus study were inconclusive. However, oral gavage treatment of rats for the in vivo micronucleus and comet assays demonstrated negative findings. The results from this battery of tests, supports that this yerba mate hydroxycinnamic acid extract is not anticipated to pose genotoxicity concerns. A high-level comparison of results to other available genotoxicity literature on yerba mate is presented with emphasis on the importance of identity when drawing conclusions on botanicals.

Factors regulating macrophage production and growth: identity of colony-stimulating factor and macrophage growth factor.

The activities of a colony-stimulating factor (CSF), which stimulates granulocyte-macrophage colony formation by mouse hemopoietic cells, and macrophage growth factor (MGF), which stimulates proliferation of activated peritoneal macrophages, have been demonstrated by various criteria to reside in the same molecular species. These criteria include occurrence in various sources and copurification of the activities in mouse L-cell-conditioned medium as well as the biological, physicochemical, and antigenic properties of the activities of L-cell-conditioned medium. CSF and MGF activities of L-cell-conditioned medium are ascribable to a glycoprotein of mol wt approximately 60,000 which migrates electrophoretically with alpha-globulin. Human urinary CSF, which also possesses MGF activity, has similar properties and can be neutralized by antiserum to highly purified L-cell medium CSF. A procedure is described for the partial purification of material from L-cell medium that has activity at 1 ng/ml in both MGF and CSF assays.

Preparation of location-specific thin foils from Fe-3% Si bi- and tri-crystals for examination in a FEG-STEM.

Bi-crystals and tri-crystals of a nominal Fe-3% Si (wt%) of well-defined orientations have been grown using a floating-zone technique with optical heating. The manufacture of these unique crystals and the preparation technique involved in harvesting thin foils from specific locations for transmission electron microscopy are described in detail. In particular, the grain boundary triple junction has been extracted from the tri-crystal and examined in high-resolution aberration-corrected FEG-STEM instruments. To achieve the necessary resolution, the foils have to be uniformly thin, in the range 50-100 nm over large areas of the specimen. For ferromagnetic materials, there are further challenges arising from the magnetic field interaction, with the electron beam placing significant demands on the aberration correction system. One way to minimise this interaction is to reduce the total mass of magnetic material. To achieve this, an in situ focused ion beam lift-out technique has been combined with an additional precision ion-polishing stage to reproducibly provide thin-foil specimens suitable for high-resolution EELS and EDX analysis. Examination of the foils reveals that the final precision ion-polishing stage removes residual damage arising from the use of focused ion beam milling procedures.

A comparison of two high spatial resolution imaging techniques for determining carbide precipitate type and size in ferritic 9Cr-1Mo steel.

Two high spatial resolution imaging techniques, focused gallium ion beam imaging in conjunction with XeF2 gas (FIB/XeF2) and high-speed atomic force microscopy (HS-AFM), were used to analyse 9Cr-1Mo ferritic steel samples, which had been exposed for extended periods to hot CO2 gas containing traces of CO, H2, H2O and CH4. The carbide precipitates embedded in the metal matrix were observed and their morphology, size and spatial distribution were quantified using these two techniques. The lower resolution of the FIB/XeF2 imaging technique suggested that small carbide precipitates (<50 nm) may be missed, while the existence of a limited flow layer introduced by sample preparation may influence the HS-AFM results. The gallium ion beam was used to remove a thin oxide layer of approximately 50 nm from sample surfaces prior to FIB/XeF2 imaging, avoiding the influence of surface contamination. HS-AFM provided higher resolution (∼5 nm) than FIB/XeF2 imaging. A quantitative comparison of the experimental data confirmed the value of both FIB/XeF2 and HS-AFM for imaging carbide precipitates, while clarifying their strengths and limitations.

The Impact of Alkaliphilic Biofilm Formation on the Release and Retention of Carbon Isotopes from Nuclear Reactor Graphite.

14C is an important consideration within safety assessments for proposed geological disposal facilities for radioactive wastes, since it is capable of re-entering the biosphere through the generation of 14C bearing gases. The irradiation of graphite moderators in the UK gas-cooled nuclear power stations has led to the generation of a significant volume of 14C-containing intermediate level wastes. Some of this 14C is present as a carbonaceous deposit on channel wall surfaces. Within this study, the potential of biofilm growth upon irradiated and 13C doped graphite at alkaline pH was investigated. Complex biofilms were established on both active and simulant samples. High throughput sequencing showed the biofilms to be dominated by Alcaligenes sp at pH 9.5 and Dietzia sp at pH 11.0. Surface characterisation revealed that the biofilms were limited to growth upon the graphite surface with no penetration of the deeper porosity. Biofilm formation resulted in the generation of a low porosity surface layer without the removal or modification of the surface deposits or the release of the associated 14C/13C. Our results indicated that biofilm formation upon irradiated graphite is likely to occur at the pH values studied, without any additional release of the associated 14C.

The role of the surface chemistry of CoCr alloy particles in the phagocytosis and DNA damage of fibroblast cells.

Surface chemistry of CoCr particles is demonstrated to be fundamental to the process of phagocytosis by fibroblast cells in vitro. Particles preincubated in serum for 5 days and washed in water before addition to cell cultures were phagocytosed less readily than were particles preincubated in minimal essential medium (MEM) for 1 h and washed in water. This was explained by the coating of calcium phosphate and protein on the serum-immersed particles investigated by time-of-flight secondary ion mass spectroscopy. The cells incubated with the serum-immersed particles had a reduced mitotic index when compared with the MEM-immersed particles, indicating that the phagocytosed particles were causing cell cycle arrest. The release of soluble ions measured by electrothermal atomic absorption spectroscopy within the first hour of particle immersion in MEM was identified as the most likely cause for the DNA damage measured by single cell gel electrophoresis ("Comet" assay). Cryofocused ion beam SEM with a spatial resolution of 8 nm was used to cross section cells, to investigate the location of the phagocytosed particles, some of which were found within the nuclear membrane. This paper demonstrated that consideration of the surface chemistry is essential to understand the processes of the effects of orthopedic wear debris.

The entrapment of corrosion products from CoCr implant alloys in the deposits of calcium phosphate: a comparison of serum, synovial fluid, albumin, EDTA, and water.

Physical wear of orthopedic implants is inevitable. CoCr alloy samples, typically used in joint reconstruction, corrode rapidly after removal of the protective oxide layer. The behavior of CoCr pellets immersed in human serum, foetal bovine serum (FBS), synovial fluid, albumin in phosphate-buffered saline (PBS), EDTA in PBS, and water were studied using X-ray Photoelectron Spectroscopy (XPS) and Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS). The difference in the corrosive nature of human serum, water, albumin in PBS and synovial fluid after 5 days of immersion was highlighted by the oxide layer, which was respectively 15, 3.5, 1.5, and 1.5 nm thick. The thickness of an additional calcium phosphate deposit from human serum and synovial fluid was 40 and 2 nm, respectively. Co and Cr ions migrated from the bulk metal surface and were trapped in this deposit by the phosphate anion. This may account for the composition of wear debris from CoCr orthopedic implants, which is known to consist predominantly of hydroxy-phosphate compounds. Known components of synovial fluid including proteoglycans, pyrophosphates, phospholipids, lubricin, and superficial zone protein (SZP), have been identified as possible causes for the lack of significant calcium phosphate deposition in this environment. Circulation of these compounds around the whole implant may inhibit calcium phosphate deposition.

Attitudes to fluorosis and dental caries by a response latency method.

BACKGROUND: Understanding socially relevant attitudes to fluorosis and dental caries is important. Previous studies have concentrated mainly on aesthetic implications. AIMS: To investigate social judgements beyond the aesthetic, made when viewing digitally manipulated extraoral images of dental fluorosis of varying degrees of severity and images of dental caries. METHODOLOGY: Using a response latency technique, which allowed both the direction and strength of attitudes to be measured, 40 volunteers made judgements on 144 image/characteristic combinations. RESULTS: Participants made social judgements which extended beyond the aesthetic to factors such as sociability, reliability and cleanliness. Judgements on mild fluorosis were not markedly different from those made about the same individual with normal enamel, but severe fluorosis had a significant negative impact on social judgements. Untreated dental caries was judged less favourably than normal enamel and mild fluorosis. CONCLUSIONS: Attribution of characteristics that go beyond the aesthetic are significantly influenced by altered tooth appearance.

The effects of calcium phosphate deposition upon corrosion of CoCr alloys and the potential for implant failure.

Physical wear of orthopedic implants is inevitable. CoCr metal samples, typically used in joint reconstruction, corrode rapidly after removal of the protective oxide layer. The behavior of CoCr pellets immersed in human serum, fetal bovine serum (FBS), synovial fluid, and water were studied using time-of-flight secondary ion mass spectroscopy (ToF-SIMS). The differences in the corrosive nature of human serum, FBS, synovial fluid, and water after 5 days immersion were highlighted by the oxide/hydroxide layer, which was, respectively, 25, 10, 1.5, and 3-3.5 nm thick. The thickness of calcium phosphate deposit from human serum, FBS, and synovial fluid was, respectively, 30, 20, and 2 nm. Co and Cr ions migrated from the bulk metal surface and were trapped in the serum deposits, where chromium existed as oxides, hydroxides, and phosphates, whereas the cobalt chemistry was dominated only by phosphates and hydroxides. This may account for the composition of wear debris from CoCr orthopedic implants, which are predominantly hydroxyphosphate compounds. From the literature, proteoglycans, pyrophosphates, phospholipids, lubricin, and superficial zone protein (SZP) have been identified as possible causes for the insignificant deposit of calcium phosphate from synovial fluid. Circulation of these compounds around the whole implant may inhibit calcium phosphate deposition and therefore contribute to osteolysis.

Evaluation of the effect of smokeless tobacco purified products and extracts on latent Epstein-Barr virus.

Numerous chemical tumor promoters induce latent Epstein Barr virus (EBV) to active replication. The effect of smokeless tobacco purified products N-nitrosonornicotine (NNN), 4-(N-methyl-N-nitrosamine)-1-3-pyridinyl)-1-butanone (NNK), benzo(a)pyrene (BaP), and smokeless tobacco extracts (dry snuff, moist snuff, and loose leaf tobacco) was tested for induction of latent EBV in Raji cells using fluorescence-activated cell sorter flow cytometry detection of the restricted component of EBV early antigen (EA-R). Concentrations of smokeless tobacco purified products or preparations were used that have carcinogenic effects in animal cell lines. There was no discernible effect for the 6-7-day duration of treatment on viability of Raji cells, or on induction of latent EBV in Raji cells. Results were comparable using paraformaldehyde- or acetone-fixed cells. There does not appear to be an in vitro effect of smokeless tobacco constituents on EBV-infected lymphocytes that may contribute to development of oral cancers.

Transmission of a newly recognized virus (Bunyaviridae, Bunyavirus) isolated from Aedes albopictus (Diptera: Culicidae) in Potosi, Missouri.

Aedes albopictus (Skuse) mosquitoes collected in Potosi, Mo., were tested for their ability to transmit a newly recognized Bunyamwera sero group virus isolated from the same mosquito population. Mosquitoes were fed artificial blood meals containing 4.5-6.2 log10 TCID50 of virus per ml. After 7-29 d at 25 degrees C, 79-99% of the mosquitoes had disseminated infections and 0-26% transmitted virus to fluid-filled capillary tubes. Transmission was first observed after 7 d of extrinsic incubation. Tests failed to detect transovarial transmission in 5,145 progeny from ovarian cycles 2-4. Following parenteral inoculation with 5.3-6.0 log10 TCID50 of virus, four of nine adult hamsters developed viremia. Ten of 16 suckling mice died following intracerebral inoculation of 5.0 log10 TCID50 of virus (fifth Vero cell passage); the average survival time was 8.8 d (SD, 3.5). No mortality occurred in 10 suckling mice inoculated with 3.6 log10 TCID50 of virus (second Vero cell passage).

Isolation of Jamestown Canyon virus (California serogroup) from Aedes mosquitoes in an enzootic focus in Michigan.

Twenty isolates of Jamestown Canyon virus were obtained from adult females of 5 Aedes species collected at the Houghton Lake Wildlife Research Area, Missaukee County, in north-central Michigan between 1985 and 1989. Fourteen were from Aedes provocans, and 6 were from 4 other snowmelt Aedes species. One isolate of trivittatus virus and one Cache Valley-like virus were also obtained. Seasonal succession patterns for numerous mosquito species were recorded over 4 years. The temporal association of adult mosquito emergence, virus isolations, and infection and seroconversion of sentinel deer suggest that Ae. provocans is a primary enzootic vector of Jamestown Canyon virus in that focus. We hypothesize that Ae. provocans provides an overwintering reservoir for Jamestown Canyon virus at the study site. A large dry ice-baited "tent trap" was the most productive method for collecting numerous aedine and other mosquito species.

Laboratory transmission of Jamestown Canyon and snowshoe hare viruses (Bunyaviridae: California serogroup) by several species of mosquitoes.

The ability of 14 species of mosquitoes to biologically transmit Jamestown Canyon virus was tested. Four species not previously described as vectors of that virus transmitted to suckling mice. Among membrane-fed mosquitoes with disseminated infections, field-collected Aedes canadensis (1/3), Anopheles punctipennis (1/12), Coquillettidia perturbans (2/14) and a laboratory strain of Ae. epactius (19/67) transmitted virus. Two species were tested for their ability to transmit snowshoe hare virus: field-collected Ae. provocans (4/20) and Ae. abserratus-punctor (2/20) successfully transmitted to suckling mice. Evidence regarding the role of these species as field vectors is summarized.

Transmission of La Crosse virus by four strains of Aedes albopictus to and from the eastern chipmunk (Tamias striatus).

Eastern chipmunks were successfully infected with La Crosse virus by bites of 3 New World strains of Aedes albopictus infected orally or transovarially. The virus was subsequently passed from the chipmunks to Ae. albopictus, POTOSI strain, and Ae. triseriatus. The chipmunks developed viremias of 1-4 days duration and antibody titers were similar in intensity and duration to those reported in chipmunks infected by Ae. triseriatus. After feeding on viremic chipmunks, Ae. albopictus became infected and transmitted La Crosse virus at rates similar to the native vector, Ae. triseriatus. Aedes albopictus transmitted La Crosse virus transovarially to first gonotrophic cycle offspring.

Antibodies to La Crosse virus in eastern chipmunks in Indiana near an Aedes albopictus population.

Blood samples of 34 Eastern chipmunks trapped in the vicinity of an Aedes albopictus population near New Alsace, IN, were tested for neutralizing antibodies to La Crosse (LAC) virus and other California group viruses. Two samples were positive for LAC antibodies. Analysis of mosquito bloodmeals demonstrated that both Ae. albopictus and Ae. triseriatus from the site fed on chipmunks. This note documents the first record of LAC antibodies in sylvan rodents from Indiana, the presence of LAC virus in the vicinity of Ae. albopictus and that wild Ae. albopictus feed on Eastern chipmunks.

The oxidative corrosion of carbide inclusions at the surface of uranium metal during exposure to water vapour.

The reaction between uranium and water vapour has been well investigated, however discrepancies exist between the described kinetic laws, pressure dependence of the reaction rate constant and activation energies. Here this problem is looked at by examining the influence of impurities in the form of carbide inclusions on the reaction. Samples of uranium containing 600 ppm carbon were analysed during and after exposure to water vapour at 19 mbar pressure, in an environmental scanning electron microscope (ESEM) system. After water exposure, samples were analysed using secondary ion mass spectrometry (SIMS), focused ion beam (FIB) imaging and sectioning and transmission electron microscopy (TEM) with X-ray diffraction (micro-XRD). The results of the current study indicate that carbide particles on the surface of uranium readily react with water vapour to form voluminous UO(3) · xH(2)O growths at rates significantly faster than that of the metal. The observation may also have implications for previous experimental studies of uranium-water interactions, where the presence of differing levels of undetected carbide may partly account for the discrepancies observed between datasets.

A new detection system for extremely small vertically mounted cantilevers.

Detection techniques currently used in scanning force microscopy impose limitations on the geometrical dimensions of the probes and, as a consequence, on their force sensitivity and temporal response. A new technique, based on scattered evanescent electromagnetic waves (SEW), is presented here that can detect the displacement of the extreme end of a vertically mounted cantilever. The resolution of this method is tested using different cantilever sizes and a theoretical model is developed to maximize the detection sensitivity. The applications presented here clearly show that the SEW detection system enables the use of force sensors with sub-micron size, opening new possibilities in the investigation of biomolecular systems and high speed imaging. Two types of cantilevers were successfully tested: a high force sensitivity lever with a spring constant of 0.17 pN nm(-1) and a resonant frequency of 32 kHz; and a high speed lever with a spring constant of 50 pN nm(-1) and a resonant frequency of 1.8 MHz. Both these force sensors were fabricated by modifying commercial microcantilevers in a focused ion beam system. It is important to emphasize that these modified cantilevers could not be detected by the conventional optical detection system used in commercial atomic force microscopes.