Influence of solvent choice and operating conditions on Chlorzoxazone crystal shape and size

Abstract Solubility of pharmaceutical drugs in organic solvents is one of the important parameters to understand the equilibrium concentration of solute-solvent, which helps optimize and design crystallization conditions to obtain the desired product crystals. In the present study, Chlorzoxazone (CHZ) is used as a model pharmaceutical compound to investigate the equilibrium solubility, the influence of solvent and the operating conditions on the shape, and the size distribution. The solubility of CHZ is determined in organic solvents like Isopropanol, Ethanol, and 2-Ethoxyethylacetate, Ethylacetate and Ethyllactate using shake flask method from -5ºC to 60ºC. The solubility of CHZ in these solvents shows an increasing trend as the temperature increases in the following order: ethyllactate + water (0.5+0.5) < ethylacetate < isopropanol < ethanol < 2-ethoxyethylacetate < ethyllactate + water (0.75+0.25). The solvents, isopropanol, ethanol, and ethyl lactate, produce needle-shaped crystals, while 2-ethoxyethylacetate and ethyl acetate tend to produce plate shaped crystals. CHZ crystals obtained from 2-ethoxyethylacetate tend to have plate shaped crystals with a lower aspect ratio and are selected for batch cooling crystallization experiments performed at different cooling rates, and agitation. It is found that the agitation at 300 rpm and the cooling rate 0.2ºC/min produce more uniform crystal size distribution

Classification of biphasic solvent systems according to Abraham descriptors for countercurrent chromatography.

The method development of liquid-liquid chromatography, either countercurrent chromatography or centrifugal partition chromatography, is slowed down by the selection of the biphasic solvent system that constitutes its column. This paper introduces a classification of 19 solvent systems, including the most popular systems based on heptane/ethyl acetate/methanol/water, some non-aqueous systems and some greener systems. This classification is based on Abraham descriptors determined through the partition coefficients of 43 probes. Among 21 determined models, nine of them allow an accurate prediction of partition coefficients from solute descriptors and another ten provide a description of the chromatographic interactions at the 5% significance level. A graphical tool (spider diagram) is built for the comparison of the chromatographic columns previously characterized with the solvation parameter model. The position of a solvent system in this spider diagram relates to the interactions at stake, thus the selection of columns offering similar or orthogonal interactions is facilitated, with no previous knowledge of the solute required. This semi-empirical strategy cannot fully predict the retention behavior but can judiciously orientate the user towards a limited number of solvent systems to be experimentally tested.

Multi-dimensional in vitro bioactivity profiling for grouping of glycol ethers.

High-content screening data derived from physiologically-relevant in vitro models promise to improve confidence in data-integrative groupings for read-across in human health safety assessments. The biological data-based read-across concept is especially applicable to bioactive chemicals with defined mechanisms of toxicity; however, the challenge of data-derived groupings for chemicals that are associated with little or no bioactivity has not been explored. In this study, we apply a suite of organotypic and population-based in vitro models for comprehensive bioactivity profiling of twenty E-Series and P-Series glycol ethers, solvents with a broad variation in toxicity ranging from relatively non-toxic to reproductive and hematopoetic system toxicants. Both E-Series and P-Series glycol ethers elicited cytotoxicity only at high concentrations (mM range) in induced pluripotent stem cell-derived hepatocytes and cardiomyocytes. Population-variability assessment comprised a study of cytotoxicity in 94 human lymphoblast cell lines from 9 populations and revealed differences in inter-individual variability across glycol ethers, but did not indicate population-specific effects. Data derived from various phenotypic and transcriptomic assays revealed consistent bioactivity trends between both cardiomyocytes and hepatocytes, indicating a more universal, rather than cell-type specific mode-of-action for the tested glycol ethers in vitro. In vitro bioactivity-based similarity assessment using Toxicological Priority Index (ToxPi) showed that glycol ethers group according to their alcohol chain length, longer chains were associated with increased bioactivity. While overall in vitro bioactivity profiles did not correlate with in vivo toxicity data on glycol ethers, in vitro bioactivity of E-series glycol ethers were indicative of and correlated with in vivo irritation scores.

Tuning Alginate-Gelatin Bioink Properties by Varying Solvent and Their Impact on Stem Cell Behavior.

Bioink optimization is considered as one of main challenges in cell-laden 3D bioprinting. Alginate-Gelatin (Alg-Gel) hydrogel have been extensively used as bioink. However, its properties could be influenced by various parameters, and little is known about the evidence featuring the impact of solvent. Here we investigated four Alg-Gel bioink by varying solvent ionic strength (named B-1, B-2, B-3 and B-4). Mechanical properties and printability of bioink samples and their impacts on behaviors of encapsulated epidermal stem cells (ESCs) were tested. Bioink with increased ionic strength of solvent showed decreased stiffness and viscosity, and increased swelling and degradation by printability and mechanical property tests. Due to the increased swelling and degradation was associated with shape-maintenance of post-printing constructs, B-3 and B-4 were hardly observable after 14 days. Cellular behaviors were assessed through viability, proliferation, aggregation and differentiation tests. B-2 with optimal properties resulted in higher viability and proliferation of ESCs, and further facilitated cellular aggregation and lineage differentiation. We demonstrated that the solvent can be tuned by ionic strength to control the properties of Alg-Gel bioink and post-printing constructs, which represented a promising avenue for promotion of therapeutic stem cell behaviors in 3D bioprinting.

Pre-selection and assessment of green organic solvents by clustering chemometric tools.

The study presents the result of the application of chemometric tools for selection of physicochemical parameters of solvents for predicting missing variables - bioconcentration factors, water-octanol and octanol-air partitioning constants. EPI Suite software was successfully applied to predict missing values for solvents commonly considered as "green". Values for logBCF, logKOW and logKOA were modelled for 43 rather nonpolar solvents and 69 polar ones. Application of multivariate statistics was also proved to be useful in the assessment of the obtained modelling results. The presented approach can be one of the first steps and support tools in the assessment of chemicals in terms of their greenness.

The Influence of Different Solvents on Systemic Sclerosis: An Updated Meta-analysis of 14 Case-Control Studies.

BACKGROUND: Several studies have collected detailed data to examine which specific solvents account for the association between solvents and risk of systemic sclerosis (SSc). These studies generally reported elevated risks associated with many of the specific solvents examined, such as toluene, xylene, and trichloroethylene. The previous meta-analysis was not able to conduct separate analyses for specific solvent subtypes. OBJECTIVE: The aims of the new meta-analysis were to investigate a more comprehensive estimate and to consider the effect of different solvents on SSc. METHODS: We searched PubMed, Biosis Previews, China National Knowledge Infrastructure, and Wanfang for all articles published before July 2015. Fourteen case-control studies (1657 patients and 3838 controls) were included. The quality of studies was scored according to the Newcastle-Ottawa scale. The final odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by a fixed- or random-effects model according to heterogeneity test. Publication bias was assessed using Begg test. RESULTS: The risk of SSc was significantly different among sex, age, and exposure assessment methods. Separate analyses for specific solvent subtypes indicated that SSc was associated with aromatic solvents (OR, 2.72; 95% CI, 1.21-6.09), trichloroethylene (OR, 2.07; 95% CI, 1.34-3.17), halogenated solvents (OR, 1.49; 95% CI, 1.12-1.99), and ketones (OR, 4.20; 95% CI, 2.19-8.06). CONCLUSIONS: Exposure to identified types solvents does seem to be a risk factor for developing SSc. Needed efforts to decrease such exposures are discussed.

Hydration Properties and Solvent Effects for All-Atom Solutes in Polarizable Coarse-Grained Water.

Due to the importance of water in chemical and biological systems, a coarse-grained representation of the solvent can greatly simplify the description of the system while retaining key thermodynamic properties of the medium. A multiscale solvation model that couples all-atom solutes and polarizable Martini coarse-grained water (AAX/CGS) is developed to reproduce free energies of hydration of organic solutes. Using Monte Carlo/free energy perturbation (MC/FEP) calculations, results from multiscale and all-atom simulations are compared. Improved accuracy is obtained with the AAX/CGS approach for hydrophobic and sulfur- or halogen-containing solutes, but larger deviations are found for polar solute molecules where hydrogen bonding is featured. Furthermore, solvent effects on conformational and tautomeric equilibria of AA solutes were investigated using AA, CG, and GB/SA solvent models. It is found that the CG solvent model can reproduce well the medium effects from experiment and AA simulations; however, the GB/SA solvent model fails in some cases. A 7-30-fold reduction in computational cost is found for the present AAX/CGS multiscale simulations compared to the AA alternative.

A dopant for improved sensitivity in easy ambient sonic-spray ionization mass spectrometry.

Recently, 3-nitrobenzonitrile (3-NBN) has been used to improve sensitivity of sonic-spray ionization mass spectrometry. Easy ambient sonic-spray ionization (EASI) is one of the simplest, gentlest and most used spray-based desorption/ionization ambient techniques, but limited sensitivity has been commonly taken as its major drawback. Herein we investigate the use of 3-NBN as a dopant in EASI-MS for improved sensitivity. Using a few typical EASI samples as test cases, the presence of 10 ppm (µg ml(-1) ) of 3-NBN in the spray solvent showed two to fourfold gains in EASI-MS sensitivity as measured both by total ion current and S/N ratios, accompanied with significant reductions in chemical noise. Sensitivity for DESI using 3-NBN as a dopant also improved and dopant DESI versus dopant EASI sensitivities were compared. The use of solvent dopants seems therefore to be a promising strategy to improve sensitivity for spray-based ambient MS techniques. Copyright © 2015 John Wiley & Sons, Ltd.

Natural Origin Lycopene and Its "Green" Downstream Processing.

Lycopene is an abundant natural carotenoid pigment with several biological functions (well-known for its antioxidant properties) which is under intensive investigation in recent years. Lycopene chemistry, its natural distribution, bioavailability, biological significance, and toxicological effects are briefly outlined in the first part of this review. The second, major part, deals with various modern downstream processing techniques, which are assessed in order to identify promising approaches for the recovery of lycopene and of similar lipophilic compounds. Natural lycopene is synthesized in plants and by microorganisms, with main representatives of these two categories (for industrial production) tomato and its by-products and the fungus Blakeslea trispora, respectively. Currently, there is a great deal of effort to develop efficient downstream processing for large scale production of natural-origin lycopene, with trends strongly indicating the necessity for "green" and mild extraction conditions. In this review, emphasis is placed on final product safety and ecofriendly processing, which are expected to totally dominate in the field of natural-origin lycopene extraction and purification.

Characterization of the toxicological hazards of hydrocarbon solvents.

Hydrocarbon solvents are liquid hydrocarbon fractions derived from petroleum processing streams, containing only carbon and hydrogen atoms, with carbon numbers ranging from approximately C5-C20 and boiling between approximately 35-370°C. Many of the hydrocarbon solvents have complex and variable compositions with constituents of 4 types, alkanes (normal paraffins, isoparaffins, and cycloparaffins) and aromatics (primarily alkylated one- and two-ring species). Because of the compositional complexity, hydrocarbon solvents are now identified by a nomenclature ("the naming convention") that describes them in terms of physical/chemical properties and compositional elements. Despite the compositional complexity, most hydrocarbon solvent constituents have similar toxicological properties, and the overall toxicological hazards can be characterized in generic terms. To facilitate hazard characterization, the solvents were divided into 9 groups (categories) of substances with similar physical and chemical properties. Hydrocarbon solvents can cause chemical pneumonitis if aspirated into the lung, and those that are volatile can cause acute CNS effects and/or ocular and respiratory irritation at exposure levels exceeding occupational recommendations. Otherwise, there are few toxicologically important effects. The exceptions, n-hexane and naphthalene, have unique toxicological properties, and those solvents containing constituents for which classification is required under the Globally Harmonized System (GHS) are differentiated by the substance names. Toxicological information from studies of representative substances was used to fulfill REACH registration requirements and to satisfy the needs of the OECD High Production Volume (HPV) initiative. As shown in the examples provided, the hazard characterization data can be used for hazard classification and for occupational exposure limit recommendations.

Comparison of sensor characteristics of three real-time monitors for organic vapors.

OBJECTIVE: Sensor characteristics and performance of three real-time monitors for volatile organic compounds (VOC monitor) equipped with a photo ionization detector (PID), a sensor using the interference enhanced reflection (IER) method and a semiconductor gas sensor were investigated for 52 organic solvent vapors designated as class 1 and class 2 of organic solvents by the Ordinance of Organic Solvent Poisoning Prevention in Japan. METHODS: Test vapors were prepared by injecting each liquid solvent into a 50 l Tedlar® bag and perfectly vaporizing it. The vapor concentration was from one-tenth to twice the administrative control level for all solvents. The vapor concentration was measured with the monitors and a gas chromatograph equipped with a flame ionization detector simultaneously, and the values were compared. RESULTS: The monitor with the PID sensor could measure many organic vapors, but it could not detect some vapors with high ionization potential. The IER sensor could also detect many vapors, but a linear response was not obtained for some vapors. A semiconductor sensor could detect methanol that could not be detected by PID and IER sensors. CONCLUSIONS: Working environment measurement of organic vapors by real-time monitors may be possible, but sensor characteristics and their limitations should be known.

[Extraction of flutamide from aqueous solutions].

The objective of the present study was to elucidate peculiarities of flutamide extraction from aqueous solutions using he liquid-liquid extraction technique and spectrophotometry in the visible spectrum. The results of flutamide extraction were shown to depend on the chemical nature of the extractant, pH of the aqueous phase as well as its saturation with water and electrolytes. The study has demonstrated that the optimal conditions for flutamide extraction by ethylacetate are created by using the aqueous phase with the pH values in the range from 1.0 to 12.0. We have calculated extraction factor necessary for the luberation of the desired amount of flutamide from the aqueous solutions with the use of a concrete solvent.

Grouping of residual solvents present in pharmaceuticals using experimental planning and chemometric methods.

The main effects of six experimental factors on the efficiency of HS (headspace) extraction in headspace gas chromatography--flame ionization detector (HS-GC-FID) determination of twenty organic solvents routinely used in production of pharmaceuticals were obtained on the basis of the results of experiments carried out according to the Plackett-Burman factorial design. The effects were used as a basis for grouping the solvents into five groups, the solvents belonging to a group responded similarly to changes of HS conditions. To this end, visualization approaches were used as well as chemometric methods: cluster analysis (CA) and principal component analysis (PCA). Moreover, the most important HS experimental factors were selected for further optimization of the HS-GC determination procedure.

Use of organic solvents in large research institutions in Japan.

OBJECTIVES: Laboratories in research institutions use organic solvents in research and development. Nevertheless, the types of solvents in use have been seldom reported. This study was initiated to elucidate types of organic solvents used in large research institutions in Japan, with a focus on possible different use among research fields. METHODS: In 2010-2011, 4517 laboratories in seven large research institutions were visited. In accordance with legal stipulations, air in each laboratory was collected in polyvinyl fluoride bags and analyzed by direct injection into a gas-chromatograph for 47 types of organic solvents. In evaluation, the laboratories were grouped by 5 research fields, i.e., agriculture, biology, medicine, natural science, and technology and engineering. RESULTS: Types of organic solvents commonly used in research activities were not diverse. Those commonly used were chloroform and 1,2-dichloroethane out of 7 Group 1 organic solvents (with high toxicities); 6 organic solvents, i.e., acetone and methyl alcohol in general, ethyl acetate, hexane and toluene in technology and engineering laboratories; and xylenes in medical fields out of 40 Group 2 organic solvents (with relatively low toxicities). Judging from solvent vapor concentrations, work environments in more than 99 % of laboratories were considered adequate. Nevertheless, use of chloroform in high-performance liquid chromatography (HPLC) resulted in inadequate environments in 30 laboratories (0.7 %). CONCLUSIONS: Organic solvents commonly used were not very diverse. Work environments in research laboratories were generally good, but the environment with use of chloroform in HPLC analysis remained yet to be improved.

Improved dissolution and micromeritic properties of naproxen from spherical agglomerates: preparation, in vitro and in vivo characterization

Naproxen, an anti-inflammatory drug, exhibits poor aqueous solubility, which limits the pharmacological effects. The present work was carried out to study the effect of agglomeration on micromeritic properties and dissolution. Naproxen agglomerates were prepared by using a three solvents system composed of acetone (good solvent), water (non-solvent) and dichloromethane (bridging liquid). Differential Scanning Calorimetry (DSC) results showed no change in the drug after crystallization process. X-Ray Powder Diffraction (XRPD) studies showed the sharp peaks are present in the diffractograms of spherical agglomerates with minor reduction in height of the peaks. The residual solvents are largely below the tolerated limits in the agglomerates. Scanning Electronic Microscopy (SEM) studies showed that agglomerates were spherical in structure and formed by cluster of small crystals. The agglomerates exhibited improved solubility, dissolution rate and micromeritic properties compared to pure drug. Anti-inflammatory studies were conducted in Wistar strain male albino rats and naproxen agglomerates showed more significant activity than the pure drug.

Naproxeno, fármaco anti-inflamatório, apresenta baixa solubilidade em água, o que limita os efeitos farmacológicos. O presente trabalho foi realizado para estudar o efeito da aglomeração nas propriedades micromeríticas e na dissolução. Aglomerados de naproxeno foram preparados por meio da utilização de sistema de três solventes composto de acetona (bom solvente), água (não-solvente) e diclorometano (líquido de ligação). A DSC não resulta mostrou nenhuma mudança na droga depois de processo de cristalização. Estudos de difração de Raios X do Pó (XRPD) mostraram picos agudos nos difratogramas de aglomerados esféricos, com redução mínima dea altura dos picos. Os solventes residuais estão amplamente abaixo dos limites tolerados nos aglomerados. Os estudos de Microscopia Eletrônica de Varredura (SEM) mostraram que esses aglomerados eram de estrutura esférica e formados por grupos de pequenos cristais. Os aglomerados apresentaram solubilidade, taxa de dissolução e propriedades micromeríticas aprimoradas em comparação com o fármaco puro. Estudos anti-inflamatórios foram conduzidos em ratos Wistar albinos masculinos e os aglomerados de naproxeno mostraram atividade mais significativa do que o fármaco puro.

Sulfur, oxygen, and nitrogen mustards: stability and reactivity.

Mustard gas, bis(ß-chloroethyl) sulfide (HD), is highly toxic and harmful to humans and the environment. It comprises one class of chemical warfare agents (CWAs) that was used in both World Wars I and II. The three basic analogues or surrogates are: the monochloro derivative, known as the half mustard, 2-chloroethyl ethyl sulfide (CEES); an oxygen analogue, bis(ß-chloroethyl) ether (BCEE); and several nitrogen analogues based on the 2,2'-dichlorodiethylamine framework (e.g., HN1, HN2, and HN3). The origin of their toxicity is considered to be from the formation of three-membered heterocyclic ions, a reaction that is especially accelerated in aqueous solution. The reaction of these cyclic ion intermediates with a number of important biological species such as DNA, RNA and proteins causes cell toxicity and is responsible for the deleterious effects of the mustards. While a number of studies have been performed over the last century to determine the chemistry of these compounds, early studies suffered from a lack of more sophisticated NMR and X-ray techniques. It is now well-established that the sulfur and nitrogen mustards are highly reactive in water, while the oxygen analog is much more stable. In this study, we review and summarize results from previous studies, and add results of our own studies of the reactivity of these mustards toward various nonaqueous solvents and nucleophiles. In this manner a more comprehensive evaluation of the stability and reactivity of these related mustard compounds is achieved.

Therapeutic effects of various solvent fractions of Alstonia boonei (apocynaceae) stem bark on Plasmodium berghei-induced malaria.

Malaria, the most important parasitic disease afflicting man is the leading cause of mortality and morbidity in the world. Chemotherapy remains the mainstay for the treatment and prevention of the disease in the absence of an effective vaccine. The incidence of resistance of malaria parasites to chemotherapy is increasing and complicated. This study was therefore undertaken in order to evaluate the therapeutic effects of fractions of the stem bark of A. boonei on P. berghei-induced malaria using chloroquine as control. Different doses (200 mg/kg and 400 mg/kg body weight) of methanolic extract (ME), n-hexane (HF), chloroform (CF), ethylacetate(EF) and aqueous (AF) fractions of the stem bark of A. boonei were administered orally to albino mice. Five milligrammes chloroquine base per kilogramme body weight (5 mg/kg bw) was used as positive control while the negative control mice received only the vehicle (5% v/v tween 80). The results obtained showed that the 400 mg/kg bw dose was more effective with respect to the parasite clearance than the 200 mg/kg bw dose. The 400 mg/kg bw dose of ME gave 68.1% percent parasite clearance. The CF gave the highest clearance of 98.4% at 400 mg/kg bw after 7 days treatment while chloroquine at 5 mg/kg bw gave 100% parasite clearance. The order of increasing potency of the fractions (parasite clearance) was (EF 50.0% < AF 60.3% < HF 63.1%, < CF 98.4%) indicating that the active principle in the stem bark was highest in the CF. Percentage parasitemia following exposure to these fractions also decreased in all groups in the same order and was only significant (p < 0.05) in CF (0.11%) compared to the untreated control group. The ME of A. boonei also caused increase in PCV by 15.5%. Purification enhanced PCV value as the HF and CF fractions gave 19.0% and 24.5% increases, respectively. However, 31.5% increase in PCV was obtained in the albino mice treated with chloroquine. The EF and AF gave increase of 10.0% and 11.0% increase relative to the negative control treated mice. The high bioactivity of CF and HF indicate that the putative compound(s) in A. boonei are lipophillic and further purification could enhance greater activity. Further work is required to isolate the bioactive compound for a promising antimalarial drug from the chloroform fraction.

Toxicity ranking and toxic mode of action evaluation of commonly used agricultural adjuvants on the basis of bacterial gene expression profiles.

The omnipresent group of pesticide adjuvants are often referred to as "inert" ingredients, a rather misleading term since consumers associate this term with "safe". The upcoming new EU regulation concerning the introduction of plant protection products on the market (EC1107/2009) includes for the first time the demand for information on the possible negative effects of not only the active ingredients but also the used adjuvants. This new regulation requires basic toxicological information that allows decisions on the use/ban or preference of use of available adjuvants. In this study we obtained toxicological relevant information through a multiple endpoint reporter assay for a broad selection of commonly used adjuvants including several solvents (e.g. isophorone) and non-ionic surfactants (e.g. ethoxylated alcohols). The used assay allows the toxicity screening in a mechanistic way, with direct measurement of specific toxicological responses (e.g. oxidative stress, DNA damage, membrane damage and general cell lesions). The results show that the selected solvents are less toxic than the surfactants, suggesting that solvents may have a preference of use, but further research on more compounds is needed to confirm this observation. The gene expression profiles of the selected surfactants reveal that a phenol (ethoxylated tristyrylphenol) and an organosilicone surfactant (ethoxylated trisiloxane) show little or no inductions at EC(20) concentrations, making them preferred surfactants for use in different applications. The organosilicone surfactant shows little or no toxicity and good adjuvant properties. However, this study also illustrates possible genotoxicity (induction of the bacterial SOS response) for several surfactants (POEA, AE, tri-EO, EO FA and EO NP) and one solvent (gamma-butyrolactone). Although the number of compounds that were evaluated is rather limited (13), the results show that the used reporter assay is a promising tool to rank commonly used agricultural adjuvants based on toxicity and toxic mode of action data.

Assessing the human carcinogenic potential of tetrahydrofuran: I. Mode of action and human relevance analysis of the male rat kidney tumor.

In 1998, the National Toxicology Program concluded that inhalation exposure to tetrahydrofuran resulted in increased incidences of renal adenomas and carcinomas (combined) in male F344 rats and of hepatocellular adenomas and carcinomas (combined) in female B6C3F1 mice. In the present paper, the bioassay results and additional information are evaluated using the IPCS/ILSI Mode of Action/Human Relevance Framework to determine if the data are sufficient to describe the possible mode(s) of action (MOA) underlying the reported results for the rat renal tumor and to determine if any of these modes of action could be operative in humans. Preliminary analysis of the rat renal tumor data and related information suggested that a MOA could be described, but questions remained concerning the role that chronic progressive nephropathy (CPN) may play in the development of the lesions. In 2009, a Pathology Working Group concluded that the rat renal lesions resulted primarily from regenerative processes associated with advanced CPN. The renal tumor finding is considered not relevant to humans and should not be considered in any further risk assessment efforts on this chemical. A companion paper describes a similar analysis of the female mouse liver tumor finding.

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.