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Following adoption of the new OECD test guideline (TG) 474 for the in vivo mammalian erythrocyte micronucleus (MN) test (29 July 2016), demonstration of exposure of target tissue (bone marrow) is required, if the test result is negative i.e. no cytogenetic damage. It implies that for many active ingredients, relevant metabolites or significant impurities with existing in vivo MN tests resulting in negative genotoxicity findings, evidence of target tissue exposure may be lacking and is considered a data gap in regulatory reviews. We present here toxicokinetic (TK) testing strategies for the design and conduct of studies that would demonstrate evidence of delivery of the test substance to the bone marrow. To illustrate this, three examples are presented with methods utilized under each scenario. We also propose a decision tree that may help design suitable TK studies to establish evidence of bone marrow exposure.

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Bifenthrin, a pyrethroid insecticide, undergoes oxidative metabolism leading to the formation of 4'-hydroxy-bifenthrin (4'-OH-BIF) and hydrolysis leading to the formation of TFP acid in rat and human hepatic microsomes. In this study, age-dependent metabolism of bifenthrin in rats and humans were determined via the rates of formation of 4'-OH-BIF and TFP acid following incubation of bifenthrin in juvenile and adult rat (PND 15 and PND 90) and human (<5years and >18years) liver microsomes. Furthermore, in vitro hepatic intrinsic clearance (CLint) of bifenthrin was determined by substrate consumption method in a separate experiment. The mean Vmax(±SD) for the formation of 4'-OH-BIF in juvenile rat hepatic microsomes was 25.0±1.5pmol/min/mg which was significantly lower (p<0.01) compared to that of adult rats (86.0±17.7pmol/min/mg). However, the mean Km values for juvenile (19.9±6.6µM) and adult (23.9±0.4µM) rat liver microsomes were similar. On the other hand, in juvenile human hepatic microsomes, Vmax for the formation of 4'-OH-BIF (73.9±7.5pmol/min/mg) was significantly higher (p<0.05) than that of adults (21.6±0.6pmol/min/mg) albeit similar Km values (10.5±2.8µM and 8.9±0.6µM) between the two age groups. The trends in the formation kinetics of TFP acid were similar to those of 4'-OH-BIF between the species and age groups, although the differences between juveniles and adults were less pronounced. The data also show that metabolism of bifenthrin occurs primarily via oxidative pathway with relatively lesser contribution (~30%) from hydrolytic pathway in both rat and human liver microsomes. The CLint values for bifenthrin, determined by monitoring the consumption of substrate, in juvenile and adult rat liver microsomes fortified with NADPH were 42.0±7.2 and 166.7±20.5µl/min/mg, respectively, and the corresponding values for human liver microsomes were 76.0±4.0 and 21.3±1.2µl/min/mg, respectively. The data suggest a major species difference in the age dependent metabolism of bifenthrin. In human liver microsomes, bifenthrin is metabolized at a much higher rate in juveniles than in adults, while the opposite appears to be true in rat liver microsomes.

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The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [14C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [14C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 h of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes.

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The present study examined the bioconcentration of 2 basic pharmaceuticals: verapamil (a calcium channel blocker) and clozapine (an antipsychotic compound) in 2 fresh water fishes, fathead minnow and channel catfish. In 4 separate bioconcentration factor (BCF) experiments (2 chemicals × 1 exposure concentration × 2 fishes), fathead minnow and channel catfish were exposed to 190 µg/L and 419 µg/L of verapamil (500 µg/L nominal) or 28.5 µg/L and 40 µg/L of clozapine (50 µg/L nominal), respectively. Bioconcentration factor experiments with fathead consisted of 28 d uptake and 14 d depuration, whereas tests conducted on catfish involved a minimized test design, with 7 d each of uptake and depuration. Fish (n = 4-5) were sampled during exposure and depuration to collect different tissues: muscle, liver, gills, kidneys, heart (verapamil tests only), brain (clozapine tests only), and blood plasma (catfish tests only). Verapamil and clozapine concentrations in various tissues of fathead and catfish were analyzed using liquid chromatography-mass spectrometry. In general, higher accumulation rates of the test compounds were observed in tissues with higher perfusion rates. Accumulation was also high in tissues relevant to pharmacological targets in mammals (i.e. heart in verapamil test and brain in the clozapine test). Tissue-specific BCFs (wet wt basis) for verapamil and clozapine ranged from 0.7 to 75 and from 31 to 1226, respectively. Tissue-specific concentration data were used to examine tissue-blood partition coefficients.

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Solid bases, such as SBA-15-oxynitrides, have attracted considerable interest for potential applications as catalysts in important industrial processes. Reported herein is that by simply tuning the temperature of nitridation (ammonolysis), the catalytic activity of these solid bases can be enhanced. Solid-state NMR spectroscopy and XPS studies provided the reasoning behind this change in activity.

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The vaccination planning tool for avian influenza supports evidence-based planning and preparedness for vaccinating poultry at national and regional levels. This study describes the development, testing, and application of a vaccination planning tool for H5N1 highly pathogenic avian influenza (HPAI) used in two South Asian countries. The tool consists of eight planning clusters, 37 planning elements, and 303 referenced planning criteria. Both countries attained a score of 52% among planning clusters as a measure of preparedness. The highest and lowest planning cluster scores included vaccination strategies and financial readiness, respectively. The comprehensive vaccination program was identified as the most-useful planning cluster for assessing preparedness, and 86% of participants indicated that the objectives of the planning tool were achieved. Based on these results, the planning tool provides a structured approach for decision makers to develop their national vaccination program for HPAI as part of an overall strategy for the progressive reduction and control of endemic influenza viruses in poultry.

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The environmental presence of the oral contraceptive norethindrone (NET) has been reported and shown to have reproductive effects in fish at environmentally realistic exposure levels. The current study examined bioconcentration potential of NET in fathead minnow (Pimephales promelas) and channel catfish (Ictalurus punctatus). Fathead minnows were exposed to 50 µg/l NET for 28 days and allowed to depurate in clean water for 14 days. In a minimized 14-day test design, catfish were exposed to 100 µg/l NET for 7 days followed by 7-day depuration. In the fathead test, tissues (muscle, liver, and kidneys) were sampled during the uptake (days 1, 3, 7, 14, and 28) and depuration (days 35 and 42) phases. In the catfish test, muscle, liver, gill, brain, and plasma were collected during the uptake (days 1, 3, and 7) and depuration (day 14) stages. NET tissue levels were determined by gas chromatography-mass spectrometry (GC-MS). Accumulation of NET in tissues was greatest in liver followed by plasma, gill, brain, and muscle. Tissue-specific bioconcentration factors (BCFs) ranged from 2.6 to 40.8. Although NET has been reported to elicit reproductive effects in fish, the present study indicated a low potential to bioconcentrate in aquatic biota.

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Pharmaceutical products and their metabolites are being widely detected in aquatic environments and there is a growing interest in assessing potential risks of these substances to fish and other non-target species. Ibuprofen is one of the most commonly used analgesic drugs and no peer-reviewed laboratory studies have evaluated the tissue specific bioconcentration of ibuprofen in fish. In the current study, fathead minnow (Pimephales promelas) were exposed to 250 µg L(-1) ibuprofen for 28 d followed by a 14 d depuration phase. In a minimized bioconcentration test design, channel catfish (Ictalurus punctatus) were exposed to 250 µg L(-1) for a week and allowed to depurate for 7 d. Tissues were collected during uptake and depuration phases of each test and the corresponding proportional and kinetic bioconcentration factors (BCFs) were estimated. The results indicated that the BCF levels were very low (0.08-1.4) implying the lack of bioconcentration potential for ibuprofen in the two species. The highest accumulation of ibuprofen was observed in the catfish plasma as opposed to individual tissues. The minimized test design yielded similar bioconcentration results as those of the standard test and has potential for its use in screening approaches for pharmaceuticals and other classes of chemicals.

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Legal, procedural, and institutional restrictions on safe abortion services-such as laws forbidding the practice or policies preventing donors from supporting groups who provide legal services-remain a major access barrier for women worldwide. However, even when abortion services are legal, women face social and cultural barriers to accessing safe abortion services and preventing unwanted pregnancy. Interpersonal communication interventions play an important role in overcoming these obstacles, including as part of broad educational- and behavioral-change efforts. This article presents results from an interpersonal communication behavior change pilot intervention, Dialogues for Life, undertaken in Nepal from 2004 to 2006, after abortion was legalized in 2002. The project aimed to encourage and enable women to prevent unplanned pregnancies and unsafe abortions and was driven by dialogue groups and select community events. The authors' results confirm that a dialogue-based interpersonal communication intervention can help change behavior and that this method is feasible in a low-resource, low-literacy setting. Dialogue groups play a key role in addressing sensitive and stigmatizing health issues such as unsafe abortion and in empowering women to negotiate for the social support they need when making decisions about their health.

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We examined the acute effects of triclosan (TCS) exposure, a common antimicrobial found as a contaminant in the field, on survival and physiology of amphibian larvae. LC50 values were determined after 96h for North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a developmental model: Xenopus laevis. Amphibian larvae were most sensitive to TCS exposure during early development based upon 96-h LC50 values. Heart rates for X. laevis and North American larvae exposed to TCS were variable throughout development. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to TCS were significantly affected in larvae exposed to [50% LC50] and [LC50]. Tissue uptake and tissue bioconcentration factor (BCF) of TCS were investigated in X. laevis, B. woodhousii woodhousii, and R. sphenocephala. In general, a significant increase was observed as exposure concentration increased. Tissue BCF values were dependent upon stage and species. While TCS concentrations used here are higher than environmental concentrations, exposure to TCS was dependent upon species and developmental stage, with early developmental stages being most sensitive to TCS exposure.

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Pancreas of Beagle dogs deriving from 166 male and 166 female animals were examined microscopically and were found to show nesidioblastosis-like structural alteration in 29 dogs. The lesion was represented by endocrine and ductular epithelial cell proliferation. The incidence profile and the severity of the changes observed were closely associated with the age of the dogs, younger dogs being more often and more seriously affected than older dogs. No link with altered insulin function has been established as serum glucose levels were found to be within the normal range. The pathology of spontaneous extra-islet endocrine cell proliferation in the young Beagle dogs, described in this study, has some similarities to that of nesidioblastosis of human neonates and infants.

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The lymphoid organs/tissues are widely distributed throughout the body. However, a study of the primary and secondary lymphoid organs provides an adequate assessment of immune status. A histopathological approach to investigate immunotoxicity of various agents is described. The advantages and limitations of this approach are discussed. The usefulness of immunocytochemistry as an aid to study the various sub-populations of lymphocytes are stressed. Histopathological assessments are useful in evaluating immunosuppressive responses. Their value is somewhat less obvious in studying the potentials of test compounds for allergic and hypersensitive reactions. Variable factors affecting the structure and function of lymphoid organs such as stress, nutrition and age are discussed. Pathological lesions produced by various agents in lymphoid organs such as thymus, bone marrow, spleen, and lymph nodes are described.

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Oncogenicity studies of ramosetron ((R)-5-[(1-methyl-3-indolyl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazol e hydrochloride, CAS 132907-72-3, YM060), a new compound having serotonin (5-HT)3 receptor antagonist activity, were carried out in male and female mice and rats. Six groups (two control and four treated) of B6C3F1 mice and F344 rats were given YM060, dissolved in distilled water, once daily by oral intubation at doses of 0, 1, 10, 30 and 100 mg/kg/d. Toxicokinetics indicated that sufficient exposure of the animals to the test material was achieved during the oncogenicity studies. Cmax and AUC of YM060 at 100 mg/kg/d were in the range of 3-5 micrograms/ml and 8 micrograms.h/ml in mice, 1-5 micrograms/ml and 7-16 micrograms.h/ml in rats, respectively. The administration of YM060 resulted in a slightly increased mortality rate among female rats treated with 30 or 100 mg/kg/d, particularly during the Weeks 38-87. Body weights of the high-dosed male and female rats during the Weeks 36 to 96 were significantly decreased when compared to controls. An approximately 30% suppression of body weight gain was recorded during Weeks 36-96 for both male and female rats, and 15% suppression of body weight gain was recorded during Weeks 0-104 for male mice. There was no evidence of a treatment-related effect on the incidence of any tumor or tumor type, and there were no non-neoplastic findings considered to be related to the administration of YM060. All microscopic changes seen in mice and rats were of the usual type commonly occurring in untreated aged B6C3F1 mice and F344 rats. In conclusion, there was no evidence of an oncogenic effect of YM060 in mice and rats.

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A repeated dose toxicity study of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was conducted in beagle dogs. The dogs were given the drug intravenously for 4 weeks at doses of 0 (control), 0.0002, 0.002, 0.02, 0.2, 2 and 20 mg/kg in males and 0, 0.2, 2 and 20 mg/kg in females. After discontinuation of the treatment, a 4-week recovery test was also conducted in the 0 and 20 mg/kg groups. No deaths related to the treatment were observed. There were no changes in body weight gain, and food and water consumptions. Nasal discharge was seen in all dose groups. Salivation, emesis and hypoactivity were observed in the 0.2 mg/kg group and over. Licking chops were seen in the 2 and 20 mg/kg groups. Trembling and agitated/restless behavior were seen in the 20 mg/kg group. Electrocardiographic examination revealed elevated heart rate in the 0.2 mg/kg group and over. Ophthalmoscopic and hematologic examinations, and urinalysis failed to show any abnormalities attributable to the treatment. Blood chemical examination disclosed increases in T3 level in the 2 and 20 mg/kg groups of males and in T4 level in the 0.2 mg/kg group and over of males. There were no pathological findings attributable to the treatment. The changes mentioned above were satisfactorily reversible. The nasal discharge seen in the 0.02 mg/kg group and below was considered to be of no toxicological significance. These results show that the NOAEL of montirelin hydrate is 0.02 mg/kg for 4-week repeated dose toxicity in dogs.

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A 52-wk toxicity study by dietary administration was performed in Sprague-Dawley rats and in pure-bred beagle dogs with beta-cyclodextrin, a starch derivative that acts as a molecular inclusion agent. Doses of 0 (control), 12,500, 25,000 and 50,000 ppm were selected for the rat study, and 0 (control), 6200, 12,500 and 50,000 ppm were selected for the dog study. The liver and kidney were identified at the histopathological examination as target organs for toxicity in the rat at doses of 50,000 and 25,000 ppm, with the hepatic changes associated with increased plasma liver enzyme and reduced plasma triglyceride concentrations. In the dog study, there was no pathological evidence of systemic toxicity, although there were minor changes in urinalysis and biochemical parameters and a slightly higher incidence of liquid faeces. These changes were considered to be of no toxicological importance. The results in these studies, therefore, indicate that the non-toxic effect level was 12,500 ppm in the rat (equivalent to 654 or 864 mg/kg/day for males or females, respectively) and 50,000 ppm in the dog (equivalent to 1831 or 1967 mg/kg/day for males or females, respectively).

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A 52-week toxicity study by oral gavage administration was performed in Sprague-Dawley rats with nefiracetam (N-(2,6-dimethylphenyl)-2-(2-oxo-1-pyrrolidinyl) acetamide, DM-9384, CAS 77191-36-7), a new cognition-enhancing agent, as a part of a safety evaluation program. Dosages of 0 (control), 10, 30, 100 and 300 mg/kg/d were selected for this study. Treatment-related findings were confined to the 300 mg/kg/d level and, to a lesser extent, the 100 and 30 mg/kg/d levels, with the investigations indicating the kidney as the main target organ for toxicity. The microscopic pathology examination of this organ showed papillary epithelial hyperplasia and/or collecting duct epithelial hyperplasia, with cortical scarring and occasional mineralisation in the papilla. Histopathological changes in the liver, centrilobullar hepatocyte enlargement (accompanied by fine vacuolation) and foci/areas of eosinophilic hepatocytes were considered to reflect the induction of drug-metabolising enzymes in the liver. Other tissues showing treatment-related findings included the salivary glands, urinary bladder, spleen, pancreas and adrenals. Additionally, other notable findings included (in the high dosage males only) a decline in body weight (from week 34), lower erythrocytic characteristics and slightly higher plasma urea nitrogen and alkaline phosphatase values. The results in this study, therefore, indicated that the non-toxic effect level was 10 mg/kg/d of nefiracetam.

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A 52-week toxicity study by oral administration (capsule) was performed in beagle dogs with nefiracetam (N-(2,6-dimethylphenyl)-2-(2- oxo-1-pyrrolidinyl) acetamide, DM-9384, CAS 77191-36-7), a new cognition-enhancing agent, as a part of a safety evaluation program. Dosages of 0 (control), 10, 30 and 90 mg/kg/d were selected for this study. Treatment-related findings were confined to the 90 mg/kg/d level and indicated the kidney and the testis as the main target organs for toxicity. Signs of systemic toxicity, as indicated by the laboratory investigations, were not apparent until the second half of the study and included the principal findings of higher urea nitrogen, and creatinine, with higher urinary volumes and corresponding lower specific gravity, osmolarity and protein values. The microscopic pathology examination showed various changes at the renal papilla, collecting ducts, and medullary and cortical scarring. This examination also revealed decreased spermatogenesis in the testes, with associated decreased numbers/absence of spermatozoa in the epididymides. At the 30 mg/kg/d level, the minor microscopic pathology changes seen in the kidneys of one male animal were considered to be of equivocal toxicological importance. There were no treatment-related findings at the low dosage level (10 mg/kg/d) and, therefore, this level was considered as the non-toxic effect level of nefiracetam.

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A preliminary dose-range finding study and a 13-week toxicity study were performed in male cynomolgus monkeys with catena-(S)-[mu-[N a-(3-aminopropionyl) histidinato (2-)-N1,N2,O:N tau]-zinc] (Z-103, CAS 107667-60-7), a novel anti-peptic ulcer agent, as part of a safety evaluation program. In the preliminary ascending dose study emesis was observed in animals treated at 625 mg/kg and transient reductions in food intake with associated body weight loss in a male treated at 625 or 312.5 mg/kg. Plasma zinc levels were also increased in all animals treated at 625 or 312.5 mg/kg. As a result dosages of 0, 20, 63 and 200 mg/kg/day were selected for the 13-week toxicity study. In this study, treatment-related changes were confined to the 200 mg/kg/day dosage and consisted of emesis, piloerection and transient body weight loss in one animal, increased plasma zinc concentrations, and zinc and copper deposition in the liver and kidneys without any associated morphological change. The no observed effect level was estimated to be 63 mg/kg/day in this study.

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