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Botulinum neurotoxins (BoNTs) are important therapeutic agents. The in vivo median lethal dose (LD50) assay has been commonly used to measure the potency of BoNT commercial preparations. As an alternative, we developed cell-based assays for abobotulinumtoxinA in both powder (Dysport®, Azzalure®) and liquid (Alluzience®) formulations using the in vitro BoCell® system. The assays demonstrated linearity over 50–130% of the expected relative potency, with a correlation coefficient of 0.98. Mean recoveries of 90–108% of the stated potency were observed over this range. The coefficients of variation for powder and liquid formulations, respectively, were 3.6% and 4.0% for repeatability and 8.3% and 5.0% for intermediate precision. A statistically powered comparability assessment of the BoCell® and LD50 assays was performed. Equivalence was demonstrated between the assays for the liquid formulation at release and end of shelf life using a paired equivalence test with predefined equivalence margins. For the powder formulation, the assays were also shown to be equivalent for release samples and when determining loss of potency following thermal degradation. The BoCell® assay was approved for establishing the potency of abobotulinumtoxinA for both powder and liquid formulations in Europe and for the powder formulation only in the USA.

Graphical abstract, Highlights • CNCs have potential to stabilize curcuminoid content for at least 12-months. • CNCs are safe in appropriate dose as low and medium doses of toxicity testing. • High dose of CNCs could produce toxicity in blood parameters and histopathology. • Most altered parameters in chronic toxicity test could recover within 28 days. • CNCs have very low toxicity in non-clinical trials and are ready for clinical study., We recently developed a modified solid dispersion of curcumin-loaded nanocomplexes (CNCs) in gums which promoted the prolonged and sustained release of curcumin. However, its safety assessment has not yet been investigated. Here, acute and chronic toxicities of CNCs were assayed using mice and hamsters. CNCs were orally administered to the animals. Doses of CNCs used for acute toxicity testing were 0.1, 1.1, 11.0 g/kg body weight for mice and 0.2, 2.1 and 21.4 g/kg body weight for hamsters. Doses of CNCs for chronic toxicity testing were 0.09, 0.27, 0.8 g/kg body weight/day for mice and 0.18, 0.54 and 1.61 g/kg body weight/day for hamsters. This regimen was followed daily for 6 months. Low and medium doses of CNCs did not induce any side effects in acute and chronic toxicity tests in either animal species. However, in acute toxicity testing, the organ-weight to body-weight ratio of spleen was significantly increased in mice treated with 11 g/kg body weight along with elevated levels of some biochemical parameters. There was a significant increase in organ-weight to body-weight ratios of stomach, liver and heart in hamsters treated with 21.4 g/kg body weight, but no elevated levels of biochemical parameters. Oral LD50 of CNCs in mice and hamsters were 8.9 and 16.8 g/kg body weight (equivalent to 2.5 and 4.7 g curcumin/kg body weight), respectively. Daily CNCs high-dose treatment for 6 months significantly increased organ-weight to body-weight ratios of stomach and intestine in mice and of lung and heart in hamsters. Elevated levels of glucose, total protein, ALT, AST and globulin in mice, and increased levels of AST, but decrease in cholesterol, in hamsters were concurrently observed with inflammation in liver and lung. These abnormalities were resolved within 28 days after cessation of treatment. The no-observed-adverse-effect level of CNCs was determined at 0.27 and 0.54 g/kg body weight/day in mice and hamsters. In conclusion, toxicity of high-dose CNCs treatment was graded as very low, possibly due to the components of the nanocomplex.

Scorpionism is a global health concern, with an estimation of over one million annual envenomation cases. Despite this, little is known regarding the drivers of scorpion venom potency. One widely held view is that smaller scorpions with less-developed chelae possess the most potent venoms. While this perception is often used as a guide for medical intervention, it has yet to be tested in a formal comparative framework. Here, we use a phylogenetic comparative analysis of 36 scorpion species to test whether scorpion venom potency, as measured using LD50, is related to scorpion body size and morphology. We found a positive relationship between LD50 and scorpion total length, supporting the perception that smaller scorpions possess more potent venoms. We also found that, independent of body size, scorpion species with long narrow chelae have higher venom potencies compared to species with more robust chelae. These results not only support the general perception of scorpion morphology and potency, but also the presence of an ecology trade-off with scorpions either selected for well-developed chelae or more potent venoms. Testing the patterns of venom variations in scorpions aids both our ecological understanding and our ability to address the global health burden of scorpionism.

Understanding the toxicity and production rates of the various secondary metabolites produced by Gambierdiscus and cohabitating benthic dinoflagellates is essential to unravelling the complexities associated with ciguatera poisoning. In the present study, a sulphated cyclic polyether, gambierone, was purified from Gambierdiscus cheloniae CAWD232 and its acute toxicity was determined using intraperitoneal injection into mice. It was shown to be of low toxicity with an LD50 of 2.4 mg/kg, 9600 times less toxic than the commonly implicated Pacific ciguatoxin-1B, indicating it is unlikely to play a role in ciguatera poisoning. In addition, the production of gambierone and 44-methylgambierone was assessed from 20 isolates of ten Gambierdiscus, two Coolia and two Fukuyoa species using quantitative liquid chromatography–tandem mass spectrometry. Gambierone was produced by seven Gambierdiscus species, ranging from 1 to 87 pg/cell, and one species from each of the genera Coolia and Fukuyoa, ranging from 2 to 17 pg/cell. The production of 44-methylgambierone ranged from 5 to 270 pg/cell and was ubiquitous to all Gambierdiscus species tested, as well as both species of Coolia and Fukuyoa. The relative production ratio of these two secondary metabolites revealed that only two species produced more gambierone, G. carpenteri CAWD237 and G. cheloniae CAWD232. This represents the first report of gambierone acute toxicity and production by these cohabitating benthic dinoflagellate species. While these results demonstrate that gambierones are unlikely to pose a risk to human health, further research is required to understand if they bioaccumulate in the marine food web.

Graphical abstract, Highlights • Toxicity was studied for Hydrocotyle sibthorpioides. • LD50 was found to be greater than 2000 mg/kg of body weight. • Petroleum ether extract showed increase in somatomotor activity for short time. • Methanolic and aqueous extract exhibited some change in Haematological parameters., Hydrocotyle sibthorpioides (Family: Apiaceae) is a medicinal plant and is been a part of traditional cuisine. Hence, an acute toxicity study was performed to confirm its safety profile. Female rats (n=5) were randomly divided into 5 groups. One group served as the control and the other groups were been administered with the petroleum ether, chloroform, methanolic and aqueous extract of Hydrocotyle sibthorpioides at 2000 mg/kg body weight. Blood was collected at certain intervals to measure the haematological and biochemical parameters. The vital organs were isolated and preserved for histopathological examinations. The result suggested that the LD50 of all the extracts are greater than 2000 mg/kg body weight. There was no significant change in body weight, Behaviour, Renal Function test, Liver function test and Lipid profile. In haematological evaluation, there was an increase in RBC count and haemoglobin production post administration of methanolic and aqueous extract. The study suggested that the LD50 of the plant is greater than 2000 mg/kg body weight of rats in all the extracts.

Palytoxin (PLTX) is one of the most poisonous substances known to date and considered as an emergent toxin in Europe. Palytoxin binds to the Na+-K+ ATPase, converting the enzyme in a permeant cation channel. This toxin is known for causing human fatal intoxications associated with the consumption of contaminated fish and crustaceans such as crabs, groupers, mackerel, and parrotfish. Human intoxications by PLTX after consumption of contaminated fishery products are a serious health issue and can be fatal. Different reports have previously explored the acute oral toxicity of PLTX in mice. Although the presence of palytoxin in marine products is currently not regulated in Europe, the European Food Safety Authority expressed its opinion on PLTX and demanded assessment for chronic toxicity studies of this potent marine toxin. In this study, the chronic toxicity of palytoxin was evaluated after oral administration to mice by gavage during a 28-day period. After chronic exposure of mice to the toxin, a lethal dose 50 (LD50) of 0.44 &micro, g/kg of PLTX and a No-Observed-Adverse-Effect Level (NOAEL) of 0.03 &micro, g/kg for repeated daily oral administration of PLTX were determined. These results indicate a much higher chronic toxicity of PLTX and a lower NOAEL than that previously described in shorter treatment periods, pointing out the need to further reevaluate the levels of this compound in marine products.

Piper capense Linn is a plant used in Cameroon to treat cancer and several other diseases such as urinary tract disorder, fever, stomach-ache and to improve appetite. The methanol extract of Piper capense has been reported for its antiproliferative activity towards several human cancer cell lines. The aim of this work was to evaluate the acute and subchronic oral toxicities of a methanol extract from P. capense fruits on rats. The acute oral toxicity assay was carried out by administration of a single dose of 5000 mg/kg body weight of methanol extract of the Piper capense to five female rats, after which the behavior of the animals and the number of deaths were noted after 48 h. The animals were then kept for observation for 14 days. On the 15th day, the rats were sacrificed and macroscopic observation of the organs was made. Concerning the subchronic toxicity study, the rats composed of males and females received three doses (250, 500 and 1000 mg/kg body weight/day) for a period of 28 days by oral gavage. General animal behavior, food intake, weight gain, organ weights, haematological parameters, serum, and urinary biochemical parameters, and histological sections of liver and kidneys, were evaluated. Methanol extract from the Piper capense fruits did not cause any death in rats that were administered a single dose of 5000 mg/kg body weight of extract and therefore, the letal dose 50 (LD50) of the extract is greater than 5000 mg/kg body weight. Subchronic administration of the methanol extract of Piper capense fruits showed significant variations (P > 0.05) after analysis of certain biochemical parameters: serum urea, urinary urea, alanine aminotransferase (ALAT), aspartate aminotransferase, (ASAT), serum protein; in both male and female rats that received the dose of 1000 mg/kg body weight/day. No major signs of toxicity were observed in the liver and kidneys of animals after analysis of the histological sections performed. Beside, some signs of toxicity were observed, including cell lysis and inflammation on the liver and kidney organs at a dose of 1000 mg/kg body weight/day. Finally, the methanol extract of Piper capense fruits is safe at lower doses, but could cause some damages at doses as high as 1000 mg/kg body weight/day. Consequently, it should be taken with caution when used in therapy., Piper capense; Toxicity; Acute; Sub-acute; LD50; Biochemical parameters; Alternative medicine; Biological sciences; Cancer research; Diet; Evidence-based medicine; Health sciences; Hematological system; Toxicology.

Graphical abstract Toxicological study of aqueous-methanol solvent fraction of methanol extract of Dacryodes edulis leaves., Highlights • Toxicity of aqueous-methanol solvent fraction of methanol extract of Dacryodes edulis leaves was evaluated. • The oral median lethal dose (LD50) was greater than 5000 mg/kgbw –practically non-toxic. • Erythrocytic indices – RBC, HB, HCT – were significantly decreased. • No renal or liver toxicity observed on treating (200−600 mg/kgbw) for 28- days. • Mitotic depression and chromosomal aberrations were observed in the Allium cepa model., Dacryodes edulis (G. Don) H.J. Lam) is the most popular species under the genus Dacryodes. It is well known for its nutritional and ethno-medicinal uses in South-eastern and South-western Nigeria. This study was aimed to evaluate the toxicity of the aqueous-methanol fraction of crude methanol extract of Dacryodes edulis leaves (AMDE). The test rats were randomized to groups of single oral treatment of AMDE (10−5000 mg/kgbw) for the acute toxicity study. They were monitored for obvious signs of behavioural change and mortality. For the subacute toxicity study, the rats were randomized to three daily treatment groups (of 200, 400 and 600 mg/kgbw of AMDE) for 28 days. The fourth group (control) received 2.5 %v/v DMSO. At the end of the experiment, blood samples were collected for hematology and clinical chemistry evaluation. The histopathology of the livers and kidneys were assessed using the excised organs. The cytotoxicity and genotoxicity of AMDE were also evaluated using Allium cepa model. The result showed that acute administration of AMDE, up to a dose of 5000 mg/kgbw did not result in mortality of the test rats. The observed median lethal dose (LD50) was greater than 5000 mg/kgbw. The subacute oral administration of AMDE for 28 days showed no significant (p > 0.05) effect on liver function, kidney function indices, organ - body weight ratio, but significantly (p

Venoms are best known for their ability to incapacitate prey. In predatory groups, venom potency is predicted to reflect ecological and evolutionary drivers relating to diet. While venoms have been found to have preyspecific potencies, the role of diet breadth on venom potencies has yet to be tested at large macroecological scales. Here, using a comparative analysis of 100 snake species, we show that the evolution of prey-specific venom potencies is contingent on the breadth of a species&rsquo, diet. We find that while snake venom is more potent when tested on species closely related to natural prey items, we only find this prey-specific pattern in species with taxonomically narrow diets. While we find that the taxonomic diversity of a snakes&rsquo, diet mediates the prey specificity of its venom, the species richness of its diet was not found to affect these prey-specific potency patterns. This indicates that the physiological diversity of a species&rsquo, diet is an important driver of the evolution of generalist venom potencies. These findings suggest that the venoms of species with taxonomically diverse diets may be better suited to incapacitating novel prey species and hence play an important role for species within changing environments.

The venom of predators should be under strong selection pressure because it is a costly substance and prey may potentially become resistant. Particularly in prey-specialized predators, venom should be selected for its high efficiency against the focal prey. Very effective venom paralysis has been observed in specialized predators, such as spiders preying on dangerous prey. Here, we compared the toxicity of the venoms of two prey-specialized species, araneophagous Palpimanus sp. and myrmecophagous Zodarion nitidum, and their related generalist species. We injected different venom concentrations into two prey types&mdash, the prey preferred by a specialist and an alternative prey&mdash, and observed the mortality and the paralysis of the prey within 24 h. We found that the venoms of specialists were far more potent towards the preferred prey than alternative prey. The venoms of generalists were similarly potent towards both prey types. In addition, we tested the efficacy of two venom fractions (smaller and larger than 10 kDa) in araneophagous Palpimanus sp. Compounds larger than 10 kDa paralyzed both prey types, but smaller compounds (&lt, 10 kDa) were effective only on preferred prey, suggesting the presence of prey-specific compounds in the latter fraction. Our results confirm that prey-specialized spiders possess highly specific venom that allows them to subdue dangerous prey.

The safe and effective dosing of botulinum neurotoxins (BoNTs) requires accurate and reliable methods to measure their potency. Several novel methods have been introduced over the past decade; however, only few studies have compared the potency of BoNT products with that of the LD50 and other alternative assays. Therefore, the objective of this study was to comparatively evaluate widely used BoNT products using various test methods. Four types of BoNTs (prabotulinumtoxin A, onabotulinumtoxin A, neubotulinumtoxin A, and letibotulinumtoxin A) were used in this study. The estimated potency was assessed using the LD50 assay, and the total BoNT type A protein levels were measured using the enzyme-linked immunosorbent assay (ELISA). The in vitro efficacy of the BoNTs was determined using fluorescence resonance energy transfer (FRET) and surface plasmon resonance (SPR) assays. The results showed differences in the total amount of BoNT protein and the cleavage activity of SNAP-25 within all types of BoNTs. The SPR study seemed to be useful for evaluating the potency by specifically measuring intact 19S neurotoxin, and these results provide new insights for assessing different BoNT products.

The purpose of our work was the further exploration of the new 5-(furan-2-yl, 2-metylfuran-3-yl)-4-amino-1,2,4-triazole-3-thiol alkyl derivatives’ acute toxicity, setting some patterns of alkyl substituents influence by the Sulfur atom on the acute toxicity. Research materials and methods. In this study we used first time synthesized 5-(furan-2-yl, 2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole-3-thione derivatives. Acute toxicity was conducted on white rats weighing 160–250 g, which were injected once intraperitoneally with the investigated substances. The rats were received from the nursery of the Pharmacology and Toxicology Institute of Ukraine Medical Sciences Academy. The animals were kept on a standard diet with natural light mode "day-night". Results and their discussion. After the acute toxicity studies in a group of 5-(furan-2-yl, 2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole 3-alkyl derivatives it was found that among all studied structures the most toxic was 2e , LD 50 of which was 263 mg/kg, and the least toxic compound was 2a , LD 50 of which was 1570 mg/kg, that belongs to the V toxicity class. After comparing the acute toxicity of well-known antimycotic agent fluconazole with the studied compounds it can be argued that most compounds are less toxic than the comparison drug fluconazole with the index of LD 50 ˃320 mg/kg. It was found that the transition in a group from butyl to decyl, octyl, ventyl, propyl, nonyl and heptyl substituents in the molecule of 3-alkylthio 5-(furan-2-yl)-4-amino-4 H -1,2,4-triazole is accompanied by the toxicity increasing. Speaking about the 5-(2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole 3-alkylthio derivatives we can find that this dependence is observed in a number from propyl, pentyl, nonil, butyl, heksyl, octyl and heptyl hydrocarbon chains. Conclusions. The investigated 3-alkylthio 5-(furan-2-yl, 2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole derivatives belong to the IV-V toxicity class. The toxicity of 5-(furan-2-yl, 2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole alkyl derivatives varies depending on the hydrocarbon substituents, so the presence of 3-heptylthiol substituent in the С 3 atom at 5-(furan-2-yl, 2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole leads to the toxicity increase. Introduction of 3-butylthiol to the molecule of 5-(furan-2-yl)-4-amino-4 H -1,2,4-triazole, and the 3-propylthiol substituents in the 5-(2-metylfuran-3-yl)-4-amino-4 H -1,2,4-triazole results the formation of lowest acute toxicity

The aim of this study was to develop a biological early warning system for the detection of aquatic toxicity and test it with five toxicants with distinct chemical nature. This was done in order to verify the spectrum of sensitivities of the proposed system, as well as the potential identification capability of the tested contaminants, using only the analysis of zebrafish’s behavior. Six experimental conditions were tested: negative control and five toxicants (bleach, lindane, tributyltin, mercury, and formaldehyde). The exposure time was 45 min, and the concentrations used corresponded to 9% of LC50’s-96 h for the tested compounds, to ensure ecologically relevant results. A total of 108 fish were used, with each individual experimental condition being tested 18 times. A statistical model of diagnosis was used, combining self-organizing map and correspondence analysis. The values of sensitivity, specificity, accuracy, false positive, false negative, positive predictive value (PPV), and negative predictive value (NPV) were calculated. The objectives of the work were accomplished and the system showed a good overall diagnostic performance with 79% in accuracy, 77% in sensitivity, and 88% in specificity. The lowest result of the predictive values was 78% (lindane and mercury), in the case of the NPV, and 86% (bleach and lindane), in the case of the PPV. The best result of the predictive values was 100% (bleach and tributyltin), for the NPV, and 89% (tributyltin), for the PPV. Regarding the five tested toxicants, the system was able to correctly identify the agent responsible for the contamination in 40% of the positive diagnoses. © 2017, Springer-Verlag Berlin Heidelberg. This article is a result of project INNOVMAR-Innovation and Sustainability in the Management and Exploitation of Marine Resources (reference NORTE-01-0145-FEDER-000035), supported by North Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This study complied with the European Directive 2010/63/EU (2010) and National Guidelines Decreto-Lei 113/2013 (2013) on animal experiments as well as with the principles of the 3R guidelines (refinement, replacement, and reduction of animal use). No animals were sacrificed in the course of this study. The authors declare that they have no conflict of interest. This research was partially funded by UID/Multi/04423/2013 project from Funda??o para a Ci?ncia e Tecnologia. The funding source had no involvement in any process of the experience.

Fungicide is one of the common pesticide applies in agriculture. Even though fungicide is specifically designed to prevent growth of fungi, some studies reported neurotoxic effects of fungicide to some tested animals. Therefore there are concerns about the possible determintal effects of fungicide to beneficial non target organisms, like pollinating insects. This study was conducted to determine the toxicity of commercial fungicide to pollinator, Trigona ( Tetrgonula ) laeviceps Smith, by determining the LD 50 and hazard quotient (HQ). The fungicides tested were triazole (tridimefon and difenoconazole), propamorcab (propamocarb HCl), and dithiocarbamate (mancozeb and propineb). Forager bees were collected and anasthesized by exposing the bees to low temperature (-10 o C) for 1 min. About 1 µl of tiadimefon (6,25; 12,5; 25; 37,25; 50 µg/bee), difenoconazole (1,25; 2,5; 5; 6,25; 12,5; 25 µg/bee), propamocarb HCl (72.,25; 144,5; 216,6; 361,25; 433,5 µg/bee), mancozeb (1,6; 3,2; 4,8; 6,4; 8 µg/bee), and propineb (0.9; 1.8; 2.7; 6; 8.4; 16.8; 25.2 µg/bee) were applied using topical application on dorsal area of bees. Ten bees were used for each concentration and replicated 3 times. Mortality of bees was recorded for 48 hours. Results showed that LD 50 and HQ for each tested fungicide were as follow: triadimefon LD 50 13.43 ±1.78 µg/bee, HQ = 18.62; difenoconazole LD 50 9.25 ± 1.21 µg/bee, HQ = 27.03; propamocarb HCl LD 50 270.45 ± 19.48 µg/bee, HQ = 14.34; mancozeb LD 50 3.75±0.47 µg/bee, HQ = 512; and propineb LD 50 9.71 ±2.20 µg/bee, HQ = 144.18. Among all 5 tested fungicide, two fungicide, mancozeb and propineb, has intermediate level of toxicity to foragers of T. laeviceps and could produce determintal effect to colony.

Graphical abstract, Highlights • Saccharum munja Roxb. roots extract was subjected to acute oral toxicity evaluation. • No animal died at limit dose (2000 mg/kg), this is indicative of LD50 is greater than 2000 mg/kg. • But extract showed moderate toxicity signs on vital organs., Background S. munja roots have been used in ethno medicines for the treatment of different ailments. Despite its beneficial uses no studies on its toxicity potential have been reported. Objective The study was designed to evaluate acute toxic potential of aqueous ethanolic extract of S. munja roots according to OECD TG No. 425. Material and methods Female mice were divided into two groups (n = 5). One group served as control while the other as treated group that received 2000 mg/kg b.w. of S. munja roots ethanolic extract orally. Then both groups were observed for 14 days. Then the blood samples were collected by cardiac puncture, under chloroform general anesthesia and were subjected to hematological and biochemical analyses. The vital organs of anesthetized animals were preserved for histopathological examination. Results The the data revealed that LD50 of the extract was greater than 2000 mg/kg b.w. There was no significant alteration found in body weight and organ to body mass index. In comparison with control group, there was significant increase in levels of ALT, AST, total proteins, globulin levels, serum urea, cholesterol, triglycerides, LDL, platelet count, MCV, MCH, WBC count and lymphocytes whereas ALP and MCHC levels were reduced significantly. Conclusions From the data obtained in this study, It can be concluded that though LD50 is greater than 2000 mg/kg b.w. but moderate toxicity signs appeared in liver, kidney, lipid profile and CBC also showed blood dyscresias at limit dose.

Organochlorine (OC) pesticides are synthetic pesticides widely used all over the world. They belong to the group of chlorinated hydrocarbon derivatives, which have vast application in the chemical industry and in agriculture. These compounds are known for their high toxicity, slow degradation and bioaccumulation. Even though many of the compounds which belong to OC were banned in developed countries, the use of these agents has been rising. This concerns particularly abuse of these chemicals which is in practice across the continents. Though pesticides have been developed with the concept of target organism toxicity, often non-target species are affected badly by their application. The purpose of this review is to list the major classes of pesticides, to understand organochlorine pesticides based on their activity and persistence, and also to understand their biochemical toxicity.

Ethanolic extract of bacang mango leaves (Mangifera foetida L.) was reported could decrease blood iron concentration of iron overloaded-Sprague dawley rats. The aim of this experiment was to determine acute toxicity (LD50) value of ethanolic extract of M. foetida by weil method and its effect to blood component. This study was conducted by employing a complete random design using 25 male and 25 female mice of DDY strain which was divided into 5 groups. The first until fourth group were administered by the aquadest suspension of ethanolic extract of M. foetida with dose variation, orally. The fifth group was control group that administered by aquadest only. The LD50 was determined by the total of death on all group. LD50 value of the extract showed no death in the biggest doses (13.013 g/kg). The examination was continued with measured blood count (erythrocytes, trombocytes, leukocytes, and haemoglobin concentration). It was shown that the extract at dose 1.626; 3.253; 6.506 and 13.013 g/kg unchanged the blood count measurement. It is concluded that Mangifera foetida L. leaf was not toxic, but could not change the blood count measurement.

A strong argument can be made that the European Union has the most rigorous regulatory system for pesticides in the world, and that modern pesticide use poses fewer environmental threats than older regimes. Nevertheless, the impacts of pesticides on bees and other non-target organisms are much debated in Europe as elsewhere. Here we document changing patterns of pesticide use in arable and horticultural crops in Great Britain from 1990 to 2015. The weight of pesticides used has approximately halved over this period, but in contrast the number of applications per field nearly doubled. The total potential kill of honeybees (the total number of LD50 doses applied to the 4.6 million hectares of arable farmland in Great Britain each year) increased six-fold to approximately 3 × 1016 bees, the result of the increasing use of neonicotinoids from 1994 onwards which more than offset the effect of declining organophosphate use. It is important to stress that this does not mean that this number of bees will be killed, and also to acknowledge that our simple analysis does not take into account many factors such as differences in persistence, and timing and mode of application of pesticides, which will affect actual exposure of non-target organisms. Nonetheless, all else being equal, these data suggest that the risk posed by pesticides to non-target insects such as bees, other pollinators and natural enemies of pests, has increased considerably in the last 26 years.

Acute systemic toxicity testing provides the basis for hazard labeling and risk management of chemicals. A number of international efforts have been directed at identifying non-animal alternatives for in vivo acute systemic toxicity tests. A September 2015 workshop, Alternative Approaches for Identifying Acute Systemic Toxicity: Moving from Research to Regulatory Testing, reviewed the state-of-the-science of non-animal alternatives for this testing and explored ways to facilitate implementation of alternatives. Workshop attendees included representatives from international regulatory agencies, academia, nongovernmental organizations, and industry. Resources identified as necessary for meaningful progress in implementing alternatives included compiling and making available high-quality reference data, training on use and interpretation of in vitro and in silico approaches, and global harmonization of testing requirements. Attendees particularly noted the need to characterize variability in reference data to evaluate new approaches. They also noted the importance of understanding the mechanisms of acute toxicity, which could be facilitated by the development of adverse outcome pathways. Workshop breakout groups explored different approaches to reducing or replacing animal use for acute toxicity testing, with each group crafting a roadmap and strategy to accomplish near-term progress. The workshop steering committee has organized efforts to implement the recommendations of the workshop participants.

Prior findings showed that serum from DBA/2 mice that had been given whole-body irradiation for 1 hour at a low dose rate (LDR) of 30 cGy/h induced protection against radiation in reporter cells by a mechanism depending on transforming growth factor β3 and inducible nitric oxide synthase activity. In the present study, the effect of the 1 hour of LDR irradiation on the response of the preirradiated mice to a subsequent lethal dose and on the life span is examined. These DBA/2 mice were prime irradiated for 1 hour at 30 cGy/h. Two experiments with 9 and 9.5 Gy challenge doses given 6 weeks after priming showed increased survival in primed mice compared to unprimed mice followed up to 225 and 81 days after challenge irradiation, respectively. There was no overall significant difference in life span between primed and unprimed mice when no challenge irradiation was given. The males seemed to have a slight increase in lifespan after priming while the opposite was seen for the females.

Spiders rely on venom to catch prey and few species are even capable of capturing vertebrates. The majority of spiders are generalist predators, possessing complex venom, in which different toxins seem to target different types of prey. In this study, we focused on the trophic ecology and venom toxicity of Phoneutria boliviensis F. O. Pickard-Cambridge, 1897, a Central American spider of medical importance. We tested the hypothesis that its venom is adapted to catch vertebrate prey by studying its trophic ecology and venom toxicity against selected vertebrate and invertebrate prey. We compared both trophic ecology (based on acceptance experiments) and toxicity (based on bioassays) among sexes of this species. We found that P. boliviensis accepted geckos, spiders, and cockroaches as prey, but rejected frogs. There was no difference in acceptance between males and females. The venom of P. boliviensis was far more efficient against vertebrate (geckos) than invertebrate (spiders) prey in both immobilization time and LD50. Surprisingly, venom of males was more efficient than that of females. Our results suggest that P. boliviensis has adapted its venom to catch vertebrates, which may explain its toxicity to humans.

Background: Endosulfan, a neurotoxic organochlorine insecticide and cypermethrin, a synthetic pyrethroid insecticide used to control pests in domestic, industrial, and agricultural situations. Materials and Methods: The present study was carried out to investigate the acute oral toxicity, behavioral and histopathological changes of combination of endosulfan and cypermethrin in albino rats. According to Miller and Tainter analysis method, at 48 h, LD50 value of combination of endosulfan and cypermethrin (ratio 1:1) in rats was found to be 691.83 mg/kg bw by oral gavage. Results: When combination of both these pesticides was administered orally at concentration of 103.72 mg/kg bw, 172.95 mg/kg bw and 207.50 mg/kg bw, respectively, as a single dose, no significant changes in behavior of rats was observed, neither in dosed nor in control group of rats. Combination of endosulfan- and cypermethrin-treated rats showed mild histopathological changes in liver and kidney in group IV (207.50 mg/kg BW) as compared to the control. However, no significant changes were observed in brain and small intestine at either dose of combination of endosulfan and cypermethrin with respect to control. Conclusion: Thus, the present study, first of its kind in India, demonstrated the oral toxicity, behavioral, and histo-architectual alterations after induction of combination of endosulfan and cypermethrin at acute doses in Wistar rats.

Background Snakebite incidence in southwestern China is mainly attributed to one of the several venomous snakes found in the country, the white-lipped green pit viper Trimeresurus albolabris. Since antivenom produced from horses may cause numerous clinical side effects, the present study was conducted aiming to develop an alternative antivenom antibody (immunoglobulin Y - IgY) from leghorn chickens. Methods IgY in egg yolk from white leghorn chicken previously injected with T. albolabris venom was extracted by water, precipitated by ammonium sulfate and purified by affinity chromatographic system. IgY was identified by SDS-PAGE, ELISA and Western blot, and its neutralizing assay was conducted on mice. Results Chickens injected multiple times with T. albolabris venom elicited strong antibody responses, and from their egg yolk IgY was isolated and purified, which exhibited a single protein band on SDS-PAGE and two bands (about 65 and 35 kDa, respectively) under reduced conditions. Immunoblot analysis revealed that these IgY are polyclonal antibodies since they bind with most venom components. In the neutralizing assay, all mice survived while the ratios of IgY/venom reached up to 3.79 (50.0 mg/13.2 mg). Conclusions IgY antibody response was successfully conducted in white leghorn chicken injected with T. albolabrisvenom. IgY against T. albolabris venom was obtained for the first time, and it exhibited strong neutralizing potency on mice. These results may lay a foundation for the development of IgY antivenom with clinical applications in the future.

Background Maintenance of scorpions under laboratory conditions is ideal for long-term venom collection to explore the therapeutic applications of scorpion venom. Collection of venom by electrical stimulation requires a reliable stimulator and effective restrainer. Thus, the present study was conducted to develop a convenient method to maintain scorpions and to extract their venom for toxicity studies via a modified restrainer and stimulator. Methods Four different scorpion species were collected, among which three species were maintained in the laboratory in containers that mimic their natural habitat. Venom was extracted from Hottentotta rugiscutis by electrical stimulation at 8 V for 18 months and LD50 was estimated by the graphic method of Miller and Tainter. Results A total of 373 scorpions including Hottentotta rugiscutis, Hottentotta tamulus, Lychas tricarinatus and Heterometrus swammerdami were collected, identified and maintained successfully, achieving a 97 % survival rate. Hottentotta rugiscutis yielded 6.0 mL of venom by electrical stimulation. The LD50 of H. rugiscutis venom was estimated to be 3.02 mg/kg of body weight in female Swiss albino mice. Conclusions Scorpions were successfully maintained for 18 months. Herein we have also documented a simple, cost-effective method of venom extraction by electrical stimulation using a modified restrainer. Furthermore, Hottentotta rugiscutis was reported for the first time in Karnataka.

Neonicotinoids, systemic neuro-active pesticides similar to nicotine, are widely used in agriculture and are being investigated for a role in honey bee colony losses. We examined one neonicotinoid pesticide, imidacloprid, for its effects on the foraging behavior of free-flying honey bees (Apis mellifera anatoliaca) visiting artificial blue and white flowers. Imidacloprid doses, ranging from 1/5 to 1/50 of the reported LD50, were fed to bees orally. The study consisted of three experimental parts performed sequentially without interruption. In Part 1, both flower colors contained a 4 mu L 1 M sucrose solution reward. Part 2 offered bees 4 mu L of 1.5 M sucrose solution in blue flowers and a 4 mu L 0.5 M sucrose solution reward in white flowers. In Part 3 we reversed the sugar solution rewards, while keeping the flower color consistent. Each experiment began 30 min after administration of the pesticide. We recorded the percentage of experimental bees that returned to forage after treatment. We also recorded the visitation rate, number of flowers visited, and floral reward choices of the bees that foraged after treatment. The forager return rate declined linearly with increasing imidacloprid dose. The number of foraging trips by returning bees was also affected adversely. However, flower fidelity was not affected by imidacloprid dose. Foragers visited both blue and white flowers extensively in Part 1, and showed greater fidelity for the flower color offering the higher sugar solution reward in Parts 2 and 3. Although larger samples sizes are needed, our study suggests that imidacloprid may not affect the ability to select the higher nectar reward when rewards were reversed. We observed acute, mild effects on foraging by honey bees, so mild that storage of imidacloprid tainted-honey is very plausible and likely to be found in honey bee colonies. Süleyman Demirel Üniversitesi National Science Foundation (NSF) (1263327)

Carbon capture and storage (CCS) technologies are considered vital and economic elements for achieving global CO2 reduction targets, and is currently introduced worldwide (for more information on CCS, consult for example the websites of the International Energy Agency (http://www.iea.org/topics/ccs/) and the Global CCS Institute (http://www.globalccsinstitute.com/)). One prominent CCS technology, the amine-based post-combustion process, may generate nitrosamines and their related nitramines as by-products, the former well known for their potential mutagenic and carcinogenic properties. In order to efficiently assess the carcinogenic potency of any of these by-products this paper reviews and discusses novel prediction approaches consuming less time, money and animals than the traditionally applied 2-year rodent assay. For this, available animal carcinogenicity studies with N-nitroso compounds and nitramines have been used to derive carcinogenic potency values, that were subsequently used to assess the predictive performance of alternative prediction approaches for these chemicals. Promising cancer prediction models are the QSARs developed by the Helguera group, in vitro transformation assays, and the in vivo initiation-promotion, and transgenic animal assays. All these models, however, have not been adequately explored for this purpose, as the number of N-nitroso compounds investigated is yet too limited, and therefore further testing with relevant N-nitroso compounds is needed.

A k-nearest neighbor (k-NN) classification model was constructed for 118 RDT NEDO (Repeated Dose Toxicity New Energy and industrial technology Development Organization, currently known as the Hazard Evaluation Support System (HESS)) database chemicals, employing two acute toxicity (LD50)-based classes as a response and using a series of eight PaDEL software-derived fingerprints as predictor variables. A model developed using Estate type fingerprints correctly predicted the LD50 classes for 70 of 94 training set chemicals and 19 of 24 test set chemicals. An individual category was formed for each of the chemicals by extracting its corresponding k-analogs that were identified by k-NN classification. These categories were used to perform the read-across study for prediction of the chronic toxicity, i.e., Lowest Observed Effect Levels (LOEL). We have successfully predicted the LOELs of 54 of 70 training set chemicals (77%) and 14 of 19 test set chemicals (74%) to within an order of magnitude from their experimental LOEL values. Given the success thus far, we conclude that if the k-NN model predicts LD50 classes correctly for a certain chemical, then the k-analogs of such a chemical can be successfully used for data gap filling for the LOEL. This model should support the in silico prediction of repeated dose toxicity.

We investigate the procedure of checking for overlap between confidence intervals or standard error intervals to draw conclusions regarding hypotheses about differences between population parameters. Mathematical expressions and algebraic manipulations are given, and computer simulations are performed to assess the usefulness of confidence and standard error intervals in this manner. We make recommendations for their use in situations in which standard tests of hypotheses do not exist. An example is given that tests this methodology for comparing effective dose levels in independent probit regressions, an application that is also pertinent to derivations of LC50S for insect pathogens and of detectability half-lives for prey proteins or DNA sequences in predator gut analysis.

Polyhexamethylene biguanide (PHMB) is an antiseptic with antiviral and antibacterial properties used in a variety of products including wound care dressings, contact lens cleaning solutions, perioperative cleansing products, and swimming pool cleaners. There are regulatory concerns with regard to its safety in humans for water treatment. We decided to assess the safety of this chemical in Sprague-Dawley rats. PHMB was administered in a single dose by gavage via a stomach tube as per the manufacturer’s instruction within a dose range of 2 mg/kg to 40 mg/kg. Subchronic toxicity studies were also conducted at doses of 2 mg/kg, 8 mg/kg and 32 mg/kg body weight and hematological, biochemical and histopathological findings of the major organs were assessed. Administration of a dose of 25.6 mg/kg, i.e. 1.6 mL of 0.4% PHMB solution (equivalent to 6.4×103 mg/L of 0.1% solution) resulted in 50% mortality. Histopathological analysis in the acute toxicity studies showed that no histopathological lesions were observed in the heart and kidney samples but 30% of the animals had mild hydropic changes in zone 1 of their liver samples, while at a dosage of 32 mg/kg in the subchronic toxicity studies, 50% of the animals showed either mild hepatocyte cytolysis with or without lymphocyte infiltration and feathery degeneration. Lymphocyte infiltration was, for the first time, observed in one heart sample, whereas one kidney sample showed mild tubular damage. The acute studies showed that the median lethal dose (LD50) is 25.6 mg/kg (LC50 of 1.6 mL of 0.4% PHMB. Subchronic toxicological studies also revealed few deleterious effects on the internal organs examined, as seen from the results of the biochemical parameters evaluated. These results have implications for the use of PHMB to make water potable.

WOS: 000305761800011, The venomous Levantine viper, Macrovipera lebetina lebetina is endemic to Cyprus. The objective of this study was to investigate in vitro cytotoxicity, in vivo lethality, and antivenom production followed by a re-immunization schedule in mice against Macrovipera lebetina lebetina venom. The LD50 value was estimated as 7.58mg/kg within 24 hours by different venom doses administrated intraperitoneally in mice. Freund's complete and incomplete adjuvants were used for first and second immunization of mice in antivenom production. A cell-based assay was performed to determine the effects of Macrovipera lebetina lebetina venom and antivenom neutralizing potency on L929 cell viability. The snake venom toxicity and cytotoxicity were examined and comparison of results showed good correlation, the LD50 value was tenfold higher than the IC50 value. The IC50 value was 0.62 +/- 0.18 mu g/mL after 48 hours treatment while the calculated value was 1.62 +/- 0.25 mu g/mL for the culture media totally refreshed after two hours treatment with venom. The in vitro efficacy of antivenom against Macrovipera lebetina lebetina venom was found to be low. This is the first report that describes the in vivo and in vitro toxic effects of Macrovipera lebetina lebetina venom and antivenom production against this species.

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD50 determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.

Among the various methods for evaluating animal venom toxicity, the calculation of the median lethal dose (LD50) is the most widely used. Although different protocols can be used to calculate the LD50, the source of the venom and the method of extraction, as well as the strain, age, and sex of the animal model employed, should be taken into consideration. The objective of the present study was to evaluate the influence of sex and age on the toxicity of Tityus serrulatus scorpion venom in Swiss mice. Although the symptoms of envenomation were similar in male and female animals, female mice proved to be more resistant to the venom. In females, age had no impact on the susceptibility to scorpion envenomation. Male mice were more sensitive to T. serrulatus venom. Moreover, in males, age was an important parameter since sensitivity to the venom increased with age.

Tityus trivittatus is the Argentinean scorpion reported to cause the majority of human fatalities in the country, however no systematic studies have been conducted with the venom of this species. This communication describes a general biochemical and immunological characterization of the venom obtained from T. trivittatus scorpions collected in the city of Buenos Aires and various provinces of Argentina: Catamarca, Cordoba, Entre Rios, La Rioja, Santa Fe and Santiago del Estero. These are places where human accidents were reported to occur due to this scorpion. For comparative purposes two types of samples were assayed: whole soluble venom obtained by electrical stimulation and supernatant from homogenized venomous glands. Two strains of mice (NIH and CF-1) were used for LD50 determinations by two distinct routes of administration (intravenously and intraperitoneally). Important variations were found that goes from 0.5 to 12 mg/kg mouse body weight. Samples of soluble venom were always more potent than Telson homogenates. More complex pattern was observed in homogenates compared to soluble venom, as expected. This was supported by gel electrophoretic analysis and high performance liquid chromatographic (HPLC) separations. Additionally, the HPLC profile was enriched in proteins resolved at similar elution times as other known toxins from scorpion venoms studied. Immune enzymatic assays were also conducted comparatively, using four different anti-venoms commercially available for treatment of scorpion stings (Argentinean antidote from INPB, two anti-venoms from Butantan Institute of Brazil and Alacramyn from the Mexican Bioclon Institute). Cross-reactivities were observed and are reported among the various venoms and anti-venoms used. Lung, heart, liver and pancreas pathological modifications were observed on tissues of intoxicated mice. It seems that there are important variations on the venom compositions of the various samples studied and reported here, depending on the geographical area where the scorpions were captured. The results reported here are important for the clinical outcome of human accidents. Fil: de Roodt, Adolfo Rafael. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Coronas, Fredy I.V.. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México Fil: Lago, Néstor Rubén. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Patología; Argentina Fil: González, María E.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Laskowicz, Rodrigo D.. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Beltramino, Juan C.. Provincia de Santa Fe. Ministerio de Salud. Hospital de Niños “Orlando Alassia”; Argentina Fil: Saavedra, Silvina. Provincia de Entre Ríos. Ministerio de Salud. Dirección de Zoonosis; Argentina Fil: López, Raúl A.. Provincia de Catamarca. Ministerio de Salud. Departamento de Zoonosis; Argentina Fil: Reati, Gustavo J.. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Centro de Zoología Aplicada; Argentina Fil: Vucharchuk, Miriam G.. Provincia de Santiago del Estero. Ministerio de Salud. Instituto de Animales Venenosos “Jorge W. Abalos"; Argentina Fil: Bazán, Eduardo. Provincia de La Rioja. Ministerio de Salud. Dirección de Epidemiología; Argentina Fil: Varni, Liliana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Salomón, Oscar Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; Argentina. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud “Dr. C. G. Malbrán”; Argentina Fil: Possani, Lourival D.. Universidad Nacional Autónoma de México. Instituto de Biotecnología; México

9 páginas, 5 tablas. 5 figuras, Nowadays, a variety of protocols are applied to quantitate palytoxin. However, there is not desirable agreement among them, the confidence intervals of the basic toxicological parameters are too wide and the formal descriptions lack the necessary generality to establish comparisons. Currently, the mouse bioassay is the most accepted one to categorize marine toxins and it must constitute the reference for other methods. In the present work, the mouse bioassay for palytoxin is deeply analyzed and carefully described showing the initial symptoms of injected mice which are presented here in the first time. These symptoms clearly differ from the more common marine toxins described up to now. Regarding to the toxicological aspects two considerations are taking into account: (i) the empiric models based in the dose–death time relationships cause serious ambiguities and (ii) the traditional moving average method contains in its regular use any inaccuracy elements. Herein is demonstrated that the logistic equation and the accumulative function of Weibull’s distribution (with the modifications proposed) generate satisfactory toxicological descriptions in all the respects

Toxicity tests were carried out on two Bezethiazine and Benzoxazine derivatives. For this experiment, a total of sixty (60) Albino mice with average weight range 18-26g were employed. For each test compound, thirty (30) mice were distributed in five cages and fed ad libitum for 14 days for acclimatization. The test compounds were suspended in a non-toxic solvent (Tween 80) and intraperitoneally administered into the mice at doses of 450mg/kg, 500mg/kg, 550mg/kg and 600mg.kg. The results showed that the compound pyrrolobenzothiazine ring system has LD 50 value of 500.00mg/kg, while the compound pyrrolobenzoxazine ring system has an LD 50 value of 517.60mg/kg. These results are indicative of low toxicity values for the two compounds. KEY WORDS: Toxicity, LD50, Mice, Benzothiazine, Benzoxazine. J.Expt. & Clin. Anat. 2(1) 2003: 34-37