New Class Of Insecticides Research Articles

Neonicotinoid insecticides: an emerging cause of acute pesticide poisoning

Neonicotinoids are a new class of insecticides widely applied for crop protection. Information on human exposures to neonicotinoids is limited. The most common routes of exposure were ingestion (51%), dermal (44%), and ocular (11%). These insecticides act as agonists at nicotinic acetylcholine receptors, which cause insect paralysis and death the high specificity for receptors in insects was considered to possess highly selective toxicity to insects and relative sparing of mammals. However, an increasing number of cases of acute neonicotinoid poisoning have been reported in recent years. Present study report three cases presented to us with acute neonicotinoid poisoning with different manifestations including acute myocardial infarction, central nervous system (CNS) depression, and acute kidney injury, who recovered subsequently with supportive care. A detailed literature review found that respiratory, cardiovascular and certain neurological presentations are warning signs of severe neonicotinoid intoxication. Supportive treatment and decontamination are the practical methods for the management of all neonicotinoid-poisoned patients.

Electrocatalytic activity of the metallic silver electrode for thiamethoxam reduction: application for the detection of a neonicotinoid in tomato and orange samples.

Thiamethoxam (3-[(2-chloro-5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadiazin-4-imine) belongs to a relatively new class of insecticides known as neonicotinoids, which can block irreversibly nicotinic acetylcholine receptors of the nervous system of insects. Its determination at trace levels is an acute analytical problem. Therefore, chromatography, spectroscopy, and electrochemical technics were reported. These last have several advantages (simple sensitive and less expensive). This work investigated the electro-reduction of Thiamethoxam using simple and rapid method in real samples using metallic silver electrode. Silver particles, known as effective catalysts in the reduction of nitro groups, were studied to explore their roles in the shift of thiamethoxam peak potential. Cyclic voltammetry, Tafel plot and electrochemical impedance spectroscopy analysis suggest that metallic silver electrodes have a significantly greater electrocatalytic activity in thiamethoxam electroreduction than glassy carbon electrodes and carbon paste electrodes. The electrocatalytic activity of metallic silver electrodes in thiamethoxam reduction was investigated by cyclic voltammetry and chronoamperometry. A linear variation in cathodic current with the square root of the scan rate was observed, suggesting that the process is diffusion controlled. Several supporting electrolytes were tested, and the best results were obtained with Britton-Robinson (BR) buffer, pH10.4. A linear relationship between peak current and concentration was found in the range from 1.0 × 10-5 to 1.0 × 10-4 mol L-1 using square wave voltammetry (SWV) with a correlation coefficient of 0.994. The detection limit and quantification limit were 5.49 × 10-6 and 1.83 × 10-5 mol L-1 , respectively. Silve metallic electrode exhibits efficient catalytic activity towards the Thiamethoxam reduction. The proposed electrode was then used for the determination of thiamethoxam in tomato anad orage juice samples. © 2019 Society of Chemical Industry.

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes.

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Degradation of neonicotinoids by UV irradiation: Kinetics and effect of real water constituents

Neonicotinoids are a new class of insecticides whose potential side-effects on the human health and the environment have not yet been well investigated. Recently, the EU approved the Decision 2015/495 establishing a watch list of substances for Union-wide monitoring in the field of water policy, which includes five neonicotinoids: thiamethoxam (TMX), imidacloprid (ICP), clothianidin (CTD), thiacloprid (TCP) and acetamiprid (AMP). The photodegradation of neonicotinoids by monochromatic UV irradiation (254 nm) has been investigated. The photolysis rate followed pseudo-first order kinetics, with rate constant values depending on the nature of the specific compound, pH, and the presence or absence of radical scavengers. A method based on atrazine actinometry provided the following quantum yields at pH = 7: (77 ± 3) × 10−3, (43 ± 2) × 10−3, (48 ± 1) × 10−3, (17 ± 2) × 10−3 and (5 ± 2) × 10−3 mol E−1 for TMX, CTD, ICP, TCP and AMP, respectively. The effects of the presence of bicarbonate, nitrate, nitrite and chloride ions on the simultaneous photodegradation of nicotinoids were not very significant, and only the removal of AMP, the least photosensitive neonicotinoid, was negatively affected. However, the photodegradation of neonicotinoids was clearly inhibited by the addition of humic acids. The results obtained in experiments performed in the presence of hydrogen peroxide or persulfate indicated that while direct UV photolysis was the main degradation pathway for TMX and even for CTD, ICP and TCP, the contribution of indirect photolysis (radical pathway) was important in the case of pollutants with low values of quantum yield such as AMP. Although the presence of natural organic and inorganic matter in real water matrices decreased the degradation rate of the selected neonicotinoids, UV photolysis and the AOPs UV/H2O2 and UV/PS processes constitute efficient technologies for the removal of these pollutants from contaminated waters.

In situ liquid cell crystallization and imaging of thiamethoxam by helium ion microscopy

Pest insect control is an essential component for crop protection and public health. Neonicotinoids are a relatively new class of insecticides developed in the last four decades. Thiamethoxam, a member of the neonicotinoid class, has shown outstanding potency for crop protection against a variety of piercing-sucking pests. However, its use in industrial-volume packing and transportation is complicated by crystallization dynamics. In this work, a helium ion microscope (HIM) with a Protochips liquid cell was utilized to crystallize and image thiamethoxam in situ. The results of the study illustrate the growth and morphology of the thiamethoxam crystals at different He+ exposure doses, which is markedly different from what has been typically observed. Energy-dispersive x-ray spectroscopy results confirm the presence of the thiamethoxam on the liquid cell membrane. This imaging study illustrates the HIM ability to image and induce the crystallization in soft materials in liquid environments, and attempts to shed light onto the key processes involved in liquid imaging.

Molecular Mechanism of Action and Selectivity of Sodium Ch annel Blocker Insecticides.

Sodium channel blocker insecticides (SCBIs) are a relatively new class of insecticides that are represented by two commercially registered compounds, indoxacarb and metaflumizone. SCBIs, like pyrethroids and DDT, target voltage-gated sodium channels (VGSCs) to intoxicate insects. In contrast to pyrethroids, however, SCBIs inhibit VGSCs at a distinct receptor site that overlaps those of therapeutic inhibitors of sodium channels, such as local anesthetics, anticonvulsants and antiarrhythmics. This review will recount the development of the SCBI insecticide class from its roots as chitin synthesis inhibitors, discuss the symptoms of poisoning and evidence supporting inhibition of VGSCs as their mechanism of action, describe the current model for SCBI-induced inhibition of VGSCs, present a model for the receptor for SCBIs on VGSCs, and highlight differences between data collected from mammalian and insect experimental models.

A Voucher System To Speed Review Could Promote A New Generation Of Insecticides To Fight Vector-Borne Diseases.

Many in the scientific community are concerned about the potential increase in prevalence of insect-borne diseases such as Chagas disease, Chikungunya, dengue fever, malaria, and Zika in the United States and around the world. Beyond vaccines and drugs to prevent and treat these diseases, a comprehensive approach to fighting these diseases should include control of disease-carrying vectors, such as mosquitoes. Vector-control methods, such as using insecticides to treat bed nets and spray the walls of homes, have prevented millions of deaths from malaria. However, mosquitoes are becoming resistant to insecticides, and no new class of insecticides for vector control has been introduced in decades. We recommend the creation of a new type of incentive for the development and commercialization of safe new insecticides: a Vector Expedited Review Voucher, to be awarded to a sponsor that introduces a novel insecticide for public health use. The voucher could be redeemed to expedite registration of a second, more profitable, product by the US Environmental Protection Agency.

Insecticidal activities of histone deacetylase inhibitors against a dipteran parasite of sheep, Lucilia cuprina

Histone deacetylase inhibitors (HDACi) are being investigated for the control of various human parasites. Here we investigate their potential as insecticides for the control of a major ecto-parasite of sheep, the Australian sheep blowfly, Lucilia cuprina. We assessed the ability of HDACi from various chemical classes to inhibit the development of blowfly larvae in vitro, and to inhibit HDAC activity in nuclear protein extracts prepared from blowfly eggs. The HDACi prodrug romidepsin, a cyclic depsipeptide that forms a thiolate, was the most potent inhibitor of larval growth, with equivalent or greater potency than three commercial blowfly insecticides. Other HDACi with potent activity were hydroxamic acids (trichostatin, CUDC-907, AR-42), a thioester (KD5170), a disulphide (Psammaplin A), and a cyclic tetrapeptide bearing a ketone (apicidin). On the other hand, no insecticidal activity was observed for certain other hydroxamic acids, fatty acids, and the sesquiterpene lactone parthenolide. The structural diversity of the 31 hydroxamic acids examined here revealed some structural requirements for insecticidal activity; for example, among compounds with flexible linear zinc-binding extensions, greater potency was observed in the presence of branched capping groups that likely make multiple interactions with the blowfly HDAC enzymes. The insecticidal activity correlated with inhibition of HDAC activity in blowfly nuclear protein extracts, indicating that the toxicity was most likely due to inhibition of HDAC enzymes in the blowfly larvae. The inhibitor potencies against blowfly larvae are different from inhibition of human HDACs, suggesting some selectivity for human over blowfly HDACs, and a potential for developing compounds with the inverse selectivity. In summary, these novel findings support blowfly HDAC enzymes as new targets for blowfly control, and point to development of HDAC inhibitors as a promising new class of insecticides.

Studies on Aphis gossypii cytochrome P450s CYP6CY22 and CYP6CY13 using an in vitro system.

A field-collected Aphis gossypii clone [Kushima resistant (KR) clone] was resistant to neonicotinoid insecticides (23.8- to 394-fold). RNA-seq and next-generation sequence analyses were conducted to identify nine cytochrome P450 (CYP) genes that were significantly upregulated in the KR clone as compared with those in the insecticide-susceptible clone. A. gossypii P450s were transiently and efficiently expressed in S2 cell to show that CYP6CY22 (c21228) and CYP6CY13 (c21368), which were the most upregulated of the nine P450s in the KR clone, did not degrade sulfoxaflor, a new class of insecticides acting on insect nAChRs, but markedly metabolized all of the neonicotinoids tested. Hence, P450s are likely to underpin neonicotinoid resistance in other aphids as well in the future, and the P450 expression protocol established here will prompt studies on P450-medidated insecticide resistance and structural analyses of relevant metabolites.

Trichothecenes Production by Entomopathogenic Fungus Conidiobolus Coronatus

Entomopathogenic fungi are of particular interest now as the likely source of a new class of insecticides. This interest stems from the fact that these organisms naturally present in the environment, in most cases selectively affect specific organisms, and metabolites produced by them do not pass into the food chain.

Effects of three diamides (chlorantraniliprole, cyantraniliprole and flubendiamide) on life history, embryonic development and oxidative stress biomarkers of Daphnia magna

The diamides have become one of the most promising new classes of insecticides. In this study, we evaluated the toxicity of three diamides (chlorantraniliprole, cyantraniliprole and flubendiamide) to Daphnia magna. The acute toxicity test showed that the 48-h EC50 of chlorantraniliprole, cyantraniliprole and flubendiamide were 8.5, 23.9 and 63.5 μg/L, respectively. Biochemical measurements revealed a significant increase in reactive oxygen species (ROS) in D. magna after acute exposure to the three diamides. A significant decrease in activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) was observed, which was consistent with the down-regulated transcription of antioxidant genes sod and gpx. Catalase (CAT) activity exhibited a significant increase while the related gene cat showed no obvious change in daphnids acutely exposed to the three diamides. The chronic test revealed that the three diamides could cause lethal and sub-lethal effects on daphnids within constricted range of concentrations at μg/L level. The 21-d EC50 of chlorantraniliprole, cyantraniliprole and flubendiamide for mobility were 5.0, 13.6 and 36.8 μg/L, respectively. The chronic LOEC of chlorantraniliprole, cyantraniliprole and flubendiamide based on survival, growth and reproduction of D. magna were 4.05, 10.24 and 19.36 μg/L, respectively. Moreover, these three diamides can induce severe developmental abnormalities in D. magna embryos including underdeveloped second antennae, curved tail spine and abnormal body region after acute exposure and the 48-h EC50 were 6.2, 14.1 and 30.8 μg/L for chlorantraniliprole, cyantraniliprole and flubendiamide respectively. Our findings indicate that even low levels of diamides can pose ecological risks to aquatic ecosystems.

An insecticide resistance-breaking mosquitocide targeting inward rectifier potassium channels in vectors of Zika virus and malaria.

Insecticide resistance is a growing threat to mosquito control programs around the world, thus creating the need to discover novel target sites and target-specific compounds for insecticide development. Emerging evidence suggests that mosquito inward rectifier potassium (Kir) channels represent viable molecular targets for developing insecticides with new mechanisms of action. Here we describe the discovery and characterization of VU041, a submicromolar-affinity inhibitor of Anopheles (An.) gambiae and Aedes (Ae.) aegypti Kir1 channels that incapacitates adult female mosquitoes from representative insecticide-susceptible and -resistant strains of An. gambiae (G3 and Akron, respectively) and Ae. aegypti (Liverpool and Puerto Rico, respectively) following topical application. VU041 is selective for mosquito Kir channels over several mammalian orthologs, with the exception of Kir2.1, and is not lethal to honey bees. Medicinal chemistry was used to develop an analog, termed VU730, which retains activity toward mosquito Kir1 but is not active against Kir2.1 or other mammalian Kir channels. Thus, VU041 and VU730 are promising chemical scaffolds for developing new classes of insecticides to combat insecticide-resistant mosquitoes and the transmission of mosquito-borne diseases, such as Zika virus, without harmful effects on humans and beneficial insects.

Synthesis and biological activity of a new class of insecticides: the N-(5-aryl-1,3,4-thiadiazol-2-yl)amides.

Optimization studies on a high-throughput screening (HTS) hit led to the discovery of a series of N-(6-arylpyridazin-3-yl)amides with insecticidal activity. It was hypothesized that the isosteric replacement of the pyridazine ring with a 1,3,4-thiadiazole ring could lead to more potent biological activity and/or a broader sap-feeding pest spectrum. The resulting N-(5-aryl-1,3,4-thiadiazol-2-yl)amides were explored as a new class of insecticides. Several methods for 2-amino-1,3,4-thiadiazole synthesis were used for the preparation of key synthetic intermediates. Subsequent coupling to variously substituted carboxylic acid building blocks furnished the final targets, which were tested for insecticidal activity against susceptible strains of Aphis gossypii (Glover) (cotton aphid), Myzus persicae (Sulzer) (green peach aphid) and Bemisia tabaci (Gennadius) (sweetpotato whitefly). Structure-activity relationship (SAR) studies on both the amide tail and the aryl A-ring of novel N-(5-aryl-1,3,4-thiadiazol-2-yl)amides led to a new class of insecticidal molecules active against sap-feeding insect pests. © 2016 Society of Chemical Industry.

Rapid selection for resistance to diamide insecticides in Plutella xylostella via specific amino acid polymorphisms in the ryanodine receptor

Diamide insecticides, such as flubendiamide and chlorantraniliprole, are a new class of insecticide with a novel mode of action, selectively activating the insect ryanodine receptor (RyR). They are particularly active against lepidopteran pests of cruciferous vegetable crops, including the diamondback moth, Plutella xylostella. However, within a relatively short period following their commercialisation, a comparatively large number of control failures have been reported in the field. In this review we summarise the current body of knowledge regarding the molecular mechanisms of diamide resistance in P. xylostella. Resistant phenotypes collected from different countries can often be linked to specific target-site mutation(s) in the ryanodine receptors’ transmembrane domain. Metabolic mechanisms of resistance have also been proposed. Rapid resistance development is probably a consequence of over-reliance on this one class of chemistry for diamondback moth control.

Residue level and dissipation pattern of spiromesifen in cabbage and soil from 2-year field study.

Spiromesifen is a new class of insecticide used for the control of whiteflies and mites which have developed resistance to the more commonly used neonicotinoids. Dissipation pattern of spiromesifen on cabbage was evaluated over 2 years by conducting supervised field studies as per good agricultural practices. Cabbage and soil samples were extracted and purified using modified QuEChERS method and analyzed through gas chromatography (GC). Confirmatory studies were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The recoveries of spiromesifen from cabbage and soil were between 85.44 and 103.37% with the relative standard deviation (RSD) between 3.2 and 9.4% (n = 6). The limit of detection (LOD) and limit of quantification (LOQ) were 0.003 μg mL(-1) and 0.01 mg kg(-1), respectively. The measurement uncertainties (MUs) were within 9.9-14.9%. Initial residues of spiromesifen on cabbage were 0.640 and 1.549 mg kg(-1) during 2013 and 0.723 and 1.438 mg kg(-1) during 2014 from treatments at standard and double doses of 125 and 250 g active ingredient (a.i.) ha(-1), respectively. Spiromesifen residue dissipation followed first-order rate kinetics, and it degraded within the half-lives of 2.9 and 3.9 days during 2013 and 3.2 and 4.5 days during 2014. The residue levels reached below the maximum residue limit (MRL; 0.02 mg kg(-1)) within 15-17 days at the standard dose and 24-27 days at the double dose. The field soil analyzed at harvest (30 days) was free from spiromesifen residues. Metabolite spiromesifen-enol was not detected in any sample which was confirmed through LC-MS/MS analysis.

Michael Elliott CBE. 30 September 1924 — 17 October 2007

Michael Elliott was the leader of work at Rothamsted that invented and subsequently commercially developed the pyrethroids, a new class of insecticides. Michael made probably the greatest individual contribution to the control of insect pests that not only constrain global food production but also affect the health of ourselves and our livestock. In one of the first pioneering structure–activity relationship studies, Michael led the multidisciplinary team that invented the major pyrethroid insecticides bioresmethrin, permethrin, cypermethrin and deltamethrin. In the 1980s these represented two-thirds of the global pyrethroid market; at that time pyrethroids captured more than 25% of the total insecticide market and were used on 33 million hectares of crops (Wirtz et al . 2009). In 2002 deltamethrin was the world's largest-selling pyrethroid, with annual sales worth $208 million (information from Cropnosis Ltd). In terms of human health, in 2009 it was estimated that pyrethroid-treated bednets significantly decreased the number of deaths due to malaria among children under five years of age by about one-fifth as well as reducing all incidents of malaria, and in 2011 the World Health Organization recommended its vastly expanded use. Reference Wirtz, K., Bala, S., Amann, A. & Elbert, A. 2009 A promise extended—future roles of pyrethroids in agriculture. Bayer CropSci. J. 62 , 145–158.

A new class of insecticide for malaria vector control: evaluation of mosquito nets treated singly with indoxacarb (oxadiazine) or with a pyrethroid mixture against Anopheles gambiae and Culex quinquefasciatus.

BackgroundUniversal coverage with long-lasting insecticidal mosquito nets (LLIN) or indoor residual spraying (IRS) of houses remain the primary strategies for the control of mosquito vectors of malaria. Pyrethroid resistant malaria vectors are widespread throughout sub-Saharan Africa and new insecticides with different modes of action are urgently needed if malaria vector control is to remain effective. Indoxacarb is an oxadiazine insecticide that is effective as an oral and contact insecticide against a broad spectrum of agricultural pests and, due to its unique site of action, no cross-resistance has been detected through mechanisms associated with resistance to insecticides currently used in public health.MethodsWHO tunnel tests of host seeking mosquitoes were carried out as a forerunner to experimental hut trials, to provide information on dosage-dependent mortality, repellency, and blood-feeding inhibition. A dosage range of indoxacarb treated netting (100–1000 mg/m2) was tested against a pyrethroid susceptible strain of Anopheles gambiae. In addition, efficacy of indoxacarb 500 mg/m2 was compared with a standard pyrethroid formulation against pyrethroid susceptible and resistant Culex quinquefasciatus. Dosages between 25 and 300 mg/m2 indoxacarb were tested in tunnel tests and in ball-frame bioassays as mixtures with alphacypermethrin 25 mg/m2 and were compared with singly applied treatments against an insectary reared pyrethroid resistant strain of Cx. quinquefasciatus originally collected in Cotonou, Benin.ResultsThere was a dosage-dependent response in terms of indoxacarb induced mortality, with dosages >100 mg/m2 producing the best mortality response. In tunnel tests indoxacarb 500 mg/m2 exceeded WHOPES thresholds with >80 % mortality of adult An. gambiae and blood-feeding inhibition of 75 %. No cross-resistance to indoxacarb was detected through mechanisms associated with resistance to pyrethroid insecticides and was equally effective against susceptible and resistant strains of Cx. quinquefasciatus. Indoxacarb 500 mg/m2 killed 75 % of pyrethroid resistant Cx. quinquefasciatus compared with only 21 % mortality with alphacypermethrin 40 mg/m2. Mixtures of indoxacarb with pyrethroid produced an additive response for both mortality and blood-feeding inhibition. The best performing mixture (indoxacarb 200 mg/m2 + alphacypermethrin 25 mg/m2) killed 83 % of pyrethroid resistant Cx. quinquefasciatus and reduced blood-feeding by 88 %, while alphacypermethrin only killed 36 % and inhibited blood-feeding by 50 %.ConclusionsNew insecticides with different modes of action to those currently used in mosquito vector control are urgently needed. Indoxacarb shows great promise as a mixture with a pyrethroid and should be evaluated in experimental hut trials to determine performance against wild free-flying, pyrethroid resistant An. gambiae and wash-resistant formulations developed.

Toxicity of Cephalaria Species and their Individual Constituents against Aedes aegypti

Crude acetone and ethanol extracts of the aerial parts of 21 Cephalaria species collected from Turkey were investigated for larvicidal and adult topical activity against Aedes aegypti. The ethanol extracts from C. elazigensis var. purpurea, C. anatolica, and C. elmaliensis possessed the highest mortality against first instar Ae. aegypti larvae. Luteolin-7-O-β-D-glycoside (1), isolated from C. elmaliensis ethanol extract, demonstrated 33% and 53% mortality at 0.1 μg/mL concentration against first instar ORL (susceptible) and PR (pyrethroid resistant) strains, respectively. C. scoparia acetone extract showed 100% mortality against adult Ae. aegypti. From this extract compounds 2-8 were isolated. Compound 2 (isoorientin) possessed the highest toxicity with 31.7% and 65% mortality at a 10 μg/mL concentration against adult ORL and PR strains, respectively. This is the first screening report of potential insecticides from Cephalaria species against the yellow fever mosquito, Ae. aegypti, and the active compounds (1 and 2) could lead to the development of a new class of insecticide.

New Class Of Insecticides

New Class Of Insecticides

Peculiarities of toxicodynamics and evaluation of hazard of the new avermectin class insecticides–abamectin and emamectin.

Based on the expert-analytical study of literature and websites on the results of toxicological experiments of varying duration, a hygienic evaluation of the new class of insecticides – macrocyclic lactones: abamectin and emamectin was done. It was found that by the parameters of acute oral and inhalation toxicity abamectin isreferred to a highly dangerous pesticides (class 1), by acute percutaneous toxicity – to moderately dangerous (3), by irritating and allergenic action – to low dangerous (4). According to LD50 emamectin in single administration into the stomach and on the skin belongs to class 3, by acute inhalation toxicity and irritation of the mucous membranes – to class 2 (dangerous), by skin irritation – to 3-4 class, having allergenic potential – 4 class of danger. CF-1 mice are animals most sensitive to the effects of avermectins due to polymorphism of their mdr 1a gene, coding P-glycoprotein. The main target organ to avermectin action was recognized to be the central nervous system. ADI of abamectin for humans was established at the level of 0,0002 mg/kg (the lowest NOAEL by systemic toxicity for parent and offspring generation was set in the experiment studying reproductive toxicity on rats – 0,12 mg/kg, a safety factor – 600) and emamectin – 0,001 mg/kg (the lowest NOEL was established in 16-day experiment on CF-1 mice – 0,1 mg/kg and a safety factor – 100).