Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 11-19, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1013 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Dilek Akyil (2021) Mutagenic and cytotoxic activity of insecticide Napole- on 4EC in Allium cepa and Ames test. Caryologia 74(2): 11-19. doi: 10.36253/ caryologia-1013 Received: July 06, 2020 Accepted: July 20, 2021 Published: October 08, 2021 Copyright: © 2021 Dilek Akyil. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Mutagenic and cytotoxic activity of insecticide Napoleon 4EC in Allium cepa and Ames test Dilek Akyil Department of Molecular Biology and Genetic, Faculty of Arts and Sciences, Afyon Kocatepe University, Afyonkarahisar/Turkey E-mail: dilekakyil9@gmail.com Abstract. The objective of this study was to explore the mutagenic and cytotoxic effects of Napoleon 4EC pesticide used in Turkey to control insect pest by using two stand- ard assays. The Allium cepa test was used for determined the cytotoxic effects of this pesticide. For this test, onion seeds were exposed to Napoleon 4EC (100, 200, and 400 ppm) for 24, 48, and 72 hours. For each test group root tip cells were stained with Feulgen and five slides were prepared for each concentration and counted microscopi- cally. The concentrations Napoleon 4EC was compared with the value for the nega- tive control using Dunnet-t test, 2 sided. The results indicated that mitotic index was clearly decreased with increasing the concentration of Napoleon 4EC in each treatment group as compared to the controls. The percentage of mitotic phases has been marked- ly impacted. Five different doses of the pesticide (50, 100, 200, 400, 800 μg/plate) were examined with Ames test using Salmonella typhimurium strains TA98 and TA100 with and without S9 metabolic activation for mutagenic activity. Ames test results showed a dose dependent effect, but not twice the negative control for S. typhimurium TA98 and TA100, with or without S9 mix except 800 μg/plate doses. In 800 μg/plate doses, colony numbers are two-fold increase according to colony number of control group. So, this places the this compound as a weak mutagen according to the parameters. Keywords: Allium test, Ames test, cytotoxicity, mutagenicity, Napoleon 4EC. INTRODUCTION The identification of chemicals capable of inducing mutations has become an important procedure in safety estimation and such substances can potentially induce to fertility problems in future generations. Mutagen- ic materials are also capable of inducing cancer, and this problem has been increased of the importance the mutagenicity testing systems (Kumar et al. 2013). Earlier studies have showed that some pesticides are clastogenic and mutagenic in different biological test systems (Siroki et al. 2001; Celik 2003; Stivaktakis et al. 2010; Moulas et al. 2013; Akyıl et al. 2014; Akyıl and Konuk 2015; Özkara et al. 2015a). Organophosphorus pesticides (OPs) are one of the most important groups of pesticides which have been broadly used in indus- try, hygiene and agriculture (Bello-Ramı́ rez et al. 2000; Ballesteros and Par- 12 Dilek Akyil rado 2004; Wu et al. 2007). Napoleon 4EC is also an OPs which has an important role to control crops. OPs are strong inhibitors of cholinesterase enzymes and they were improved to replace organohalide pesti- cides in the late 1950’s due to relatively easier to degrade via microbial or environmental processes (Obare et al. 2010). On the other hand, these substances or their derivatives can accumulate in the living organisms and induce mutagenicity, teratogenicity, immunotoxicity and carcinogenicity. Short-term test methods have been used for many years from past to present to identify the genotoxic activities of pesticides (Miao et al. 2017; Özkara 2017; Khallef et al. 2018; Özkara 2019; Akyıl 2019). The short- term test systems can detect different types of genetic DNA damage: (i) gene or point mutations; (ii) primary DNA damage and repair; (iii) chromosomal alterations. One of these test systems is the Ames test which is used to evaluate the mutagenic activity of chemicals; it is a short-term bacterial reverse mutation assay (Mortel- mans and Zeiger 2000; Liman et al. 2010; Arriaga-Alba et al. 2013; Escobar et al. 2013). The other test system is the Allium test which is one of the well-known and reli- able test systems to determine the toxicity in the labora- tories (Konuk et al. 2007; Liman et al. 2010; Özkara et al. 2015; Bonciu et al. 2018). To analyze the effects of dif- ferent substances, higher plants (Vicia faba, Tradescan- tia paludosa, Pisum sativum, Hordeum vulgare, Crepis capillaris and Allium cepa, etc.) have proven to be useful when used as bioindicators (Enan 2009; Siddiqui and Al- Rumman 2020). However, Allium cepa has as an advan- tage due to its large chromosomes, easily observed with a light microscope; also in relation to the features that may reveal an effect even at relatively low level of inter- action of the tested substance with the genetic material, besides its long history of use as cytotoxicological test. As it is an in vivo test, the data can be used for assess- ment of genotoxicity on plants and even for eukaryotes in general, including humans (Bonciu et al. 2018). The use of insecticides has become increasingly widespread throughout the world so additional stud- ies are necessary to determinate the potential toxic risk of insecticides on non-target organisms through bacte- rial mutational/ames test and Allium test, a plant assay (Yaduvanshi et al. 2012). To our knowledge, there is no study on the cytotoxicity and mutagenicity of Napoleon 4EC except in the present paper. The aim of this experi- ment was to evaluate both the mutagenic and cytotoxic effects of different doses of Napoleon 4EC by the bacte- rial reverse mutation assay in S. typhimurium TA98 and TA100 strains with or without S9 mix and Allium cepa test, respectively. MATERIALS AND METHODS Chemicals and test strains The LT-2 TA98 and TA100 histidine demanding auxotrophs of S. typhimurium were kindly obtained from Prof. B.N. Ames (University of California, Berke- ley). These strains were incubated for 16 h in liquid nutrient broth and kept at -80°C. Their genetic markers and other properties, such as the numbers of spontane- ous revertants and responses to positive controls, were controlled as described by Maron and Ames (1983). The test substance Napoleon 4EC was purchased from a local market in Afyonkarahisar/Turkey and dissolved in ster- ille distilled water. Allium cepa onion bulbs, 25–30 mm diameter, were obtained from a local market without any treatments. The other chemicals were obtained from Merck and Riedel. Ames plate incorporation test The mutagenicity of the Napoleon 4EC was deter- mined using the standard plate incorporation assay. Salmonella typhimurium strains TA98 and TA100 were used with or without S9 mix in this test (Maron and Ames 1983). The tester strains were tested for the pres- ence of the strain-specific markers as described by Maron and Ames (1983). Before the experiment, deter- mination of the cytotoxic doses of the substance to be used during the experiment is carried out as a prelimi- nary step. Cytotoxic doses of Napoleon 4EC (10.000, 1.000, 100, 10, 1 and 0.1 µg/plate) were determined by the method of Dean et al. (1985). The strains were selected according to the strategies of Mortelmans and Zeiger (2000). The stock solutions of the test materi- als were dissolved in sterile distilled water and stored at 4°C. The S. typhimurium strains were incubated in nutrient broth at 37°C for 16h with shaking. A specific positive control was always used to test the experimen- tal defect, if any, for each tester strain. Positive controls were 4-nitro-o-phenylenediamine (NPD) for TA98 and sodium azide (SA) for TA100, applied without metabol- ic activation, and 2-aminofluorene (AF) for TA98 and 2-aminoanthracene (2AA) for TA100 used with meta- bolic activation. Determination of cytotoxic doses For the test of cytotoxic doses were prepared by add- ing 0.1 ml of the test suspension for each concentration and 0.1 ml bacterial suspension of TA100 from an over- 13Mutagenic and cytotoxic activity of insecticide Napoleon 4EC in Allium cepa and Ames test night culture to 2 ml top agar which kept in 45°C water bath. The mixture was shaken for 3 s with a vortex mix- er, and added into the nutrient agar. All test plates were incubated for 24 h at 37°C and then the revertant colo- nies were counted for each plate and determinated toxic and non-toxic doses which used in the experiment. For the test of without S9 mix were prepared by adding 0.1 ml of the test suspension for each concentra- tion, 0.1 ml bacterial suspension from an overnight cul- ture, and 0.5 ml phosphate buffer to 2 ml top agar which kept in 45°C water bath. The mixture was shaken for 3 s with a vortex mixer, and added into the minimal agar. For the test plates with S9 mix were prepared by add- ing 0.5 ml of S9 mix instead of the phosphate buffer. All test plates were incubated for 72 h at 37°C and then the revertant colonies were counted for each plate. Samples were evaluated on triplicate plates in two independent parallel experiments and all results of the experiment were analysed by the statistical analysis. Allium test The root inhibition test procedure was performed as described by Fiskesjo (1985). Preliminary experiments were conducted to determine the concentrations of each pesticide to be used in the actual cytotoxicity experi- ments. The pesticides were dissolved in distilled water. Outer scales of the bulbs and the dry bottom plates were removed without damage the root primordia. The onions were germinated in freshly distilled water for the first 24 h and then exposed for 96 h to the different doses of Napoleon 4EC (25, 50, 100, 200 and 400 ppm, respectively). The roots from each group including con- trol group were cut off on the fifth day and length of each root was measured in order to determine the EC50 values. EC50 value was determined as the concentration which retards the growth of root 50% less when com- pared with the control group. Root Growth Inhibition Test (EC50 determination) The onions were grown in freshly made distilled water for 24 h and then exposed for four days to the control group and other concentrations of extracts. In order to determine efficient concentration (EC50) values, ten roots from each onion were cut off at the end of the treatment period, and the root’s length was measured. The concentration that decreased root growth about 50% when compared to the negative control group (distilled water), was accepted as EC50 value. To deter- mine the possible toxic effects on roots, EC50/2, EC50 and EC50×2 concentrations of root were used in Allium mitotic index test. The EC50 value was approximately 200 ppm for Napoleon 4EC. In order to show possible concentra- tion-dependent effects of this pesticide, the root tips were treated with 100 ppm (EC50/2), 200 ppm (EC50) and 400 ppm (EC50x2) concentrations of Napoleon 4EC and all application groups were performed 24, 48 and 72 h treatment periods. After treatment, the roots were washed in distilled water and fixed in 3:1 ethanol:glacial acetic acid for 24 h and then roots were taken in 70% alcohol and stored +4°C. Feulgen was used for staining root tip cells. Slides were randomly coded and scored blindly. For mitotic index (MI), the different stages of mitosis were counted in a total of 5000–6000 cells (1000 cells/slide) per concentration, and expressed as a per- centage. Statistical Analysis Root length and MI datas were given as percent- ages. The levels of difference in treatment groups were analyzed statistically by SPSS 15.0 version for Windows. Dunnett-t test (2 sided) was used on both the Allium and Ames tests in the analyses. RESULTS Ames test results was carried out for mutagenic- ity determination of the tested material. For this test histidine mutant strains of S. typhymurium, TA98 and TA100 were used, and control group colony numbers were compared with the test material. The concentra- tions which caused two-fold increase in the colony num- ber of control group were accepted as mutagenic ones. A compound is considered a weak mutagen if it produces a reproducible, dose-related increase in the number of revertant colonies in one or more strains but the num- ber of revertants is not double the background number of colonies (Mortelmans and Zeiger 2000). It was found that only 1000 μg/plate concentration was cytotoxic against S. typhymurium strains among six tested concentrations for cytotoxicity tests (1000, 800, 400, 200, 100, 50 and 25 μg/plate). So this toxic concen- tration was not applied in Ames test. In Ames test posi- tive controls were 4-nitro-o-phenylenediamine (NPD) for TA98 and sodium azide (SA) for TA100, used with- out metabolic activation, and 2-aminofluorene (AF) for TA98 and 2-aminoanthracene (2AA) for TA100 used with metabolic activation, while distilled water was used as a negative control group. Most of the results, whether 14 Dilek Akyil increasing or decreasing relative to the negative control group, were not statistically significant at P<0.05 (Dun- nett-t test, 2 sided) except for in the 800 μg/plate doses of the Napoleon 4EC in the TA98 without S9 mix. Ames test results showed a dose dependent effect, but not twice the negative control for S. typhimurium TA98 and TA100, with or without S9 mix except 800 μg/ plate doses. In 800 μg/plate doses, colony numbers are two-fold increase according to colony number of con- trol group. So, this places the this compound as a weak mutagen according to the parameters. When S9 was added, revertant colony numbers in TA98 and TA100 became stronger and Ames test data’s is summarized in Table 1. Table 2 are summarized in Allium root growth test results. The effective concentration (EC50) was deter- mined as 200 ppm. Table 3 gives the effect of Napoleon 4EC on MI and mitotic phase in the root meristematic cells of A. cepa treated for 24, 48 and 72 h. At all con- centrations treated in the incubations of root diminished MI compared to negative control at each exposure time. The highest values were showed from 24 h examination of 100 ppm, and the lowest one in 72 h application of 400 ppm concentrations of Napoleon 4EC. The reduced of MI indicates statistically significant results (p < 0.05) all concentrations and all treatment time. All doses of Napoleon 4EC applied in the experiment caused changes in the percentage of particular phases’ distribution in comparison to the control. DISCUSSION While the use of pesticides are planned to elimi- nate pests and develop the quality and quantity of yield in agriculture, there is concern about their use because some have cytotoxic/mutagenic and carcinogenic effects and harm non-target organisms (Asita and Mokobo 2013). Earlier studies have reported that some pesticides have mutagenic and clastogenic activities in several bio- logical test systems (Yaduvanshi et al. 2012; Topcu et al. 2013; Asita and Mokobo 2013; Özkara et al. 2015b; Karaismailoğlu 2016; Khallef et al. 2017). The bacterial reverse mutation test uses amino-acid requiring strains of S. typhimurium to identify point mutations, which include substitution, deletion or addi- tion of one or a few DNA base pairs. The cause of many human genetic diseases are originated point mutations and their occurrence in oncogenes and tumor suppres- sor genes of somatic cells are caused in tumor formation Table 1. The mutagenicity assay results of Napoleon 4EC for S. tyhimurium TA98 and TA100 strains. Test Substance Concentration (µg/ plate) No of His+ revertants/plate, mean±SD TA98 TA100 - S9 + S9 - S9 + S9 Napoleon 4EC 800 70.20±4.14* 83.24±6.25 110.54±6.04 197.54±11.58 400 52.10±3.21 83.40±3.46 102.21±4.54 168.21±6.52 200 45.21±3.56 76.45±4.32 90.54±6.87 154.31±10.25 100 40.90±3.68 65.20±6.25 91.21±9.62 142.30±11.54 50 31.62±1.83 46.70±5.54 83.02±7.04 138.12±12.26 Neg. Control 100 32.70±4.68 45.21±2.57 81.21±11.24 126.32±5.83 SA 10 2824.56±68.31* 2AA 5 2468.24±70.15* 2AF 200 988.50±17.57* NPD 200 1445.60±22.23* *Mean statistically significant at p<0.05 (Dunnett t-test), SA:Sodium azide, NPD: 4-nitro-o-phenylendiamine, 2AF: 2-aminofluorene, 2AA: 2-aminoanthracene, SD: Standard deviation, Negative control: distilled water. Table 2. Allium root growth inhibition test. Test Substance Concentrations (ppm) Mean of root length±SD Negative Control - 3.86±0.41 Positive Control - 1.12±0.14* Napoleon 4EC 25 3.41±0.26* 50 2.20±0.14* 100 1.94±0.31* 200 1.62±0.21* 400 1.27±0.14* *Significantly different from negative control (p<0.05 Dunnet-t test, 2-sided) SD: Standart deviation. 15Mutagenic and cytotoxic activity of insecticide Napoleon 4EC in Allium cepa and Ames test in experimental animals and humans (Malev 2012). The Salmonella/microsome assay is broadly used for evaluating the mutagenicity of chemicals including pesticides (Yaduvanshi et al. 2012 ). In the present stud- ies, Ames plate incorporation assay with the different concentrations of Napoleon 4EC showed a mutagenic response only TA98 tester strain. In order to character- ize the possible mechanism of mutagenicity, the impor- tant bacterial strains, sensitive to different mutational events due to their specific geno-types, were used. There are much reports on the mutagenic effects of OPs deter- mined with the Ames test (Aufderheide and Gressmann 2007; Coral et al. 2009; Wu et al. 2012; Akyıl and Konuk 2014). However, no study has yet reported on the in vitro mutagenicity of Napoleon 4EC using the Ames assay. S. typhimurium TA98 strain is defined by the -1 frameshift deletion hisD3052, which effects the reading frame of a close by repetitive –C–G– bases and can be reverted by frameshift mutagens. TA100 contains the marker hisG46, which causes from a base-pair substitu- tion of a leucine (GAG/CTC) by a proline (GGG/CCC): this mutation is reverted by mutagens causing base sub- stitutions at G-C sequences (Di Sotto et al. 2008). In this context these bacterial properties, our results can be said that Udimo 75 WG mutagenicity in TA98 strain is caused by frameshift mutations and that of TA100 strain is due to base change (Di Sotto et al. 2008). All concentrations of this pesticide were weak muta- genic in the TA98 and TA100 strains, with or without the S9 fraction except 800 μg/plate doses of the Napo- leon 4EC in the TA98 without S9 mix. In 800 μg/plate doses, colony numbers are two-fold increase accord- ing to colony number of control group. Exposure to the pesticide induced G–C base pair mutations (Maron and Ames, 1983) causing a frameshift reversion of the his- tidine-dependent tester strain (TA98) to the wild type (his+). However, the addition of S9 mixture resulted in a reduction of the mutagenic effect of Napoleon 4EC but not significant according to negative control. Due to biotransformation, a compound that is active biologi- cally can be changed to an inactive metabolite. Simi- larly an inactive compound can be changed to an active metabolite (Paolini and Forti 1997). In other words, the presence of an eukaryote enzyme in S9 fraction resulted in eliminate of the mutagenic activity of the tested sub- stance. Therefore, it is vital to use the S9 fraction in the Ames test. Yaduvanshi et al. (2012) reported that negative results for chlorpyrifos-an organophosphate pesticide- with all three tester strains (TA97, TA98, TA102) of Sal- monella used in the presence or absence of metabolic activation. However, chlorpyrifos was toxic in TA98 tester strain at the dose of 5000 μg/plate in absence of metabolic activation while reduction in toxicity was seen on addition of S9 mixture. In another study, five differ- ent concentrations of the Chlorthiophos were tested by Ames test using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without S9 meta- bolic activation. No concentrations of Chlorthiophos showed mutagenic activity on the TA97, TA100, and Table 3. The effects of Napoleon 4EC on MI and mitotic phases in the root cells of A. cepa. Concentration (ppm ) Treatment Time Counted Cell Number Mitotic Index ± SD Mitotic Phases (%) ± SD Prophase Metaphase Anaphase Telophase Negative control 24 hour 5102 87.40±9.68 83.21±10.66 1.86±0.23 1.01±0.45 0.88±0.75 Positive control 4802 43.36±4.76* 38.27±4.26* 0.62±0.17* 0.55±0.54 0.50±0.28 100 4685 41.45±4.06* 41.10±2.71* 1.02±0.27* 0.71±0.22 1.05±0.46 200 5002 38.21±4.25* 38.44±4.02* 0.98±0.24* 1.00±0.53 1.27±0.24 400 5012 39.26±2.85* 36.11±2.45* 0.87±0.14* 0.45±0.12 0.62±0.47 Negative control 48 hour 5045 78.34±4.54 75.21±6.47 1.70±0.12 1.34±0.42 1.26±0.18 Positive control 5065 38.22±3.12* 36.85±3.54* 0.60±0.19* 0.64±0.31* 0.72±0.31 100 5145 31.54±3.65* 37.62±3.53* 0.79±0.49* 0.76±0.12* 1.21±0.24 200 5214 28.42±3.06* 32.54±2.08* 0.62±0.14* 0.60±0.20* 1.02±0.23 400 5162 23.40±1.48* 30.54±2.78* 0.45±0.23* 0.51±0.11* 0.92±0.41 Negative control 72 hour 5252 65.45±2.23 62.25±3.14 1.54±0.41 1.50±0.24 0.94±0.25 Positive control 5265 31.12±3.16* 32.02±3.69* 0.42±0.24* 0.86±0.24* 0.85±0.23 100 5189 28.17±5.44* 30.27±3.58* 0.65±0.19* 0.66±0.54* 0.52±0.14 200 4989 25.01±2.14* 24.24±2.45* 0.53±0.14* 0.62±0.27* 0.54±0.47 400 4980 18.17±2.25* 17.45±2.07* 0.38±0.47* 0.49±0.14* 0.61±0.51 * Significantly different from negative control (p< 0.05 Dunnet-t test. 2-sided) SD: Standart deviation. 16 Dilek Akyil TA102 strains, with and without S9 fraction, but were all mutagenic to the TA98 strain without S9 (Akyıl and Konuk 2014). Many researchers who have studied Ames test systems with OPs also reported mutagenic or non- mutagenic result (Aiub et al. 2002; Kumar et al. 2013; Arroyo et al. 2015; Akyıl et al. 2017). Chemicals which tested with different test methods can be genotoxic or not genotoxic depending on a number of factors such as chemical structure, biological activity, the positions of the binding location and having rings in the struc- ture (Kutlu et al. 2011). Furthermore, it might be related to differences in test conditions, such as exposure time, concentrations of substances, the dispersal of the mate- rials in the cell and physico-chemical characteristics of the chemicals (Ema et al. 2012; Kaur et al. 2014). There- fore, it could be explained why some studies find an increase of genetic damage while in others result as neg- ative. It is well known that plants are direct recipients of toxins and the Allium cepa assay is one of the plant assay method used broadly to study the genotoxicity of pesti- cides (Fernandes et al. 2007). Also, Allium cepa showed a good correlation with the results from other estab- lished test systems using eukaryotic as well prokaryotic cells (Yıldız et al. 2009). Mitotic index is a parameter that allows to estimate the frequency of cellular division (Marcano et al. 2004) and the reduction of mitotic activities has been used fre- quently to determine the cytotoxicity (Linnainmaa et al. 1978). Many investigators have reported the change mitotic index following the treatment of test organ- isms with pesticides (Panda and Sahu 1985; Amer and Farah 1974). In this experiment, mitotic index mostly decreased with increase Napoleon 4EC concentrations at each treatment times in comparison with control groups (p<0.05). When the phase frequencies are compared with control in different treatment groups, significant outcomes were obtained statistically (p<0.05). The per- centages of the mitotic phases were clearly influenced in totally almost all applications (p<0.05). Chlorpyrifos, the active ingredient in Napoleon 4EC, was shown to a dose-dependent increase in DNA damage in the liver and brain of rats using the sin- gle cell gel electrophoresis (or comet) assay (Mehta et al. 2008). Chlorpyrifos is known to generate oxidative stress, induce lipid peroxidation, and cause depletion of reduced glutathione (GSH), increase in oxidized glu- tathione (GSSG), and decrease in the ratio of GSH/GSSG in rat erythrocytes and tissues (Gultekin et al. 2001; Ver- ma and Srivastava, 2003). Chlorpyrifos exposure causes inhibition of antioxidant enzyme activities and increase in the levels of hydrogen peroxide (H2O2) in rat brain and liver (Gultekin et al. 2001; Verma and Srivastava 2003). Additionally, Chlorpyrifos was not mutagenic in the Ames Salmonella mutagenicity assay and mammali- an cell cultures (CHO/HGPRT assay), cytogenetic abnor- malities in mammalian cells both in vitro (rat lympho- cyte chromosomal aberration test, RLCAT) and in vivo (mouse bone marrow micronucleus test) and induction of DNA damage and repair in rat hepatocytes in vitro (Gollapudi et al. 1995). Dursban 4 (Chlorpyrifos-ethyl) was decreased mitotic index and induced chromosome aberrations in the root meristem cells of Allium cepa (Topcu et al. 2013). In the present study with Allium cepa root tip mer- istem cells, the three doses of Napoleon 4EC tested induced cytotoxicity thus corroborating the findings of these studies but not the in vitro studies with S. typh- imurium cited above. There are some possible mecha- nisms for chemically decreased MI in plant cells. The significant decline in the mitotic index could be due to the inhibition of the DNA synthesis or the blocking of the G1 suppressing the DNA synthesis or effecting the test compound at the G2 phase of the cell cycle (Sudha- kar et al. 2001; Majewska et al. 2003). When a pesticide penetrates the cells and reaches a critical dose, it could be an active form, causing lesions during several follow- ing cellular cycles (Marcano et al. 2004). The decrease of the mitotic index in our study can be related to this. In this study, all the concentrations of Napoleon 4EC caused the changes in the percentage of the par- ticular phases’ distribution when compared to the con- trol group. Pesticides accumulate in the cell due to this substance not being able to emerge out of the cell easily after once penetrating the cell and it may be highly toxic in the cell (Antunes-Madeira and Madeira 1979). The safety evaluation of a fragrance material includes a broad range of toxicological information, both for the substance itself and for structurally related chem- icals belonging to the same chemical group (Bickers et al. 2003). Among toxicological information, genotoxicity is a systemic consideration, as it can be related to carci- nogenicity (Di Sotto et al. 2008). Normally, to evaluate a potential genotoxic risk due to a chemical exposition, in vitro assays for detecting point mutations (Ames test) and extended treatment (e.g., micronucleus assay, Allium test, single cell gel electrophoresis assay or comet assay) are used in the first instance (EMEA 2008; Di Sotto et al. 2013). If the results of these studies are positive, in vivo studies, for example a mammalian cytogenetic study, are performed (EFSA 2014). In conclusion, Napoleon 4EC was determined to be cytotoxic due to reducing of MI in Allium test and weak mutagenic in Ames test. For this reason, further inves- 17Mutagenic and cytotoxic activity of insecticide Napoleon 4EC in Allium cepa and Ames test tigations are needed to determine the toxicity of this compound using other in vivo and in vitro biological test systems. A single test system is not enough to deter- minate a compound whether it is toxic or non-toxic. In this study we performed two different test methods. However, for the reliable results, additional mutagenic- ity studies should be conducted. For example, the umu test may be used in conjunction with the Ames test, as it indicates carcinogens as those substances that induce the expression of the umu operon (Reifferschied and Heil 1996). The other in vivo or in vitro tests would help confirm the findings of the results and give predictions for the chemical’s effect in an organism. 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