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 Table of Contents  
Year : 2018  |  Volume : 10  |  Issue : 1  |  Page : 21-28  

Safety evaluation of various vector repellents in combination with deltamethrin in Wistar rats

Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu, India

Date of Web Publication20-Mar-2018

Correspondence Address:
Mrs. Anitha Magesh
Department of Research and Development, Saveetha Institute of Medical and Technical Sciences, Chennai, Tamil Nadu 602105
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpbs.JPBS_219_17

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Background: Vector repellents are used globally to avoid insect bites and control transmission of diseases. It is important to ensure safety levels of these compounds, although they are noxious to insects. Vector repellents when used in combination are known to bring additional benefits in getting rid of the harmful insects. Unfortunately, the safety levels of various vector repellents such as Deet (N,N-diethyl-m-toluamide), Depa (N,N-diethyl phenylacetamide), and Deb (N,N-diethylbenzamide) are not studied in combination with the widely used pyrethroid deltamethrin (Del). Materials and Methods: In this study, safety evaluation of various vector repellents by oral route in combination with Del was studied by assessing body weight and organ weight changes, hematological parameters, and biochemical parameters in Wistar rats. Results: The results revealed significant changes in liver marker enzymes in Del, Deb, Del + Depa, and Del + Deet groups. Urea levels were significantly altered after treatment with various repellents and in combination with Del, except Deb-alone group. Hematological and rest of the biochemical parameters were found to be unaltered in all the treatment groups. Significant changes in the liver weight were found in Deb, Depa, Deet, Del + Deb, and Del + Deet groups. Conclusions: Taken together, the overall results of this study indicate that single, high oral dose of various insect repellents did not show any additive toxicity.

Keywords: Biochemical studies, hematology, organ weight, pyrethroid, safety evaluation, vector repellents, Wistar rats

How to cite this article:
Magesh A , Sivanesan S , Rajagopalan V , Geetha RV, Roy A . Safety evaluation of various vector repellents in combination with deltamethrin in Wistar rats. J Pharm Bioall Sci 2018;10:21-8

How to cite this URL:
Magesh A , Sivanesan S , Rajagopalan V , Geetha RV, Roy A . Safety evaluation of various vector repellents in combination with deltamethrin in Wistar rats. J Pharm Bioall Sci [serial online] 2018 [cited 2022 May 20];10:21-8. Available from:

   Introduction Top

The concept of using insect repellents to restrict vectors and pests dates back longer with the application of various plant oils, smokes, and tars. Repellents are widely used as inexpensive chemicals to protect humans from the bites of insect pests and also to manage many vector-borne diseases.[1],[2] For more than five decades, Deet (N,N-diethyl-m-toluamide) has been widely used as a standard component in mosquito repellents. However, Depa (N,N-diethyl phenylacetamide) has been recognized as a safe and effective alternative to Deet in India, Malaysia, and other Asian countries.[3] The acute and subacute inhalation toxicity studies of Depa revealed a low mammalian toxicity. In a comparative inhalation toxicity study using aerosols of insect repellents such as Deb (N,N-diethylbenzamide), Depa, and Deet in mice, Deet and Depa did not show any adverse effect on respiratory parameters.[4] Permethrin-containing products are not intended for dermal application; however, they are recommended for use on clothing, shoes, bednets, and camping gear. Although pyrethroids can kill mosquitoes and ticks, Depa and Deet can only avoid mosquitoes from biting. On the basis of reports, both Deet and Depa revealed efficient repellency when tested against Aedes aegypti.[2]

Deltamethrin (Del), a pyrethroid, which is more toxic to insects than mammals, has been used as a solution in mosquito nets in tropical countries to protect from malaria and vector-borne viral fevers. Its mode of action is by targeting sodium and chloride channels.[5] The net reduction in resting membrane chloride conductance by Del is expected to amplify the effect of prolonged sodium current, thereby eliciting its action on excitable membranes in mammalian skeletal muscle and nonmyelinated nerve fibers.[6] The neurotoxic effect of Del shows degeneration and apoptotic cell death in rat brain.[7] The neurobehavioral studies conducted in animals using Del had revealed decreased grip strength and poor coordination in rotarod test,[8] and poor performance in 16 figure-eight maze test,[9] thereby implicating poor motor coordination and activity.

It is an encouraging aspect that repellents are formulated in the form of lotions and creams, which are well tolerated on bare skin. Therefore, repellents such as Del, Deb, Deet, and Depa are marketed in the form of aerosols, creams, solids (sticks), pump sprays, and liquids. Notably, the repellent Depa was evaluated in comparison with dimethyl phthalate (DMP) and Deet under field condition on army personnel against mosquitoes, black flies, and land leeches. Both Depa and Deet had proven their efficacy for repellency when evaluated in army personnel. Strikingly, Depa was found to be better than DMP against various organisms tested. On the basis of recent report from Centers for Disease Control and Prevention, two active ingredients were reported as safe and effective repellents, picaridin and oil of lemon eucalyptus.[10] Although the neurotoxic potentials of Deet[11],[12],[13] are well known, there are no reports on neurotoxicity associated with Depa. Del and the other insect repellents are used independently as vector-control measures indoor and outdoor. Although there are reports that revealed the lethal dose toxicity of Del, pyrethroid, Deet, and Depa independently, their toxicities in combination with Del have not been studied so far. In this study, we attempted to evaluate the combined toxicity by assessing various hematological and biochemical parameters.

   Materials and Methods Top


Del, Deb, and Depa were obtained from M/s Tagros Chemicals India Ltd. (Chennai, India), and Deet from Alkyl Amine Chemicals Ltd. (Mumbai, India). All other chemicals used were of analytical grade.


Adult female Wistar rats (Rattus norvegicus) weighing between 150 and 200g from Centre for Laboratory and Animal Research, Saveetha Institute of Medical and Technical Sciences, Chennai, were used. The animals were housed as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. The rats were fed with standard pellet diet and given filtered water ad libitum. Feed was withdrawn 3h before oral feeding of the test chemicals. The rats were maintained at natural light and dark cycle. The study protocol was approved by the Institutional Animal Ethics Committee of Saveetha Medical College (SU/BRULAC/RD/009/2014).

Experimental design

Forty-eight rats were divided into eight groups, each group containing six rats.

  • Group I: Olive oil administered orally and served as control group

  • Group II: 0.5 LD50 of Del (64.9mg/kg body weight [b.w.]) administered orally in olive oil

  • Group III: 0.5 LD50 of Deb (517mg/kg b.w.) administered orally in olive oil

  • Group IV: 0.5 LD50 of Depa (166.7mg/kg b.w.) administered orally in olive oil

  • Group V: 0.5 LD50 of Deet (947mg/kg b.w.) administered orally in olive oil

  • Group VI: A combination of 0.25 LD50 Del (32.4mg/kg b.w.) + 0.25 LD50 Deb (258.8mg/kg b.w.) administered orally in olive oil

  • Group VII: A combination of 0.25 LD50 Del (32.4mg/kg b.w.) + 0.25 LD50 Depa (83.3mg/kg b.w.) administered orally in olive oil

  • Group VIII: A combination of 0.25 LD50 Del (32.4mg/kg b.w.) + 0.25 LD50 Deet (473.5mg/kg b.w.) administered orally in olive oil

The study was conducted in two phases with three animals each per group and the data were pooled.

Sample collection

After 24h of treatment, all the animals were weighed, anesthetized with isoflurane (Raman and Weil, Mumbai, India), and blood was collected from the orbital plexus using fine capillary into Vacutainer tubes with (K3-EDTA) or without anticoagulant. After the collection of blood, animals were killed, and their vital organs such as liver, kidney, spleen, and lungs were removed and washed with saline, weighed, and preserved in 10% formalin solution.

Hematological parameters

Blood samples collected with anticoagulant (K3-EDTA) were analyzed using an automated hematology analyzer (Beckman Coulter India, Miami, Florida, USA). that calculates hemoglobin (Hb) concentration, packed cell volume (PCV), red blood cell (RBC) count, white blood cell (WBC) count, and platelet count.

Biochemical parameters

The serum was separated from the blood by centrifugation and used for the estimation of glucose, urea, creatinine, uric acid, total protein, albumin, total bilirubin, triglycerides, and cholesterol. The enzymes such as aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), and acetylcholinesterase (AChE) were also estimated. The samples were fed into the autoanalyzer (Hitachi 912; Roche Diagnostics, Almere, the Netherlands) after programming. Glucose was estimated by o-toluidine method;[14] urease and uricase method was adopted to estimate urea[15] and uric acid,[16] respectively. Creatinine was estimated by Jaffe’s method.[17] Both Cholesterol and triglycerides were estimated using Trinder method.[18],[19] Biuret method was adopted for protein estimation[20] and Bromocresol green method for albumin estimation.[21] Total bilirubin was estimated by diazo method.[22] ALT, AST, and ALP activity were assayed by International Federation of clinical chemistry kinetic method.[23],[24] GGT activity was determined by coupled enzyme assay.[25] AChE level was estimated using Beckman Coulter automated analyzers by established procedures.

Statistical analysis

The results were analyzed using one-way analysis of variance, and comparison between the treated groups and the control group was carried out by Dunnett’s test. The level of statistical significance was kept as P ≤ 0.05. The analysis and plotting of graphs were carried out using SigmaPlot 13, Systat Software, Cranes Software International Ltd., (Bangalore, Karnataka).

   Results Top

Changes in body weight and organ-to-body weight ratio

Percent change in body weight and organ-to-body weight ratio following 24-h treatment with various repellents is shown in [Table 1]. Only small changes in body weight were detected with respect to individual treatment (Del, Deb, and Depa). With combinations such as Del + Deb and Del + Depa, a 6% loss was observed and with Del + Deet, approximately 8% loss of body weight was observed. On the basis of present results, from organ-to-body weight ratio, significant changes in liver (P < 0.01) occurred following treatments such as Deb, Depa, Deet, Del + Deb, and Del + Deet. Del-alone group and Del + Depa combination group did not show any significant changes in the liver weight. Other organs such as lung, spleen, kidney, and heart did not reveal any significant alterations in weight change.
Table 1: Twenty-four-hour body weight (%) and organ-to-body weight ratio (mean ± standard error [SE])

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Hematological studies

Hematological results did not show any significant changes in the levels of PCV, Hb concentration, RBC, WBC, and platelet count between control and all treatment groups [Figure 1]. This indicates that treatment with Del, Deb, Depa, and Deet as either alone or in combination with Del did not alter the hematological profile in rats.
Figure 1: Hematological parameters in control (A), Del (B), Deb (C), Depa (D), Deet (E), Del + Deb (F), Del + Depa (G), and Del + Deet (H) groups. The values are expressed as mean + standard error (SE), n = 5–6; P > 0.05 versus control (not significant). Single drug dose: 0.5 LD50 (B–E); combination drug dose: each 0.25 LD50 (F–H)

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Biochemical parameters

Results of the various biochemical parameters such as glucose, bilirubin, triglycerides, and cholesterol analyzed using respective serum samples are shown in [Figure 2]. No statistical significance was seen between control and treatment groups for these parameters. Data obtained from protein and albumin measurements also revealed no statistical difference between control and treatment groups [Figure 3]. Likewise, serum creatinine and uric acid [Figure 4] did not show any significant change between control and treatment groups, whereas the urea parameter [Figure 4] revealed a different pattern of results. However, for urea, in comparison with the control group, the levels were found to be significantly decreased in Del, Depa, Deet, and Del + Deb groups. By contrast, the Del + Depa and Del + Deet combinations showed elevated urea levels in comparison with control. Interestingly, the urea levels in Deb group were slightly decreased with respect to control group, and hence were not significant.
Figure 2: Serum glucose, cholesterol, triglycerides, and bilirubin levels in control (A), Del (B), Deb (C), Depa (D), Deet (E), Del + Deb (F), Del + Depa (G), and Del + Deet (H) groups. The values are expressed as mean + standard error (SE) (n = 5–6); P > 0.05 versus control (not significant). Single drug dose: 0.5 LD50 (B–E); combination drug dose: each 0.25 LD50 (F–H)

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Figure 3: Serum protein and albumin levels in control (A), Del (B), Deb (C), Depa (D), Deet (E), Del + Deb (F), Del + Depa (G), and Del + Deet (H) groups. The values are expressed as mean + standard error (SE) (n = 5–6); P > 0.05 versus control (not significant). Single drug dose: 0.5 LD50 (B to E); combination drug dose: each 0.25 LD50 (F–H)

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Figure 4: Serum urea, creatinine, and uric acid levels in control (A), Del (B), Deb (C), Depa (D), Deet (E), Del + Deb (F), Del + Depa (G), and Del + Deet (H) groups. The values are expressed as mean + standard error (SE), n = 5–6; P > 0.05 versus control (not significant). Single drug dose: 0.5 LD50 (B–E); combination drug dose: each 0.25 LD50 (F–H)

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Results of the marker enzymes of liver diseases such as AST, ALT, and ALP are shown in [Figure 5]. It was found that the AST activity increased significantly in Deb, Del + Depa, and Del + Deet combination groups in comparison with control. Quite interestingly, in comparison with control, ALT activity decreased significantly only in Del-alone group, whereas Del + Depa and Del + Deet combination groups showed significantly increased activity. Notably, no significant changes in the activities of GGT and AChE were seen between control and rest of the groups [Figure 6].
Figure 5: Serum aspartate aminotransferase (AST), serum alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels in control (A), Del (B), Deb (C), Depa (D), Deet (E), Del + Deb (F), Del + Depa (G), and Del + Deet (H) groups. The values are expressed as mean + standard error (SE) (n = 5 or 6); *P < 0.05 versus control (significant). Single drug dose: 0.5 LD50 (B–E); combination drug dose: each 0.25 LD50 (F–H)

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Figure 6: Serum γ-glutamyl transferase (GGT) and acetylcholinesterase (AChE) levels in control (A), Del (B), Deb (C), Depa (D), Deet (E), Del + Deb (F), Del + Depa (G), and Del + Deet (H) groups. The values are expressed as mean + standard error (SE) (n = 5 or 6); P > 0.05 versus control (not significant). Single drug dose: 0.5 LD50 (B–E); combination drug dose: each 0.25 LD50 (F–H)

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   Discussion Top

So far, the toxicological evaluations of various insecticides were studied only alone, and therefore, we lack precise work showing combination toxicity with repellents. As the human society is exposed to both repellents and insecticides in their day-to-day life, it is important to know the safety and limitations of those chemicals in combinations. The reason for selecting Del in this study could be justified owing to wider application of this chemical in community settings (used as a pest control measure for both household and agricultural purposes).[26] The idea of using Deb, Deet, and Depa in this study implies that they are the most commonly used vector repellents for mosquito control. However, Del and Deet are the chemicals that are preferably used to control various pests and insects including mosquitoes.[27],[28] The other insect repellents such as Deb (used as creams and gels) and Depa are widely used across Asian regions, especially India.[29],[30],[31] It is a notifying feature that people living in tropical areas are very prone to mosquito-borne diseases, such as malaria, dengue, Japanese encephalitis, and filariasis. There are several studies that report the resistance developed by mosquitoes against these agents.[28],[32] The most commonly used mosquito-repellent formulations contain Deet, Depa, and Deb, which possess excellent repellency and protection time against mosquitoes and other biting insects. It has been suggested that Deet could mediate neurotoxicity by targeting synapses of octopaminergic nerves and also by blocking sodium and potassium channels.[33] Mild neurological effects, such as reduced motor activity, changes in heat sensitivity, loss of balance, incoordination, and tremors, were reported at 500mg/kg dose of Deet by oral or inhalation route. However, twitching, convulsions or seizures, unconsciousness, prostration, and neuropathological lesions in the brain were seen at near-lethal doses (>1000mg/kg).

There are no detailed effects of Depa, but it can stimulate the central nervous system (CNS).[34] In this study, acute single oral toxicity of Del, Deb, Depa, and Deet as alone (0.5 LD50 each) and in combinations (0.25 LD50 each) as high dose was studied by assessment of body weight, hematological parameters, and biochemical parameters. There was no change in the body weight after 24h in all treatment groups, except Del + Deet group, which clearly shows the safety aspects of the dose used in this study. There was an 8% loss of body weight in Del + Deet group within 24h, which could be due to reduced consumption of feed and water by rats for a shorter duration of 1 or 2 days. Later, the rats recovered. The significant increase in liver weight observed in Deb group of rats could be possibly due to acute hepatotoxic effect as evidenced with elevated AST levels. The insect repellents tested either as alone or in combinations with the respective proportions did not alter various hematological parameters (Hb concentration, PCV, WBC, RBC, and platelet count). Likewise, the activity of enzymes such as ALP, GGT, and AChE did not alter significantly between all the groups. Other serum parameters such as glucose, bilirubin, triglycerides, cholesterol, creatinine, and uric acid did not change significantly between all groups. Obviously, in comparison with control, serum urea levels were found to decrease in Del-, Depa-, and Deet-alone groups, and Del + Deb combination group. In fact, no changes were seen in total serum protein and albumin levels between all the groups. In an earlier work, it was reported that there was a significant alteration in various biochemical parameters in Del (0.1 LD50)-treated rats only after repeated administration for 30 days.[35] In another work, Del (0.05 LD50) given orally for 30 days to Wistar rats caused a marked increase in serum enzymes such as ALT, AST, and ALP, and renal markers such as urea and creatinine with significantly decreased Catalase, Glutathione S-transferase, and AChE[36] activities. In this study, single oral dose of Del and other vector repellents (as alone [0.5 LD50 each] and in combinations [0.25 LD50 each]) did not have any significant effect on alterations of all hematological and almost all of the biochemical parameters, which implicated the safety of the higher dose used. At 0.5 LD50 dose of Del, Hb levels did not change significantly with respect to control, and this finding was consistent with that of similar studies conducted on male mice.[37]

Evaluation of Deet toxicity (1.0 µg/L, 0.1mg/L, and 1.0mg/L) in fish such as common carp (Cyprinus carpio L.) using a set of biochemical, hematological, and inflammatory parameters revealed that the selected dose were within the safety limits.[38] Deet is easily absorbed into many organs and tissues after oral administration by various species including rats. On the basis of other studies, it was reported that administration of Deet at 100mg/kg/day did not have any effect, although at high-dose level (400mg/kg/day), reduced body weights and slightly elevated serum cholesterol levels were observed in female rats.[39] In fact, a still higher dose of Deet (500mg/kg) by single intraperitoneal injection had considerably decreased the respiratory and heart rate in rats.[40] However, it was revealed that acute neurotoxicity complications including ataxia, tremors, convulsions, and abnormal head movements are very common after chronic Deet toxicity. Subchronic (90 day) toxicity (both oral and dermal) in male rats caused renal lesions, decreased body weight, and increased liver weights in both sexes.[41] In rats, combination of Deet with permethrin caused a striking increase of markers of DNA damage and oxidative stress.[42] On the basis of previous literature, it appears that even the minimum adverse effects seen with Deet treatment arise from only very large doses; therefore, the present results from Deet on the changes in body weight and biochemical studies are implicative of the safety level of administered dose. However, recent studies suggest that Deet is an AChE inhibitor and that this action may result in neurotoxicity and pose a risk to humans from its use as an insect repellent.[33]

Oral intoxication of Depa in rats caused a wide organ distribution of this repellent including lung, liver and stomach.[43] On the basis of many toxicological studies performed in different species, it was found that Depa is a safe repellent.[2] However, it was revealed that median lethal dose of Depa (635mg/kg body weight) when administered orally into male rats caused intoxication affecting CNS.[34] Unfortunately, we lack biochemical and hematological studies so far from rats subjected to Depa and Deb acute oral toxicity; hence, this study bridges this gap. Most importantly, the reason for decreased urea levels noticed in Depa-alone group and such elevated levels in Del + Depa combination group remains quite elusive. Taken together, this study indicates that there could be chances of hepatotoxicity with Del, Deb, Del + Depa, and Del + Deet combinations. Single high dose of vector repellents when administered orally into rats did not cause any adverse effect, but may cause hepatotoxicity in combination with Del. As this study was focused with limited exposure time of such test chemicals and was found to be safe based on hematological and biochemical studies, future work can establish the safety profile of the chemicals after exposure for a long time. In future, this study will be extended for the assessment of neurotoxicity of these chemicals along with histopathology and oxidative stress parameters.

   Conclusions Top

The safety evaluation of insect repellents such as Deb, Depa, and Deet alone and in combination with insecticide, Del, was assessed by hematological and biochemical parameters and it did not reveal any additive toxic effect in Wistar rats.


We acknowledge M/s Tagros Chemicals India Ltd., Chennai, for gifting the chemicals such Del, Deb, and Depa used in this study.

Financial support and sponsorship

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest

There are no conflicts of interest.

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1]


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