Neurobehavioral and Systemic Toxicity of Sub-Chronic Transfluthrin Exposure in Rats: An Exploratory In Vivo Study

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Objective: This study aimed to evaluate the sub-chronic effects of transfluthrin exposure via oral and inhalation routes on neurobehavior, systemic toxicity, and muscarinic acetylcholine receptor (mAchR) gene expression in rats. Methods: Adult male Sprague Dawley rats were exposed to transfluthrin for 90 days either orally (500 ppm in chow) or via inhalation (0.88% transfluthrin vaporizers, 12 h/day). Neurobehavioral assessments were performed using the Elevated Plus Maze (EPM), Open Field Test (OFT), and Morris Water Maze (MWM). Hematological and biochemical parameters were measured, and histopathological analysis of lung and liver tissues was conducted. mAchR gene expression was evaluated via quantitative RT-PCR, and blood transfluthrin levels were quantified using gas chromatography-mass spectrometry (GC-MS). Results: Transfluthrin-exposed rats exhibited significantly increased anxiety-like behavior (p=0.003), hyperactivity (p=0.04), and impaired spatial learning and memory (p=0.04). Elevated levels of liver enzymes alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and renal markers (urea, creatinine) indicated systemic toxicity. Histopathological findings included hepatic dysplastic foci and pulmonary inflammation.Additionally, a non-significant downward trend in mAChR gene expression was observed in exposed groups.Transfluthrin was detected in the bloodstream following both exposure routes, with significantly higher concentrations in orally exposed rats. Conclusion: Sub-chronic transfluthrin exposure induces neurobehavioral impairments, systemic toxicity, and organ damage in rats. While mAChR downregulation may contribute to cognitive deficits, further mechanistic studies are needed. Considering the widespread domestic use of transfluthrin, these findings underscore the need for comprehensive toxicological evaluations to inform public health policies. Toxicology Transfluthrin Neurobehavior Memory Toxicity Muscarinic Acetylcholine receptor (mAchR) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION Vector-borne diseases (VBDs), such as malaria and dengue, pose a significant global health challenge, contributing to high morbidity and mortality—particularly in regions like South East Asia, including India. Their prevention and control are complex due to the multifaceted nature of vector transmission, which is influenced by ecological, biological, social, and economic factors, including human migration [ 1 ]. Among the most widely adopted preventive measures is the use of commercial household insecticides. In urban settings, where individuals spend approximately 80% of their time indoors, exposure to these chemicals is significantly heightened [ 2 ]. While these products help mitigate disease transmission, the increasing and prolonged use of insecticides raises concerns about their potential toxicity to non-target organisms, including humans. These concerns are amplified by changing lifestyles and the expanding use of insecticide-based products. Transfluthrin is a synthetic type-I pyrethroid insecticide known for its rapid knockdown effect against flying insects such as mosquitoes and flies, as well as its utility in agriculture for crop protection [ 3 ]. It is commonly found in household repellents, including vaporizers, coils, and creams, as well as in outdoor sprays for vector control. However, transfluthrin's widespread use increases the risk of environmental contamination and human exposure, especially among vulnerable groups such as children, pregnant women, and the elderly. Evidence indicates that pyrethroid insecticides, including transfluthrin, may cause neurotoxic effects, disrupt immune and reproductive systems, and potentially exert carcinogenic effects [ 4 , 5 , 6 ]. Pyrethroids are known to modulate cholinergic neurotransmission, particularly through their interaction with muscarinic acetylcholine (mACh) receptors, which play a critical role in cognitive functions [ 7 , 8 ]. Animal studies have shown that exposure to these compounds can impair learning and memory, with oxidative stress hypothesized as a contributing mechanism [ 9 ]. Toxicological data indicate that transfluthrin exhibits low acute toxicity in rats, with an LD₅₀ exceeding 5000 mg/kg for oral, inhalation, and dermal exposures, and a NOAEL of 100 mg/kg/day [ 10 , 11 ]. However, sub-chronic and chronic exposure studies report various organ toxicities, including hepatocellular degeneration, interstitial pneumonia, renal hemorrhages, and gliosis in brain tissue [ 11 ]. Behavioral abnormalities such as hyperactivity, tremors, and poor grooming have also been observed in animal models [ 11 ]. Additionally, carcinogenicity studies link transfluthrin exposure to urinary bladder tumors, thyroid abnormalities, and other neoplastic changes [ 12 ]. The Indian mosquito repellent market is growing at an annual rate of 7–10%, currently valued between Rs 500–600 crores (US $ 12–15 million) [ 13 ]. This growth is driven by rising urbanization, climate change, and increased consumer affordability. Despite their widespread use, significant knowledge gaps remain, particularly regarding the molecular and neurological impacts of pyrethroids such as transfluthrin. In particular, there is limited understanding of how these compounds affect acetylcholine receptor gene expression and cholinergic signaling pathways in vivo [ 14 ]. Hypothesis and Study Objective This study hypothesizes that exposure to transfluthrin (0.88% w/w) via inhalation and oral routes impairs neurobehavioral functions by disrupting cholinergic neurotransmission. To test this, we conducted behavioral assessments, analyzed muscarinic acetylcholine receptor (mAChR) gene expression, and evaluated systemic toxicity markers in Sprague Dawley (SD) rats. 2. MATERIALS AND METHODS 2.1. Study Animal Adult male Sprague Dawley (SD) rats (4–6 months old; 150–250 g) were obtained from the Central Animal Facility at Maulana Azad Medical College (MAMC), New Delhi, following approval by the Institutional Animal Ethics Committee (IAEC; Ref No. EC/2021/04). All procedures adhered to the guidelines of the Committee for the Control and Supervision of Experimental Animals (CCSEA) for laboratory animal care and use [ 15 ]. Rats were housed under standard conditions: temperature 22 ± 2°C, relative humidity 70 ± 2%, and a 12-hour light/dark cycle. Animals were allowed a 7-day acclimatization period prior to experimentation and had free access to standard chow and water throughout [ 16 ]. 2.2. Experimental design Eighteen rats were randomly divided (n = 6 per group) using a computer-generated table: 2.2.1. Control group : Received standard rat chow (Ashirwad Industries, Punjab) for 12 weeks without transfluthrin exposure. Inhalation group : Exposed to transfluthrin vapor in a 10 × 10 × 10 ft chamber with five electric mosquito repellent vaporizers (0.88% transfluthrin), operated 12 hours daily (7 AM–7 PM) for 90 days [ 3 , 4 ]. Oral exposure group : Received transfluthrin (PESTANAL™, Sigma-Aldrich, USA) mixed into chow at a sub-chronic dose of 500 ppm for 90 days. At baseline and after the 90-day exposure period, blood samples were collected, and animals were euthanized via intraperitoneal injection of thiopental sodium (90 mg/kg body weight). Tissue samples from brain, liver, lungs, kidneys, and intestines were harvested for histopathological analysis. 2.3. Neurobehavioral Assessments Behavioral evaluations were conducted at baseline and after 90 days of exposure: General and Stereotypic Behavior : Animals were observed for 2 hours. Parameters assessed included sniffing, grooming, gnawing, catalepsy, salivation, gait, reflexes, and other stereotypies. The General Behaviour Stereotype Score (GBSS) was recorded [ 7 ]. Anxiety (Elevated Plus Maze, EPM) : Each rat was placed at the maze center facing an open arm and allowed to explore for 5 minutes under dim light. Sessions were video-recorded, and the maze was cleaned with 70% ethanol between trials. Parameters included arm entry count and time spent in open vs. closed arms [ 7 , 17 , 18 ]. Locomotor Activity (Open Field Test, OFT) : Rats were placed near the wall of the arena, and line crossings were recorded over 5 minutes [ 7 , 17 ]. Memory (Morris Water Maze, MWM) : Rats were trained for 3 days to locate a submerged platform. On day 4 (test day), the platform was removed, and the latency to reach the former platform location was recorded to assess spatial memory [ 19 ]. 2.4. Hematological and Biochemical Parameters At baseline and day 90, 2 mL of blood was collected via retro-orbital plexus under isoflurane anesthesia and allowed to sit at room temperature for 1 hour. The following parameters were analyzed using standard diagnostic kits: 2.4.1. Hematology : Hemoglobin (Hb), total leukocyte count (TLC), differential leukocyte count (DLC), and platelet count (PC). 2.4.2. Liver function : Alanine transaminase (ALT), aspartate transaminase (AST), and bilirubin. 2.4.3. Renal function : Blood urea and serum creatinine. 2.5. Histopathology Post-euthanasia, organs were fixed in 10% neutral buffered formalin. Tissue sections were processed and stained using standard histological procedures. Histopathological features were examined under a light microscope, and representative images were captured for documentation. 2.6. Muscarinic Acetylcholine Receptor (mAChR) Gene Expression Following euthanasia, brain tissue (amygdala and adjacent areas) was rapidly isolated, rinsed in ice-cold physiological saline, and homogenized. Total RNA was extracted using the Aurum Total RNA Mini Kit. Complementary DNA (cDNA) synthesis was performed using the iScript™ cDNA Synthesis Kit (Bio-Rad, USA). Quantitative reverse transcription PCR (qRT-PCR) was used to assess mAChR gene expression, normalized to β-actin as the reference gene. 2.7. Quantification of Transfluthrin Levels in Blood Transfluthrin residues in whole blood were quantified using gas chromatography–mass spectrometry (GC-MS) in electron ionization mode with selective ion monitoring. The setup included: Instrument: Agilent 8890 GC with 5977B GC/MSD and Agilent 7963A auto-injector, controlled via OpenLab CDS. Column: HP-5MS UI capillary column (30 m × 0.25 mm × 0.25 µm). Extraction solvent: Hexane: acetone (80:20 v/v). Carrier gas: Helium at 1.0 mL/min. GC oven temperature program : Initial: 60°C (3 min) Ramp 1: 10°C/min to 200°C (hold 5 min) Ramp 2: 5°C/min to 240°C (hold 45 min) Injector/interface: 290°C/300°C 2.7.1. Analytical Method Validation A well-resolved peak for transfluthrin (0.10 ppm) appeared at a retention time of 21.38 minutes. Calibration standards 2.0–10.0 ppm (Table 1 ) showed linearity in peak area response (Figs. 1 A, 1 B and 1 C). Table 1 Peak area values of known concentration of transfluthrin following estimation by GC-MS (Mean values of 3 replication). Concentration (ppm) Average area 2.0 530066 4.0 720351 6.0 1334510 8.0 1649052 10 1874576 2.7.2. Recovery Experiment Blood samples fortified with 10 ppb transfluthrin demonstrated recovery rates between 90–99%. The method showed a limit of detection (LOD) of 0.01 ppb, indicating high sensitivity and reliability (Fig. 1 B). 3. Statistical Analysis Data were analyzed using Statistical Package for Social Sciences (SPSS) version 25.0. Results are presented as Mean ± SD or Median ± SD, with significance set at p < 0.05. For group comparisons, one-way ANOVA with Tukey’s HSD post hoc for normally distributed data, while the Kruskal-Wallis test and Wilcoxon signed-rank tests were used for non-parametric data. A p -value < 0.05 was considered statistically significant. 4. RESULTS At baseline, the three groups were comparable with respect to the GBSS. The change from baseline to day 90 in GBSS was not statistically significant for the inhalation group [0.50 (0.00–1.00)] and the oral group [0.50 (0.00–1.00)]. However, at day 90, the inhalation group exhibited a median GBSS of 1.00 (0.25–1.00), and the oral group showed a median of 1.00 (1.00–1.75) (Fig. 2A). 4.1. Anxiety : Significant differences were observed among the groups in the time spent in the closed arm of the Elevated Plus Maze (EPM) at day 90 compared to baseline. The mean time spent in the closed arm decreased in both the control and inhalation groups but increased significantly in the oral group, rising from 3.47 ± 0.26 minutes at baseline to 3.84 ± 0.45 minutes at day 90 ( p = 0.003) (Fig. 2B). 4.2. Locomotor Activity : The mean number of line crossings in the Open Field Test increased significantly in both exposed groups by day 90. In the inhalation group, it rose from 33.17 ± 5.0 at baseline to 46.67 ± 6.31 (p = 0.04), and in the oral group, from 34.00 ± 5.37 to 51.83 ± 7.0 (p = 0.04) (Fig. 2C). 4.3. Learning and Memory Assessment : The mean latency to find the platform increased significantly in both exposure groups at day 90 compared to baseline. In the inhalation group, latency increased from 87.83 ± 12.37 seconds to 106.83 ± 10.96 seconds (p = 0.04). In the oral group, it increased from 87.50 ± 10.50 seconds to 115.33 ± 6.28 seconds (p = 0.04) (Fig. 2D). 4.4. Hematological Parameters : A significant decrease in mean hemoglobin (Hb) was observed in the oral group ( p = 0.005). Although Hb levels in the inhalation group decreased slightly from 14.10 g/dL at day 0 to 13.97 g/dL at day 90, this change was not statistically significant ( p = 0.824). Both inhalation and oral exposure groups exhibited a significant increase in total leukocyte count (TLC) ( p = 0.036) (Figs. 3A & 3B). 4.5. Biochemical Parameters : Rats exposed to transfluthrin exhibited elevated hepatic enzyme levels. Alanine aminotransferase (ALT) increased significantly in both the inhalation ( p = 0.001) and oral ( p = 0.024) groups. Aspartate aminotransferase (AST) levels increased significantly in all groups: control ( p = 0.036), inhalation ( p = 0.031), and oral ( p = 0.031). Alkaline phosphatase (ALP) was significantly elevated in the inhalation ( p = 0.019) and oral ( p = 0.001) groups. Additionally, the oral group showed a significant increase in blood urea ( p < 0.003) and serum creatinine ( p < 0.001) (Fig. 4: A. ALT; B. AST; C. ALP; D. Blood urea; E. Serum creatinine). 4.6. Histopathology 4.6.1. Lungs : Histological examination of lung tissues revealed acute bronchial inflammation in both the inhalation and oral exposure groups after 90 days of transfluthrin exposure. Key findings included bronchial dilation, patchy cellular infiltration, and features suggestive of bronchiectasis. Control lung tissue appeared normal (Fig. 5A), whereas pathological changes are shown in Figs. 5B (inhalation group) and 5C (oral group). 4.6.2. Liver : Dysplastic foci, indicative of hepatic cellular alteration or damage, were observed in rats exposed to transfluthrin via the oral route (Figs. 5D and 5E), suggesting possible early pre-neoplastic changes or liver stress responses. 4.7. Muscarinic Acetylcholine Receptor (mAChR) Gene Expression The mean fold change in mAChR gene expression was 1.00 (± 0.15) in the control group, 0.91 (± 0.12) in the inhalation group, and 0.88 (± 0.10) in the oral group. Although a downward trend in expression was observed in both exposed groups, the difference was not statistically significant ( p = 0.286) (Fig. 6A). 4.8. Blood Transfluthrin Levels At day 90, serum transfluthrin concentrations were significantly higher in the oral group, with a median (IQR) of 1.43 (1.25–1.59) µg/mL, compared to the inhalation group, which recorded 0.24 (0.22–0.30) µg/mL (Fig. 6B). These results confirm systemic absorption of transfluthrin via both exposure routes, with notably greater bioavailability following oral administration. 5. DISCUSSION Transfluthrin, a fast-acting synthetic pyrethroid widely used in mosquito control, has been shown to enter systemic circulation via both dietary and inhalational exposure routes [ 19 ]. Although its toxicological profile has been partially characterized [ 20 ], data on its sub-chronic impact on cognitive function, neurobehavior, and systemic physiology in mammalian models remain limited. This study is among the first comprehensively evaluate the effects of sub-chronic transfluthrin exposure via both inhalation and oral routes in rats. We assessed neurobehavioral outcomes, hematological and biochemical parameters, histopathological changes, systemic transfluthrin levels, and muscarinic acetylcholine receptor (mAChR) gene expression to elucidate potential mechanisms underlying observed effects. For inhalational exposure, commercial transfluthrin-based vaporizers (0.88%, SC Johnson Products Pvt. Ltd., Baddi, Solan, HP, India) were deployed in a 1000 ft³ space. While one vaporizer typically suffices for a similarly sized human-occupied area, five vaporizers were used to account for rats’ higher metabolic rate—approximately five times that of humans—thus achieving physiologically relevant exposure levels [ 20 ]. Oral dosages were adapted from prior toxicological studies [ 21 , 22 ]. Transfluthrin was detected in the bloodstream following both inhalational and oral exposure, confirming systemic absorption through both routes. While systemic presence following ingestion is expected, its detection via inhalation underscores potential human health concerns, especially considering the widespread indoor use of vaporizer-based repellents. Behavioral testing revealed that transfluthrin-exposed rats spent significantly more time in the closed arms of the Elevated Plus Maze (EPM), suggesting heightened anxiety. Increased line crossings were also observed across both exposure groups (p < 0.005), consistent with enhanced exploratory behavior typically associated with anxiogenic states [ 23 – 25 ]. These behavioral outcomes align with known neurotoxic mechanisms of pyrethroids, such as the prolonged opening of voltage-gated sodium channels, which enhances neuronal excitability [ 25 , 26 ]. In the Morris Water Maze (MWM), transfluthrin-exposed rats showed increased latency to locate the hidden platform, indicating impairments in spatial learning and memory. Given the hippocampus's reliance on cholinergic signaling via mAChRs for memory consolidation [ 24 – 29 ], we hypothesized that cognitive deficits could be linked to mAChR dysregulation. Quantitative RT-PCR analysis of midbrain tissue revealed a downward trend in mAChR mRNA expression in orally exposed rats, though the changes were not statistically significant in either group. These results suggest that mAChR disruption may contribute to, but not fully explain, the cognitive impairments observed. Moreover, the lack of assessment in the amygdala—a key region in emotion-related memory—represents a limitation that warrants further exploration. Importantly, pesticide exposure has been implicated in neurodegenerative diseases such as Alzheimer’s, characterized by cholinergic dysfunction and progressive memory loss. Thus, the potential link between chronic transfluthrin exposure and such conditions should be evaluated through long-term and epidemiological studies. Hematologically, a significant increase in total leukocyte count (TLC) was observed in both exposure groups, suggesting an immune response to transfluthrin. While previous studies have primarily reported immunosuppressive effects [ 29 ], the leukocytosis noted here may reflect either a compensatory activation of the immune system or a chemical stress-induced response. Alternatively, this may indicate transient leukocyte mobilization or dysregulation, commonly reported under xenobiotic stress conditions [ 30 – 33 ]. Hemoglobin levels were significantly reduced in the orally exposed group, indicating potential transfluthrin-induced anemia. Interestingly, red blood cell (RBC) and platelet counts remained unchanged, suggesting that the anemia may not stem from impaired erythropoiesis. Instead, hemolytic activity or disruptions in iron metabolism may be responsible, though further studies are required to confirm the same [ 34 – 38 ]. Biochemical markers of liver and kidney function (ALT, AST, ALP, bilirubin, urea, creatinine) were significantly elevated in both exposure groups, with effects more pronounced in orally exposed rats. These findings, corroborated by histopathological evidence—such as hepatic dysplastic foci and pulmonary inflammation—are consistent with prior studies linking transfluthrin to oxidative stress and tissue damage [ 38 – 40 ]. Previous work from our group has also identified detrimental effects of transfluthrin on reproductive health [ 41 ], emphasizing the broader toxicological implications of prolonged exposure. 6. Limitations of the Study Limited Sample Size : The modest number of animals per group may limit the statistical power to detect subtle or borderline effects. Future studies with larger cohorts are necessary to validate and expand on these findings. Lack of Brain Tissue Quantification : Transfluthrin levels were not quantified in brain tissue using gas chromatography–mass spectrometry (GC-MS). Determining its ability to cross the blood–brain barrier is critical for elucidating direct neurotoxic effects. Future studies are needed for further mechanistic studies to confirm the findings of our study. Absence of Biochemical Cholinergic Markers : Biochemical assays such as acetylcholinesterase (AChE) activity in specific brain regions (e.g., hippocampus, prefrontal cortex) were not conducted. These could have provided stronger mechanistic support for the observed behavioral impairments. Animal Model Constraints : As the study was conducted in a rodent model, extrapolation of results to humans should be done cautiously. Additional studies—including human epidemiological research and in vitro cellular assays—are required for better risk assessment. 7. Conclusion Collectively, our findings indicate that sub-chronic exposure to transfluthrin—via both inhalation and oral routes—can adversely affect neurobehavior, cognitive performance, immune responses, hematological parameters, and vital organ function in rats. The detection of transfluthrin in systemic circulation following inhalation is particularly concerning, given its widespread domestic use. Although changes in mAChR expression may contribute to cognitive deficits, additional molecular mechanisms likely play a role. These results underscore the necessity for comprehensive toxicological evaluations, including long-term animal studies and human epidemiological research, to inform safety guidelines and public health policies concerning transfluthrin exposure. Declarations Ethical clearance The study obtained ethical clearance from IAEC (No. EC/2021/05 dated 24/05/2021) Conflicts of interests: The authors declare that they have no conflicts of interests. Funding: None Acknowledgement We acknowledge the Government of National Capital Territory of Delhi (GNCTD) for funding to address idea of concepts by conducting research. Multidisciplinary Research Unit, MAMC, New Delhi acknowledged for experiments facilities. 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04:46:11","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92391,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/4aed506120e75d580bd71101.html"},{"id":91943399,"identity":"a486687f-a685-43ab-9739-3ee2c641c826","added_by":"auto","created_at":"2025-09-23 04:46:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":52165,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection and quantification of transfluthrin using gas chromatography-mass spectrometry (GC-MS):\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Chromatogram of analytical-grade transfluthrin standard at 0.10 ppm. \u003cstrong\u003eB.\u003c/strong\u003eChromatogram of transfluthrin extracted from rat blood spiked with 0.10 ppm transfluthrin, demonstrating efficient recovery and detection. \u003cstrong\u003eC.\u003c/strong\u003eCalibration curve showing a linear relationship between peak area and known transfluthrin concentrations, validating the accuracy and reliability of the quantification method.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/fedf0ab8148dc81bbc3e5913.png"},{"id":91944327,"identity":"5cf15ea1-adaf-48ba-9ddf-42e7c11caeff","added_by":"auto","created_at":"2025-09-23 04:54:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":48242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eEffect of transfluthrin exposure on neurobehavioral parameters: \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eA.\u003c/strong\u003eGeneral behavioral observations. \u003cstrong\u003eB.\u003c/strong\u003e Anxiety-like behavior assessed by time spent in the closed arms of the EPM test. \u003cstrong\u003eC.\u003c/strong\u003eLocomotor activity evaluated by the number of line crossings in the OFT. \u003cstrong\u003eD.\u003c/strong\u003eCognitive performance assessed by latency to locate the hidden platform in the MWM test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/63fdcdcdb69e7e19c4f2f852.png"},{"id":91943407,"identity":"0c775602-be7d-4e91-9101-325f2a0c986b","added_by":"auto","created_at":"2025-09-23 04:46:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":31040,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of transfluthrin on Hematological parameters: \u003cstrong\u003eA. \u003c/strong\u003eHemoglobin level; \u003cstrong\u003eB.\u003c/strong\u003eTotal Leucocyte Count (TLC)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/92d324980794a279abe1d12d.png"},{"id":91943406,"identity":"0e30d59f-e5b6-49cb-8260-21213c180169","added_by":"auto","created_at":"2025-09-23 04:46:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":103277,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of transfluthrin on liver and renal parameters: \u003cstrong\u003eA.\u003c/strong\u003e ALT; \u003cstrong\u003eB.\u003c/strong\u003e AST; \u003cstrong\u003eC.\u003c/strong\u003eALP; \u003cstrong\u003eD.\u003c/strong\u003e Total Bilirubin; \u003cstrong\u003eE. \u003c/strong\u003eBlood urea; \u003cstrong\u003eF\u003c/strong\u003e. Serum creatinine\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/20de867fb2752585fd6ae59a.png"},{"id":91943402,"identity":"dc739aeb-6127-4e0c-8313-f17afc575d60","added_by":"auto","created_at":"2025-09-23 04:46:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological changes in lung and liver tissues of transfluthrin-exposed rats (Hematoxylin \u0026amp; Eosin stain).\u003c/strong\u003e \u003cstrong\u003eA.\u003c/strong\u003e Lung parenchyma from control group showing normal architecture (×200). \u003cstrong\u003eB.\u003c/strong\u003e Oral exposure group: Dilated bronchi with ulceration and suppurative inflammation involving the bronchial wall and surrounding lung parenchyma (×100). \u003cstrong\u003eC.\u003c/strong\u003e Inhalation exposure group: Dilated bronchus with intense peribronchial inflammation (×100). \u003cstrong\u003eD.\u003c/strong\u003eOral exposure group: Liver tissue showing a dysplastic focus with loss of normal trabecular architecture; hepatocytes display enlarged nuclei and prominent nucleoli (×200). \u003cstrong\u003eE.\u003c/strong\u003e Inhalation exposure group: Liver section with a small focus of dysplastic change (×100)\u003c/p\u003e","description":"","filename":"placeholderimage.png","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/a4b510d79f83bf2ed74d5802.png"},{"id":91944768,"identity":"d267980a-eca6-4eca-b895-69a920f60a95","added_by":"auto","created_at":"2025-09-23 05:02:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":37308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular and pharmacokinetic effects of sub-chronic transfluthrin exposure (90 Days): A.\u003c/strong\u003eFold change in muscarinic acetylcholine receptor (mAchR) gene expression in the midbrain of rats. \u003cstrong\u003eB.\u003c/strong\u003e Blood concentrations of transfluthrin (µg/mL), higher systemic levels were observed in the orally exposed group compared to the inhalation group.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/e6ae9950940560272904402f.png"},{"id":91945329,"identity":"41455bbc-b0f9-450d-96c2-7072d45df4a1","added_by":"auto","created_at":"2025-09-23 05:10:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1488824,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/e8117cf2-b755-41fb-91af-aeb8c4bd08fd.pdf"},{"id":91944767,"identity":"7996af5d-9db7-43e7-9a66-5c8d732dac5b","added_by":"auto","created_at":"2025-09-23 05:02:11","extension":"ppt","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":113664,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.ppt","url":"https://assets-eu.researchsquare.com/files/rs-7645422/v1/ac2c4e684c3feef0e039cee2.ppt"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eNeurobehavioral and Systemic Toxicity of Sub-Chronic Transfluthrin Exposure in Rats: An Exploratory In Vivo Study\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eVector-borne diseases (VBDs), such as malaria and dengue, pose a significant global health challenge, contributing to high morbidity and mortality\u0026mdash;particularly in regions like South East Asia, including India. Their prevention and control are complex due to the multifaceted nature of vector transmission, which is influenced by ecological, biological, social, and economic factors, including human migration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAmong the most widely adopted preventive measures is the use of commercial household insecticides. In urban settings, where individuals spend approximately 80% of their time indoors, exposure to these chemicals is significantly heightened [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. While these products help mitigate disease transmission, the increasing and prolonged use of insecticides raises concerns about their potential toxicity to non-target organisms, including humans. These concerns are amplified by changing lifestyles and the expanding use of insecticide-based products.\u003c/p\u003e\u003cp\u003eTransfluthrin is a synthetic type-I pyrethroid insecticide known for its rapid knockdown effect against flying insects such as mosquitoes and flies, as well as its utility in agriculture for crop protection [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is commonly found in household repellents, including vaporizers, coils, and creams, as well as in outdoor sprays for vector control. However, transfluthrin's widespread use increases the risk of environmental contamination and human exposure, especially among vulnerable groups such as children, pregnant women, and the elderly.\u003c/p\u003e\u003cp\u003eEvidence indicates that pyrethroid insecticides, including transfluthrin, may cause neurotoxic effects, disrupt immune and reproductive systems, and potentially exert carcinogenic effects [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Pyrethroids are known to modulate cholinergic neurotransmission, particularly through their interaction with muscarinic acetylcholine (mACh) receptors, which play a critical role in cognitive functions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Animal studies have shown that exposure to these compounds can impair learning and memory, with oxidative stress hypothesized as a contributing mechanism [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eToxicological data indicate that transfluthrin exhibits low acute toxicity in rats, with an LD₅₀ exceeding 5000 mg/kg for oral, inhalation, and dermal exposures, and a NOAEL of 100 mg/kg/day [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, sub-chronic and chronic exposure studies report various organ toxicities, including hepatocellular degeneration, interstitial pneumonia, renal hemorrhages, and gliosis in brain tissue [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Behavioral abnormalities such as hyperactivity, tremors, and poor grooming have also been observed in animal models [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, carcinogenicity studies link transfluthrin exposure to urinary bladder tumors, thyroid abnormalities, and other neoplastic changes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe Indian mosquito repellent market is growing at an annual rate of 7\u0026ndash;10%, currently valued between Rs 500\u0026ndash;600 crores (US \u003cspan\u003e$\u003c/span\u003e12\u0026ndash;15\u0026nbsp;million) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This growth is driven by rising urbanization, climate change, and increased consumer affordability. Despite their widespread use, significant knowledge gaps remain, particularly regarding the molecular and neurological impacts of pyrethroids such as transfluthrin. In particular, there is limited understanding of how these compounds affect acetylcholine receptor gene expression and cholinergic signaling pathways in vivo [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eHypothesis and Study Objective\u003c/strong\u003e\u003cp\u003eThis study hypothesizes that exposure to transfluthrin (0.88% w/w) via inhalation and oral routes impairs neurobehavioral functions by disrupting cholinergic neurotransmission. To test this, we conducted behavioral assessments, analyzed muscarinic acetylcholine receptor (mAChR) gene expression, and evaluated systemic toxicity markers in Sprague Dawley (SD) rats.\u003c/p\u003e\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study Animal\u003c/h2\u003e\u003cp\u003e Adult male Sprague Dawley (SD) rats (4\u0026ndash;6 months old; 150\u0026ndash;250 g) were obtained from the Central Animal Facility at Maulana Azad Medical College (MAMC), New Delhi, following approval by the Institutional Animal Ethics Committee (IAEC; Ref No. EC/2021/04). All procedures adhered to the guidelines of the Committee for the Control and Supervision of Experimental Animals (CCSEA) for laboratory animal care and use [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRats were housed under standard conditions: temperature 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, relative humidity 70\u0026thinsp;\u0026plusmn;\u0026thinsp;2%, and a 12-hour light/dark cycle. Animals were allowed a 7-day acclimatization period prior to experimentation and had free access to standard chow and water throughout [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Experimental design\u003c/h2\u003e\u003cp\u003eEighteen rats were randomly divided (n\u0026thinsp;=\u0026thinsp;6 per group) using a computer-generated table:\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1. \u003cb\u003eControl group\u003c/b\u003e: Received standard rat chow (Ashirwad Industries, Punjab) for 12 weeks without transfluthrin exposure.\u003c/h2\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eInhalation group\u003c/b\u003e: Exposed to transfluthrin vapor in a 10 \u0026times; 10 \u0026times; 10 ft chamber with five electric mosquito repellent vaporizers (0.88% transfluthrin), operated 12 hours daily (7 AM\u0026ndash;7 PM) for 90 days [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eOral exposure group\u003c/b\u003e: Received transfluthrin (PESTANAL\u0026trade;, Sigma-Aldrich, USA) mixed into chow at a sub-chronic dose of 500 ppm for 90 days.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eAt baseline and after the 90-day exposure period, blood samples were collected, and animals were euthanized via intraperitoneal injection of thiopental sodium (90 mg/kg body weight). Tissue samples from brain, liver, lungs, kidneys, and intestines were harvested for histopathological analysis.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Neurobehavioral Assessments\u003c/h2\u003e\u003cp\u003eBehavioral evaluations were conducted at baseline and after 90 days of exposure:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eGeneral and Stereotypic Behavior\u003c/b\u003e: Animals were observed for 2 hours. Parameters assessed included sniffing, grooming, gnawing, catalepsy, salivation, gait, reflexes, and other stereotypies. The General Behaviour Stereotype Score (GBSS) was recorded [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAnxiety (Elevated Plus Maze, EPM)\u003c/b\u003e: Each rat was placed at the maze center facing an open arm and allowed to explore for 5 minutes under dim light. Sessions were video-recorded, and the maze was cleaned with 70% ethanol between trials. Parameters included arm entry count and time spent in open vs. closed arms [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eLocomotor Activity (Open Field Test, OFT)\u003c/b\u003e: Rats were placed near the wall of the arena, and line crossings were recorded over 5 minutes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eMemory (Morris Water Maze, MWM)\u003c/b\u003e: Rats were trained for 3 days to locate a submerged platform. On day 4 (test day), the platform was removed, and the latency to reach the former platform location was recorded to assess spatial memory [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Hematological and Biochemical Parameters\u003c/h2\u003e\u003cp\u003eAt baseline and day 90, 2 mL of blood was collected via retro-orbital plexus under isoflurane anesthesia and allowed to sit at room temperature for 1 hour. The following parameters were analyzed using standard diagnostic kits:\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. \u003cb\u003eHematology\u003c/b\u003e: Hemoglobin (Hb), total leukocyte count (TLC), differential leukocyte count (DLC), and platelet count (PC).\u003c/h2\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. \u003cb\u003eLiver function\u003c/b\u003e: Alanine transaminase (ALT), aspartate transaminase (AST), and bilirubin.\u003c/h2\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. \u003cb\u003eRenal function\u003c/b\u003e: Blood urea and serum creatinine.\u003c/h2\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Histopathology\u003c/h2\u003e\u003cp\u003ePost-euthanasia, organs were fixed in 10% neutral buffered formalin. Tissue sections were processed and stained using standard histological procedures. Histopathological features were examined under a light microscope, and representative images were captured for documentation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Muscarinic Acetylcholine Receptor (mAChR) Gene Expression\u003c/h2\u003e\u003cp\u003eFollowing euthanasia, brain tissue (amygdala and adjacent areas) was rapidly isolated, rinsed in ice-cold physiological saline, and homogenized. Total RNA was extracted using the Aurum Total RNA Mini Kit. Complementary DNA (cDNA) synthesis was performed using the iScript\u0026trade; cDNA Synthesis Kit (Bio-Rad, USA). Quantitative reverse transcription PCR (qRT-PCR) was used to assess mAChR gene expression, normalized to β-actin as the reference gene.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Quantification of Transfluthrin Levels in Blood\u003c/h2\u003e\u003cp\u003eTransfluthrin residues in whole blood were quantified using gas chromatography\u0026ndash;mass spectrometry (GC-MS) in electron ionization mode with selective ion monitoring. The setup included:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eInstrument: Agilent 8890 GC with 5977B GC/MSD and Agilent 7963A auto-injector, controlled via OpenLab CDS.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eColumn: HP-5MS UI capillary column (30 m \u0026times; 0.25 mm \u0026times; 0.25 \u0026micro;m).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eExtraction solvent: Hexane: acetone (80:20 v/v).\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eCarrier gas: Helium at 1.0 mL/min.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGC oven temperature program\u003c/b\u003e:\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eInitial: 60\u0026deg;C (3 min)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eRamp 1: 10\u0026deg;C/min to 200\u0026deg;C (hold 5 min)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eRamp 2: 5\u0026deg;C/min to 240\u0026deg;C (hold 45 min)\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eInjector/interface: 290\u0026deg;C/300\u0026deg;C\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section3\"\u003e\u003ch2\u003e2.7.1. Analytical Method Validation\u003c/h2\u003e\u003cp\u003eA well-resolved peak for transfluthrin (0.10 ppm) appeared at a retention time of 21.38 minutes. Calibration standards 2.0\u0026ndash;10.0 ppm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed linearity in peak area response (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePeak area values of known concentration of transfluthrin following estimation by GC-MS (Mean values of 3 replication).\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eConcentration (ppm)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAverage area\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e530066\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e720351\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e6.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1334510\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e8.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1649052\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1874576\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.7.2. Recovery Experiment\u003c/h2\u003e\u003cp\u003eBlood samples fortified with 10 ppb transfluthrin demonstrated recovery rates between 90\u0026ndash;99%. The method showed a limit of detection (LOD) of 0.01 ppb, indicating high sensitivity and reliability (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Statistical Analysis","content":"\u003cp\u003eData were analyzed using Statistical Package for Social Sciences (SPSS) version 25.0. Results are presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or Median\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, with significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. For group comparisons, one-way ANOVA with Tukey\u0026rsquo;s HSD post hoc for normally distributed data, while the Kruskal-Wallis test and Wilcoxon signed-rank tests were used for non-parametric data. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"4. RESULTS","content":"\u003cp\u003eAt baseline, the three groups were comparable with respect to the GBSS. The change from baseline to day 90 in GBSS was not statistically significant for the inhalation group [0.50 (0.00\u0026ndash;1.00)] and the oral group [0.50 (0.00\u0026ndash;1.00)]. However, at day 90, the inhalation group exhibited a median GBSS of 1.00 (0.25\u0026ndash;1.00), and the oral group showed a median of 1.00 (1.00\u0026ndash;1.75) (Fig.\u0026nbsp;2A).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.1. Anxiety\u003c/strong\u003e: Significant differences were observed among the groups in the time spent in the closed arm of the Elevated Plus Maze (EPM) at day 90 compared to baseline. The mean time spent in the closed arm decreased in both the control and inhalation groups but increased significantly in the oral group, rising from 3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 minutes at baseline to 3.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 minutes at day 90 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003) (Fig.\u0026nbsp;2B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2. Locomotor Activity\u003c/strong\u003e: The mean number of line crossings in the Open Field Test increased significantly in both exposed groups by day 90. In the inhalation group, it rose from 33.17\u0026thinsp;\u0026plusmn;\u0026thinsp;5.0 at baseline to 46.67\u0026thinsp;\u0026plusmn;\u0026thinsp;6.31 (p\u0026thinsp;=\u0026thinsp;0.04), and in the oral group, from 34.00\u0026thinsp;\u0026plusmn;\u0026thinsp;5.37 to 51.83\u0026thinsp;\u0026plusmn;\u0026thinsp;7.0 (p\u0026thinsp;=\u0026thinsp;0.04) (Fig.\u0026nbsp;2C).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Learning and Memory Assessment\u003c/strong\u003e: The mean latency to find the platform increased significantly in both exposure groups at day 90 compared to baseline. In the inhalation group, latency increased from 87.83\u0026thinsp;\u0026plusmn;\u0026thinsp;12.37 seconds to 106.83\u0026thinsp;\u0026plusmn;\u0026thinsp;10.96 seconds (p\u0026thinsp;=\u0026thinsp;0.04). In the oral group, it increased from 87.50\u0026thinsp;\u0026plusmn;\u0026thinsp;10.50 seconds to 115.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.28 seconds (p\u0026thinsp;=\u0026thinsp;0.04) (Fig.\u0026nbsp;2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4. Hematological Parameters\u003c/strong\u003e: A significant decrease in mean hemoglobin (Hb) was observed in the oral group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.005). Although Hb levels in the inhalation group decreased slightly from 14.10 g/dL at day 0 to 13.97 g/dL at day 90, this change was not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.824). Both inhalation and oral exposure groups exhibited a significant increase in total leukocyte count (TLC) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036) (Figs.\u0026nbsp;3A \u0026amp; 3B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5. Biochemical Parameters\u003c/strong\u003e: Rats exposed to transfluthrin exhibited elevated hepatic enzyme levels. Alanine aminotransferase (ALT) increased significantly in both the inhalation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and oral (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.024) groups. Aspartate aminotransferase (AST) levels increased significantly in all groups: control (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.036), inhalation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031), and oral (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.031). Alkaline phosphatase (ALP) was significantly elevated in the inhalation (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019) and oral (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) groups. Additionally, the oral group showed a significant increase in blood urea (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.003) and serum creatinine (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;4: A. ALT; B. AST; C. ALP; D. Blood urea; E. Serum creatinine).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6. Histopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6.1. Lungs\u003c/strong\u003e: Histological examination of lung tissues revealed acute bronchial inflammation in both the inhalation and oral exposure groups after 90 days of transfluthrin exposure. Key findings included bronchial dilation, patchy cellular infiltration, and features suggestive of bronchiectasis. Control lung tissue appeared normal (Fig.\u0026nbsp;5A), whereas pathological changes are shown in Figs.\u0026nbsp;5B (inhalation group) and 5C (oral group).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6.2. Liver\u003c/strong\u003e: Dysplastic foci, indicative of hepatic cellular alteration or damage, were observed in rats exposed to transfluthrin via the oral route (Figs.\u0026nbsp;5D and 5E), suggesting possible early pre-neoplastic changes or liver stress responses.\u003c/p\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.7. Muscarinic Acetylcholine Receptor (mAChR) Gene Expression\u003c/h2\u003e\n\u003cp\u003eThe mean fold change in mAChR gene expression was 1.00 (\u0026plusmn;\u0026thinsp;0.15) in the control group, 0.91 (\u0026plusmn;\u0026thinsp;0.12) in the inhalation group, and 0.88 (\u0026plusmn;\u0026thinsp;0.10) in the oral group. Although a downward trend in expression was observed in both exposed groups, the difference was not statistically significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.286) (Fig.\u0026nbsp;6A).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.8. Blood Transfluthrin Levels\u003c/h2\u003e\n\u003cp\u003eAt day 90, serum transfluthrin concentrations were significantly higher in the oral group, with a median (IQR) of 1.43 (1.25\u0026ndash;1.59) \u0026micro;g/mL, compared to the inhalation group, which recorded 0.24 (0.22\u0026ndash;0.30) \u0026micro;g/mL (Fig.\u0026nbsp;6B). These results confirm systemic absorption of transfluthrin via both exposure routes, with notably greater bioavailability following oral administration.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"5. DISCUSSION","content":"\u003cp\u003eTransfluthrin, a fast-acting synthetic pyrethroid widely used in mosquito control, has been shown to enter systemic circulation via both dietary and inhalational exposure routes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Although its toxicological profile has been partially characterized [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], data on its sub-chronic impact on cognitive function, neurobehavior, and systemic physiology in mammalian models remain limited.\u003c/p\u003e\u003cp\u003eThis study is among the first comprehensively evaluate the effects of sub-chronic transfluthrin exposure via both inhalation and oral routes in rats. We assessed neurobehavioral outcomes, hematological and biochemical parameters, histopathological changes, systemic transfluthrin levels, and muscarinic acetylcholine receptor (mAChR) gene expression to elucidate potential mechanisms underlying observed effects.\u003c/p\u003e\u003cp\u003eFor inhalational exposure, commercial transfluthrin-based vaporizers (0.88%, SC Johnson Products Pvt. Ltd., Baddi, Solan, HP, India) were deployed in a 1000 ft\u0026sup3; space. While one vaporizer typically suffices for a similarly sized human-occupied area, five vaporizers were used to account for rats\u0026rsquo; higher metabolic rate\u0026mdash;approximately five times that of humans\u0026mdash;thus achieving physiologically relevant exposure levels [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Oral dosages were adapted from prior toxicological studies [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTransfluthrin was detected in the bloodstream following both inhalational and oral exposure, confirming systemic absorption through both routes. While systemic presence following ingestion is expected, its detection via inhalation underscores potential human health concerns, especially considering the widespread indoor use of vaporizer-based repellents.\u003c/p\u003e\u003cp\u003eBehavioral testing revealed that transfluthrin-exposed rats spent significantly more time in the closed arms of the Elevated Plus Maze (EPM), suggesting heightened anxiety. Increased line crossings were also observed across both exposure groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), consistent with enhanced exploratory behavior typically associated with anxiogenic states [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These behavioral outcomes align with known neurotoxic mechanisms of pyrethroids, such as the prolonged opening of voltage-gated sodium channels, which enhances neuronal excitability [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn the Morris Water Maze (MWM), transfluthrin-exposed rats showed increased latency to locate the hidden platform, indicating impairments in spatial learning and memory. Given the hippocampus's reliance on cholinergic signaling via mAChRs for memory consolidation [\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], we hypothesized that cognitive deficits could be linked to mAChR dysregulation. Quantitative RT-PCR analysis of midbrain tissue revealed a downward trend in mAChR mRNA expression in orally exposed rats, though the changes were not statistically significant in either group. These results suggest that mAChR disruption may contribute to, but not fully explain, the cognitive impairments observed. Moreover, the lack of assessment in the amygdala\u0026mdash;a key region in emotion-related memory\u0026mdash;represents a limitation that warrants further exploration.\u003c/p\u003e\u003cp\u003eImportantly, pesticide exposure has been implicated in neurodegenerative diseases such as Alzheimer\u0026rsquo;s, characterized by cholinergic dysfunction and progressive memory loss. Thus, the potential link between chronic transfluthrin exposure and such conditions should be evaluated through long-term and epidemiological studies.\u003c/p\u003e\u003cp\u003eHematologically, a significant increase in total leukocyte count (TLC) was observed in both exposure groups, suggesting an immune response to transfluthrin. While previous studies have primarily reported immunosuppressive effects [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], the leukocytosis noted here may reflect either a compensatory activation of the immune system or a chemical stress-induced response. Alternatively, this may indicate transient leukocyte mobilization or dysregulation, commonly reported under xenobiotic stress conditions [\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHemoglobin levels were significantly reduced in the orally exposed group, indicating potential transfluthrin-induced anemia. Interestingly, red blood cell (RBC) and platelet counts remained unchanged, suggesting that the anemia may not stem from impaired erythropoiesis. Instead, hemolytic activity or disruptions in iron metabolism may be responsible, though further studies are required to confirm the same [\u003cspan additionalcitationids=\"CR35 CR36 CR37\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBiochemical markers of liver and kidney function (ALT, AST, ALP, bilirubin, urea, creatinine) were significantly elevated in both exposure groups, with effects more pronounced in orally exposed rats. These findings, corroborated by histopathological evidence\u0026mdash;such as hepatic dysplastic foci and pulmonary inflammation\u0026mdash;are consistent with prior studies linking transfluthrin to oxidative stress and tissue damage [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePrevious work from our group has also identified detrimental effects of transfluthrin on reproductive health [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], emphasizing the broader toxicological implications of prolonged exposure.\u003c/p\u003e"},{"header":"6. Limitations of the Study","content":"\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eLimited Sample Size\u003c/b\u003e: The modest number of animals per group may limit the statistical power to detect subtle or borderline effects. Future studies with larger cohorts are necessary to validate and expand on these findings.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eLack of Brain Tissue Quantification\u003c/b\u003e: Transfluthrin levels were not quantified in brain tissue using gas chromatography\u0026ndash;mass spectrometry (GC-MS). Determining its ability to cross the blood\u0026ndash;brain barrier is critical for elucidating direct neurotoxic effects. Future studies are needed for further mechanistic studies to confirm the findings of our study.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAbsence of Biochemical Cholinergic Markers\u003c/b\u003e: Biochemical assays such as acetylcholinesterase (AChE) activity in specific brain regions (e.g., hippocampus, prefrontal cortex) were not conducted. These could have provided stronger mechanistic support for the observed behavioral impairments.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eAnimal Model Constraints\u003c/b\u003e: As the study was conducted in a rodent model, extrapolation of results to humans should be done cautiously. Additional studies\u0026mdash;including human epidemiological research and in vitro cellular assays\u0026mdash;are required for better risk assessment.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e"},{"header":"7. Conclusion","content":"\u003cp\u003eCollectively, our findings indicate that sub-chronic exposure to transfluthrin\u0026mdash;via both inhalation and oral routes\u0026mdash;can adversely affect neurobehavior, cognitive performance, immune responses, hematological parameters, and vital organ function in rats. The detection of transfluthrin in systemic circulation following inhalation is particularly concerning, given its widespread domestic use. Although changes in mAChR expression may contribute to cognitive deficits, additional molecular mechanisms likely play a role. These results underscore the necessity for comprehensive toxicological evaluations, including long-term animal studies and human epidemiological research, to inform safety guidelines and public health policies concerning transfluthrin exposure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cb\u003eEthical clearance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe study obtained ethical clearance from IAEC (No. EC/2021/05 dated 24/05/2021)\u003c/p\u003e\u003cp\u003e\u003ch2\u003eConflicts of interests:\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no conflicts of interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eNone\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the Government of National Capital Territory of Delhi (GNCTD) for funding to address idea of concepts by conducting research. Multidisciplinary Research Unit, MAMC, New Delhi acknowledged for experiments facilities.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSoderlund DM (2012) Molecular mechanisms of pyrethroid insecticide neurotoxicity: recent advances. Arch Toxicol 86(2):165\u0026ndash;181\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAbhilash PC, Singh N (2009) Pesticide use and application: an Indian scenario. J Hazar Mater 165(1\u0026ndash;3):1\u0026ndash;12\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVesin A (2013) Transfluthrin indoor air concentration and inhalation exposure during application of electric vaporizers. Environ Int 60:1\u0026ndash;6\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNazimek T, Wasak M, Zgrajka W (2011) Content of transfluthrin in indoor air during the use of electro-vaporizers. 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Alter Lab Anim 32(1suppl):411\u0026ndash;415\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMisslin R, Belzung C, Vogel E (1989) Behavioural validation of a light/dark choice procedure for testing anti-anxiety agents. Behav. Proc. ;18(1\u0026ndash;3):119\u0026thinsp;\u0026ndash;\u0026thinsp;32\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrut L, Belzung C (2003) The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. EJP 463(1\u0026ndash;3):3\u0026ndash;3\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEnnaceur A, Chazot PL (2016) Preclinical animal anxiety research\u0026ndash;flaws and prejudices. Pharmacol R Per 4(2):e00223\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eD\u0026rsquo;Hooge R, De Deyn PP (2001) Applications of the Morris water maze in the study of learning and memory. Brain R Reviews 36(1):60\u0026ndash;90\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLichtenberg E, Zimmerman R (1999) Information and farmers\u0026rsquo; attitudes about pesticides, water quality, and related environmental effects. Agri Eco Environ 73(3):227\u0026ndash;236\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShafer TJ, Meyer DA, Crofton KM (2004) Developmental Neurotoxicity of Pyrethroid Insecticides; Critical Review and Future Research Needs. Environ H Pers 113(2):123\u0026ndash;136\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTransfluthrin Liquid Electric (2020) Product type 18 Transfluthrin Case Number in R4BP: BC-CH021598-44 Evaluating Competent Authority: Netherlands Date: 22 October\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEspejo EF (1997) Structure of the mouse behaviour on the elevated plus-maze test of anxiety. Behav Brain Res 86(1):105\u0026ndash;112\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRicci EL (2013) Behavioral and neurochemical evidence of deltamethrinanxiogenic-like effects in rats. Braz J Vet Res Anim Sci 50(1):33\u0026ndash;42\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDel-Rahman A (2004) Neurological deficits induced by malathion, DEET, and permethrin, alone or in combination in adult rats. J Toxicol Environ Health Part A 67(4):331\u0026ndash;356\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMandhane SN, Chopde CT (1997) Neurobehavioral effects of low level fenvalerate exposure in mice. Indian J Exp Biol 35(6):623\u0026ndash;627\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCagen SZ (1984) Pyrethroid-mediated skin sensory stimulation characterized by a new behavioral paradigm. Toxicol Pharmacol 76(2):270\u0026ndash;279\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMorgan MK (2012) Children\u0026rsquo;s exposures to pyrethroid insecticides at home: a review of data collected in published exposure measurement studies conducted in the United States. Int J Environ Res Public Health 9(8):2964\u0026ndash;2985\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBromley-Brits K, Deng Y, Song W Morris water maze test for learning and memory deficits in Alzheimer's disease model mice. JoVE 2011 Jul 20(53):e2920\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoniz AC (1999) Perinatal fenvalerate exposure: behavioral and endocrinology changes in male rats. Neurotoxicol Teratol 21(5):611\u0026ndash;618\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSuresh PS, Koner BC (2012) Effect of acute and chronic stress on leucocyte count: modulation by chlordiazepoxide. Immunopharmacol Immunotoxicol 34(4):586\u0026ndash;589\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAndini A The Effect of Insect Repellent Exposure on Leukocyte Profile and Histopathologic Findings in Lungs\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhurana R, Chauhan RS (2005) Immunopathological effects of pesticides on lymphoid organs in sheep. J Immunol Immunopathol 7(1):64\u0026ndash;68\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKale M, Rathore N, John S (1999) Lipid peroxidative damage on pyrethroid exposure and alterations in antioxidant status in rat erythrocytes: a possible involvement of reactive oxygen species. ToxicolLett 105(3):197\u0026ndash;205\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGarba SH (2007) Toxicological Effects of Inhaled Mosquito Coil Smoke on the Rat Spleen: A Haematological and Histological Study SH Garba, MM Shehu and AB Adelaiye. J Med Sci 7(1):94\u0026ndash;97\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShringi KL, Dulara SC, Ansari RK (2015) Uncontrolled seizures and unusual rise in leucocytes count: transfluthrin, liquid mosquito repellent suicidal poisoning. Indian J Anaesth 59(1):47\u0026ndash;49\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePrasanthi K, Rajini PS (2005) Morphological and biochemical perturbations in rat erythrocytes following in vitro exposure to Fenvalerate and its metabolite. Toxicol Vitro 19(4):449\u0026ndash;456\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdelaiye SG (2007) Toxicological Effects of Inhaled Mosquito Coil Smoke on the Rat Spleen: A Haemat. J Med Sci 7(1):94\u0026ndash;97\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan A, Ahmad L, Khan MZ (2012) Hemato-Biochemical Changes Induced by Pyrethroid Insecticides in Avian, Fish and Mammalian Species. Int J Agri\u0026amp; Biol. ;14(5)\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYamada T (2009) Case study: an evaluation of the human relevance of the synthetic pyrethroidmetofluthrin-induced liver tumors in rats based on mode of action. Toxicol Sci 108(1):59\u0026ndash;68\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMishra A, Dewangan G, Dhakad MS, Sonkar SC, Dalal J, Pradhan S, Sharma D, Roy V, Koner BC (2025) Exploration of the transfluthrin effects on fertility and pregnancy outcomes: An in-vivo study in rat. Pestic Biochem Physiol 207:106220\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Maulana Azad Medical College","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Transfluthrin, Neurobehavior, Memory, Toxicity, Muscarinic Acetylcholine receptor (mAchR)","lastPublishedDoi":"10.21203/rs.3.rs-7645422/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7645422/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Transfluthrin,a fast-acting synthetic pyrethroid widely used in household insecticides, associated with potential neurotoxic and systemic health effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective:\u003c/strong\u003e This study aimed to evaluate the sub-chronic effects of transfluthrin exposure via oral and inhalation routes on neurobehavior, systemic toxicity, and muscarinic acetylcholine receptor (mAchR) gene expression in rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Adult male Sprague Dawley rats were exposed to transfluthrin for 90 days either orally (500 ppm in chow) or via inhalation (0.88% transfluthrin vaporizers, 12 h/day). Neurobehavioral assessments were performed using the Elevated Plus Maze (EPM), Open Field Test (OFT), and Morris Water Maze (MWM). Hematological and biochemical parameters were measured, and histopathological analysis of lung and liver tissues was conducted. mAchR gene expression was evaluated via quantitative RT-PCR, and blood transfluthrin levels were quantified using gas chromatography-mass spectrometry (GC-MS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e Transfluthrin-exposed rats exhibited significantly increased anxiety-like behavior (p=0.003), hyperactivity (p=0.04), and impaired spatial learning and memory (p=0.04). Elevated levels of liver enzymes alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and renal markers (urea, creatinine) indicated systemic toxicity. Histopathological findings included hepatic dysplastic foci and pulmonary inflammation.Additionally, a non-significant downward trend in mAChR gene expression was observed in exposed groups.Transfluthrin was detected in the bloodstream following both exposure routes, with significantly higher concentrations in orally exposed rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Sub-chronic transfluthrin exposure induces neurobehavioral impairments, systemic toxicity, and organ damage in rats. While mAChR downregulation may contribute to cognitive deficits, further mechanistic studies are needed. Considering the widespread domestic use of transfluthrin, these findings underscore the need for comprehensive toxicological evaluations to inform public health policies.\u003c/p\u003e","manuscriptTitle":"Neurobehavioral and Systemic Toxicity of Sub-Chronic Transfluthrin Exposure in Rats: An Exploratory In Vivo Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-23 04:46:07","doi":"10.21203/rs.3.rs-7645422/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"851c2c72-59be-442c-812f-1582e939b021","owner":[],"postedDate":"September 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54918642,"name":"Toxicology"}],"tags":[],"updatedAt":"2025-09-23T04:46:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-23 04:46:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7645422","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7645422","identity":"rs-7645422","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}