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. 2026 Jul 8;118(7):e70086. doi: 10.1002/bdr2.70086

Combined Effects of Dinotefuran and Piperonyl Butoxide on Behavioral Development in an F1 ‐Generation Toxicity Study in Mice

Toyohito Tanaka 1,✉, Motoki Hojo 1, Akiko Inomata 2
PMCID: PMC13470652  PMID: 42420751

ABSTRACT

Background

Few published studies have reported on the reproductive and neurobehavioral toxicity of combined exposure to neonicotinoid insecticides and synergists in mammals. This study aimed to evaluate the reproductive and neurobehavioral effects of a combined treatment with dinotefuran (DIN) and piperonyl butoxide (PBO) in an F1‐generation toxicity study in mice.

Methods

DIN and PBO were given in the diet to provide levels of 0% (control), DIN 0.005% + PBO 0.03%, DIN 0.01% + PBO 0.03%, and DIN 0.02% + PBO 0.03% from 5 weeks of age of the F0 generation to 11 weeks of age of the F1 generation in mice. Selected reproductive and neurobehavioral parameters were measured in the F1 generation.

Results

For behavioral development during the lactation period, surface righting on PND 4 and olfactory orientation route on PND 14 indicated a significantly high score in the DIN 0.02% + PBO 0.03% group in male offspring. In female offspring, olfactory orientation route and time on PND 14 indicated a significantly high score in the DIN 0.02% + PBO 0.03% group. For exploratory behavior in the F1‐generation offspring, several variables indicated significant effects in male and female offspring. In the spontaneous behavior of males in the F1 generation, the parallel lines among the control and treatment groups indicated significant distances in total distance, movement time, and number of rearing.

Conclusions

The dose levels of DIN with PBO in the present study produced several adverse effects on neurobehavioral parameters in F1 generation mice at lower dose levels than DIN alone.

Keywords: behavioral development, dinotefuran (DIN), exploratory behavior, mice, neurobehavioral toxicity, piperonyl butoxide (PBO), reproductive toxicity, spontaneous behavior

1. Introduction

Currently, the risk assessment of pesticide residues in food is conducted on a compound‐by‐compound basis. Nevertheless, consumers are frequently exposed to more than one pesticide residue simultaneously or within a short period (Boobis et al. 2008). Mixed exposures to different pesticide residues could therefore occur as a consequence of intake from a single food item containing multiple residues or from several food items each containing one or more residues (Tanaka et al. 2020). The consequence of mixed exposure has raised concerns among both consumers and regulators.

Piperonyl butoxide (PBO) is a pesticide synergist found in hundreds of pesticide products used in agricultural, commercial, and residential settings (Everson et al. 2019; Riviera‐González et al. 2021). PBO is one of the most frequently detected pesticides in infants and young children (16.7%) in a French infant total diet study (Nougadère et al. 2020).

PBO is primarily used in combination with pyrethroids, but it has also been used in conjunction with neonicotinoid insecticides (Tanaka and Inomata 2026). The combination of dinotefuran (DIN) and PBO is used to control hygiene pests such as flies and mosquitoes (Senbo 2007; Darriet and Chandre 2011) and to exterminate and prevent fleas in cats and dogs (Yannai and Kafri 2017). DIN is one of the most frequently detected pesticides in domestic crops (19.5%) in Japan (the Ministry of Health, Labour and Welfare of Japan 2020).

Dinotefuran (DIN), 1‐methyl‐2‐nitro‐3‐(oxolan‐3‐ylmethyl)guanidine [IUPAC], (CAS No. 165252–70‐0) is a third‐generation neonicotinoid insecticide available as water‐soluble granules, wettable powders, granules, and dusts (Tanaka et al. 2022). DIN is one of the nicotinic acetylcholine receptor (nAChR) competitive modulators in the mode of action (MOA) for insecticides (Insecticide Resistance Action Committee; IRAC 2026). DIN is a broad‐spectrum systemic insecticide and is effective on insect pest species such as homopterans, heteropterans, thysanopterans, lepidopterans, orthopterans, coleopterans, dipterans, dictyopterans, siphonoapterans, and isopterans (Yamada 2003; Wakita et al. 2005).

DIN is registered for agricultural use on various grain crops, legumes, vegetables, and fruits. DIN has been registered in Brazil, Canada, Japan, South Korea, and the United States of America (Food Safety Commission of Japan 2017). DIN is used for expelling termites in housing and chironomid and moth fly larvae in water stagnation in Japan (Tanaka et al. 2023). DIN is used as an active ingredient of attractive targeted sugar baits to control mosquitoes outdoors in Kenya, Mali, and Zambia (Attractive Targeted Sugar Bait Phase III Trial Group 2022).

The shipping amount of DIN manufactured in 2020 was 164.8 tons in Japan (National Institute for Environmental Studies, Japan 2022). The acceptable daily intake (ADI) of DIN for humans has been set at 0.2 mg/kg body weight (bw), and the acute reference dose (ARfD) has been set at 1 mg/kg bw (Joint FAO/WHO Meeting on Pesticide Residues [JMPR] 2013).

Piperonyl butoxide (PBO), 5‐[2‐(2‐butoxyethoxy)ethoxymethyl]‐6‐propyl‐1,3‐benzodioxole (IUPAC), (CAS No. 51‐03‐6) is a pesticide synergist used to enhance the effect of pyrethrins, rotenone, tetramethrin, and other insecticides in oil solutions, aerosols, dust, wettable powders, and slurries (Kennedy Jr et al. 1977). PBO is registered for use on various fruits, vegetables, forage, and grain crops, as well as on livestock and agricultural premises (Kennedy Jr et al. 1977; Cardy et al. 1979). PBO is used as a food additive in Japan, and its maximum allowable use level on raw cereals is 0.024 g/kg (24 ppm). The ADI of PBO for humans has been set at 0–0.2 mg/kg bw (JMPR 1996). The setting of an ARfD for PBO was not justified by the 2001 JMPR (WHO 2018).

In neurotoxicity studies of DIN (JMPR 2013), the no‐observed adverse effect level (NOAEL) in acute neurotoxicity in rats was 750 mg/kg bw/day, and the corresponding value in subchronic neurotoxicity in rats was 5000 ppm (approximately 327 mg/kg bw/day). In a developmental neurotoxicity study in rats, there was no evidence for developmental neurotoxicity, and its NOAEL was 3000 ppm for maternal toxicity (approximately 237 mg/kg bw/day).

Nevertheless, few neurobehavioral toxicity studies of DIN were carried out except for the above report. However, Tanaka et al. (2022) reported that female offspring indicated several adverse effects on behavioral development during the lactation period in a maternal exposure study in mice (0.012%–0.075% in the diets). The related variables of rearing in the exploratory behavior of female offspring indicated dose‐dependent effects. Tanaka et al. (2023) reported that DIN induced several adverse effects on neurobehavioral parameters in an F1‐generation toxicity study in mice (0.015%–0.06% in the diets). Some variables of exploratory and spontaneous behavior were affected significantly in the lower‐dose groups in both male and female mice.

Regarding the neurobehavioral toxicity of PBO, Tanaka (2003) found that total distance was significantly increased in the higher‐dose groups (≥ 0.03% in the diet), and average distance and speed were significantly increased in the high‐dose group (0.09% in the diet). These effects showed dose‐related responses (p < 0.01 in each) in adult males of the F1 generation. Tanaka et al. (2009) reported that females in the F1‐generation mice showed more activity in some variables of spontaneous behavior in the high‐dose group (0.02%–0.18% in the diet).

In maternal exposure studies of PBO, Tanaka and Inomata (2015) found that several variables in behavioral development were significantly delayed in the higher dose groups (≥ 0.03% in the diet), and those effects showed dose‐related responses (0.01%–0.09% in the diet). The rearing of spontaneous behavior increased in the high‐dose group (0.09% in the diet) of males in the F1 generation. Tanaka and Inomata (2016) reported that the average time of movement in males and the average time of rearing in females showed dose‐related effects (0.015%–0.06% in the diet) in spontaneous behavior in the F1 generation.

However, these studies have only examined the effects of DIN and PBO alone. We are frequently exposed to DIN and PBO from food, both of which are suggested to have developmental neurotoxicity. PBO is frequently found at significantly higher concentrations than the active insecticidal ingredients (Everson et al. 2019; Riviera‐González et al. 2021).

In the previous study (Tanaka and Inomata 2026), a combined exposure to DIN (0.012%–0.024%) and PBO (0.03%) induced adverse effects on exploratory and spontaneous behavior in an F1 generation toxicity study of mice. This study aimed to assess the reproductive and neurobehavioral effects of combining lower doses of DIN with a synergistic dose of PBO, by measuring exploratory and spontaneous behaviors in mice across multiple generations. The dose level of PBO was set to 0.03% because PBO induced synergistic effects with combined exposure to DIN on reproductive and neurobehavioral parameters at 0.03% in the diet in the previous study (Tanaka and Inomata 2026).

2. Materials and Methods

2.1. Materials

DIN was obtained from Tokyo Chemical Industry Co. Ltd., Tokyo, Japan (Lot no. AIBHM‐QO). The purity of DIN was > 98.0% (HPLC). DIN is stable and resistant to hydrolysis, oxidation, and heat (Japan Plant Protection Association 2016).

PBO was obtained from Tokyo Chemical Industry Co. Ltd., Tokyo, Japan (Lot no. PF4DI‐RD). The chemical characterization was a practical grade, and the purity was > 90.0% (GC). PBO is quite stable, resistant to hydrolysis, oxidation, heat, and exposure to sunlight or intense fluorescent light (IPCS 1965; Friedman and Epstein 1970).

2.2. Animals and Maintenance

Male and female mice (Crl: CD1, 4 weeks of age) were purchased from The Jackson Laboratory Japan Inc., Kanagawa, Japan. They were housed individually in polycarbonate solid‐floored cages with pulp chips (ALPHA‐dri, made from alpha cellulose) in a temperature‐controlled room maintained at 24.5°C ± 1°C with a relative humidity of 50% ± 5% on a 12‐h light/dark cycle. They were given control (CE‐2; Clea Japan Inc., Tokyo, Japan) or experimental diets, and filtered water ad libitum.

2.3. Experimental Design

DIN and PBO were given in the diet to provide levels of 0% (control), DIN 0.005% + PBO 0.03%, DIN 0.01% + PBO 0.03%, and DIN 0.02% + PBO 0.03% from 5 weeks of age of the F0 generation to 11 weeks of age of the F1 generation in mice. The middle dose level of DIN (0.01%) was set near the 100‐fold ADI of DIN (0.2 mg/kg bw). The control group (20 mice: 10/sex) was given basal diets (CE‐2) for the corresponding period. The experimental diets were prepared bimonthly (twice) in our laboratory. After mixing DIN and/or PBO with the powdered basal diet (CE‐2), pellets were formed and fed to mice.

Individual food intake of mice was measured during the following six periods (Tanaka et al. 2023): preconception (from 5 weeks of age to mating); mating (5 days); gestation (14 days: Gestation Day 1–5 to 15–19); Lactation I (from birth to the second postpartum week: Lactation Day 0 to 14–18); Lactation II (from the third postpartum week to weaning, including offspring consumptions: Lactation Day 15–19 to 24–28); and F1 generation (4–11 weeks of age). Individual food intake (g/kg bw/day) was calculated as (the difference in feed weight between the previous and present week, excepting the weights of the spilled pellets in the cage)/(average body weight between the previous and present week)/days (Tanaka et al. 2023). In Lactation II, individual food intake (g/kg bw/day) was calculated as (the difference in feed weight between the previous and present week, excepting the weights of the spilled pellets in the cage)/(average body weight of dams and their litter between the previous and present week)/days (Tanaka et al. 2023).

2.4. Reproductive Procedure

Mice from the F0 generation were 5 weeks of age at the start of the study. They were weighed individually on Experimental Days 0, 2, 4, 7, 14, 21, 28, and 30 during the preconception period. Males and females were measured on exploratory behavior at 8 weeks of age (details in Section 2.5).

At 9 weeks of age, each female was paired with one male from the same treatment group for 5 days. Females were checked twice daily for mating by the appearance of the vaginal plug (morning and evening). The males were separated from the females after 5 days and were euthanized and dissected after mating. The females were then allowed to carry their litters to term, deliver, and raise all of their offspring. The dams were weighed individually during the gestation (every day on weekdays) and lactation periods (once a week). The dams were examined for delivery three times daily (morning, early afternoon, and evening).

At birth, live and dead offspring were counted, and litter size, litter weight, and sex ratio (male/female) of live offspring were measured on postnatal day (PND) 0. Litter was not culled, and all offspring were examined for the test procedure (Paget and Thomson 1979). The non‐culling method has been used in our institute to primarily observe the developmental effects of offspring (Palmer and Ulbrich 1997). The offspring were individually weighed on PNDs 0, 4, 7, 14, and 21 during the lactation period.

The survival indices were calculated as follows: (live offspring at each period)/(live and dead offspring at birth) × 100 (%). The offspring were weaned when they were 4 weeks of age, and dams were euthanized and dissected after weaning. One male and one female from each litter were randomly selected for continued evaluation. The rest of the offspring were euthanized. Mice were weighed every week from 4 weeks to 11 weeks of age after weaning.

2.5. Neurobehavioral Procedures

The functional and behavioral developmental parameters were measured and scored for all individual offspring during the lactation period in the F1 generation (Tanaka et al. 1992) and analyzed on a whole‐litter basis (Abbey and Howard 1973). The parameters tested at the same age were measured continuously for each litter in the order listed. The measured parameters are detailed below.

  1. Surface righting on PNDs 4 and 7 (Fox 1965; Pantaleoni et al. 1988). The offspring were placed on their backs on a smooth surface, and the time required was recorded to right themselves to a position where all four limbs touched the surface. The following scoring system was employed for successful righting: 2 = righting within 1 s; 1 = righting in > 1 s but within 2 s; 0 = righting in > 2 s.

  2. Inclined plane test on PNDs 4 and 7 (Fox 1965; Altman and Sudarshan 1975; Pantaleoni et al. 1988; Motz and Alberts 2005). The offspring were placed in a head‐down position on a plane inclined at 30° and the time required to reorient to a head‐up position was recorded. The plane was made of plywood covered with sandpaper (fine grade). The following scoring system was employed: 0 = no response within 60 s; 1 = response within 60 s; and 2 = response within 30 s.

  3. Cliff avoidance on PND 7 (Fox 1965; Altman and Sudarshan 1975; Pantaleoni et al. 1988). The offspring were placed on a platform elevated 10 cm above a tabletop. Forelimbs and snout were positioned so that the platform's edge passed just behind an imaginary line drawn between the eye orbits. The following scoring system was employed: 0 = no response within 20 s; 1 = avoided backward within 20 s; and 2 = avoiding with turn.

  4. Swimming behavior on PNDs 7 and 14 (Schapiro et al. 1970; Vorhees et al. 1979; Vorhees 1986; Pantaleoni et al. 1988). The offspring were placed in a round tank (Ø 300 mm × H 150 mm) with water temperature maintained at 23°C ± 1°C. The offspring were observed for up to 20 s. Swimming behavior was rated for direction (1 = floating; 2 = circling; and 3 = straight) and head angle (1 = unable to hold head up; 2 = nose and top of the head out of water; 3 = ears half out of water; and 4 = ears out of water). Limb movement was rated as 1 = all four limbs used and 2 = only hindlimbs used.

  5. Olfactory orientation on PND 14 (Gregory and Pfaff 1971; Altman and Sudarshan 1975; Barlow et al. 1978). The offspring were placed into an arm connecting two compartments. One compartment was covered with “home” pulp chips (i.e., from their cages) and the other was covered with fresh pulp chips. The time required to enter the compartment with the home pulp chips was recorded. Olfactory orientation was scored for the time required (0 = no response within 90 s; 1 = entered within 90 s; 2 = entered within 60 s; and 3 = entered the home pulp chips compartment within 30 s) and for the route (1 = entered the home pulp chips compartment via the fresh pulp chips compartment; and 2 = entered the home pulp chips compartment directly).

  6. Exploratory behavior. The exploratory behavior of mice was measured in the animal movement analysis system SCANET CV‐40 (Melquest Ltd., Toyama, Japan) on distance (DT) mode at 8 weeks of age in the F0 generation, and 3 and 8 weeks of age in the F1 generation. The system consisted of a rectangular cage (300 × 202 × 208 mm) made from acrylate resins with two crossing sensor frames of 72 units of detectors of near‐infrared photosensors for measuring spontaneous motor activity (Mikami et al. 2002). Each sensor unit was scanned at different heights, and these steps were completed within 0.1 s and repeated (Tanaka 2015a). The measurements of behavior were carried out by four sets of measuring units. The measuring cages of four sets each male and female were used alternately and washed immediately during the next measurement (Tanaka 2020). The behavioral parameters were recorded (8:50–10:20 a.m.) for 10 min on one male and one female mouse selected randomly from each litter at 3 weeks of age in the F1 generation, and on all mice at 8 weeks of age in the F0 and F1 generation. The measurement parameters on DT mode were as follows: total distance (cm), number of horizontal activities, movement time (s), number of rearing, rearing time (s), average time of movement (s), average speed (cm/s), average time of rearing (s), number of defecations, and presence of urine (Tanaka 2015a).

  7. Maze learning. A day before the initial learning trial, each mouse acclimated to the testing apparatus by placing them in a separate straight channel where they could swim and learn to escape. To assess learning, each mouse underwent one trial per day for 3 days in a Biel‐type multiple‐T water maze adapted for mice (external dimensions: 565 × 565 × 185 mm, waterway width: 60 mm, water depth: 100 mm) at 7 weeks of age in the F1 generation (Biel 1940; Kitatani et al. 1988). The pattern of the apparatus used in the present study was a Biel's original type maze (Type‐A). The water temperature was maintained at 20°C ± 1°C. The time required and number of errors were measured from start to finish for a maximum of 120 s. The escape was defined as riding on a platform of goal. The errors were defined as a mistake at choice points including backtracking. If the time required was > 120 s, it was recorded as 120 s (Kitatani et al. 1988), and the animal reaching the 120 s was guided out goal.

  8. Spontaneous behavior. The spontaneous behavior of mice was measured in the animal movement analyzing system SCANET CV‐40 (Melquest Ltd.) on DT mode from 9 to 10 weeks of age in the F1 generation. The behavioral parameters were measured on all mice for 120 min (male: 9:00–11:00 a.m.; female: 11:20 a.m.–13:20 p.m.) at an interval of 10 min after 10 min latency. The measurement parameters were the same as those for exploratory behavior except for the urination and number of defecations.

2.6. Statistical Analyses

Food intake, litter size, litter weight, average sex ratio (male%), and body weight were assessed using the Williams test (p < 0.05). The body weight of offspring and behavioral developmental data during the lactation period were analyzed on a whole‐litter basis (Abbey and Howard 1973). Total sex ratio (male/female) and survival data were assessed using the χ2 test or the Fisher's exact test of frequency analysis.

Litter effects (litter‐to‐litter variation) on behavioral development during the lactation period were assessed using generalized linear models (GLMs). Group, litter size, and offspring weight were used as explanatory variables, and behavioral development scores were used as response variables. The explanatory variables were selected using stepwise Akaike's information criterion (AIC). Behavioral developmental data were assessed using the Shirley‐Williams test (p < 0.05) of nonparametric methods (Martin and Bateson 1990).

The exploratory behavior was assessed using the Shirley‐Williams test (p < 0.05). Regarding the spontaneous behavior data, the longitudinal pattern was assessed using profile analysis (test for equality of mean vectors, test for equality of covariance matrices and the parallelism hypothesis test; Fujikoshi 2009; Fujikoshi et al. 2008), and variables at each point of time were assessed using the Shirley‐Williams test (p < 0.05) within treatment groups.

Multiple‐T water maze performance data were assessed using the Signed Wilcoxon test for trials and assessed using the Shirley‐Williams test (p < 0.05) within treatment groups after a mixed ANOVA model for the longitudinal analysis. Dose–response effects were assessed using the Jonckheere test for ordered alternatives or the cumulative χ2 test (multi) for frequency data.

2.7. Guidelines

The design of the present study referred to the guidelines issued by ICH (Tripartite Guideline S5 (R3) 2020) and OECD (Guideline for Testing Chemicals 426 2007) adapted for mice. The major different points from ICH and OECD guidelines are animal species (rats → mice), sample size (20/group → 10/group), culling offspring (culling → non‐culling), selection of offspring for behavioral tests (selected offspring → all offspring), and the absence of neuropathological examinations (Tanaka et al. 2023). In our institute, mice are usually employed in reproductive and neurobehavioral toxicity studies to conserve resources (chemicals, diets, space, etc.), and background data for mice are sufficient for the evaluation of neurobehavioral effects (Tanaka 1998; Tanaka 2004; Tanaka 2010; Tanaka 2015a, 2015b; Tanaka 2020).

The present study was conducted in accordance under guidelines set by the National Research Council (2010) and the Science Council of Japan (2006). Animal experiments conformed with the Japanese laws and relevant regulations: “Act on Welfare and Management of Animals” (Act No. 105 of October 1, 1973, revised on Act No. 39 of June 19, 2019), Notice No. 88 of April 28, 2006, and Notice No. 84 (revised) of August 30, 2013, of the Ministry of the Environment of Japan “Standards Relating to the Care and Management of Laboratory Animals and Relief of Pain,” and Notification of June 1, 2006 (revised on February 20, 2015) of the Ministry of Health, Labor and Welfare of Japan “Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Research Institutions under the Jurisdiction of the Ministry of Health, Labor and Welfare.” This study was approved by Tokyo Metropolitan Institute of Public Health (March 5, 2025, no. 25‐14).

3. Results

3.1. Intake of Food and Chemicals

The average food intake was calculated, except for data from individuals with large spilled pellets. During the first week of treatment, the spilled pellets and powders of food increased in several individuals in the treatment groups. The average food intake per male and female during the preconception indicated no significant effects in all treatment groups (Table 1). The average food intake per pair of mice during mating indicated no significant effects in any of the treatment groups (Table 1). The average food intake per dam during the gestation period indicated no significant effects in all treatment groups (Table 1).

TABLE 1.

Average daily food intake (g/kg/day) of mice in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
F0 generation
Preconception
Male 153.03 ± 2.44 151.19 ± 3.39 155.71 ± 3.92 149.52 ± 2.77
Female 175.85 ± 5.47 186.38 ± 8.39 187.64 ± 14.07 174.56 ± 5.84
Mating 146.34 ± 5.68 147.14 ± 4.37 143.54 ± 5.16 144.86 ± 4.71
Gestation 163.57 ± 9.04 159.94 ± 6.62 156.85 ± 12.69 148.81 ± 4.85
Lactation I 417.02 ± 6.64 377.50 ± 16.44 403.71 ± 11.87 387.57 ± 13.00
Dams and their offspring
Lactation II 220.48 ± 9.39 230.28 ± 10.37 224.87 ± 11.27 220.21 ± 7.96
F1 generation
Male 184.60 ± 4.70 168.30 ± 4.83 171.32 ± 4.18 172.64 ± 1.65
Female 200.94 ± 5.74 196.71 ± 5.61 202.76 ± 8.58 214.36 ± 10.52
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Note: Each value represents daily intake during each period (mean ± SE). Lactation I is from birth to the second postpartum week, and Lactation II including offspring consumptions is from the third postpartum week to weaning.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide.

During the lactation period, no difference was indicated in the average food intake of dams in Lactation I, and the average food intake of dams and their offspring showed no significant effect in Lactation II (Table 1). After weaning, the average food intake of both sexes showed no significant effects (Table 1). During each period, the average intake of DIN increased consistently in a dose‐related manner, and that of PBO was similar in treatment groups (Table 2).

TABLE 2.

Average daily chemical intake (mg/kg/day) of mice administered in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
F0 generation
Preconception
Male
DIN — 7.56 ± 0.17 15.57 ± 0.39 29.90 ± 0.55
PBO — 45.36 ± 1.02 46.71 ± 1.18 44.86 ± 0.83
Female
DIN — 9.32 ± 0.42 18.76 ± 1.41 34.91 ± 1.17
PBO — 55.91 ± 2.52 56.29 ± 4.22 52.37 ± 1.75
Mating
DIN — 7.36 ± 0.22 14.35 ± 0.52 28.97 ± 0.94
PBO — 44.14 ± 1.31 43.06 ± 1.55 43.46 ± 1.41
Gestation
DIN — 8.00 ± 0.33 15.68 ± 1.27 29.76 ± 0.97
PBO — 47.98 ± 1.99 47.05 ± 3.81 44.64 ± 1.46
Lactation I
DIN — 18.87 ± 0.82 40.37 ± 1.19 77.51 ± 2.60
PBO — 113.25 ± 4.93 121.11 ± 3.56 116.27 ± 3.90
Dams and their offspring
Lactation II
DIN — 11.51 ± 0.52 22.49 ± 1.13 44.04 ± 1.59
PBO — 69.08 ± 3.11 67.46 ± 3.38 66.06 ± 2.39
F1 generation
Male
DIN — 8.41 ± 0.24 17.13 ± 0.42 34.53 ± 0.33
PBO — 50.49 ± 1.45 51.40 ± 1.25 51.79 ± 0.49
Female
DIN — 9.84 ± 0.28 20.28 ± 0.86 42.87 ± 2.10
PBO — 59.01 ± 1.88 60.83 ± 2.57 64.31 ± 3.16
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Note: Each value represents daily intake during each period (mean ± SE). Lactation I is from birth to the second postpartum week, and Lactation II including offspring consumptions is from the third postpartum week to weaning.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide.

3.2. F0 Generation

3.2.1. General Conditions During the Preconception Period

The average body weight of male and female mice showed no difference (p > 0.05) related to DIN and PBO treatment during the preconception period (Table 3).

TABLE 3.

Summary of average body weight (g) of F0 generation mice in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
Male
Day 0 30.08 ± 0.64 29.97 ± 0.40 30.09 ± 0.54 29.88 ± 0.42
Day 2 31.12 ± 0.67 31.11 ± 0.49 31.04 ± 0.53 30.67 ± 0.53
Day 4 32.02 ± 0.65 32.04 ± 0.52 31.91 ± 0.52 31.68 ± 0.58
Day 7 33.09 ± 0.77 33.42 ± 0.72 33.60 ± 0.63 32.81 ± 0.57
Day 14 35.21 ± 0.81 35.65 ± 0.83 35.36 ± 0.79 34.61 ± 0.60
Day 21 36.18 ± 0.90 36.71 ± 0.83 36.00 ± 0.84 35.90 ± 0.67
Day 28 37.08 ± 0.85 37.60 ± 0.80 37.02 ± 0.86 36.77 ± 0.80
Day 30 36.75 ± 0.92 37.52 ± 0.86 36.83 ± 0.90 36.53 ± 0.72
Day 35 36.64 ± 0.82 36.88 ± 092 36.06 ± 0.76 35.42 ± 0.65
Female
Day 0 23.82 ± 0.52 24.29 ± 0.30 24.31 ± 0.30 24.46 ± 0.28
Day 2 24.17 ± 0.52 24.53 ± 0.33 25.05 ± 0.35 24.97 ± 0.34
Day 4 24.51 ± 0.54 24.62 ± 0.28 25.11 ± 0.38 25.09 ± 0.35
Day 7 25.33 ± 0.51 25.82 ± 0.31 26.39 ± 0.46 26.36 ± 0.40
Day 14 27.66 ± 0.44 27.20 ± 0.36 28.31 ± 0.52 28.20 ± 0.48
Day 21 28.63 ± 0.67 28.61 ± 0.39 30.19 ± 0.68 29.62 ± 0.60
Day 28 29.61 ± 0.62 28.92 ± 0.47 31.15 ± 0.79 30.54 ± 0.51
Day 30 29.55 ± 0.66 29.29 ± 0.54 30.79 ± 0.73 30.56 ± 0.60
Day 35 31.17 ± 0.72 30.58 ± 0.90 31.98 ± 0.60 32.35 ± 0.64
Dam
Gestation
First week 37.55 ± 0.79 38.02 ± 0.82 38.72 ± 0.74 39.32 ± 1.18
Pre‐delivery day 62.84 ± 1.97 63.95 ± 1.73 66.43 ± 1.83 66.50 ± 2.21
Post‐delivery day a 37.86 ± 0.60 39.00 ± 0.82 40.05 ± 0.63* 40.69 ± 0.99*
Lactation
First week 46.18 ± 0.90 47.87 ± 1.22 48.80 ± 0.79 47.53 ± 1.07
Second week 48.34 ± 1.49 49.31 ± 1.33 49.62 ± 1.51 50.52 ± 1.41
Third week 44.12 ± 1.41 42.04 ± 0.79 45.29 ± 1.63 43.01 ± 1.21
Weaning 41.16 ± 0.76 41.05 ± 1.07 44.37 ± 1.05 41.53 ± 0.91
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Note: Each value represents the mean ± SE. Significantly different from controls: *p < 0.05.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide.

a

Significant dose‐related manner: *p < 0.05.

3.2.2. Exploratory Behavior

Regarding the movement activity of exploratory behavior at 8 weeks of age, no variables showed a difference (p > 0.05) related to treatment in either sex.

3.2.3. General Conditions of Dams

After mating, one female each from the DIN 0.01% + PBO 0.03% group, and the DIN 0.02% + PBO 0.03% group did not become pregnant (Table 4). During the gestation period, the average body weight of dams increased in a significant dose‐related manner on post‐delivery day. It significantly increased in the DIN 0.01% + PBO 0.03% group and the DIN 0.02% + PBO 0.03% group on post‐delivery day (Table 3).

TABLE 4.

Summary of data of litters at birth in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
No. of females examined 10 10 10 10
No. of pregnant females 10 10 9 9
No. of abortive birth 0 0 0 0
No. of litters 10 10 9 9
No. of live offspring 122 120 122 116
No. of dead offspring 4 2 0 1
Average litter size 12.20 ± 1.21 12.00 ± 1.13 13.56 ± 0.75 12.89 ± 0.56
Average litter weight (g) 20.75 ± 1.84 21.06 ± 1.91 23.59 ± 1.27 23.03 ± 1.12
Sex ratio (male/female) 1.218 (67/55) 1.222 (66/54) 1.033 (62/60) 1.189 (63/53)
Average sex ratio (male %) 55.61 ± 2.25 59.40 ± 5.76 52.47 ± 4.60 54.50 ± 3.80
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Note: Each value represents the mean ± SE.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide.

Two dams in the control group killed all their offspring a day after parturition. The dams indicated hyperactivity after parturition. One dam in the DIN 0.005% + PBO 0.03% group killed all her offspring on the day of parturition. No effects (p > 0.05) were indicated in the average body weight of dams in the DIN and PBO groups during the lactation period (Table 3).

3.3. F1 Generation

3.3.1. General Conditions During the Lactation Period

No difference (p > 0.05) related to the treatment of DIN and PBO was observed in average litter size, average litter weight, total sex ratio (male/female), and average sex ratio (male%) at birth (Table 4). The average body weight of offspring showed no difference (p > 0.05) related to treatment in both sexes at birth (Table 5).

TABLE 5.

Summary of average body weight (g) of offspring during the lactation period in F1 generation mice in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
Male offspring
PND 0 1.788 ± 0.065 1.813 ± 0.041 1.785 ± 0.024 1.835 ± 0.032
PND 4 3.128 ± 0.198 3.393 ± 0.143 3.315 ± 0.051 3.425 ± 0.095
PND 7 4.537 ± 0.301 4.937 ± 0.279 4.793 ± 0.103 4.917 ± 0.155
PND 14 6.231 ± 0.336 6.947 ± 0.552 6.355 ± 0.218 6.587 ± 0.220
PND 21 8.611 ± 0.884 11.151 ± 1.461 9.896 ± 0.679 10.314 ± 0.900
Female offspring
PND 0 1.692 ± 0.056 1.710 ± 0.024 1.701 ± 0.031 1.729 ± 0.026
PND 4 2.972 ± 0.189 3.159 ± 0.135 3.122 ± 0.078 3.225 ± 0.083
PND 7 4.297 ± 0.280 4.557 ± 0.247 4.621 ± 0.143 4.535 ± 0.176
PND 14 5.945 ± 0.335 6.173 ± 0.395 6.215 ± 0.260 6.147 ± 0.211
PND 21 8.134 ± 0.829 9.437 ± 1.229 9.892 ± 0.653 9.787 ± 0.809
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Note: Each value represents the mean ± SE.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide; PND, postnatal day.

Two litters of the control group were killed by their dams at PND 1. One litter was killed their dam in the DIN 0.005% + PBO 0.03% group on the day of parturition (PND 0). In one litter of the DIN 0.005% + PBO 0.03% group, all offspring except one female offspring from one litter died during the third week of lactation.

The average body weight of male and female offspring indicated no difference (p > 0.05) in all groups during the lactation period (Table 5). The survival indices, which were calculated excluding the killed litters, showed no difference (p > 0.05) related to treatment during the lactation period in either sex (Table 6).

TABLE 6.

Summary of the ratio of live offspring and survival index (%) during the lactation period in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
Male offspring
PND 0 67/68 (98.5) 66/66 (100.00) 62/62 (100.00) 63/63 (100.00)
PND 4 54/57 (94.7) 58/59 (98.3) 61/62 (98.4) 62/63 (98.4)
PND 7 52/57 (91.2) 58/59 (98.3) 61/62 (98.4) 62/63 (98.4)
PND 14 52/57 (91.2) 58/59 (98.3) 61/62 (98.4) 62/63 (98.4)
PND 21 52/57 (91.2) 51/59 (86.4) 57/62 (91.9) 59/63 (93.7)
Female offspring
PND 0 55/58 (94.8) 54/56 (96.4) 60/60 (100.00) 53/54 (98.1)
PND 4 45/50 (90.0) 48/50 (96.0) 59/60 (98.3) 53/54 (98.1)
PND 7 44/50 (88.0) 48/50 (96.0) 59/60 (98.3) 53/54 (98.1)
PND 14 44/50 (88.0) 48/50 (96.0) 59/60 (98.3) 53/54 (98.1)
PND 21 41/50 (82.0) 43/50 (86.0) 56/60 (93.3) 50/54 (92.6)
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Note: Each value represents the ratio of live offspring: survival index (%) in parentheses. Survival indices [(live offspring at each period)/(live and dead offspring at birth) × 100 (%)] are calculated except for the dead litters at each period.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide; PND: postnatal day.

3.3.2. Behavioral Development During the Lactation Period

No effects (p > 0.05) of litter size and offspring weight were observed in any variables of behavioral development in either sex. In male offspring, surface righting on PND 4 and olfactory orientation route on PND 14 indicated a significantly high score in the DIN 0.02% + PBO 0.03% group (Figure 1), and these effects indicated dose‐related responses (p = 0.0470 and 0.0452, respectively).

FIGURE 1.

FIGURE 1

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Average score for behavioral development in the lactation period in an F1‐generation toxicity study of combined exposure to dinotefuran (DIN) and piperonyl butoxide (PBO) administered to mice. Each value represents the mean ± SE. Significantly different from controls: *p < 0.05. Surface righting and olfactory orientation route of males indicated significant dose‐related manners (p = 0.0470 and 0.0452, respectively). Olfactory orientation time of females indicated a significant dose‐related manner (p = 0.0130).

In female offspring, olfactory orientation route and time on PND 14 indicated a significantly high score in the DIN 0.02% + PBO 0.03% group (Figure 1), and the effect in olfactory orientation time indicated a dose‐related response (p = 0.0130). Other variables showed no difference (p > 0.05) related to DIN and PBO treatment in either sex.

3.3.3. Exploratory Behavior of Offspring

In movement activity, exploratory behavior at 3 weeks of age indicated significant effects on several variables in both male and female offspring. In males, the number of defecations increased significantly in all treatment groups (Figure 2), with the effect indicating a dose‐related response (p = 0.0220).

FIGURE 2.

FIGURE 2

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Movement activity of exploratory behavior at 3 weeks of age of F1‐generation mice in an F1‐generation toxicity study of combined exposure to dinotefuran (DIN) and piperonyl butoxide (PBO) administered to mice. Each value represents the mean ± SE. Significantly different from controls: *p < 0.05. The number of defecations of males indicated a significant dose‐related manner (p = 0.0220). The average time of rearing of females indicated a significant dose‐related manner (p = 0.0315).

In females, the total distance significantly decreased in all treatment groups, and movement time significantly shortened in the DIN 0.01% + PBO 0.03% and DIN 0.02% + PBO 0.03% groups (Figure 2). The average time of rearing lengthened in a significant dose‐related response (p = 0.0315).

3.3.4. General Conditions After Weaning

The offspring were weaned at 4 weeks of age (each 34 males and females). One very small female (< 6 g at weaning) in the DIN 0.005% + PBO 0.03% group died at 5 weeks of age. The cause of the female's death was uncertain since no morbidity of the female was observed. The average body weight of the male mice significantly increased (p < 0.05) at 5 weeks of age in all treatment groups (Table 7). The average body weight of the female mice significantly increased (p < 0.05) at 7 weeks of age in all treatment groups (Table 7).

TABLE 7.

Summary of average body weight (g) of F1 generation mice after weaning in an F1‐generation toxicity study of combined exposure to DIN and PBO.

Dose level (%)
DIN 0 0.005 0.01 0.02
PBO 0 0.03 0.03 0.03
Male
4 weeks of age 15.10 ± 1.15 21.24 ± 1.74 18.28 ± 1.53 18.23 ± 1.21
5 weeks of age 25.87 ± 0.79 30.73 ± 1.34* 28.11 ± 0.85* 28.82 ± 0.85*
6 weeks of age 31.28 ± 0.73 34.32 ± 1.41 32.37 ± 0.63 33.49 ± 0.80
7 weeks of age 34.59 ± 0.99 36.99 ± 1.55 34.99 ± 0.58 36.09 ± 0.87
8 weeks of age 36.73 ± 1.21 39.00 ± 1.62 36.94 ± 0.67 37.48 ± 0.93
9 weeks of age 38.40 ± 1.31 40.38 ± 1.76 38.41 ± 0.60 38.94 ± 0.95
10 weeks of age 39.48 ± 1.35 41.35 ± 1.76 39.71 ± 0.60 40.02 ± 1.01
11 weeks of age 41.22 ± 1.34 42.16 ± 2.00 40.76 ± 0.78 40.60 ± 1.11
Female
4 weeks of age a 13.91 ± 0.96 17.95 ± 1.24 16.39 ± 1.03 16.23 ± 1.06
5 weeks of age a 21.99 ± 0.78 24.67 ± 1.09 24.40 ± 0.79 24.00 ± 0.64
6 weeks of age 24.57 ± 0.67 26.75 ± 1.00 26.61 ± 0.64 26.36 ± 0.57
7 weeks of age 25.95 ± 0.68 27.93 ± 1.13* 28.16 ± 0.67* 27.73 ± 0.51*
8 weeks of age 27.02 ± 0.67 29.12 ± 1.20 28.95 ± 0.88 28.47 ± 0.63
9 weeks of age 28.88 ± 0.98 30.49 ± 1.51 30.31 ± 0.81 29.93 ± 0.58
10 weeks of age 29.05 ± 1.02 31.21 ± 1.35 31.29 ± 0.89 30.40 ± 0.74
11 weeks of age 29.54 ± 1.12 32.05 ± 1.58 31.60 ± 0.86 30.84 ± 0.84
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Note: Each value represents the mean ± SE. Significantly different from controls: *p < 0.05.

Abbreviations: DIN, dinotefuran; PBO, piperonyl butoxide.

a

Excluding data from a dead female at 5 weeks of age.

3.3.5. Maze Learning

Regarding multiple‐T water maze performance, the time required shortened significantly in the second trial compared to the first trial in the control and DIN 0.02% + PBO 0.03% groups of males (Figure 3). The number of errors decreased significantly in the second trial of the DIN 0.02% + PBO 0.03% group of males compared to the control group.

FIGURE 3.

FIGURE 3

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Maze learning performance at 7 weeks of age of F1‐generation mice in an F1‐generation toxicity study of combined exposure to dinotefuran (DIN) and piperonyl butoxide (PBO) administered to mice. Each value represents the mean ± SE. Significantly different from controls: *p < 0.05. Significantly different from the first trial: # p < 0.05.

In females, the time required shortened significantly in the second and third trials in the DIN 0.02% + PBO 0.03% group compared to the control group (Figure 3). The time required shortened significantly in the third trial compared to the first trial in the control and DIN 0.02% + PBO 0.03% groups of females. The number of errors decreased significantly in the third trial of the DIN 0.02% + PBO 0.03% group of females compared to the control group. From the results, there was no significant effect caused by DIN and PBO treatment on multiple‐T water maze performance in either sex at 7 weeks of age.

3.3.6. Exploratory Behavior of Adults

In the movement activity of exploratory behavior at 8 weeks of age, the average speed significantly decreased in the DIN 0.02% + PBO 0.03% group in males (Figure 4). Other variables showed no difference (p > 0.05) related to treatment in males. In females, no variable showed a difference (p > 0.05) related to treatment.

FIGURE 4.

FIGURE 4

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Movement activity of exploratory behavior at 8 weeks of age of F1‐generation mice in an F1‐generation toxicity study of combined exposure to dinotefuran (DIN) and piperonyl butoxide (PBO) administered to mice. Each value represents the mean ± SE. Significantly different from controls: *p < 0.05.

3.3.7. Spontaneous Behavior of Adults

For the movement activity of spontaneous behavior from 9 to 10 weeks of age in the F1 generation, the longitudinal patterns of all variables measured were parallel (similar pattern) in each sex, except for the number of horizontal activities of males (Figure 5). The longitudinal pattern of the number of horizontal activities was not parallel (p = 0.0030) and indicated a significant difference (p = 0.0049) in the test for equality of mean vectors in males. The parallel lines of the control and treatment groups indicated significant differences in total distance, movement time, and number of rearing events (p = 0.0260, 0.0114, and 0.0137, respectively).

FIGURE 5.

FIGURE 5

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The longitudinal pattern of the movement activity of spontaneous behavior from 9 to 10 weeks of age of F1‐generation mice in an F1‐generation toxicity study of combined exposure to dinotefuran (DIN) and piperonyl butoxide (PBO) administered to mice. Each value represents the mean ± SE. Significantly different from controls: *p < 0.05. The longitudinal pattern of the number of horizontal activities was not parallel (p = 0.0030), and indicated a significant difference (p = 0.0049) in the test for equality of mean vectors in males. The parallel lines of the control and treatment groups indicated significant differences in total distance, movement time, and number of rearing events (p = 0.0260, 0.0114, and 0.0137, respectively).

In males, the total distance significantly lengthened at 100 min in the DIN 0.01% + PBO 0.03% group and DIN 0.02% + PBO 0.03% group, and significantly shortened at 120 min in the DIN 0.02% + PBO 0.03% group. The number of horizontal activities significantly increased in the DIN 0.01% + PBO 0.03% group and DIN 0.02% + PBO 0.03% group at 100 min and in all treatment groups at 110 min, and significantly decreased in the DIN 0.02% + PBO 0.03% group at 120 min. The movement time significantly lengthened in the DIN 0.01% + PBO 0.03% group and DIN 0.02% + PBO 0.03% group at 100 min, and significantly shortened in the DIN 0.02% + PBO 0.03% group at 120 min.

The average speed significantly increased in the DIN 0.02% + PBO 0.03% group at 90 min, and significantly decreased in the DIN 0.02% + PBO 0.03% group at 120 min. The average time of movement significantly lengthened in the DIN 0.02% + PBO 0.03% group at 100 min, and significantly shortened in the DIN 0.02% + PBO 0.03% group at 120 min.

The number of rearing significantly decreased in the DIN 0.02% + PBO 0.03% group at 120 min. The rearing time significantly lengthened in all treatment groups at 110 min, and significantly shortened in the DIN 0.02% + PBO 0.03% group at 120 min. The average time of rearing significantly lengthened in all treatment groups at 90 min, and significantly shortened in the DIN 0.02% + PBO 0.03% group at 120 min.

In females, one female in the DIN 0.01% + PBO 0.03% group indicated extremely hyperactive (> 5 times the baseline for hyperactivity: Tanaka 2020). The total distance shortened significantly at 30 min in the DIN 0.02% + PBO 0.03% group (Figure 5), and movement time significantly shortened in the DIN 0.02% + PBO 0.03% group at 30 min.

The average speed significantly decreased in the DIN 0.02% + PBO 0.03% group at 100 min. The average time of movement significantly shortened in the DIN 0.02% + PBO 0.03% group at 30 min and in all treatment groups at 100 min. The average time of rearing significantly lengthened in the DIN 0.02% + PBO 0.03% group at 90 min. Other variables showed no difference (p > 0.05) related to treatment in females.

4. Discussion

In the present study, the combined exposure to DIN and PBO produced some significant effects on neurobehavioral parameters. At birth, the average litter size showed no difference (p > 0.05) related to treatment (DIN 0.005% + PBO 0.03%–DIN 0.02% + PBO 0.03%). In a previous study on combined exposure to DIN and PBO (Tanaka and Inomata 2026), the average litter size significantly increased in the DIN 0.024% + PBO 0.03% group. Therefore, the lowest observed adverse effect level (LOAEL) for the effect of combined exposure to DIN and PBO on litter size was estimated to be 0.024% DIN + 0.03% PBO (equivalent to 38 mg/kg bw/day and 47 mg/kg bw/day, respectively), and the NOAEL was estimated to be 0.02% DIN + 0.03% PBO (equivalent to 29 mg/kg bw/day and 44 mg/kg bw/day, respectively).

About the movement activity of exploratory behavior in the F0 generation, no significant effects were indicated on any variables. In the previous study on combined exposure to DIN and PBO (Tanaka and Inomata 2026), several variables indicated significant adverse effects in both sexes. Therefore, the effects of the exploratory behavior in the F1 generation were not reproducible in the current study. In this study, mice were moved to different rooms multiple times after arrival and before measurements due to air conditioning failure, which may have affected the results.

For the behavioral developmental parameters, the development of surface righting in male offspring indicated a significantly high score in the DIN 0.02% + PBO 0.03% group on PND 4. The olfactory orientation route indicated significantly high scores in the DIN 0.02% + PBO 0.03% group on PND 14 in both sexes. The olfactory orientation time of female offspring indicated a significantly high score in the DIN 0.02% + PBO 0.03% group on PND 14. These effects had not been observed in the previous study (Tanaka and Inomata 2026).

Regarding exploratory behavior in the F1 generation, the average speed decreased significantly in the DIN 0.02% + PBO 0.03% group among adult males. Nevertheless, the total distance, movement time, average speed, and average time of movement indicated significant tendencies to increase in the treatment groups for exploratory behavior of adult males in the F1 generation in the previous study (Tanaka and Inomata 2026). Thus, the effects of combined exposure to DIN and PBO on the exploratory behavior of F1 adult mice were not reproducible.

In the present study, several variables of spontaneous behavior showed significant effects in adult males of the treatment groups in the F1 generation. The longitudinal pattern of the number of horizontal activities was not parallel, and the parallel lines of the control and treatment groups indicated significant differences in total distance, movement time, and number of rearing events.

In a previous study (Tanaka and Inomata 2026), the parallel lines of the control and treatment groups indicated significant distances in total distance, average speed, and average time of movement in adult males. Since similar variables of spontaneous behavior were impacted in both studies, combined exposure to DIN and PBO is likely to affect spontaneous behavior in males. Therefore, the present dose of DIN and PBO seems to produce different effects on spontaneous behavior in both sexes.

While neonicotinoid‐PBO mixtures are currently restricted to household products, concurrent exposure likely occurs in agriculture. This is because farmers frequently combine neonicotinoids with pyrethroids, which often contain PBO. Neonicotinoids are divided into two groups, with a cyano (acetamiprid and thiacloprid) or nitro (imidacloprid, thiamethoxam, clothianidin, DIN, and nitenpyram). Xu et al. (2021) reported that the presence of the synergist PBO altered neonicotinoid metabolism in vivo, primarily via the nitro reduction pathway, whereas a low abundance of related metabolites was observed in the conventional hydroxylation and demethylation pathways due to inhibition of CYP450 enzymes by the synergist.

Liang et al. (2026) demonstrated a link between prenatal neonicotinoid exposure and decreased neurocognitive scores in preschoolers, noting that DIN and clothianidin specifically correlated with lower Full‐Scale Intelligence Quotient (FSIQ). Horton et al. (2011) observed a significant inverse association between prenatal exposures to PBO (> 4.34 ng/m3, approximately > 1.25 ng/kg bw/day) and 36‐month neurodevelopment in human children. Therefore, DIN and PBO may affect human neurobehavioral development, and the risk is estimated to increase when they are used together.

The dose levels of DIN with PBO in the present study affected exploratory and spontaneous behavior at lower dose levels than DIN alone in the previous study (Tanaka et al. 2023). Since most affected variables in neurobehavioral parameters were only present in the DIN 0.02% + PBO 0.03% group, the LOAEL was estimated at DIN 0.02% + PBO 0.03% (equivalent to 35–44 mg/kg bw/day and 52–66 mg/kg bw/day, respectively), while the NOAEL was also estimated at DIN 0.01% + PBO 0.03% (equivalent to 17–22 mg/kg bw/day and 51–67 mg/kg bw/day, respectively). Although direct application is challenging due to the study's focus on combined exposure to two chemicals, the Benchmark dose approach (BMD) may offer a more accurate assessment of the dose–response relationship.

Funding

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

The authors would like to thank A. Nagasawa for contributing to acquiring and maintaining the data with the experiments.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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