Imidacloprid, 1[(6-chloro-3-pyridinyl)methyl]-N-nitro-2-imidazolidinimine, is a new neonicotinoid insecticide which has become an important pest control agent on many crops. The neonicotinoid insecticides are related to nicotine in their structure and action at the nicotinic acetylcholine receptor (Casida, 1998). Imidacloprid is most active against suckling insects because of their unique plant-systemic and translaminar properties. Imidacloprid poisoning in the American cockroach, Periplaneta americana, is characterized, in sequence, by loss of leg strength, leg tremors, body shaking and death. Neurophysiological studies have confirmed that imidacloprid is an agonist at the postsynaptic nicotinic acetylcholine receptor of insects. It appears to act like acetylcholine, by exciting specific nerve cells. There is also some evidence, however, that imidacloprid has multiple agonist and antagonist effects on neuronal nicotinic acetylcholine receptor channels of clonal rat phaeochromocytoma cells (Nagata et al., 1998).

The selective toxicity of imidacloprid to insects and not to mammals is attributed to differences in the binding affinity or potency at the nicotinic acetylcholine receptor (Chao & Casida, 1997). The specificity of nicotinoid insecticides appears to be related to receptor subtype, function, neuronal region and developmental stage, and their metabolic lability leads to bioactivated toxicants such as desnitro-imidacloprid in mammals. The selectivity also depends in large part on major structural differences in the neuronal nicotinic acetylcholine receptor binding sites of mammals and insects (Casida, 1998). The selectivity of the chloronicotyl compounds for insects as opposed to mammals can be partly explained by differences in the ionization of the pyrolidine nitrogen. Imidacloprid is poorly ionized in neutral media, in contrast to nicotine, and thus passes easily through insect lipophilic barriers (Sone et al., 1994; Roe et al., 1999). Imidacloprid has not previously been evaluated by the JMPR.

(a) Absorption, distribution and excretion

Imidacloprid labelled with ¹⁴C in either the methylene or the imidazolidine ring was administered in a physiological saline solution at concentrations of 0.1–2 mg/l to a total of 50 male and 20 female rats. Groups of five male and five female rats were given a single dose of 1 mg/kg bw intravenously or a single dose of 1  or 20 mg/kg bw orally. Other groups were given 14 doses of of the non-radiolabelled compound at 1 mg/kg bw orally once per day and, 24 h after the last dose, a single oral dose of 1 mg/kg bw as the radiolabelled compound. Radiolabel was determined in plasma and excreta as a function of time and, after sacrifice 48 h after treatment, the concentration of total radiolabel was determined in organs and tissues. A further group of five male rats was given a single oral dose of radiolabelled compound at 20 mg/kg bw, and ¹⁴CO₂ was measured over 48 h. In an additional test, four groups of five male rats were given a single oral dose of 20 mg/kg bw, killed after 40 min, 1.5, 3 and 6 h, and total radiolabel was determined as a function of time in single organs. A final group of five male rats with bile-duct fistulas was given a single intraduodenal dose of 1 mg/kg bw in order to determine the amount of total radiolabel absorbed and to measure the rate and extent of biliary excretion. The design of the study complied with good laboratory practice (GLP).

After oral administration of 1  or 20 mg/kg bw of [pyridinyl-¹⁴C-methylene]imidacloprid, the radiolabel was extensively absorbed from the intestinal lumen and readily distributed from the plasma into the body. The radiolabel was also readily eliminated. After intravenous administration of 1 mg/kg bw, about 92% of the recovered radiolabel was excreted in urine and faeces within 48 h. Most of the radiolabel was excreted via the kidneys, with an average ratio in urine:faeces  of 4:1. After oral administration, about 96% of the dose was excreted in urine and faeces within 48 h. No difference was found between female and male rats. More than 90% of the renal radiolabel was excreted during 24 h after dosage. The average residual radiolabel in the body, excluding the gastrointestinal tract, at sacrifice was about 0.5%, and that in the gastrointestinal tract was about 0.06% of the administered dose. The rats with bile-duct fistulas excreted only 4.7% of the administered dose with faeces, 56% in urine and about 36% with bile. These findings indicate the existence of enterohepatic circulation of the radiolabel. Significant amounts of radiolabel were not excreted in expired air (CO₂). The elimination of total radiolabel from the plasma could be approximated by a combination of two exponential terms, from which elimination half-lives were calculated. The two half-lives varied from 2.6–3.6 to 26–118 h, respectively (Klein, 1987a).

The distribution of [pyridinyl-¹⁴C-methylene]imidacloprid was investigated in male rats by conventional whole-body autoradiography on X-ray film in a study conducted according to GLP. The rats were given a single oral dose of 20 mg/kg bw, and the distribution of radiolabel was visualized at 1, 2, 4, 24 and 48 h after treatment. For visualization of the initial distribution, one animal was injected intravenously with 20 mg/kg bw and killed 5 min later.

The radiolabel was readily absorbed and rapidly distributed to the tissues and organs. The pattern of distribution showed that the radiolabel could readily permeate tissues: With the exception of fatty tissues, the central nervous system and the mineral part of bone, blackening on the autoradiogram was seen on all other parts of the body 5 min after intravenous injection and 1 h after oral dosage. Higher concentrations were seen later in the thyroid and adrenals, but, after 24 h, all other organs and tissues showed only small amounts of radiolabel. The high degree of blackening over the kidney during the first 24 h is a reflection of the high rate of renal excretion of the administered compound. The concentration of radiolabel decreased in organs and tissues with time after administration. The concentrations in the fatty tissues and in the central nervous system were very low throughout the study (Klein, 1987b).

Imidacloprid labelled with ¹⁴C in the 4 and 5 positions of the imidazolidine ring was administered orally to male and female rats at a dose of 1 mg/kg bw and additionally to male rats at a dose of 150 mg/kg bw, in a study conducted according to GLP. Radiolabel was determined in excreta and in plasma as a function of time, and single organs and tissues were assayed for total radiolabel at sacrifice 48 h after treatment. The biokinetics of [imidazolidine-4,5-¹⁴C]imidacloprid was similar to that of methylene-¹⁴C-labelled compound. The administered radiolabel was extensively absorbed from the intestinal lumen and rapidly distributed in the body. Excretion was rapid, and the renal route predominated (Klein & Brauner, 1991).

The nitroso metabolite of imidacloprid, 1-(6-chloro-3-pyridylmethyl)-N-nitroso(imidazoli-din-2-ylidene)amine (WAK 3839), has been identified as a minor constituent of edible plant commodities but was not found in the excreta of rats in the studies described above. Absorption of imidacloprid and WAK 3839 began immediately after oral administration of 1 mg/kg bw. A comparison of the curves of concentration–time in plasma suggested that the absorption was similarly extensive, and no significant differences in terminal half-times were found after administration of imidacloprid (38 h) and WAK 3839 (47 h). WAK 3839 was eliminated slightly faster, and the concentrations of total radiolabel in the organs were lower than with the parent compound. After administration of a single high dose of imidacloprid, 150 mg/kg bw, to male rats, the ratio of excretion of total radiolabel was the same as after the low dose. Pretreatment with repeated doses also did not affect the pattern of excretion (Klein, 1990).

(b) Metabolism

In the biokinetics study of Klein (1987a), urine was collected separately from each rat under cool conditions, at intervals of 0–4, 4–8, 8–24 and 24–48 h, and faeces were collected at intervals of 0–24 and 24–48 h after treatment. The metabolites were extracted from faeces, further isolated by preparative high-performance liquid chromatography (HPLC) and then purified by other HPLC methods. The metabolites were identified by chromatographic comparison with authentic reference compounds in at least two independent chromatographic systems or by ¹H-nuclear magnetic resonance and mass spectroscopic techniques.

A proposed metabolic pathway of imidacloprid in rats is shown in Figure 1. After administration of low doses, no relevant sex dependence was seen in the excretion pattern of the compound or in the metabolic profiles in excreta. All the identified metabolites were found at each dose group and in both sexes. The main metabolites were 6-chloronicotinic acid and its glycine conjugate, which were found only in urine. The amounts of monohydroxylated (5-hydroxy-imidacloprid) and olefinic metabolites were similar to those of unchanged parent compound. All other biotransformation products were quantitatively of minor significance.

Two main routes of metabolism responsible for the degradation of imidacloprid were identified. The first is oxidative cleavage, yielding 6-chloronicotinic acid, which is conjugated with glycine to form a hippuric acid-type conjugate. These two metabolites together represented most of the identified metabolites, or about 30% of the recovered radiolabel. Of minor importance in terms of quantity is dechlorination of the pyridinyl moiety, producing the 6-hydroxy nicotinic acid and its methylmercapturic acid derivative, probably as a degradation product of a glutathione conjugate. The 6-methylmercapto nicotinic acid conjugated with glycine, and the glycine conjugate constituted 5.6% of the recovered radiolabel. The second important biodegradation step starts with hydroxylation of the imidazolidine ring at the 4 or 5 position, and about 16% of the recovered radiolabel was identified as the sum of 4- and 5-hydroxy imidacloprid. The loss of water yields the olefinic compound. These biotransformation products and the unchanged parent compound were excreted in urine and faeces, while the guanidine compound was a less important metabolite and was eliminated only in faeces.

The high excretion rate of the parent compound (average, 14%) indicates rapid passage through the body, as was confirmed by the elimination of > 90% of the recovered radiolabel within 24 h after administration (Klein & Karl, 1990).

The distribution of the metabolites in liver and kidney at various times after a single oral dose was investigated in rats under the same test conditions as used by Klein (1987a). The metabolites were extracted from lyophilized organs with water and methanol, further purified by HPLC and thin-layer chromatography and identified by comparative HPLC with authentic reference compounds in at least two independent chromatographic systems and also by mass and ¹H-nuclear magnetic resonance spectroscopic techniques. The metabolites found in the kidney were identical to those identified in urine. Triazinone was not found in the excreta and may have undergone further biodegradation before elimination via the kidney or the bile. The relative amounts of those biotransformation products formed by oxidative mechanisms (e.g. 6-chloronicotinic acid) increased in the liver during the test period. In kidney, the relative amount of the more polar compounds decreased with time (6-chloronicotinic acid and its glycine conjugate), while the amounts of the olefinic metabolite and the mono-hydroxylated derivative 4-hydroxyimidacloprid showed a relative increase. The proportion of the parent compound decreased slowly as it was metabolized (Karl & Klein, 1992).

The metabolic pattern of imidacloprid and its nitroso metabolite in excreta was investigated in male rats after administration of a single oral dose of 1 mg/kg bw of imidacloprid or the nitroso compound and after administration of a high dose of 150 mg/kg bw imidacloprid. After the low dose of imidacloprid, about 82% of the renal radiolabel was detected. The main renal metabolite (about 30%) was the glycine conjugate of 6-chloronicotinic acid. The parent compound was present at about 12%, the two monohydroxylated biotransformation products (4-hydroxy and 5-hydroxyimidacloprid) at about 19% and the olefinic metabolite at about 11%. 6-Chloronicotinic acid represented 7.9% of the renal radiolabel. No nitroso compound was formed under these conditions. In the faeces, imidacloprid, the olefinic metabolite, 6-chloronicotinic acid and its glycine conjugate were identified. These findings are in good agreement with those reported by Klein & Karl (1990).

Few metabolites were found in the urine of male rats given the nitroso compound orally. Besides unchanged nitroso compound, only 8% of the renal radiolabel was attributable to the guanidine-type metabolite, some of which was also found in faeces. This finding indicates that the metabolism of the nitroso compound is completely different from that of its parent compound. In order to investigate whether the nitroso compound is formed as a biotransformation product of imidacloprid in vivo, a high single oral dose of 150 mg/kg bw imidacloprid was given to male rats. No nitroso metabolite was formed. In the urine of rats given a diet containing 1800 ppm of imidacloprid for 1 year and then one oral dose of [methylene-¹⁴C]imidacloprid, 9.3% of the radiolabel was attributable to the nitroso metabolite, corresponding to 6.8% of the administered dose. The nitroso compound is therefore formed in vivo in rats after long-term intake of imidacloprid. In order to confirm this finding, a direct isotope dilution analysis was conducted with the urine of these rats and also with the urine of mice that had been fed a diet containing 2000 ppm of imidacloprid for about 1 year. Both analyses clearly demonstrated the presence of the nitroso compound in the urine (Klein, 1990).

(a) Acute toxicity

The results of studies on the acute toxicity of imidacloprid are summarized in Table 1. The methods used in these studies complied with OECD guidelines and GLP.

Imidacloprid given orally as a single dose was moderately toxic to rats (LD₅₀, 380–650 mg/kg bw) and mice (LD₅₀, 130–170 mg/kg bw). Behavioural and respiratory signs, disturbances of motility, narrowed palpebral fissures, transient trembling and spasms were seen in rats and mice treated orally at doses > 200 mg/kg bw and > 71 mg/kg bw, respectively. The clinical signs were reversed within 6 days. Imidacloprid given intraperitoneally showed moderate to low acute toxicity in rats, the signs being similar to those after oral administration. Very little toxicity was seen after acute dermal application. The LC₅₀ for a single exposure to an aerosol could not be determined exactly, as rats tolerated inhalation for 4 h of the maximum concentration of dust that could be produced technically (0.069 mg/l of air) without signs or deaths.

Imidacloprid (purity, 94.2%) did not irritate the eyes or skin of HC New Zealand white rabbits (Pauluhn, 1988b,c) and did not sensitize the skin of DHPW guinea-pigs (Ohta, 1988).

(b) Short-term studies of toxicity

Groups of 10 male and 10 female Charles-River B6C3F₁ mice were given diets containing imidacloprid (purity, 92.8%) at a concentration of 0, 120, 600 or 3000 ppm for up to 107 days, equivalent to 17, 86 and 430 mg/kg bw per day. The study did not comply with GLP.

Seven males and seven females at 3000 ppm died; furthermore, several animals displayed a poor general condition, and rough coats were observed frequently. Body-weight gain was reduced and food consumption was increased in males at 600 ppm and males and females at 3000 ppm. At this dose, clinical chemical tests showed significantly decreased urea and cholesterol concentrations in males and lowered alanine aminotransferase activity and glucose concentration in females. Alkaline phosphatase activity was significantly increased in both sexes at 3000 ppm and in females at 120 and 600 ppm. Differences in the weights of the liver, heart, spleen, kidneys, testes and adrenals were observed at 3000 ppm. The NOAEL was 120 ppm, equivalent to 17 mg/kg bw per day (Eiben, 1988a).

Groups of 10 male and 10 female Wistar rats [Bor:WISW(SPF-Cpb)] received diets containing imidacloprid (purity, 92.8%) at a concentration of 0, 120, 600 or 3000 ppm for up to 98 days, equal to 11, 57 and 410 mg/kg bw per day for males and 15, 78 and 510 mg/kg bw per day for females. The study did not comply with GLP.

Food intake was increased in animals at 3000 ppm, and body-weight gain was decreased at 600 and 3000 ppm. Significantly elevated alkaline phosphatase activity and depressed glucose concentration were found in males and females at 3000 ppm, and the males also showed a reduced cholesterol concentration. Degenerative histological lesions in the epithelium of the testicular tubules were seen in five of 10 males at 3000 ppm, and multifocal group cell necrosis was diagnosed in the liver of one male at this dietary concentration. The NOAEL was 120 ppm, equal to 11 mg/kg bw per day (Eiben, 1988b).

Groups of 10 male and 10 female Wistar rats [Bor:WISW(SPF-Cpb)] received diets containing imidacloprid (purity, 95.3%) at a concentration of 0, 150, 600 or 2400 ppm for up to 96 days, equal to 14, 61 and 300 mg/kg bw per day for males and 20, 83 and 420 mg/kg bw per day for females. Satellite groups consisting of 10 male and 10 female rats received the test substance at a concentration of 0 or 2400 ppm over the same period, and, to study the reversibility of any effects, control diet was provided during the subsequent 4-week post-treatment observation period. The study was conducted according to GLP.

In animals at 2400 ppm, feed intake was increased during treatment and recovery. Reduced body-weight gain was observed in males at 600 ppm and in females at 2400 ppm. Slightly longer thromboplastin times and depressed thrombocyte counts were found at 2400 ppm, both of which were only partially reversible. Elevated alkaline phosphatase and alanine aminotransferase activities and depressed protein, albumin, cholesterol and triglyceride concentrations were found in males and females at 2400 ppm. Depressed protein concentrations were also found in males at 150 and 600 ppm. Males at 2400 ppm showed increased incidences of cellular necroses, round-cell infiltration, swollen cellular nuclei and cytoplasmic lesions in the liver, but these hepatotoxic effects were reversible within the subsequent 4-week post-treatment observation period. The NOAEL was 150 ppm, equal to 14 mg/kg bw per day (Eiben & Rinke, 1989).

Imidacloprid (purity, 95.3%) was administered in dust form through the nose only to groups of 10 male and 10 female Wistar rats [Bor:WISW(SPF-Cpb)] at analytically determined concentrations of 0, 20, 110 and 500 mg/m³ on 5 consecutive days for 6 h/day. The respirability of the test substance at the highest concentration was relatively low; about 20% of the administered mass of the substance was in the form of particles < 5 mg. The study was conducted according to GLP.

Body-weight gain was slightly decreased at the two higher concentrations. Elevated O-demethylase and N-demethylase activities were found in a liver homogenate from animals at 110 mg/m³ air. The NOAEL was 20 mg/ m³ air (Pauluhn, 1988a).

Imidacloprid (purity, 95.3%) was administered in dust form through the nose only to groups of 10 male and 10 female Wistar rats [Bor:WISW(SPF-Cpb)] at analytically determined concentrations of 0, 5.5, 30 and 190 mg/m³ for 6 h/day, 5 days per week for 4 weeks. The study was conducted according to GLP.

Body-weight gain was decreased in males at the two higher concentrations. Increased mixed-function oxidase activities were found in liver homogenate from females at these concentrations and in males at 190 mg/m³ air. Increased alanine aminotransferase and glutamate dehydrogenase activities were seen in both sexes at the highest concentration, and alanine aminotransferase activity was increased in females at 30 mg/m³ air. The females had increased alkaline phosphatase activity at the two higher concentrations and increased liver weights at 190 mg/m³ of air. The serum alpha1-globulin fraction was reduced in both sexes at the two higher concentrations. At 190 mg/m³ of air, blood coagulation time was increased in females and thrombocyte counts were depressed in males. The NOAEL was 5.5 mg/ m³ air (Pauluhn, 1988d)

Imidacloprid (purity, 95.0%), mixed to a paste in a physiological saline solution containing 2% Cremophor EL, was applied to the shorn dorsal and flank skin of groups of five male and five female HC New Zealand white rabbits at a dose of 0 or 1000 mg/kg bw for 6 h/day for 15 days. The study was conducted according to GLP. No treatment-related effects were observed. The NOAEL was 1000 mg/kg bw per day at the limit dose tested (Flucke, 1990).

Groups of two male and two female pure-bred beagle dogs received diets containing imidacloprid (purity, 92.8%) at a concentration of 0, 200, 1000 or 5000 ppm for up to 28 days, providing average intakes of 0, 7.3, 31 and 49 mg/kg bw per day for males and females combined. The study was not conducted according to GLP.

Two animals at 5000 ppm died, and the other two were killed in moribund condition. The symptoms observed were ataxia, tremor and occasional vomiting. Reductions in food consumption were recorded at the two higher concentrations, and body weight was markedly reduced at 5000 ppm. The results of hearing tests, ophthalmic examinations, urine analyses and haematological examiinations indicated no treatment-related changes at any dietary concentration. Cytochrome P450 activity in the liver was slightly increased in males and females at 1000 ppm. The triiodothyronine concentration in plasma was slightly decreased in one male and one female at 5000 ppm. The weight of the liver was increased in one female at 1000 ppm; one male at this concentration showed slight hepatocellular hypertrophy with slight pigmentation of Kupffer cells, and another had minimal follicular atrophy of the thyroid. The NOAEL was 200 ppm, equal to 7.3 mg/kg bw per day (Bloch et al., 1987).

Groups of four male and four female beagles [Bor:Beag] were given diets containing imidacloprid (purity, 95.3%) at a concentration of 0, 200, 600 or 1800/1200 ppm for 13 weeks. The concentration of active ingredient at the highest level was reduced at week 4 because of low food intake. The study was conducted according to GLP.

The food consumption of animals at 600 ppm was reduced. At higher concentrations, the animals showed a clinically emaciated state and transient trembling. Body-weight gain was reduced at 1800 ppm, but, once the active ingredient concentration had been reduced to 1200 ppm, a trend to normalization was apparent. The NOAEL was 200 ppm, equal to 7.5 mg/kg bw per day (Ruf, 1990).

Groups of four male and four female pure-bred beagles were given diets containing imidacloprid (purity, 94.9%) at a concentration of 0, 200, 500 or 1250/2500 ppm for 52 weeks. The concentration of 1250 ppm was increased from week 17 onwards. The mean intakes of imidacloprid were equal to 0, 6.1, 15 and 41/72 mg/kg bw per day for males and females combined. The study was conducted according to GLP.

Slight, temporary reductions in food intake were seen in both sexes at 1250 ppm and at the increased concentration of 2500 ppm. At 1250/2500 ppm, clinical chemical examination revealed an increased plasma cholesterol concentration in females after 13 and 26 weeks, increased hepatic cytochrome P450 activity and slightly increased liver weights in males and females after 52 weeks. The NOAEL was 500 ppm, equal to 15 mg/kg bw per day (Allen et al., 1989).

(c) Long-term studies of toxicity and carcinogenicity

Groups of 50 male and 50 female Charles-River B6C3F₁ mice received diets containing imidacloprid (purity, 95.3%) at a concentration of 0, 100, 330 or 1000 ppm for 24 months. In a supplemental study to determine the maximum tolerated dose, groups of 50 male and female mice were given diets containing imidacloprid (purity, 90.0%) at a concentration of 0 or 2000 ppm for 24 months. Ten additional mice of each sex were included per dose in both studies for interim sacrifice after 12 months of treatment. The mean intake of imidacloprid was equal to 20, 66, 210 and 410 mg/kg bw per day for males and 30, 100, 270 and 420 mg/kg bw per day for females. The study was conducted according to GLP.

Food intake was decreased by 24% in females at 2000 ppm, and water intake was decreased in males and females at this concentration. Mice at 1000 and 2000 ppm had significantly, dose-related reduced weight gains, particularly during the latter half of the study. At 2000 ppm, lower leukocyte counts were found in both sexes. Reduced blood cholesterol concentrations were observed at 2000 ppm after 52 weeks. An increased incidence of low-grade periacinar hepatic-cell hypertrophy was found in males at 2000 ppm. The brains of animals at the highest concentration showed more mineralization of the thalamus than in the control groups. There was no evidence of a carcinogenic effect. The NOAEL was 330 ppm, equal to 66 mg/kg bw per day (Watta-Gebert, 1991a,b).

Groups of 50 male and 50 female Wistar rats [Bor:WISW (SPF-Cpb)] received diets containing imidacloprid (purity, 94.3–95.3%; mixed batch) at a concentration of 0, 100, 300 or 900 ppm for 24 months. In a supplemental study to determine the maximum tolerated dose, groups of 50 male and female Wistar rats were given diets containing imidacloprid at a concentration of 0 or 1800 ppm for 24 months. Ten additional rats of each sex were included per dose in each study for interim sacrifice after 12 months of treatment. The mean intake of imidacloprid was equal to 5.7, 17, 51 and 100 mg/kg bw per day for males and 7.6, 25, 73 and 140 mg/kg bw per day for females. The study was conducted according to GLP.

Water intake was reduced by 13% in females at 1800 ppm. Reduced weight gain was noted in males and females at 900 ppm, the decreases reaching a maximum of 11–12% at 1800 ppm. The absolute weights of the liver and kidney in females and of the liver in males were reduced after 12 months at 900 ppm. Histological assessment of these organs afforded no evidence of treatment-related lesions. An increased incidence of mineralization in the colloid of the thyroid gland follicles, in comparison with the incidences of this lesion in controls in previous studies, was determined in males at concentrations > 300 ppm and in females at 900 ppm. At 1800 ppm, fewer colloid aggregations and parafollicular hyperplasia were observed. There was no evidence of a carcinogenic effect. The NOAEL was 100 ppm, equal to 5.7 mg/kg bw per day (Eiben, 1991; Eiben & Kaliner, 1991).

(d) Genotoxicity

Imidacloprid was investigated in an adequate range of assays for genotoxicity in vitro and in vivo (Table 2).

    See Also:
       Toxicological Abbreviations
       Imidacloprid (ICSC)