FLONICAMID. First draft prepared by K. Low 1 and C. Lambré 2. Ontario, Canada

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1 FLONICAMID First draft prepared by K. Low 1 and C. Lambré 2 1 Health Evaluation Directorate, Pest Management Regulatory Agency, Health Canada, Ottawa, Ontario, Canada 2 Dammartin-en-Goële, France Explanation Evaluation for acceptable intake Biochemical aspects Absorption, distribution and excretion Biotransformation Toxicological studies Acute toxicity (a) Lethal doses (b) Dermal irritation (c) Ocular irritation (d) Dermal sensitization Short-term studies of toxicity (a) Oral administration (b) Dermal application (c) Exposure by inhalation Long-term studies of toxicity and carcinogenicity Genotoxicity (a) In vitro studies (b) In vivo studies Reproductive and developmental toxicity (a) Multigeneration studies (b) Developmental toxicity Special studies (a) Neurotoxicity (b) Immunotoxicity (c) Studies on metabolites (d) Mode of action studies Observations in humans Comments Toxicological evaluation References Appendix 1: Mode of action Explanation Flonicamid is the International Organization for Standardization (ISO) approved common name for N-cyanomethyl-4-(trifluoromethyl)nicotinamide (International Union of Pure and Applied Chemistry), with Chemical Abstracts Service number It is a novel systemic pyridine carboxamide insecticide with selective activity against hemipterous pests, such as aphids and whiteflies, and thysanopterous pests. Flonicamid has not previously been evaluated by the Joint FAO/WHO Meeting on Pesticide Residues (JMPR) and was reviewed by the present Meeting at the request of the Codex Committee on Pesticide Residues. All critical studies contained statements of compliance with good laboratory practice (GLP).

2 Biochemical aspects Evaluation for acceptable intake The absorption, distribution, metabolism and excretion, as well as the toxicokinetics, of flonicamid have been investigated in Sprague-Dawley rats. Summaries of the relevant data are presented below. The metabolism of flonicamid was investigated using flonicamid labelled at the 3- nicotinamide position (Fig. 1). The test item was a mixture of 14 C-labelled flonicamid and unlabelled flonicamid. Radiolabelled flonicamid was administered via oral gavage in 0.75% aqueous methyl cellulose. The study design is summarized in Table 1. Fig. 1. Structure of flonicamid with radiolabel positions for the metabolism studies (* = 14 C position) Source: Neal, Savides & Dow (2002a) Table 1. Dosing groups for metabolism experiments with [ 14 C]flonicamid Test group Pilot study routes of elimination Pilot study routes of elimination Pilot study pharmacokinetics Pilot study pharmacokinetics Single oral low dose in the rat pharmacokinetics Single oral high dose in the rat pharmacokinetics Dose of labelled material (mg/kg bw) Number of rats of each sex Remarks Reference A single nominal dose of 0.9 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 168 h A single nominal dose of 21 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 168 h. 2 5 A single nominal dose of 2 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 72 h A single nominal dose of 50 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 72 h. 2 5 A single nominal dose of 2 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 72 h A single nominal dose of 400 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 72 h. Neal & Savides (2001a) Neal & Savides (2001a) Neal & Savides (2001a) Neal & Savides (2001a) Neal & Savides (2001b) Neal & Savides (2001b)

3 277 Test group Single oral low dose in the rat elimination and distribution Single oral low dose in the rat elimination and distribution Single oral high dose in the rat elimination and distribution Single oral high dose in the rat elimination and distribution Repeated dose in the rat Repeated dose in the rat Single oral low and high dose in the rat elimination in the bile Biotransformation in the rat bw: body weight Dose of labelled material (mg/kg bw) Number of rats of each sex Remarks Reference 2 3 A single nominal dose of 2 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 0.5, 6 or 24 h. 2 5 A single nominal dose of 2 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 168 h A single nominal dose of 400 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Males killed after 3, 14.5 and 24 h, and females killed after 1, 8 and 24 h A single nominal dose of 400 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 168 h mg/kg bw per day unlabelled flonicamid was administered by oral gavage for 14 days, and 2 mg/kg bw [pyridyl-3- C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage on day 15. Killed after 0.5, 6 or 24 h following labelled dose mg/kg bw per day unlabelled flonicamid was administered by oral gavage for 14 days, and 2 mg/kg bw [pyridyl-3- C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage on day 15. Killed after 168 h following labelled dose. 2 4 A single nominal dose of 2 mg/kg bw [pyridyl-3-14 C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 48 h A single nominal dose of 400 mg/kg bw [pyridyl C]flonicamid (specific activity 9.08 MBq/mg) was administered by oral gavage. Killed after 48 h. Samples of urine, faeces, liver and bile taken from Dow (2002) and Neal, Savides & Dow (2002a,b) Neal, Savides & Dow (2002a) Neal, Savides & Dow (2002a) Neal, Savides & Dow (2002a) Neal, Savides & Dow (2002a) Neal, Savides & Dow (2002b) Neal, Savides & Dow (2002b) Dow (2002) Gupta, Shah & McClanahan (2002) 1.1 Absorption, distribution and excretion The data generated indicated that flonicamid is rapidly absorbed and excreted. High-dose males exhibited a decreased rate of elimination relative to other dose groups, with serum

4 278 Table 2. Recovery of radioactivity in tissues and excreta of rats after administration of 14 C-labelled flonicamid Single low dose (2 mg/kg bw) % of radioactive dose recovered Single high dose (400 mg/kg bw) Males Females Males Females Tissues a Urine 6 h 12 h 24 h 48 h 72 h 96 h 120 h 144 h 168 h Total urine b b b c c c d d d b b b Faeces a Cage wash a Total excreted a Total recovery a bw: body weight a At 7 days post-dosing. b Ninety-four per cent of urinary excretion in 24 h. c Ninety-six per cent of urinary excretion in 24 h. d Eighty-seven per cent of urinary excretion in 24 h. Source: Neal, Savides & Dow (2002a) concentrations reaching a plateau between hours post-dosing. In all dose groups, radiolabel concentrations in the plasma decreased with time in a manner consistent with first-order kinetics. The predominant route of excretion was in the urine, accounting for 72 78% of the administered radiolabelled dose (Table 2). Faecal and biliary excretion was minor (4 7%), and no residues were detected in the expired air following single doses. For all routes, excretion was rapid, with 95% of the radioactivity excreted within the first hours. Very little flonicamid was retained in the tissues, and repeated dosing of rats did not indicate any potential for accumulation. Maximum plasma concentrations (C max ) (Table 3) and area under the plasma concentration time curve (AUC) values were directly proportional to dose level in both sexes. Time to maximum plasma concentration (T max ) was similar in low-dose males and females; however, T max was increased in high-dose males relative to females, probably in relation to the prolonged plateau observed in plasma concentrations in males at this dose. Tissue distribution of radiolabel was similar in all groups following single dosing. Radioactivity was rapidly and widely distributed throughout the tissues at levels similar to plasma concentrations; however, slightly higher concentrations of radiolabel were noted in the liver, kidneys, adrenals, thyroid and ovaries (Table 4). Males also had increased levels of radiolabel in the lungs following repeated dosing. 1.2 Biotransformation The urinary and faecal samples from Dow (2002) and Neal, Savides & Dow (2002a,b) were subjected to high-performance liquid chromatographic analysis to determine the metabolic fate of

5 279 Table 3. Plasma kinetic parameters in rats after administration of [ 14 C]flonicamid Group T max (h) C max (ng eq/g) AUC 0 (h ng eq/g) t ½ (h) 2 mg/kg bw oral M 0.4 ± ± ± ± mg/kg bw oral F 0.4 ± ± ± ± mg/kg bw oral M 0.9 ± ± ± ± mg/kg bw oral F 0.5 ± ± ± ± 2.36 AUC: area under plasma concentration time curve; bw: body weight; C max : maximum plasma 14 C concentration; eq: equivalents; F: female; M: male; t ½ : half-life for plasma elimination; T max : time to maximum 14 C plasma concentration (using times estimated by WinNonlin rather than the discrete collection times) Source: Neal & Savides (2001b) Table 4. Distribution of radioactivity in rat tissues/organs at 168 hours after administration of [ 14 C]flonicamid Single low dose (2 mg/kg bw) % of radioactive dose administered Single high dose (400 mg/kg bw) Tissue/organ Males Females Males Females Blood Bone marrow ND ND ND ND Brain ND Heart Lungs Liver Adrenals ND ND < < Kidneys Gastrointestinal tract + contents Bone ND ND Muscle Fat ND ND Testes/ovaries ND ND Uterus N/A ND N/A < Spleen ND ND < < Pancreas ND ND ND < Thyroid ND ND < < Thymus ND ND < ND Carcass Total bw: body weight; N/A: not applicable; ND: below the limit of detection Source: Neal, Savides & Dow (2002a) flonicamid (Table 5; Fig. 2). In urine, the major residue was unchanged parent, followed by the metabolite 4-trifluoromethylnicotinamide (TFNA-AM), and was generally similar between the sexes after single and repeated dosing. TFNA-AM was also the predominant metabolite in the faeces and bile. In the faeces, 4-trifluoronicotinic acid (TFNA) was found only in low-dose animals, whereas

6 280 Table 5. Identified metabolites Metabolite designation Structure Urine Faeces Bile Flonicamid TFNA-AM N-oxide conjugate + + TFNA conjugate NG + + TFNA-AM N-oxide NG + + OH-TFNA-AM + + TFNG-AM + + TFNA-AM Flonicamid N-oxide + + TFNG TFNA + + NG: structure not given Source: Gupta, Shah & McClanahan (2002) TFNA-AM N-oxide conjugate was found only in high-dose animals. Unchanged parent was the predominant residue in the bile, and N-(4-trifluoromethylnicotinoyl)glycinamide (TFNG-AM) was

7 281 Fig. 2. Proposed metabolic pathway of flonicamid in the rat Source: Gupta, Shah & McClanahan (2002) unique to the bile of high-dose animals. In the liver, the predominant residue was unchanged parent, with N-(4-trifluoronicotinoyl)glycine (TFNG) and TFNA-AM noted as minor metabolites. 2. Toxicological studies 2.1 Acute toxicity (a) Lethal doses Flonicamid is of moderate acute oral toxicity in rats. It is of low acute dermal and inhalation toxicity (Table 6). In the acute oral toxicity study, animals were given a dose of flonicamid (purity 98.7%) at 625, 1250, 2500 or 5000 mg/kg body weight (bw), with mortalities noted at 1250 mg/kg bw and above. Clinical signs of toxicity consisted of few faeces, loss of mobility, decreased activity, hunched posture, hypothermia, laboured breathing, tremors, convulsions, ataxia and prostration. Surviving animals were free of clinical signs by day 3. All surviving animals gained weight in the first week following treatment; however, one male and one female in the 625 mg/kg bw group and one female in the 2500 mg/kg bw group lost weight from day 7 to day 14. There were no gross changes at necropsy in surviving animals; however, some decedent animals exhibited dark red-black spots on the serosal surface of the stomach, and one female exhibited anogenital staining (Ridder, Yoshida & Watson, 2001a).

8 282 Table 6. Acute toxicity of flonicamid Species Strain Sex Route Purity (%) Result Reference Rat Crl:CD (SD)BR(IGS) Male and female Oral 98.7 LD 50 = 884 mg/kg bw (M) Ridder, Yoshida & Watson (2001a) LD 50 = mg/kg bw (F) Rat Crl:CD (SD)BR(IGS) Male and female Dermal 98.7 LD 50 > mg/kg bw Ridder, Yoshida & Watson (2000a) Rat Sprague-Dawley Male and female Inhalation 98.7 LC 50 > 4.90 mg/l bw: body weight; F: females; LC 50 : median lethal concentration; LD 50 : median lethal dose; M: males Paul (2000) In the acute dermal toxicity study, animals were given a limit dose of flonicamid (purity 98.7%) at 5000 mg/kg bw. There were no mortalities. Clinical signs consisting of coloured material around the nose and eyes of most animals and anogenital staining were noted from day 1 to day 4. All animals gained weight throughout the study, and there were no gross changes at necropsy (Ridder, Yoshida & Watson, 2000a). In the acute inhalation toxicity study, male and female Sprague-Dawley-derived rats were exposed to 4.90 mg/l flonicamid (purity 98.7%) via nose-only exposure for 4 hours. Clinical signs of toxicity consisted of exaggerated breathing up until day 2 and brown staining around the snout and/or jaws during exposure and up to day 2. All animals gained weight throughout the study. At gross necropsy, minimal congestion was noted in all lung lobes, dark foci were noted in the lungs of one male and severe congestion was noted in the right posterior lobe of another male (Paul, 2000). (b) Dermal irritation In a dermal irritation study, 0.5 g of flonicamid (purity 98.7%) was moistened with approximately 1 ml of distilled water and applied to the right dorsal skin site on each of six male New Zealand White rabbits. There were no dermal or abnormal clinical findings observed in the test animals. All scores at the 1-, 24-, 48- and 72-hour time points for erythema and oedema were zero, for both the control animals and the test animals. Flonicamid is considered non-irritating to the skin of the rabbit (Ridder, Yoshida & Watson, 2000b). (c) Ocular irritation In an eye irritation study, 0.1 ml of flonicamid (purity 98.7%) was instilled into one eye of each of six young male albino New Zealand White rabbits. There were no effects observed in the cornea or iris at any time. Redness (grade 2), chemosis (grades 1 2) and discharge (grades 1 3) were observed at the 1-hour time point. There was no evidence of conjunctivitis at 72 hours postinstillation. Flonicamid is considered slightly irritating to the eye of the rabbit (Ridder, Yoshida & Watson, 2000c). (d) Dermal sensitization In a dermal sensitization study using a maximization protocol, to 7-week-old male Hartley albino guinea-pigs were each injected on day 1 with three pairs of injections, consisting of 1) 0.1 ml 1:1 volume per volume (v/v) Freund s Complete Adjuvant (FCA)/sterile water; 2) 0.1 ml 10% weight per volume (w/v) flonicamid (purity 98.7%) in mineral oil; and 3) 0.1 ml 10% w/v flonicamid in 1:1 FCA/distilled water. One week later, a topical induction dose of 0.4 ml of flonicamid (10% w/v) in mineral oil was applied to a filter paper, which was placed on the application site for 48 hours. Challenge was 14 days after the topical application, with 0.4 ml 10% w/v applied to

9 283 the test site with 24-hour exposure. An additional group of 20 guinea-pigs was treated in the same manner, except that they were exposed to the test material during the challenge phase only. The concentrations of flonicamid used were based on preliminary irritation studies. At challenge, 2/20 of the previously induced guinea-pigs and 2/20 of the control animals scored 1 (mild erythema) at 48 hours. All other scores were zero. It is therefore concluded that there was no evidence of a dermal sensitization response (Ridder & Watson, 2000). 2.2 Short-term studies of toxicity (a) Mice Oral administration In an oral 13-week toxicity study, groups of 10 Crl:CD-1 (ICR) BR mice of each sex received flonicamid (purity 98.7%) in the diet at a dose level of 0, 100, 1000 or 7000 parts per million (ppm) (equal to 0, 15, 154 and 1069 mg/kg bw per day for males and 0, 20, 192 and 1248 mg/kg bw per day for females, respectively). Animals were monitored twice daily for mortality and moribundity and daily for clinical signs of toxicity, and a detailed clinical examination was performed weekly. Body weight and feed consumption were measured weekly. At study termination, select haematology and clinical chemistry parameters were evaluated; in addition, all animals were subject to gross necropsy, and the liver with gallbladder, kidneys and spleen were weighed. The bone marrow (sternum), liver and spleen were examined histopathologically in all groups, along with all macroscopic abnormalities and the kidneys for control and high-dose animals. At 7000 ppm, body weight and body weight gain were decreased in the first 30 days of treatment in males and females. Males lost body weight in this period, and females gained 23.7% of the weight gained by control females. Feed consumption was decreased in high-dose females from weeks 3 to 7. Erythrocyte, haemoglobin and haematocrit values were decreased compared with control values in males and females at 7000 ppm. Reticulocyte counts were increased in males and females at the same dose. Mean corpuscular volume and mean corpuscular haemoglobin were increased in males and females at 7000 ppm. In clinical chemistry examination, total bilirubin level was increased in males at 7000 ppm, and glucose level was increased in non-fasted males and females at 7000 ppm (statistically significant in females only). Absolute weight of liver with gallbladder was increased in males at 7000 ppm, and relative weight of liver with gallbladder was increased in males and females at 7000 ppm. Minimal to moderate centrilobular hepatocellular hypertrophy was observed in males and females at 7000 ppm, and minimal centrilobular hepatocellular hypertrophy was observed in males at 1000 ppm. Absolute and relative spleen weights were increased in both sexes at 7000 ppm. Gross and histopathological changes consisted of minimally to moderately severe extramedullary haematopoiesis in the spleen at doses of 1000 ppm and above and minimal to moderate increased pigment deposition in the spleen in all animals at 7000 ppm. In the bone marrow, there was minimal to mild hypocellularity and increased pigment deposition at 7000 ppm in males and females. As there was only histopathological examination of the bone marrow, spleen, liver and kidneys and, specifically, no examination of the lung tissue, no end-points were selected for this study (Ridder, Yoshida & Watson, 2001b). Rats In an oral 28-day toxicity study, groups of six Wistar (Jcl:Wistar) rats of each sex received flonicamid (purity 98.7%) in the diet at a dose level of 0, 50, 100, 500, 1000 or 5000 ppm in males (equal to 0, 3.61, 7.47, 36.5, 73.8 and 353 mg/kg bw per day, respectively) and 0, 100, 500, 1000, 5000 or ppm in females (equal to 0, 8.36, 41.2, 81.9, 373 and 642 mg/kg bw per day, respectively). Additionally, there was a satellite group of two males per dose given 0 or 5000 ppm (equal to 0 and mg/kg bw per day, respectively) for 28 days that were subjected to

10 284 immunostaining of the kidney for α 2u -globulin. Animals were observed daily for morbidity, moribundity and overt clinical signs. Body weight and feed consumption measurements and a detailed examination for clinical signs were performed once per week. An ophthalmoscopic examination was performed on high-dose males and females prior to treatment and during week 4. Clinical chemistry, haematological and urine analysis parameters were measured at study termination. At necropsy, the weights of the liver, kidney and spleen were recorded, assessed by gross examination and examined histopathologically. There were no effects on mortality, clinical signs of toxicity or ophthalmoscopic examination. Body weights and feed consumption were decreased compared with controls in 5000 ppm males and ppm females. Feed efficiency was also decreased in ppm females. Clinical chemistry changes occurred at 5000 ppm in males and females, with increased cholesterol and decreased calcium levels in males and decreased alanine aminotransferase activity and increased protein and albumin levels in females. At ppm in females, gamma-glutamyltranspeptidase (GGT) activity and globulin and cholesterol levels were increased, and albumin/globulin ratios and triglyceride levels were decreased. Changes to haematological parameters consisted of decreased haematocrit and red blood cells at 5000 ppm in males and increased platelet counts in females at ppm. Kidney changes were limited to males and were noted at doses at and above 100 ppm, with hyaline droplet depositions in proximal tubular cells of the kidney. At 5000 ppm, there were increased incidences of pale kidneys and increased kidney weights in main group males and hyaline droplets and granular casts in proximal tubular cells of kidneys that were positive for α 2u -globulin antibodies in the satellite group. As a result, the hyaline droplets of the kidneys were considered to be specific to male rats and not applicable to the human risk assessment. Gross and histopathological changes in the liver were noted at 5000 ppm in males and females, with hepatocellular hypertrophy and increased incidence of liver enlargement. In males, there were increased incidences of dark liver. In females at ppm, liver weights were increased, and there was an increased incidence of centrilobular hepatocellular hypertrophy. Relative spleen weights were increased in females at ppm (Kuwahara, 2002a). In a 90-day study, groups of 12 Wistar (Jcl:Wistar) rats of each sex received flonicamid (purity 98.7%) in the diet at a dose level of 0, 50, 200, 1000 or 2000 ppm in males (equal to 0, 3.08, 12.1, 60.0 and 119 mg/kg bw per day, respectively) and 0, 200, 1000 or 5000 ppm in females (equal to 0, 14.5, 72.3 and 340 mg/kg bw per day, respectively). Animals were observed daily for morbidity, moribundity and overt clinical signs. Body weight and feed consumption measurements and a detailed examination for clinical signs were performed once per week. An ophthalmoscopic examination was performed on high-dose males and females prior to treatment and during week 13. Clinical chemistry, haematological and urine analysis parameters were measured at study termination. At necropsy, the weights of selected organs were recorded, assessed by gross examination and examined histopathologically. There were no effects on mortality, clinical signs, body weight, feed efficiency or urine analysis parameters. Feed consumption was decreased in high-dose females; however, in the absence of effects on body weight, the change was not considered to be adverse. High-dose males and females showed no changes under ophthalmoscopic examination. There were no effects on the functional observational battery and no consistent, treatment-related effects on motor activity. Changes to the kidneys were noted at doses at and above 200 ppm in males, with increased kidney weights, hyaline droplets and granular casts in the tubules and tubular basophilic changes. Effects at 200 ppm were considered to be related to the rat-specific α 2u -globulin-positive hyaline droplets seen in the 28-day study. However, as there were kidney changes at higher doses in female rats and at lower doses in female dogs (see below), changes that were not specifically linked to positive α 2u -globulin staining increased kidney weights, granular casts and increased basophilic changes in the renal tubules were considered relevant to the human risk assessment. At 2000 ppm, males exhibited pale kidneys. At 5000 ppm, kidney weights were increased in females, along with increased cytoplasmic vacuolation of the proximal tubules. In the liver, centrilobular hepatocellular hypertrophy was noted at the high dose in males (2000 ppm) and females (5000 ppm). In the 5000 ppm females, liver weights were also increased. Haematocrit was decreased in 5000 ppm females.

11 285 The no-observed-adverse-effect level (NOAEL) was 200 ppm (equal to mg/kg bw per day) in males and 1000 ppm (equal to 72.3 mg/kg bw per day) in females. The lowest-observedadverse-effect level (LOAEL) was 1000 ppm (equal to 60.0 mg/kg bw per day) in males, based on increased kidney weights, granular casts in the tubules and increased basophilic changes in the renal tubules, and 5000 ppm (equal to mg/kg bw per day) in females, based on decreased haematocrit, increased liver and kidney weights, and increased cytoplasmic vacuolation of the proximal tubules of the kidneys (Kuwahara, 2002b). Dogs In a 90-day oral toxicity study in dogs, groups of four Beagle dogs of each sex received flonicamid (purity 98.7%) in capsules at a dose level of 0, 3, 8 or 20 mg/kg bw per day for males and females and 50 mg/kg bw per day for females only. Animals were observed for mortality twice daily and for clinical signs of toxicity, moribundity and feed consumption daily, and detailed physical examinations and body weight measurements were performed weekly. Clinical chemistry and haematological parameters were measured prior to the initiation of dosing, at weeks 6 7 and prior to termination. Select organs were weighed at necropsy, assessed by gross observations and examined histopathologically. One female in the 50 mg/kg bw per day group was sacrificed moribund by week 3. Clinical signs of toxicity consisted of vomiting, ataxia, decreased activity, laboured breathing, prostration and diarrhoea in both males and females at 20 mg/kg bw per day and in the female-only 50 mg/kg bw per day dose group. In the 50 mg/kg bw per day group, severity of symptoms increased and included additional observations of excessive salivation, tremors, convulsions, few/no/soft faeces, partially closed eyelids and circling behaviour. Body weights and total body weight gain were decreased in males and females at 20 mg/kg bw per day and above when compared with controls. Decreased feed consumption was noted in the 20 and 50 mg/kg bw per day female dose groups during various weeks throughout the study period. In the female high-dose group, this decrease was directly related to feed rejection. Blood analysis at week 7 and study termination revealed a decrease in red blood cells and an increased reticulocyte count in females at 50 mg/kg bw per day. Spleen weights were decreased and lung weights were increased in high-dose females. Thymus weights were decreased in high-dose males; however, the weights were still within the historical control range for Beagle dogs between 8 and 10 months of age (Nomura, 2015a). Minimal to mild tubular vacuolation of the inner cortex of the kidneys was noted in 2/4 females at 50 mg/kg bw per day. The NOAEL was 8 mg/kg bw per day in males and females, based on vomiting, ataxia, decreased activity, laboured breathing, prostration, and decreased body weight and body weight gain in both sexes as well as decreased feed consumption in females observed at 20 mg/kg bw per day (Ridder & Watson, 2001). In a 1-year oral toxicity study in dogs, groups of six Beagle dogs of each sex received flonicamid (purity 98.7%) in capsules at a dose level of 0, 3, 8 or 20 mg/kg bw per day. Animals were observed for mortality twice daily and clinical signs of toxicity, moribundity and feed consumption daily, and detailed physical examinations and body weight measurements were performed weekly. Clinical chemistry and haematological parameters were measured prior to the initiation of dosing, every 12 weeks and prior to termination. Select organs were weighed at necropsy, assessed by gross observations and examined histopathologically. There was no mortality, and no gross or histopathological changes were noted. Treatmentrelated changes were limited to the top dose. Vomiting was noted in males and females in all groups; however, an increase in incidence indicating adversity occurred at 20 mg/kg bw per day. Increased percentages of reticulocytes were noted at the 12-month time point in males and females at the high dose. Body weight gains were decreased in females at 20 mg/kg bw per day.

12 286 The NOAEL was 8 mg/kg bw per day, based on vomiting and increased reticulocytes in males and females and decreased body weight gain in females at 20 mg/kg bw per day (Ridder & Watson, 2003a). (b) Dermal application No dermal toxicity studies were submitted. (c) Exposure by inhalation No repeated-dose inhalation toxicity studies were submitted. 2.3 Long-term studies of toxicity and carcinogenicity Mice In a carcinogenicity study in mice, flonicamid (purity 98.7%) was administered in the diet to 60 CD-1 mice of each sex per dose at 0, 250, 750 or 2250 ppm (equal to 0, 29, 88 and 261 mg/kg bw per day for males and 0, 38, 112 and 334 mg/kg bw per day for females, respectively) for up to 78 weeks. Additionally, satellite groups of 10 mice of each sex from the control and 2250 ppm dose groups were killed at week 26 (interim sacrifice 1) and week 52 (interim sacrifice 2). The animals were observed twice daily on weekdays and once daily on weekends and holidays for viability, clinical signs were recorded daily and a detailed physical examination was performed weekly. Haematology and clinical chemistry samples were collected from the satellite groups at interim kill and from surviving animals at 12 months and terminal kill; however, haematological parameters were evaluated only in the control and 2250 ppm dose groups, and evaluation of the clinical chemistry samples was not performed. Liver, kidney and spleen weights were taken for all animals killed at 26, 52 and 78 weeks, although not from animals found dead or killed in extremis. Additionally, in animals killed at 78 weeks, organ weights were taken from 10 animals of each sex per dose for the adrenals, brain, heart, testes, epididymides, ovaries and uterus. All animals were necropsied, and tissues from all control and high-dose animals and from all animals found dead or moribund were examined microscopically. The liver, spleen and bone marrow from control and high-dose animals were examined in the 26- and 52-week satellite groups. Gross lesions, liver, spleen, bone marrow and lungs were examined microscopically, regardless of dose group, in the terminal kill groups. There were no treatment-related clinical signs of toxicity or effects on body weight, body weight gain or feed consumption. In females at all doses, there was a decrease in cellularity of the femoral bone marrow, whereas in males, there was an increase in extramedullary haematopoiesis in the spleen, an increase in pigment deposition in the femoral and sternal bone marrow, increased centrilobular hepatocellular hypertrophy and an increase in masses/nodules in the lung. In both sexes, there was an increase in incidences of hyperplasia/hypertrophy of the epithelial cells of the terminal bronchioles at all doses (Table 7). Hypertrophy of lung epithelial cells is considered a marker of lung injury and is seen as round to oval or cuboidal often/mostly hypertrophic cells with abundant eosinophilic cytoplasm prominently outlining alveolar walls (Renne et al., 2009). Epithelial cell hypertrophy was correlated with an increase in alveolar/bronchiolar adenomas in all treated dose groups, and the statistically significant increase in lung epithelial cell hyperplasia was correlated with alveolar/bronchiolar carcinomas at 750 and 2250 ppm in both males and females. At 750 ppm and above, there was a decrease in cellularity of the bone marrow in the femur of males and the sternum of both sexes. Females exhibited an increase in the incidence of extramedullary haematopoiesis of the spleen and an increase in pigment deposition in the sternal bone marrow. At 2250 ppm, there were increased liver weights of both sexes and increased centrilobular hepatocellular hypertrophy in females. Additionally, there was increased pigment deposition in the spleen of both sexes and increased pigment deposition in the femur of females. At the interim kills, similar effects were noted in the bone marrow, spleen and liver. In all treated groups, there was an increase in alveolar/bronchiolar adenomas and combined alveolar/bronchiolar adenomas and/or carcinomas. At

13 287 Table 7. Non-neoplastic and neoplastic lesions in mice after 78 weeks of treatment Histopathological lesion Bone marrow (sternum) 0 ppm Incidence of lesion Males (n = 60) Females (n = 60) 250 ppm 750 ppm ppm 0 ppm 250 ppm 750 ppm ppm Hypocellularity a 0 1 5* 22* ** Deposition, brown 0 7** 15** 31** ** pigment a Bone marrow (femur) Hypocellularity a 0 1 7* 24** ** Deposition, brown ** 32** ** pigment a Lungs Epithelial cells, ** 13** hyperplasia a Epithelial cells, 2 22** 46** 46** 4 20** 41** 42** hypertrophy a Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Total mice with primary lung tumours ppm: parts per million; *: P < 0.05; **: P < 0.01 (Fisher s exact test) a Values and statistical analysis clarified in Nomura (2015b). Source: Ridder & Watson (2003b) 9 26** 24** 32** 9 21* 30** 25** 2 4 9* 10* * ppm and above in males and at 2250 ppm in females, there was an increase in alveolar/bronchiolar carcinomas. A NOAEL was not identified. The LOAEL was 250 ppm (equal to 29 and 38 mg/kg bw per day for males and females, respectively), the lowest dose tested, based on hyperplasia/hypertrophy of the epithelial cells of the terminal bronchioles, increased incidence of tissue masses/nodules in the lungs and increased alveolar/bronchiolar adenomas in both sexes, as well as centrilobular hepatocellular hypertrophy, extramedullary haematopoiesis of the spleen and pigment deposition in the femoral and sternal bone marrow in males and decreased cellularity in the femoral bone marrow in females (Ridder & Watson, 2003b). In a second carcinogenicity study in mice, flonicamid (purity 98.7%) was administered in the diet to 50 CD-1 mice of each sex per dose at 0, 10, 25, 80 or 250 ppm (equal to 0, 1.2, 3.1, 10.0 and 30.3 mg/kg bw per day for males and 0, 1.4, 3.7, 11.8 and 36.3 mg/kg bw per day for females, respectively) for up to 78 weeks. The animals were observed twice daily on weekdays and once daily on weekends and holidays for viability, clinical signs were recorded daily and a detailed physical examination including palpations was performed weekly starting at week 41. Haematology and clinical chemistry samples were collected from surviving animals at terminal kill, but were not evaluated. Liver, kidney, spleen and brain weights were measured for all animals killed at 78 weeks, although not for animals found dead or killed in extremis. All animals were necropsied, and tissues from all control and high-dose animals and from all animals found dead or moribund were examined

14 288 Table 8. Non-neoplastic and neoplastic lesions in mice after 78 weeks of treatment Histopathological lesion Bone marrow (sternum) Deposition, brown pigment Bone marrow (femur) Deposition, brown pigment Lungs Hyperplasia/ hypertrophy Alveolar/bronchiolar adenoma Alveolar/bronchiolar carcinoma Total mice with primary lung tumours 0 ppm 10 ppm Incidence of lesion Males (n = 50) Females (n = 50) 25 ppm 80 ppm 250 ppm 0 ppm 10 ppm 25 ppm 80 ppm ppm ** * ** ** ppm: parts per million; *: P < 0.05; **: P < 0.01 (Fisher s exact test); : P < 0.05 (trend test) Source: Nagaoka (2004) microscopically. Gross lesions, liver, spleen, bone marrow and lungs were examined microscopically, regardless of dose group. There were no treatment-related clinical signs of toxicity or effects on body weight, body weight gain, feed consumption or organ weights. At 250 ppm, there was an increase in lung masses and an increase in hyperplasia/hypertrophy in the terminal bronchiole epithelial cells in both sexes. There was a slight, non-statistically significant, increase in brown pigment deposition in the bone marrow in 250 ppm females (Table 8). Although this finding is consistent with those seen in the previous study, there is little evidence of treatment-related change in this study. There were no treatment-related changes in any other organ system. The incidences of alveolar/bronchiolar adenomas and of combined alveolar/bronchiolar adenomas and/or carcinomas were increased in high-dose males, and there was a trend of increased incidence of combined alveolar/bronchiolar adenomas and/or carcinomas in mid-high- and high-dose females. Combined adenomas and/or carcinomas were above the historical control range in males and just under the upper range of historical controls in the top two female dose groups. The non-neoplastic NOAEL was 80 ppm (equal to 10.0 and 11.8 mg/kg bw per day for males and females, respectively). The non-neoplastic LOAEL was 250 ppm (equal to 30.3 and 36.3 mg/kg bw per day for males and females, respectively), based on an increase in lung adenomas in males, a slight lung hyperplasia/hypertrophy in the terminal bronchiole epithelial cells in males and females and an increased incidence of hyperplasia of alveolar epithelial cells in females (Nagaoka, 2004). Rats In a combined chronic toxicity and carcinogenicity study in rats, flonicamid (purity 98.7%) was administered in the diet to 52 Wistar rats of each sex per dose at 0, 50, 100, 200 or 1000 ppm (equal to 0, 1.84, 3.68, 7.32 and 36.5 mg/kg bw per day, respectively) for males and 0, 200, 1000 or

15 ppm (equal to 0, 8.92, 44.1 and 219 mg/kg bw per day, respectively) for females for up to 24 months. Additionally, satellite groups of 10 or 14 rats of each sex per dose were similarly treated at the same dose levels and sacrificed at 6 and 12 months, respectively. The animals were observed twice daily on weekdays and once daily on weekends and holidays for viability, clinical signs were recorded daily and a detailed physical examination was performed weekly. Functional observations were performed on 10 animals of each sex per dose group from one of the satellite groups at week 49. Body weights and feed consumption were recorded weekly for 13 weeks and monthly from week 16 and thereafter. Feed consumption was measured for each cage of four rats on a weekly basis for 13 weeks, in week 16 and once every 4 weeks thereafter. An ophthalmological examination was conducted on all main study animals in all groups prior to study initiation and at week 104. Urine analysis, haematological examination and clinical chemistry analysis were performed during study weeks 13/14, 26, 52, 77/78 and 103/104. Animals were selected for analysis (10 of each sex per dose) from one of the satellite groups at weeks 13 and 26. Thereafter, 10 animals of each sex per dose were selected from the main group, with animals that were showing clinical signs unrelated to treatment that could interfere with testing excluded. Organ weight analysis was also performed on 10 animals of each sex from each dose group after 6, 12 and 24 months. All animals except those in the satellite group that were not selected for scheduled kill after 52 weeks of treatment were subjected to necropsy, and their tissues were examined histopathologically. There was no treatment-related mortality in this study, and there were no effects on urine analysis. Clinical signs of toxicity consisted of a decrease in rearing and an increase in keratitis in males at 1000 ppm. At 5000 ppm, there was an increase in rhinitis and opacity, cataracts and retinal atrophy in the eyes in females. Effects on body weight were limited to the second half of the study, with decreases in body weight and body weight gain in males at doses of 1000 ppm and in females at 5000 ppm. Decreases in feed consumption were limited to females in the 5000 ppm dose group. Haematological changes consisted of decreased haematocrit, red blood cells and haemoglobin in females at 5000 ppm. Clinical chemistry changes consisted of decreased triglyceride levels in females at 1000 ppm and above and increased cholesterol level and GGT activity in females at 5000 ppm. Other liver changes occurred in females at 5000 ppm, including increased liver weights, increased dark coloration of the liver and livers with accentuated lobular patterns, increased centrilobular hepatocellular hypertrophy and increased foci of cellular alteration (eosinophilic type). In the kidneys, there was an increase in slight to severe hyaline droplet deposition in renal proximal tubular cells and an increase in kidney pelvic dilatation in males at 1000 ppm. In females at 5000 ppm, there was an increase in kidney weights, an increase in cytoplasmic vacuolation of the renal proximal tubular cells and an increase in chronic nephropathy. There was an increased incidence of striated muscle atrophy in females at 1000 ppm. At 5000 ppm, there was also an increase in pituitary anterior cell hyperplasia in females. The NOAEL was 200 ppm (equal to 7.32 and 8.92 mg/kg bw per day for males and females, respectively). The LOAEL was 1000 ppm (equal to 36.5 and 44.1 mg/kg bw per day for males and females, respectively), based on decreased body weight and body weight gain, decreased rearing, increased incidences of keratitis and pelvic dilatation in the kidneys in males and decreased triglyceride levels and increased striated muscle atrophy in females (Kuwahara, 2002c). 2.4 Genotoxicity (a) In vitro studies A range of GLP-compliant in vitro studies of the genotoxicity of flonicamid was conducted to assess its potential for inducing chromosomal aberration, gene mutation and reverse gene mutation (summarized in Table 9). There was no evidence for genotoxicity or mutagenicity in the presence or absence of metabolic activation.

16 290 Table 9. Genotoxicity studies with flonicamid End-point Test object Concentration Purity (%) Results Reference In vitro Reverse mutation Salmonella typhimurium and Escherichia coli 0, 61.7, 185, 313, 556, 625, 1 250, 1 667, and µg/plate (±S9) 98.7 Negative Matsumoto (2002a) Chromosomal aberrations Chinese hamster lung cells 573, and µg/ml (±S9) Exposure: 6, 24 and 48 h 98.7 Negative Matsumoto (2002b) Mammalian cell gene mutation L5178Y mouse lymphoma cells, TK locus 28.3, 84.8, 143, 254, 286, 573, 763, and µg/ml 98.7 Negative Matsumoto (2002c) In vivo Mouse micronucleus CD-1 mouse bone marrow, males and females 125 (F), 250, 500 and (M) mg/kg bw Harvest time: 24, 48 and 72 h 98.7 Negative Matsumoto (2002d) Unscheduled DNA synthesis SD rats primary hepatocytes, males 0, 600 and mg/kg bw Scored: 2 and 14 h 98.7 Negative Mehmood (2003) bw: body weight; F: females; M: males; S9: 9000 g supernatant fraction from liver homogenate from Aroclor-treated rats; SD: Sprague-Dawley; TK: thymidine kinase (b) In vivo studies GLP-compliant unscheduled DNA synthesis and micronucleus assays were conducted to assess the potential of flonicamid to damage DNA and impede repair in vivo (summarized in Table 9). There was no evidence of genotoxicity. Overall, flonicamid did not demonstrate any genotoxic potential. 2.5 Reproductive and developmental toxicity (a) Multigeneration studies In a range-finding multigenerational reproductive toxicity study, flonicamid (purity 98.7%) was administered continuously in the diet to Jcl:Wistar rats (eight of each sex per dose) at a dose level of 0, 50, 200, 1000 or 2000 ppm (equal to 0, 3.3, 13.1, 65.9 and 131 mg/kg bw per day for males and 0, 3.8, 14.9, 76.5 and 155 mg/kg bw per day for females, respectively). The parental animals were given test article diet formulations for 3 weeks prior to mating to produce the F 1 litters. After weaning, the study was terminated. Animals were examined daily for clinical signs and mortality, weekly for body weight and daily for feed consumption parameters. Reproductive performance was examined, with estrous cycles, mating, fertility and gestation indices, duration of gestation and number of implantation sites reported. Gross pathological examination was performed on all parental animals. Further, liver and kidney weights were measured, and a histopathological examination was performed for all parental animals. Offspring were examined for clinical signs and mortality during the lactation period, body weights, number of pups, sex ratio and viability index. A gross pathological examination was performed on culled, found dead and terminal kill pups. In parental animals, there were no treatment-related effects on survival, body weight, body weight gain or feed consumption. There were no effects on reproductive performance. Pale kidneys were noted in parental males at doses of 1000 ppm and above, and kidney weights were increased in males at 2000 ppm. In males, histopathological changes were noted at 200 ppm and above. At 200

17 291 ppm and above, there was an increase in hyaline droplet deposition in the proximal tubular cells. At 1000 ppm and above, there was an increased incidence of tubular basophilic change. At 2000 ppm, there was an increase in granular casts in the dilated tubules. In females, there were no histopathological changes. In the offspring, there were no clinical signs of toxicity and no effects on mortality. There were no effects on numbers of pups delivered, sex ratio, viability index or pup body weights. There were no treatment-related gross changes noted in weanlings or pups culled at day 4 (Takahashi, 2002a). In a multigenerational reproductive toxicity study, flonicamid (purity 98.7%) was administered continuously in the diet to Jcl:Wistar rats (24 of each sex per dose) at a dose level of 0, 50, 300 or 1800 ppm (equal to 0, 3.7, 22.3 and 133 mg/kg bw per day for males and 0, 4.4, 26.5 and 153 mg/kg bw per day for females, respectively). The parental (P) animals were given test article diet formulations for 10 weeks prior to mating to produce the F 1 litters. After weaning, F 1 animals (24 of each sex per dose) were selected to become the parents of the F 2 generation and were given the same concentration of test formulation as was administered to their parents, starting at 10 weeks prior to mating to produce the F 2 litters. In addition to the typical parameters examined in a reproductive toxicity study, serum concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH) were measured in males and females, as well as testosterone in males and 17β-estradiol and progesterone in female parents in the F 1 generation, using a radioimmunoassay method. In a supplemental in vitro study (reported in Takahashi, 2002b), flonicamid technical solutions were serially diluted (ranging from 10 2 to 10 9 mol/l), and estrogen receptor (ER) binding assays were conducted in triplicate analyses per concentration. In parental animals, there were no treatment-related effects on survival, clinical signs, body weight, body weight gain or feed consumption. At doses of 300 ppm and above, relative kidney weights were increased in F 1 males, and there was an increased incidence of hyaline droplet deposition in the proximal tubule cells in males of both generations. These changes were considered specific to the rat and not relevant to the risk assessment. At 1800 ppm, kidney weights were increased in males of both generations, as were pale kidneys. In P and F 1 males, there were increased incidences of tubular basophilic change and granular casts in dilated tubules in kidneys; in P and F 1 females, there were increased incidences of vacuolation of the proximal tubule cells. In the ER binding assay, results demonstrated that the test substance had no clear binding affinity for the ERs α and β. Because blood serum concentrations of the test substance were not determined in the main study, it was not possible to compare the concentration of the test substance that resulted in ER binding with levels of the test substance actually in the blood in animals fed up to 153 mg/kg bw per day. In the offspring, there were no treatment-related effects on body weight, anogenital distance, gross pathology or birth, live birth, viability or lactation indices and no macroscopic findings in the F 1 or F 2 pups or weanlings that could be attributed to treatment. Microscopic examinations were not performed. At 1800 ppm, there was a 4% increase in days to vaginal opening in F 1 pups, which coincided with a 4% increase in body weight at time of vaginal opening; however, this finding was not seen in the F 2 pups. Absolute and relative (to body weight) uterine weights were significantly decreased by 19% in the F 1 weanlings sacrificed at days of age. For the reproductive parameters, there were no effects of treatment on precoital or gestation intervals; mating, fertility or gestation indices; estrous cycle duration or cyclicity; sperm enumeration, motility or morphology; or number of primordial ovarian follicles. In the F 1 females, blood serum levels of LH were significantly increased at 300 ppm and higher. FSH was significantly increased and 17β-estradiol was decreased by 27% (not significantly) for the females in the 1800 ppm dose group. However, owing to the variable and cyclic nature of hormone levels and the lack of adverse effects on the reproductive parameters, these changes were not considered to be adverse.