BUPROFEZIN First draft prepared by Dr. Otto Meyer, National Food Agency, Copenhagen, Denmark EXPLANATION Buprofezin, 2-tert-butylimino-3-isopropyl-5-phenylperhydro-1,3,5- thiadiazine-4-one is an insect growth regulator with high activity against homopterous insects. The substance acts by inhibiting chitin biosynthesis and subsequent cuticle deposition. It also has effects on hormone levels of nymphs associated with moulting. In addition this compound suppresses the egg-laying in female adults with incidental inhibition of prostoglandin synthesis. Buprofezin is being reviewed for the first time at this meeting. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Biochemical aspects Absorption, distribution and excretion 14C-Buprofezin, labelled in the phenyl ring and dissolved in olive oil was administered by gavage at a dose 10 or 100 mg/kg bw (5 µCi/rat) to 4 and 3 Sprague-Dawley (SD) male rats, respectively (mean weight 200 g). Blood was taken from the tail vein 30 minutes and every hour up to 96 hours after dosing. Radio-activity was detected 30 minutes after dosing (0.122 µgram and 0.976 µgram buprofezin equivalent per ml for 10 mg and 100 mg doses, respectively). Maximum level (Cmax) of radioactivity was reached 9 hr post-dosing. The Cmax was 1.2 and 13.3 µgram buprofezin eq/ml for 10 and 100 mg, respectively. The biological half lives were 13 hours (9-24 hours) after dosing and 60 hours (24-96 hours after dosing) irrespective of the dose. Total radioactivity in blood was calculated to be 0.82 and 0.98% of 10 and 100 mg/kg dose, respectively (Sugimoto, 1982). Male SD rats (4 groups of 5) weighing 200 g were dosed with 10 or 100 mg 14C-Buprofezin/kg b.w. (5 µCi/animal) as in the above study. The rats were sacrificed 2, 5, 9, 24 or 96 hours after dosing. Following administration of the 10 mg/kg bw dose; the highest radioactivity was detected in the urinary bladder (14.27 µgram equivalent/g) 9 hours post-dosing. Cmax in the liver was 11.6 µgram eq/g after 5 hours. Other organs and tissues with Cmax higher than 1 µgram were adipose tissue, kidney, adrenal gland, pancreas and blood (4.2, 2.5, 2.3 1.3 and 1.0 µg equivalent/g respectively). Cmax of all tissues and organ occurred at 5-9 hours. Following administration of 100 mg/kg bw, the highest radioactivity was found in adipose tissue (114.7 µg equivalent/g after 9 hours). The distribution pattern was similar to that in the animals dosed at 10 mg, the level being 10 times higher. The Cmax in liver, urinary bladder, adrenal gland kidney, pancreas and spleen were 85, 71, 47, 36, 33 and 33 µg equivalent/g, respectively. The radioactivity in all tissues and organs was below 10 µg equivalent/g or ml 96 hours post-dosing. The t´ in all tissues and organs examined was 3.5-15 hours (between 9 and 24 hours after dosing) and 15-72 hours (between 24 and 96 hours). The radioactivity in blood, kidney and liver decreased relatively slowly between 24 and 96 hours after dosing (t´ was 37-72 hours) (Sugimoto, 1982). Five male SD rats were dosed with 14C-Buprofezin as in the above study, and sagittal sections taken at 2, 5, 9, 24 and 96 hours after administration of 10 mg/kg bw (20 µCi/animal). After 2 hours most the radioactivity was found in the alimentary canal. The small amount of radioactivity detected in liver and kidney indicated that absorption and excretion had already begun. The level of radioactivity in tissues peaked after 5 and 9 hours. The levels of radioactivity at 9 hours were as follows: intestine > stomach > liver > kidney > lung, brown adipose tissue and blood > skin and thymus> thyroid > heart > muscle > brain > testis > bone marrow > eyeball. At 96 hours, radioactivity in the body was below 4% (Intestine > liver > kidney > skin > lung > blood). The levels of radioactivity in liver and intestine suggest the existence of enterohepatic circulation. The radioactivity in kidneys was always less than that in liver (Sugimoto, 1982). SD male rats 6 weeks of age (a total of 27/group) were given diets containing 200 or 1000 ppm buprofezin (purity: 99%) for up to 24 weeks. At 200 ppm, the concentrations in adipose tissue were 0.6-0.9 ppm. Less than 0.1 (the limit of detection) to 0.2 ppm was found in the liver and less than 0.1 ppm in other tissues. The corresponding figures for the 1000 ppm diet were 3.4, 0.2-0.3 and less than 0.1 ppm. The results indicate that buprofezin does not accumulate in tissues (Sugaya & Uchida, 1990). Male SD rats weighing 160-170 g were dosed with 14C-buprofezin as in the previous studies. Ten mg and 100 mg/kg bw was administered to 2 and 3 rats, respectively (2 µCi/animal). At the low dose, 82% of the radioactivity was excreted within 24 hours and 93% within 48 hours. At 100 mg/kg bw, 91% was excreted within 48 hours. Totals of 74% and 70% were excreted in faeces within 4 days for the low and high dose levels, respectively. The corresponding figures for the urinary excretion were 22 and 25%, respectively. The excretion of radioactive CO2 into expired air was 0.21% in each dose. Ten mg 14C-buprofezin/kg bw (20 µCi/animal) was orally administered to 2 cannulated SD male rats weighing 210 g, and bile was collected from the bile duct for 24 hours. 38.41 and 31.66% of the dose was excreted into the bile in the two rats respectively (Sugimoto, 1982). Male and female SD rats weighing 160-240 g, 5/sex/dose received a single oral dose of 10 or 100 mg 14C-buprofezin/kg bw (0.6-0.7 ml corn oil/animal, 9.9-12 µCi/animal). Urine and faeces were collected at 6 and 24 hours and thereafter at 24 hour-intervals for the subsequent 144 hours. Expired air was examined 0-6, 6-24 and 24-48 hours after dosing in 1 male and 1 female per dose level. Blood and tissues were analyzed at the end of the study. The main route of elimination was via the faeces. Most of the elimination via urine and faeces took place within 48 hours. In male rats, 90-91% was eliminated in this period; 20-21% in urine and 69-71% in faeces. The corresponding figures for females were 87-89%, 13-14% and 73-76%, respectively. Very little radioactivity (less than 0.5%) was found in expired air. Elimination in males was faster than that in females in the first 24 hours but fairly even by 48 hours post dose. The highest concentrations were found in liver, thyroid and blood cells at both dose levels. (1.8-2.4 µg eq./g and 0.1-0.4 µg eq, per g for high and low dose respectively). A mean of less than 0.7% of the dose remained in the body at sacrifice at both dose levels with liver constituting less than 0.2% of the total, intestine including contents less than 0.04% and other tissues less than 0.01% (Caley & Cameron, 1988). Bile duct cannulated rats, 3 of each sex (see above study) were dosed with 10 mg 14C-buprofezin as above. Bile was collected frozen at hourly intervals up to 6 h and then hourly by fraction collected up to 24 h post-dosing. Urine and faeces were collected separately for the periods 0-6 and 6-24 h. GI-tract, liver and residual carcass were analyzed at the end of the study. Total radioactivity equivalent to a mean of approximately 30% of the dose for males and 38% for females was recovered in bile 24 h post dosing. Elimination via the urine accounted for a mean of 5.4% of the dose for males and 2.6% in females. The corresponding figures for faeces were 34% and 19%, respectively. The radioactivity accounted for in GI-tract, liver and residual carcass was 16.6 and 27.5%, 1.0 and 1.5% and 3.8 and 3.2% for males and females respectively (Caley & Cameron, 1988). Biotransformation Urine, faeces and bile excreted within 24 hours were obtained for TLC analysis from male SD rats weighing 220-230 g administered a single peroral dose of 10 mg 14C-buprofezin/kg bw (20 µCi/animal). Faecal and urine of 3 rats were examined. Faeces and bile were examined from 2 rats administered 2 µCi/animal. For solvent fractionation and identification of metabolites, urine and faeces obtained from 2 rats administered a single peroral dose of 100 mg/kg bw (10 µCi/animal) and 12 rats dosed with unlabelled material were used. Hydroxylation of phenyl ring and oxidation of sulfur were the main routes of metabolism. Hydroxylation of phenyl ring gave 4-hydroxy, 3,4-dihydroxy and 3-hydroxy-4-metoxy buprofezin and some of these were conjugated with glucuronic acid or sulfate. The oxidation products of sulfur were thought to form isopropyl phenyl urea through the cleavage of the thiadiazin ring. Twelve percent of the dose was excreted into faeces as parent compound. In urine and bile only more polar metabolites were detected. In rats whose bile ducts had been cannulated, more than 95% of radio-activity in faeces was composed of the parent compound. This indicates that buprofezin itself does not change in the intestinal tract. Glucoronides found in bile were not all detected in faeces. Moreover, metabolites in bile were not all found in faeces, and different metabolites which could not be detected in bile were found in faeces. Thus bile metabolites are further metabolized in the intestinal tract to form metabolites found in faeces. 14C-Buprofezin (0.3 µCi) 4ml 4 µg was added to rat liver homogenate (SD, male, average bw: 205 g), and incubated at 350 for 2 hours, with and without S-9 and co-factors (NADH, NADPH, NADP, G6P dehydrogenase, G6P, MgCl2 and KCl). The results indicated the same metabolic route as in the above in vivo study However, isopropyl phenyl urea was only found in vitro and is probably further metabolized in vivo (Sugimoto, 1982). The profile of metabolites in urine, faeces and bile was investigated in pooled samples from SD rats weighing 160-240 g administered one single oral dose 14C-buprofezin (faeces and urine from 5 rats/sex/group dosed 10 or 100 mg/kg bw; faeces and urine from 1 male and 1 female, bile duct cannulated, dosed 10 mg/kg bw and bile from 1 male and 1 female, bile duct cannulated, dosed 10 mg/kg bw (Caley & Cameron, 1988). TLC of urine and bile indicated extensive conjugation, and only a small amount of buprofezin co-chromatographing material was found in the urine in one sample. Faecal extracts indicated conjugated material and components co-chromatographing with 2-tert-butylimino-3-isopropyl-5-(4-hydroxy-phenyl) perhydro-1,3,5- thiadiazin-4-one (a minimum of 1.76% and 0.38% of the administered dose for high and low dose respectively) and possibly with 2-tert-butylimino-5-(3,4-dimethoxyphenyl)-3-isopropyl-3,4,5,6-tetrah ydro-2H-1,3,5,-thiadiazin-4-one and with buprofezin (a minimum of 7.99% and 10.24% of the administered dose for the high and low dose respectively). Faecal extracts of the bile duct cannulated rats showed only radioactivity co-chromatographing with buprofezin. The possible intestinal deconjugation of metabolites from bile may be attributable to microorganisms in the intestine (Caley & Cameron, 1988). Polar metabolites in faeces and urine from rats administered single oral doses of 10 or 100 mg 14C-buprofezin/kg bw were studied. Sulfuric acid conjugates of 1-(4-hydroxyphenyl)-3-isopropylurea, p-aminophenol and p-acetamidophenol were identified. Sulfuric acid conjugate of p-acetamidophenol was the major metabolite in urine, accounting for 3.9% of the dose (Sugimoto et al., 1982). In faeces, the sulfuric acid conjugate of p-acetamidophenol accounted for 2.7% of the dose (Uchida 1988.) Toxicological studies Acute toxicity studies The acute toxicity of buprofezin is shown in Table 1. Table 1. Acute toxicity of buprofezin Species Sex Route LC50 LC50 Reference (mg/kg/bw) (mg/l) Mouse M&F oral > 10 000 - Ebino & Shirasu (1981a) i.p. > 10 000 - Ebino & Shirasu (1981a) s.c. > 10 000 - Ebino & Shirasu (1981a) Rat Ma oral 2198 - Ebino & Shirasu (1981b) Fa 2355 Mb oral 1635 - Tsuchiya & Sugimato (1982) Fb 2015 M&Fa i.p. > 10 000 - Ebino & Shirasu (1981b) M&Fa s.c. > 10 000 - Ebino & Shirasu (1981b) M&Fa dermal > 5000 - Ebino & Shirasu (1981b) M&Fa inhalation - > 4.6 Tsuda (1984) (4 h. exp.) Hamster Mc oral > 10 000 - Tsuchiya & Sugimato (1979b) Rabbit Md oral > 5000 - Tsuchiya & Sugimato (1979c) Buprofezin of greater than 99% purity was used. a SPF Fischer strain c Golden hamsters SPF ICR strain d Japanese albino b SD rats Short-term studies Rats Groups of Sprague-Dawley rats (Jcl: SD), 5 weeks of age received buprofezin (purity: >99%) in the diet for 90 days at concentrations of 0, 40, 200, 1000 or 5000 ppm (10 animals/sex/group). The actual concentrations were 46, 185, 982 and 4555 ppm; the compound was shown to be stable up to 18 weeks after mixing. All animals were observed daily for clinical signs and mortality. Body weights and food consumption were recorded weekly. In the 13th week, daily water intake and urine volume were recorded and urinalysis performed. Haematological examinations (on day 90) and assessment of blood chemistry at terminal sacrifice were performed. Gross pathological examination was performed on all animals at necropsy and selected organs were weighed. No effects on mortality nor clinical appearance were observed. Body weight gains were suppressed in males and females at 5000 ppm and in females at 1000 ppm. Food intake was slightly lowered in the 200, 1000 and 5000 ppm groups, but feed efficiencies were not affected. Water intake was significantly increased in top dosed males. Haematocrit, haemoglobin and red blood cell count in top-dosed males and haematocrit in top-dosed females were decreased. Activated partial thromboplastin times were prolonged in the 5000 ppm group. In animals receiving the 5000 ppm diet, serum glucose and triglyceride concentrations were lowered and the concentrations of total cholesterol and phospholipids were elevated. In males at this level, there were increases in serum beta-globulin, alpha 1-globulin and albumin and a decrease in gamma-globulin. In females at the highest dose level, an increase was recorded in albumin, alpha 1-globulin, alpha 2-globulin, alpha 3-globulin and beta-globulin. Serum glucose was lowered in males dosed at 200 ppm and above and beta-globulin and alpha 1-globulin was elevated in females receiving 1000 ppm. Both absolute and relative liver and thyroid weights were increased at 5000 ppm. In the 1000 ppm group, the relative thyroid weight was increased in males and relative liver weight in females. Enlargement of the hepatocytes in the central to the middle zone of the lobules and thickening and hyperplasia of the follicular epithelial cells of the thyroids were observed in top-dosed animals and in some animals in 1000 ppm group. No compound-related adverse effects were observed at 40 ppm in the diet, equivalent to 3.4 mg/kg bw/day for males and 4.1 mg/kg bw/day for females (range 5.7-2.5 mg/kg bw/day and 5.8-3.2 mg/kg bw/day for males and females respectively). The liver and thyroid were identified as target organs. The NOAEL was 40 ppm (Watanabe, 1986). Dogs Beagle dogs, 15-17 weeks of age (4/sex/group) received buprofezin (purity: 99.0%) orally in gelatin capsules at dosages of 0, 2, 10, 50 or 300 mg/kg bw/day for 13 weeks. Individual observations of all animals and food consumption were recorded daily. Water consumption was measured twice before commencement and during weeks 4, 8 and 13 of treatment. Each animal was weighed before feeding and weekly during the treatment period. Clinical examination was performed before commencement and after 4, 8 and 12 weeks of treatment. Ophthalmo-logical examination on each dog was carried out before commencement and after 12 weeks of treatment. Blood was sampled before commencement, and after 6 and 12 weeks of treatment for haematology and blood chemistry. Overnight urine was also collected on the same sample schedule as blood sampling for urine analysis. On completion of the treatment period, all dogs were sacrificed and subjected to necropsy. Organ weights were recorded and histopathological post-mortem examination was performed. No mortalities were observed during the study. Subdued behavior was seen in the 50 and 300 mg/kg/day dose groups and signs of slight ataxia and slight abdominal distension were noted in the 300 mg/kg/day dose group. Food consumption and body weight gains of dogs receiving 300 mg/kg/day were lower than controls. Water consumption of one high-dose female was reduced during the treatment. No treatment- related effects were revealed by clinical nor ophthalmological examination. A slight increase in prothrombin times was seen in females receiving 300 mg/kg/day. No effects of treatment were apparent on haematological parameters. Plasma alkaline phosphatase activity was raised in males receiving 50 mg/kg/day and in all dogs receiving 300 mg/kg/day. The normal decline in alkaline phosphatase activity with maturity was less evident in animals receiving 10 mg/kg/day and in females dosed at 50 mg/kg/day. Slightly elevated alanine aminotransferase activity was recorded in dogs receiving 300 mg/kg/day. No treatment-related effects were noted in urine analysis. The following changes in absolute and relative organ weights were noted; high liver weight in one male receiving 2 mg/kg/day, two males receiving 10 mg/kg/day, all dogs receiving 50 or 300 mg/kg/day, high kidney weight in dogs receiving 300 mg/kg/day, high thyroid weight in males receiving 50 mg/kg/day and males and females receiving 300 mg/kg/day. However, the changes of liver weight in the 2 and 10 mg/kg/day dose groups were considered to be of no toxicological significance. At necropsy no treatment-related lesions were noted. Homogeneity of cytoplasm and intracytoplasmic eosinophilic bodies in the hepatocytes were seen in dogs receiving 50 or 300 mg/kg/day. The NOAEL for this study was 10 mg/kg/day based on changes in the liver (Broadmeadow, 1986). Beagle dogs (6/sex/group), 16-17 weeks of age were administered 0, 2, 20 and 200 mg/kg bw/day of buprofezin (purity, >99%) by oral gelatin capsules for 107 weeks. Body weight, food consumption and clinical observations were recorded. Ophthalmoscopy was performed prior to the test and during the dosing period. Smears of bone marrow were obtained by biopsy at week 104 of treatment. During week 104 of treatment, alkaline phosphatase isozymes were separated by electrophoresis, and determined densitometrically. Blood samples were taken from each dog before, and periodically during the experiment to examine thyroid function, haematology and clinical chemistry. After 53 and 103 weeks of treatment, BSP excretion test was conducted for assessment of liver function. At week 107 all dogs were subjected to pathological examination. There were no effects of treatment on mortality or clinical signs (including ophthalmoscopy) of toxicity. Body weight gain was suppressed for dogs receiving 200 mg/kg bw/day, and food consumption was marginally lower in top-dosed males when compared to controls. Apart from occasional increases in mean cell volume and mean cell haemoglobin in males receiving 20 and 200 mg/kg bw/day, no effect of treatment on haematology (peripheral blood and bone marrow) nor urinalysis was noted. Increased plasma alkaline phosphatase activity was seen in animals in high and intermediate dose groups, and an increase in plasma alanine amino-transferase activity was recorded in top-dosed animals. Reduced serum thyroxin was noted in dogs receiving the high dose, while tri-iodothyronine and protein bound iodine concentrations were unaffected. BSP (bromsulfophthalein) retention after 103 weeks of treatment was increased in females receiving 20 or 200 mg/kg bw/day. Both absolute and relative liver and thyroid weights were increased in top-dosed females and relative liver and thyroid weight was increased in top-dosed males. Absolute liver weight was elevated in females dosed with 20 mg/kg bw/day, and the relative liver weight was increased in females in all dosage groups. No treatment-related changes were noted in macroscopic examination. Microscopic examination revealed enlargement of centrilobular hepatocytes and bile duct hyperplasia in the liver of dogs receiving 20 and 200 mg/kg bw/day. The NOAEL of buprofezin administered orally to dogs over a period of 2 years is 2 mg/kg bw/day (Cummins, 1982). Long-term/carcinogenicity studies Mice In an oral long-term toxicity and oncogenicity study, groups of 70 male and 70 female SPF ICR mice (Crj: CD-1), 5 weeks of age were exposed to buprofezin (purity: 99.5%) as a dietary admixture at concentrations of 0, 20, 200, 2000 or 5000 ppm, equivalent to 0, 1.8, 17, 190 and 481 mg/kg bw/day for males and 0, 1.9, 18 191 and 493 mg/kg bw/day for females for 104 weeks. Satellite groups of 10 animals/sex/group were treated in a similar manner for 52 weeks. Analysis of the diet showed that the test compound was stable at ambient conditions for one month. Determination of concentration and homogeneity of test compound in the diet was made at intervals of three months. Diet analysis revealed satisfactory consistency in concentration and homogeneity of the test compound. Ten animals of each sex/group were subjected to urinalysis, haematology and blood chemistry and sacrificed for pathological examinations at week 52 and 104. Gross pathological examination was performed on all animals. Organ weights were recorded in each animal subjected to interim or terminal kill. In the 5000 ppm group, retarded growth, decreased specific gravity of urine, reduced levels of protein in the urine, elevation in platelet and lymphocyte count (after 104 weeks of treatment), increased absolute and relative liver weight and an increased incidence of hepatocellular swelling (centrilobular and diffuse) and hepatocellular hyperplasia were seen in both sexes. The total blood cholesterol was increased at week 52 for females and week 104 for males. In the 2000 ppm group, slightly retarded growth and an increased incidence of centrilobular hepatocellular swelling were recorded in both sexes. The absolute and relative liver weights were increased in both sexes in the same animals after 52 weeks. An increased incidence of hepatocellular hyperplasia was observed in females. In the 200 ppm group an increase in liver weight was noted in males at week 52. The incidence of hepatocellular adenoma was increased in top-dosed females, but the combined incidence of these adenomas and hepatocellular carcinomas was not statistically significantly different from controls (12/80 and 5/80, respectively). However, the denominator should be 70, as no tumours were found at 52 weeks. The overall incidence of lung adenoma and carcinoma in males of the 200 and 5000 ppm groups were significantly higher than those of the controls. The combined incidences were 30/80, 23/80, 29/80, 26/80 and 17/80 for 5000, 2000, 200, 20 and 0 ppm, respectively. Adenomas were seen in dosed animals in an incidence of 2/10 after 52 weeks compared to 0 in controls. The incidence of hepatocellular carcinoma in the females receiving 2000 and 5000 ppm, 4/80, falls outside the historical range of 0/80-3/80. However, the control incidence was 3/80. It is concluded that the data does not indicate a carcinogenic potential. The NOAEL is 20 ppm (male) equal to 1.82 mg buprofezin/kg bw/day (Yoshida, 1983). In a long-term toxicity study, groups of 40 male and 40 female Jcl: SD rats, 5 weeks of age were exposed to buprofezin (purity:>99%) mixed in a powdered CE-2 chow in doses 0, 5, 20, 200 and 2000 ppm for 24 months. Additional groups of 5 and 10 rats of each sex were exposed in a similar manner for 6 and 12 months respectively. Analysis of test diets showed that the actual concentrations were 3.9-6.5 ppm (first 2 months, 12 ppm), 16.5-21 ppm (first 2 months, 26 ppm), 175-210 ppm and 1700-2000 ppm (last 2 month, 2300 ppm) respectively. The compound was stable in the diet up to 120 days. The daily intake (total mean) of the test compound was equal to 0.26, 0.90, 8.71 and 89.46 mg/kg bw/day for males and 0.33, 1.12, 11.19 and 114.71 mg/kg bw/day for females. Haematology, blood chemistry and urine analysis were performed at 6, 12 and 24 months of treatment. Necropsy was performed on all animals including those found dead or killed in extremis. Organ weights were measured at necropsy. Phenolsulfophthalein (PSP) and bromsulfophthalein (BSP) excretion tests were performed in 5 males and 5 females from control and 2000 ppm groups at 24 month of treatment. All animals were subjected to histopathological examination. Electron microscopic examination was performed on liver samples which showed treatment-related changes. No effect of treatment on clinical observations was observed, and survival in all groups was > 40%. Body weights were lowered in top-dosed animals of both sexes in the first months, and in addition in the females scattered during the last period of dosing. No treatment-related changes were noted in food consumption, food efficiency, urinalysis, haematology, blood chemistry, PSP and BSP excretion test. Increased absolute and relative liver and thyroid weights were seen in males and females in the 2000 ppm group. An increase in relative kidney and heart weights was noted in females dosed 200 and 2000 ppm at 24 months. Hypertrophy of hepatocytes was seen in males and females in the 2000 ppm group. The hypertrophy was associated with proliferation of smooth endoplasmatic reticulum by electron microscopy. The incidence of hyperplastic nodules was slightly higher in the 2000 ppm group at 24 months. Thickening and hyperplasia of the follicular epithelial cells in thyroids were observed in most of the animals in the 2000 ppm group and in a few animals in the 200 ppm group. In addition, increased incidence of cystitis, chronic nephrosis and interstitial oedema in the heart was observed in top-dosed females. No effect of treatment was observed on any type of tumour. The NOEL was 20 ppm, equivalent to 0.9 mg/kg bw/day and 1.12 mg/kg bw/day for males and females, respectively (Watanabe et al., 1982.) Reproduction studies Groups of 30 male and 30 female Wistar-Imamichi rats (SPF), 4 weeks of age (F0) were exposed to buprofezin (purity: 99.1%) in a pelleted diet in concentrations of 0, 10, 100 or 1000 ppm. Analysis of the diets was reported to be satisfactory. Data on stability of the chemical in the diet were not given. Rats were dosed for 13 weeks before mating (1:1). Ten days after weaning of F1a pups, the F0 were mated a second time to obtain F1b pups. The resulting F1b pups from 10 pregnant dams in each group underwent teratological examination. F1b pups, 30 of each sex, from the remaining pregnant dams (born within 4 days) were selected for production of the F2 generation. The above procedure was repeated to produce F2 animals. Fifteen rats of each sex were selected from weaned F2b pups and reared for 12 weeks. In each generation, the litters were reduced to 8, 4 males and 4 females, on day 4 post-partum. Observations in F1b and F2b pups included pinna unfolding, hair growth, incisor eruption and eye opening, righting reflex, pain reflex, auditory reflex, grasping reflex and posture and gait on day 21. The high-dose animals exhibited lower body weight gain compared to the controls; this was most pronounced in F1 and F2 males. Depressed weight gain was also seen at lower doses, especially in F1 animals the first weeks after weaning. No consistent effect was observed in food and water consumption. The actual compound intakes were determined to be 0.6-0.8, 6-8.1 and 62.5-86.5 mg/kg bw/day for males and 0.8-0.9, 7.9-8.9 and 82.6-85.2 mg/kg bw/day for females in the low, medium and high dose-groups, respectively. Litter data revealed a decrease in survival of F0 pups during day 0-4 of the lactation period in 10 and 1000 ppm group and lower mean live pups weight in all dosed groups (first mating), a decrease in number of delivered and live pups in 100 and 1000 ppm groups from F1 2nd mating (F2b), and a decrease in body weights of the pups during lactation in top-dose, most pronounced in F1a and F2a animals, and in all dose-groups in F2a. Decreased fetal weight was seen in second mating of F0 (F1b) but not in the analogous F2 animals. The absolute liver weight was increased in high-dose females in F0 and F1 and in F1 males, while the relative liver weight in the same group was increased in females in all generations and in males in all generations except the F0. At doses up to 1000 ppm buprofezin has no influence on reproductive performance in rats (Takeshima, 1982). However, the study does not clearly indicate a NOAEL for decreased body weight gain. Groups of 25 Wistar-Imamichi rats, 4 weeks of age (F0) were exposed to buprofezin (purity: 99.1%) in a pelleted diet in concentrations 0, 10, 100 or 1000 ppm (chemical analysis of the diet reported satisfactory) in a study design similar to the above study (however, no examination for teratological effects was performed). The study was initiated as an additional study to the above and it is entitled "two-generation reproduction study". However, the study design is of a one-generation study with 2 litters in the F1-generation. The actual dose for males was measured as 0.6, 6.4 and 65.6 mg/kg bw/day and for females 0.9, 8.9 and 82.8 mg/kg bw/day in the low, medium and high dose groups. Decreased body weights during lactation were observed in F1 pups of both sexes in the 1000 ppm group. Relative liver weight was increased in top-dosed animals. The NOAEL is 100 ppm, equal to 6.4 and 8.9 mg/kg bw/day for males and females, respectively (Takeshima et al., 1985). Special studies on teratogenicity and embryotoxicity Rats Four groups of 22 mated CD female rats (of Sprague-Dawley origin), 8 to 10 weeks of age were dosed with buprofezin (purity: 99.0%) in 2% (w/v) aqueous gum arabic mucilage by oral gavage at doses of 0 (vehicle), 50, 200 or 800 mg/kg bw/day from day 6 to day 15 of gestation inclusive (dosage volume: 10 ml/kg bw). Samples of each concentration of the test mixtures taken during first and last weeks of treatment showed that the low, medium and high level dose were 38.3-37.3, 188-166 and 776-819 mg/kg bw/day. Maternal toxicity was evidenced by reduced food intake, decreased body weight, loose faeces, urogenital staining, lethargy, hunched posture, thin appearance and piloerection, only in top-dosed animals. A transient reduction in food intake was also recorded on day 14 of gestation in the group receiving 200 mg. Water consumption during the treatment period was significantly increased in the groups receiving 200 or 800 mg. At 800 mg/kg bw/day, 4 females showed total resorption and in females which carried live young to term, increased early post-implantation loss and reduced litter size and fetal weight were recorded. Fetuses in the highest dosage group showed a significantly increased incidence of subcutaneous oedema and signs of slight fetal immaturity including reduced mean fetal weight. At 200 mg there was a slight reduction in the degree of ossification of supra-occipital and interparietal bones (within the ranges of historical control data) with other ossification parameters unaffected. Treatment with buprofezin at dosages up to 800 mg/kg bw/day did not give rise to any gross morphological changes (Tesh et al., 1987). The NOAEL was 50 mg/kg bw/day for maternal toxicity and less than or equal to 166 to 188 mg/kg bw/day for embryotoxicity. Rabbits Groups of 17 mated New Zealand White rabbits, 21-27 weeks old, were dosed with buprofezin (99% purity) in 2% (w/v) aqueous gum arabic by oral gavage at dosages 0, 10, 50, or 250 mg/kg bw/day from day 6 to 19 of the gestation period inclusive (dosage volume: 5 ml/kg bw). Samples of each concentration of the test mixtures taken during first and last weeks of treatment showed the low, medium and high doses were 8.4-9.5, 51-46 and 231-224 mg/kg bw/day respectively. Animals in the high dose group lost weight initially following onset of treatment, and overall body weight gain during gestation was reduced. Food consumption was reduced from the commencement of the treatment period until day 13 of gestation in the 250 mg-group, whereas water intake was unaffected. One female receiving 50 mg/kg bw/day aborted, and two females receiving 250 mg showed resorption of the entire litter. No other treatment-related effects were found. Buprofezin at dose levels up to 50 mg/kg bw/day showed no adverse effect on either maternal condition or upon the progress and outcome of pregnancy, or upon fetal morphogenesis (Tesh et al., 1986). Special studies on genotoxicity Buprofezin did not have mutagenic properties when tested in a number of in vitro and in vivo systems, which included assessment of gene mutations, DNA damage, and clastogenic effects. Table 2. Summary of special studies on the mutagenicity of buprofezin Test system Test object Concentration Purity Results Reference of buprofezin Ames test (with and S. typhimurium 10-5000 99.3% Negative Moriya (1980) without metabolic TA98, TA100, µg/plate (1) activation) TA1535, TA1537, dissolved in and TA1538 DMSO Reverse mutation (with E. coli WP2 uvr A 10-5000 99.3% Negative Moriya (1980) and without metabolic µg/plate (1) activation) dissolved in DMSO Ames test (with and S. typhimurium 1.6-5000 99.8% Negative Callander (1988) without metabolic TA98, TA100, µg/plate (2) activation TA1535, TA1537 dissolved in and TA 1538 DMSO Mouse lymphoma forward Mouse L5178Y 13.3-100 µg/ml 99.8% Negative Cross (1988) mutation assay (with TK +/- cells dissolved in (3) and without metabolic DMSO activation DNA repair assay (with B. subtilis 20-5000 µg/disk 99.3% Negative Moriya (1980) and without metabolic H17 & M45 dissolved in (4) activation) (rec +/-) DMSO In vitro cytogenetics Human 10, 60 and 100 99.8% Negative Howard & (with and without metabolic lymphocytes µg/ml dissolved (5) Richardson activation) in DMSO (1988) Table 2 (contd). Test system Test object Concentration Purity Results Reference of buprofezin Unscheduled DNA synthesis Primary hepatocytes 10-9-10-3M 99.8% Negative Trueman (1988) from male. Alpk: dissolved in (6) APfSD rats DMSO Mouse micronucleus test BDF1 mice 6400, 8000 and 99.5% Negative Sasaki (1983) 10 000 mg/kg (7) (administered orally single or 4 times) (1) The positive controls, 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide; AF-2, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine and 2-aminoanthracene gave the expected positive results. (2) The positive controls, acridine mutagen ICR191, 2-aminoanthracene, daunomycin hydrochloride, 4-nitro-o-phenylenediamine and N-methyl-N-methyl-N'-nitro-N-nitrosoguanidine gave the expected positive results. (3) The positive controls, ethylmethanesulphonate and N-nitrosodimethylamine gave the expected positive results. (4) The positive control, mitomycin C gave the expected positive results. (5) The positive controls, mitomycin C and cyclophosphamide gave the expected positive results. (6) The positive control, 6-p-dimethylaminophenylazobenthiazole gave the expected positive results. (7) The positive control, mitomycin C gave the expected positive results. Special study on duodenal ulcer formation This study was performed in order to investigate in detail duodenal ulceration seen in the acute oral toxicity study of buprofezin in rats. Groups of 10 male and 10 female SPF Fischer rats 5 weeks of age were administered a single dose of buprofezin (purity: 99.5%) at doses of 0, 613, 1036, 1751, 2959 or 5000 mg/kg by gavage (vehicle, olive oil, 1.2-10 ml/kg bw). Mortality and clinical signs were observed during 4 days after the treatment. The observation period was determined to examine the generation of duodenal ulceration in accordance with the results obtained from a previous pilot study. Body weight was recorded just before treatment, at death or at the end of the study. At day 4 all surviving animals were killed. All animals were subjected to necropsy and histopathological examination of the duodenum. Four males and 8 females in the 2959 mg/kg group and 9 males and 10 females in the 5000 mg/kg group were found dead or killed in extremis. Tremor (in animals dosed at 613 mg/kg bw and 1036 mg/kg bw or more) subdued behavior, diarrhoea, gait disturbance, piloerection, lacrimation, soiled fur, chromodacryorrhoea and eyelid closing (in animals dosed at 1751 g/kg bw or more) were observed. At 1751 mg/kg and above decreased body weight was shown in surviving animals. At necropsy there was congestion, red or white spots and/or perforated foci in the proximal duodenum in males treated at 2959 mg/kg and above and in females at 1751 mg/kg and above. There were no gross abnormalities in other tissues or organs of all treated animals. At histopathological examination, ulcerous lesions in the duodenum were detected in one male treated at 1751 mg/kg, in 5 males and 8 females at 2959 mg/kg and in all males and females at 5000 mg/kg. These lesions were not observed in males treated at 1036 mg/kg and below nor in females treated at 1751 mg/kg and below. The NOAEL of buprofezin for the formation of duodenal ulceration was 1036 mg/kg in males and 1751 mg/kg in females (Ueda, 1985). Special studies on thyroid function In male Sprague-Dawley rats treated orally with buprofezin for about one month, weight increase and morphological changes were observed in thyroid. Further studies were conducted to investigate the effects of Buprofezin on the thyroid function using rats (male S-D rats), mice (ddY mice), hamsters (golden), guinea pigs (Mortely) and rabbits (Japanese white) (number of animals not given). In male rats treated orally with buprofezin (purity: > 99%) at doses of 100, 300 or 1000 mg/kg/day (in olive oil, 5 ml/kg b.w.) for 7 days, dose-related decreases in serum tri-iodothyronine (T3) and thyroxine (T4) concentrations were observed. Rats were fed a diet containing buprofezin in concentrations of 200, 1000, or 5000 ppm propyl-thiouracil (PTU) for 1, 3 or 6 months. At 5000 ppm of buprofezin the levels of serum T3 and T4 decreased to 30% and 70% of each control level, respectively and then recovered. No effects of buprofezin were observed in rats treated with buprofezin at 1000 ppm. In the rats treated with PTU, however, the decreases of the serum levels of T3 and T4 were more marked than those in rats treated with buprofezin at 5000 ppm and the recoveries of those thyroid hormone levels were not observed. Rats were dosed with 500 mg buprofezin/kg bw/day or 30 mg PTU/kg bw/day by oral gavage for 15, 30 or 60 days and sacrificed 24 hours after the final dose. Body weight gain was strongly depressed by 15 days after commencement of the treatment in buprofezin and PTU dosed rats. Both absolute and relative thyroid weights were increased in the treated groups when compared to the control group. Serum T4 was lowered and thyroidal peroxidase activity was elevated in treated animals when compared to control. For all the above parameters the changes in PTU dosed animals were more pronounced than those of the buprofezin dosed animals. Vacuolation of cells in anterior pituitary was scored moderate to severe in buprofezin and PTU dosed animals, while that of the controls was graded normal to slight. In an in vitro study of buprofezin, PTU and potassium cyanide, KCN (PTU and KCN are known inhibitors to thyroidal peroxidase activity) were added to a reaction mixture of thyroidal peroxidase. No effect of Buprofezin was observed even at concentrations up to 7.2 x 10-5 M (greater than aqueous solubility). A comparison of the effect of Buprofezin treatment on serum protein-binding iodine (PBI) in rats (0, 100, 300, 500 and 1000 mg/kg bw/day), mice (0, 100, 300, 500 and 1000 mg/kg bw/day), hamsters (0, 300 and 500 mg/kg bw/day), guinea pigs (0, 300 and 500 mg/kg bw/day) and rabbits (0, 300 and 500 mg/kg bw/day) showed that the most marked decrease of serum PBI was observed in rats among the species tested. In guinea pigs and rabbits the decreases of serum PBI caused by Buprofezin were less than that in rats. No effects of Buprofezin on serum PBI were observed in mice and hamsters. In summary, buprofezin has a lower potency for thyroid inhibition than PTU and likely acts by a different mechanism. The rat appears to be more sensitive to this effect than mice, hamsters, guinea pigs and rabbits (Konaka & Nokata 1989). Observations in humans The effects of buprofezin in humans have been reported by a manufacturer of technical material. Medical surveillance of workers who routinely handled buprofezin in a factory has been undertaken. The survey revealed no effects which could be attributable to exposure to buprofezin (Nokata, 1990). COMMENTS The toxicokinetics and metabolism of buprofezin have been studied in rats. It is rapidly absorbed and excreted after oral administration. After oral administration of 14C-buprofezin, radioactivity was widely distributed in the tissues. After 48 hours, 91-93% of the administered radioactivity had been eliminated, 70-80% via faeces and 22-25% via urine. Up to 38% had been excreted in the bile after 24 hours. Buprofezin is metabolized by hydroxylation of the phenyl ring and oxidation of sulfur. The acute oral toxicity of buprofezin was low in mice, rats, hamsters and rabbits. WHO has classified buprofezin as "unlikely to present an acute hazard in normal use". In a 90-day study in rats, buprofezin was administered at dietary concentrations of 0, 40, 200, 1000 or 5000 ppm. The NOAEL was 40 ppm, equal to 3.4 mg/kg bw/day for males and 4.1 mg/kg bw/day for females. At higher concentrations an increase in organ weights and histological changes were noted in the liver and thyroid. In a 13-week study in dogs, buprofezin was administered orally in gelatin capsules at doses of 0, 2, 10, 50 or 300 mg/kg bw/day. The NOAEL was 10 mg/kg bw/day. Raised plasma alkaline phosphatase activity and elevated liver weights were seen at both higher doses. In a two-year study in dogs buprofezin was administered orally in gelatin capsules at doses of 0, 2, 20 or 200 mg/kg bw/day. The NOAEL was 2 mg/kg bw/day. Increased liver weight (associated with enlargement of centrilobular hepatocytes and bile duct hyperplasia) was seen at higher doses. Teratogenic studies in rats at doses of buprofezin of 0, 50, 200 or 800 mg/kg bw/day and in rabbits at 0, 10, 50 or 250 mg/kg bw/day gave no indication of teratogenic potential. The NOAEL for maternal toxicity in both species was 50 mg/kg bw/day. Two reproduction studies were performed in rats with buprofezin at dietary concentrations of 0, 10, 100 or 1000 ppm. The first of these was a two-generation study in which no clear NOAEL could be identified for possible adverse effects upon pup-weight gain; consistent treatment-related effects were observed only at 1000 ppm in both litters of each generation. No effects were noted in the F2 generation that were not also present in the F1 generation. The second study was a single-generation study in which there were two matings. The only effects attributable to treatment were reproducible reductions in pup-weight gain at 1000 ppm. The NOAEL in this study was 100 ppm, equal to 6.4 mg/kg bw/day and 8.9 mg/kg bw/day for males and females respectively. In a long-term/carcinogenicity study in mice at dietary concentrations of 0, 20, 200, 2000 or 5000 ppm the NOAEL was 20 ppm, equal to 1.82 and 1.89 mg/kg bw/day for males and females respectively. Absolute liver weight was increased at 200 ppm and above. The incidence of hepatocellular adenoma was increased in females at 5000 ppm. There was an increased overall incidence of lung adenoma and carcinoma in males dosed at 200 and 5000 ppm, but not at 1000 ppm. However, the incidences were within the historical control range for the strain used. In rats fed dietary concentrations of 0, 5, 20, 200 or 2000 ppm buprofezin for 24 months, the NOAEL was 20 ppm equal to 0.9 and 1.1 mg/kg bw/day for males and females respectively. The absolute thyroid weight and incidence of hypertrophy and hyperplasia of the epithelial cells in thyroid increased at 200 ppm and above. The incidence of hepatic hyperplastic nodules was slightly increased in rats at 2000 ppm. The Meeting concluded that there was no convincing evidence of carcinogenicity in rodents. After reviewing the available in vitro and in vivo genotoxicity assays, the Meeting concluded that there was no evidence of genotoxicity. An ADI, which was based on the NOAEL in the 2-year study in rats using a safety factor of 100, was allocated. TOXICOLOGICAL EVALUATION Level causing no toxicological effect Mouse: 20 ppm equal to 1.82 mg/kg bw/day Rat: 20 ppm equal to 0.9 mg/kg bw/day Dog: 2 mg/kg bw/day Estimate of acceptable daily intake for humans 0-0.01 mg/kg bw Studies which will provide information valuable in the continued evaluation of the compound Observations in humans. REFERENCES Broadmeadow, A. (1986) Buprofezin: Toxicity study by oral (capsule) administration to beagle dogs for 13 weeks. Unpublished report No. 85/NHH001/452 from Life Science Research Ltd., Suffolk, UK. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Caley, C.Y. & Cameron, B.D. (1988) The metabolism of 14C Buprofezin in the rat. Unpublished report No. 4556 from Inveresk Research International Ltd., Musselburgh, Scotland. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Callander, R.D. (1988) Buprofezin: An evaluation in the Salmonella mutagenicity assay. Unpublished report No. CTL/P/2009 from ICI Central Toxicology Laboratory, Cheshire, UK. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Cross, M.F. (1988) Buprofezin: Assessment of mutagenic potential using L5178Y mouse lymphoma cells. Unpublished report No. CTL/P/2195 from ICI Central Toxicology Laboratory, Cheshire, UK. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Cummins, H.A. (1982) IET 7907 (Buprofezin): Toxicity in oral administration to beagle dogs for 107 weeks. Unpublished report No. 81/IET026/615 from Life Science Research Ltd., Essex, UK. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Ebino, K. & Shirasu, Y. 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Unpublished report No. T-1033 from Institute of Life Science Research, Nihon Nohyaku Co. Ltd., Osaka, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Takeshima, T. (1982) Two-generation reproduction study and teratogenicity studies in rats with Buprofezin. Unpublished report No. T-1011 from The Institute for Animal Reproduction, Ibaraki, Japan and The Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Takeshima, T. (1985) Two-generation reproduction study in rats with Buprofezin (additional study). Unpublished report No. T-1017 from The Institute for Animal Reproduction, Ibaraki, Japan and The Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Tesh, J.M., McAnulty, P.A., Wightman, T.J., Goodsir, D.M., Wilby, O.K. & Tesh, S.A. (1986) Buprofezin: Teratology study in the rabbit. Unpublished report No. 86/NHH003/090 from Life Science Research Ltd., Suffolk, UK. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Tesh, J.M., McAnulty, P.A., Wightman, T.J., Goodsir, D.M., Wilby, O.K. & Tesh, S.A. (1987) Buprofezin: Teratology study in the rat. Unpublished report No. 87/NHH020/108 from Life Science Research Ltd., Suffolk, UK. Submitted to WHO by Nihon Nokyaku Co., Ltd., Tokyo, Japan. Trueman, R.W. (1988) Buprofezin: Assessment for the induction of unscheduled DNA synthesis in the primary rat hepatocyte cultures. Unpublished report No. CTL/P/2098 from ICI Central Toxicology Laboratory, Cheshire, UK. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Tsuchiya, K. & Sugimoto, T. (1979b) Acute oral toxicity study on Buprofezin in hamsters. Unpublished report No. T-1004 from Institute of Life Science Research, Nihon Nohyaku Co. Ltd., Osaka, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Tsuchiya, K. & Sugimoto, T. (1979c) Acute oral toxicity study on Buprofezin in rabbits. Unpublished report No. T-1005 from Institute of Life Science Research, Nihon Nohyaku Co. Ltd., Osaka, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Tsuchiya, K. & Sugimoto, T. (1982) Acute oral toxicity study on Buprofezin in rats. Unpublished report No. T-1003 from Institute of Life Science Research, Nihon Nohyaku Co. Ltd., Osaka, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Tsuda, S. (1984) Acute inhalation toxicity of Buprofezin (applaudR) to rats. Unpublished report No. T-1016 from the Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Uchida, M. (1988) Fate of 14C-Buprofezin in animal (rat)-Addendum. Unpublished report No. T-1058 from Institute of Life Science Research, Nihon Nohyaku Co. Ltd., Osaka, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Ueda, H. (1985) Buprofezin: Study on duodenal ulcer induction in rats. Unpublished report No. T-1060 from The Institute of Environmental Toxicology, Tokyo, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Watanabe, M. (1986) A 90-day oral subacute toxicity on Buprofezin in rats. Unpublished report No. ML-274A from preclinical Research Laboratories, Central Institute for Experimental Animals, Kanagawa, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Watanabe, M. Koizumi, H. & Yanagita, T. (1982) (received in 1989). 24-month toxicity study on ST-29285 (Buprofezin) in rats. Unpublished report No. ML-185 from preclinical Research Laboratories, Central Institute for Experimental Animals, Kanagawa, Japan. Submitted to WHO by Nihon Nohyaku Co., Ltd., Tokyo, Japan. Yoshida, A. (1983) (received in 1990). NNI-750 (Buprofezin): 24-month oral toxicity and oncogenicity study in mice. Unpublished report T-1019 from The Institute of Environmental Toxicology, Tokyo, Japan. 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See Also: Toxicological Abbreviations