QUINOXYFEN. First draft prepared by I. Dewhurst 1 and V. Dellarco 2

Transcription

1 QUINOXYFEN First draft prepared by I. Dewhurst 1 and V. Dellarco 2 1 Pesticides Safety Directorate, Department for Environment, Food and Rural Affairs, Mallard House, Kings Pool, York, England; and 2 United States Environmental Protection Agency, Washington DC, United States of America Explanation Evaluation for acceptable daily intake Biochemical aspects Absorption, distribution and excretion (a) Oral administration (b) Dermal administration (c) Exposure by inhalation Metabolism Toxicological studies Acute toxicity (a) General toxicity (b) Ocular irritation, dermal irritation and dermal sensitization Short-term studies of toxicity (a) Oral administration (b) Dermal administration (c) Inhalational administration Long-term studies of toxicity and carcinogenicity Genotoxicity Reproductive toxicity (a) Multigeneration studies (b) Developmental toxicity Special studies (a) Induction of liver enzymes (b) Neurotoxicity Studies on metabolites Observations in humans Comments References Explanation Quinoxyfen is the International Organization of Standardization (ISO) approved name for 5,7-dichloro-4-(4-fluorophenoxy) quinoline, a halogenated quinoline fungicide that acts against powdery mildew in cereal crops. The proposed fungicidal mechanism of action is disruption of signal transduction by targeting of G-proteins.

2 368 Quinoxyfen has not been evaluated previously by the JMPR and was reviewed at the present Meeting at the request of the Codex Committee on Pesticide residues (CCPR). In dietary studies with quinoxyfen, variable levels of incorporation were used to achieve an approximately constant level of exposure throughout the study. All the pivotal studies contained certificates of compliance with good laboratory practice (GLP). The 2005 CCPR selected quinoxyfen as the second compound for a FAO/WHO/OECD pilot project on work-sharing (see Annex 1, reference 107). The text of the working paper was based extensively on existing documents prepared by regulatory authorities in Australia, the European Union and the United States of America (USA). Evaluation for acceptable daily intake Toxicity and toxicokinetic studies with quinoxyfen have been performed over a period of approximately 15 years starting in Most studies contained documentation stating compliance with the principles of GLP and the GLP status of the individual studies is identified in the summary text. All studies were considered to have complied with the basic requirements of the applicable Organisation for Economic Co-operation and Development (OECD) or equivalent national test guidelines, unless a comment to the contrary is given in the summary text. Quinoxyfen is also known by the development codes DE-795 or XDE Biochemical aspects 1.1 Absorption, distribution and excretion (a) Oral administration Rats The absorption, distribution, excretion and metabolism of quinoxyfen were investigated in Fisher 344 rats. The rats were aged 7 8 weeks and body weights were g in males and g in females. A series of tests were conducted using uniformly phenyl ring-labelled 14 C-quinoxyfen (radiochemical purity, 98.5%) or quinoline ring-labelled (radiochemical purity, > 99%) 14 C-quinoxyfen (Figure 1). The radiolabelled quinoxyfen was suspended in 0.5% aqueous methylcellulose and administered by gavage at a dose of 10 or 500 mg/kg bw to groups of male and female rats, including bile-duct cannulated animals. The doses were based on the findings of the 13-week dietary study in rats (Szabo et al., 1992). The stability and concentrations of quinoxyfen in the dosing solutions were confirmed analytically. Radioactivity in urine, faeces, cage wash, blood, plasma, organs and tissues were measured using liquid scintillation counting (LSC) after appropriate preparatory treatment. Metabolites in the urine, faeces, bile and blood were quantified, characterized and identified using high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) techniques. Reference standards were used as appropriate for the identification of metabolites. Enzymatic incubation techniques (using β-glucuronidase and sulfatase) and acid hydrolysis were used to separate and identify metabolites. Only a limited number of metabolites were identified.

3 369 Figure 1. Chemical structure of quinoxyfen and position of radiolabels 14 C-Phenyl-labelled quinoxyfen 14 C-Quinoline-labelled quinoxyfen F F * C l O C l O C l N C l N * * Position of radiolabel. In a preliminary study that complied with GLP, two groups of Fisher F344 rats (one male and one female) were administered by gavage a target dose of 10 mg/kg bw of either 2-14 C-quinoline ring-labelled or uniformly labelled phenyl ring-labelled 14 C-quinoxyfen. Urine, faeces, and expired air were collected at several time-points and analysed for radioactivity. At 72 h after dosing, the rats were killed and the radioactivity in tissues and remaining carcass was quantified. The distribution of radiolabel in excreta and in tissues and carcass is shown in Table 1. The total amount of radiolabel recovered by 72 h after administration of phenyl ring-labelled or quinoline ring-labelled 14 C-quinoxyfen was approximately 80-90% of the total administered radiolabel. For the phenyl ring-labelled treatment, renal excretion of radiolabel (45 49%) was greater than excretion in faeces (38 40%). However, quinoline ring-labelled 14 C-quinoxyfen was excreted predominantly in faeces (66 68%) and renal excretion (15%) was the minor route of excretion. The amount of radiolabel expired was minimal ( %) for phenyl and quinoline ring-labelled 14 C-quinoxyfen. Retention of radiolabel in tissues/carcass was low ( 2%) for phenyl- and quinoline-labelled 14 C-quinoxyfen. The findings suggest a rapid and almost complete absorption, distribution and metabolism of quinoxyfen. There were no apparent sex differences in the pattern of elimination of radiolabel. The differences in the distribution of radiolabel in the faeces between phenyl and quinoline-labelled quinoxyfen and urine suggests the separation of the phenyl and quinoline rings and that the metabolites produced have significantly different molecular weights and properties, hence the different routes of elimination. (Schumann et al., 1995) Table 1. Balance of radioactivity and excretion patterns in rats 72 h after dosing with 14 C-phenylor 14 C-quinoline-labelled quinoxyfen Sample Phenyl ring-labelled quinoxyfen at 10 mg/kg bw Radioactivity (% of administered dose) Quinoline ring-labelled quinoxyfen at 10 mg/kg bw Male Female Male Female Urine Faeces Final cage wash Expired 14 CO Tissues/carcass Total From Schumann et al. (1995)

4 370 In the main study, which complied with GLP, the balance and excretion patterns of 2-quinoline ring-labelled quinoxyfen and the amount of residual radioactivity in blood, organs and tissues were determined after the following dosing regimen: a single oral dose at 10 mg/kg bw after jugular-vein cannulation; a single oral dose at 500 mg/kg bw after jugular-vein cannulation; 14 repeated daily oral doses of unlabelled quinoxyfen at 10 mg/kg bw followed by a single dose of radiolabelled quinoxyfen at at 10 mg/kg bw, after jugular-vein cannulation; a single oral dose at 10 mg/kg bw after bile-duct cannulation; or a single oral dose at 500 mg/kg bw after bile-duct cannulation. The jugular-vein cannulated rats (five males and five females per group) were killed at 48 h after dosing. The bile-duct cannulated rats (three males per group) were killed at 24 h after dosing. Blood samples were taken from the jugular cannulae at 0.25, 0.5, 0.75, 1, 1.5, 3, 6, 12, 24 and 48 h. Urine, faeces and bile were collected regularly throughout the study. Table 2 Balance of radioactivity and excretion patterns in rats 48 h after dosing with 14 C-quinoline-labelled quinoxyfen Sample Radioactivity (% of administered dose) Single dose (mg/kg bw) Repeated doses a (mg/kg bw) Male Female Male Female Male Female Urine Faeces Final cage wash Expired 14 CO 2 NA NA NA NA Tissues/carcass Contents of g astrointestinal tract Total From Schumann et al. (1995) NA, not analysed. a Fourteen doses of unlabelled quinoxyfen followed by a single dose of radiolabelled quinoxyfen. Peak plasma radioactivity (C max ) was detected at approximately 0.5 h (2 3 µg equiv./g) at 10 mg/kg bw and approximately 1.5 h (80 90 mg equiv./g) at 500 mg/kg bw. The elimination of plasma radioactivity followed a biphasic pattern with half-lives (t½) for the rapid and slow phases for the dose at 10 mg/kg bw of < 1 h and h respectively, and 2 3 h and h for the dose at 500 mg/kg bw. There were no significant differences between the repeat-dose and single-dose groups. The elimination of radioactivity from blood followed a similar pattern. The area-under-the-curve (AUC) of the plasma versus time graph for the elimination of radiolabel in plasma (0 48 h) was 22.3, 27.3 and 922 µg equiv. h/g in males and 30.4, 29.6 and 963 µg equiv. h/g in females after treatments with a single radiolabelled dose, a single radiolabelled dose after repeated dosing and 500 mg/kg bw 14 C-quinoxyfen respectively. By 24 h after dosing, 68 85% of the administered radioactivity had been recovered in the faeces and urine, indicating rapid elimination from the body. After 48 h, 90-96% of the administered 14 C-quinoline ring-labelled quinoxyfen was recovered in the urine, faeces, cage wash and tissues (Table 2). The faeces represented the major route of elimination as 68 78% of the administered dose was eliminated via this route in 48 h, while 13 20% was eliminated in the urine. Urinary half-lives

5 371 ranged from 6 to 10 h. The tissues and carcass accounted for 1 7%, contents of the gastrointestinal tract for < 3% and final cage wash for < 1% of the administered dose. There were no sex differences in the distribution of radiolabel. Repeated administration of quinoxyfen did not affect the distribution of the radiolabelled dose. Comparison of the relative concentrations in bile and urine of intact and bile-duct cannulated rats (Tables 2 & 3) indicated that enterohepatic recirculation is extensive with quinoxyfen at a dose of 10 mg/kg bw. In bileduct cannulated rats, there was a marked difference in the amount of radiolabel in the faeces of rats at 10 mg/kg bw (14.3%) compared with those at 500 mg/kg bw (57.3%) and in the amount of radiolabel in the bile of rats at 10 mg/kg bw (54.4%) compared with those at 500 mg/kg bw (21.4%). These findings indicated that absorption of quinoxyfen at a dose of 500 mg/kg bw dose was saturated. Table 3. Distribution of radiolabel in bile-duct cannulated male rats 24 h after dosing with 14 C-quinoline-labelled quinoxyfen Sample Radioactivity (% of administered dose) 10 mg/kg bw 500 mg/kg bw Urine Faeces Final cage wash Carcass Contents of the g astrointestinal tract Skin Bile Total From Schumann et al. (1995) The distribution of radiolabel in organs and tissues (percentage of administered dose/g tissue) 48 h after treatment with a single dose of quinoline ring-labelled quinoxyfen at 10 mg/kg bw showed that the highest amounts of radiolabel were present in perirenal fat (0.12 in males and 0.35 females) > ovaries (0.07) > liver (0.027 in males and in females) and kidneys (0.014 in males and in females). Significant levels were found in the skin (Table 3). Similar amounts were obtained after repeated dosing and a similar pattern after the higher dose of 500 mg/kg bw. There were no data on tissue levels at times approximating to the plasma C max (Schumann et al., 1995) Goats The metabolism, distribution, and elimination of 14 C-quinoxyfen, labelled either in the phenoxy ring or the quinoline ring, was investigated in lactating dairy goats (n = 5; kg bw). Two goats were orally dosed with phenoxy 14 C-quinoxyfen (purity, > 98%), twice per day for five consecutive days, at a concentration of 10.7 mg/kg feed. Similarly, two goats were treated with quinoline 14 C-quinoxyfen, twice per day for five consecutive days, at a concentration of 11.7 mg/kg feed. The remaining goat was used as the untreated control animal. Urine and faeces were collected at intervals of 24 h until sacrifice. Milk samples were collected before the first treatment with quinoxyfen, and then twice daily throughout the study period until animals were sacrificed. The weights of urine, faeces, cage wash and milk samples were recorded, and total radioactivity was measured using LSC. Faecal samples were homogenized with water and subjected to combustion analysis before analysis by LSC. Goats were sacrificed 16 h after the final dose, and samples of the following fluids/tissues were collected for total radiolabelled residue (TRR) analysis: whole blood, plasma, liver, kidney, skeletal muscle, subcutaneous fat, omental fat, perirenal fat, gastrointestinal tract and contents, and carcass. Tissue samples were analysed for their TRR content using combustion analysis and LSC.

6 372 Concentrations of total radioactivity in tissues from individual goats for the phenoxy label were: liver, 1.03 and 0.93 mg/kg; kidney, 0.29 and 0.34 mg/kg; muscle, and mg/kg; perirenal fat, 0.18 and 0.19 mg/kg; omental fat, 0.20 and 0.17 mg/kg; and subcutaneous fat, 0.12 and 0.12 mg/kg. Concentrations of total radioactivity in tissues for the quinoline label were: liver, 0.94 and 1.5 mg/kg; kidney, 0.22 and and 0.17 mg/kg; muscle, and mg/kg; perirenal fat, 0.13 and 0.32 mg/kg; omental fat, 0.12 and 0.26 mg/kg; and subcutaneous fat, and 0.19 mg/kg. Concentrations of radioactivity in milk at 16 h after the final dose were and 0.11 mg/kg for the phenoxy label and and mg/kg for the quinoline label (Dunsire & Paul, 1995). (b) Dermal administration No data were available. (c) Exposure by inhalation No data were available. 1.2 Metabolism A proposed metabolic pathway for quinoxyfen is given in Figure 2. HPLC separation of pooled 0 12 h urine samples from the preliminary study in rats given phenyl-ring-labelled 14 C-quinoxyfen produced eight peaks that were designated P 1 P7 and P10. Peak P5 was the major urinary fraction in the unhydrolysed urine sample (80% and 77.4% in the male and female respectively) followed by P3 (7.9% and 9.5%), P 1 (4.2% and 4.7%) and P6 (3.1% and 3.0%). The remaining peaks contained less than 3% of the urinary radiolabel. Acid hydrolysis of the urine produced a significant change in the HPLC profile. The major fraction in unhydrolysed urine was reduced to only 2.4% of the urine fraction; instead, P8 (not detectable in the unhydrolysed sample) was the major fraction (73.6%). This suggested that P5 might be a conjugate of P8. The P8 fraction was found to co-elute with the standard for 4-fluorophenol. The remaining minor peaks after acid hydrolysis were P3 (11.7%), P9 (7.1%) and P 1 (5.2%), but these metabolites were not identified. The standards used were 4-fluorophenol, 2-hydroxy-quinoxyfen and parent quinoxyfen. Parent quinoxyfen and 2-hydroxy-quinoxyfen retention times did not correspond to that of any of the HPLC urinary fractions. Faecal metabolites from phenyl- 14 C quinoxyfen showed a similar pattern to those from quinoline- 14 C quinoxyfen No further data were provided on the metabolism of the quinoline ring-labelled 14 C-quinoxyfen treated rats from the preliminary study (Schumann et al., 1995). Analysis of blood samples showed that quinoxyfen was rapidly metabolized in rats, with the AUC for total radioactivity being approximately 30 times that for the quinoxyfen. HPLC separation of pooled urine samples from groups receiving quinoline- 14 C quinoxyfen in the main study before and after acid hydrolysis produced up to 16 radiolabelled peaks that were designated Q1 Q16. In male rats receiving a single dose at 10 mg/kg bw, eight peaks were identified in unhydrolysed urine: Q3, Q4, Q7, Q8, Q9, Q11, Q12 and Q13. In females rats receiving a single dose at 10 mg/kg bw, four additional peaks were detected: Q2, Q5, Q10 and Q15 (repeated-dose only). For the males and females at 500 mg/kg bw, the peaks in the radiochromatogram of pooled 0 12 h urine samples were Q7, Q8, Q9, Q10 (female only), Q11, Q12 and Q13. The major peaks were Q11 (13 33%), Q8 (13 24%), Q9 (9 24%), Q12 (10 18%), Q13 (6 15%), Q7 (4 11%) and Q4 (< 13%). The only clearly identified urinary metabolite was 5,7 dichloro-4-hydroxyquinoline (DCHQ), which was found to co-elute with peak Q13. Acid hydrolysis resulted in a two- to four-times increase in Q13 (24 65%). In the males rats receiving a single dose at 10 mg/kg bw, increases were observed in

7 373 peaks Q3, Q6 and Q14, while peaks Q1 and Q2 were increased after acid hydrolysis. In rats receiving a single dose at 500 mg/kg bw, peak Q14 was increased in males. Concomitantly, peaks Q4, Q5, Q7, Q8 and Q10 were observed to disappear from the respective radiochromatograms, while Q3 and Q9 were markedly reduced. Enzyme hydrolysis did not affect the metabolite profile. HPLC separation of bile samples (taken at various time-points, 0 24 h) from male rats receiving quinoline ring-labelled quinoxyfen at 10 mg/kg bw or 500 mg/kg bw produced six peaks (B2 B7). Peaks B6 and B7 constituted 20 66% and 26 59% of biliary excretion at various time-points. For the 10 mg/kg bw rats, the amount of radiolabel in the peaks at various time-points were B2 6 15%; B5 4 15%; B3 and B4 < 5%. Only peaks B6 and B7 were detected in the samples from rats at 500 mg/kg bw, but this was attributed to the higher detection limits required by the investigating laboratory. Additional peaks B1, B8, B9 and B10 were detected after enzyme hydrolysis. Peak B7 was noted to disappear with the appearance of peak B10 (approximately 25%) suggesting that B7 is the glucuronide or sulfatase of B10. Acid hydrolysis of the bile samples from rats at 10 or 500 mg/kg bw resulted in an increase in B6 by 20% of the biliary radioactivity compared with the control. Peaks B1, B8 and B9 appeared in the sample from rats at 10 mg/kg bw, but were not detected at 500 mg/kg bw. B10 eluted with quinoxyfen, but MS showed that the key ions were 16 mass units greater than the corresponding fragment ions from quinoxyfen. It was concluded that two metabolites associated with peak B10 were isomers of fluorophenyl-ring hydroxylated quinoxyfen. Faecal samples exhibited incomplete extraction, typically < 65%. At 500 mg/kg bw, the main faecal metabolite was F8, which co-eluted with quinoxyfen. The primary faecal metabolite at 10 mg/kg bw was F6 which was shown to consist of two hydroxylated isomers, equivalent to the bile metabolite B10. The pattern of metabolites in faeces was essentially independent of radiolabel position (Schumann et al., 1995) In a GLP-compliant study conducted in 2001, a group of four male Fischer F344 rats received quinoxyfen (purity, 97.4%; lot DECO ) at a dose of 500 mg/kg bw by gavage in 0.5% methylcellulose. Excreta were collected at 24-h intervals for 48 h after dosing, when the animals were sacrificed. The primary aim was to investigate hydroxylation at the 3- and 6- positions of the quinoline ring of quinoxyfen. Excreta samples were pooled, treated with sulfatase/glucuronidase, extracted and analysed using electrospray ionization LC/MS and LC/MS/MS to determine levels of 3-hydroxy and 6-hydroxy-quinoxyfen. Only limited data were presented in the report owing to the very low levels of hydroxy-metabolites detected. In the 0 24 h faecal sample, 0.012% of the administered dose was present as 3-hydroxy-quinoxyfen, primarily as a conjugate. Initially, low levels of 3-hydroxy-quinoxyfen were detected in urine samples, but this was not confirmed in subsequent analyses. Further analytical work by Pearson & Reeves (2005) indicated that the compound thought to be 3-hydroxy-quinoxyfen was actually 2-oxo-quinoxyfen. No detectable levels of 6-hydroxy-quinoxyfen were found in the urine or faeces, representing < % and < 0.004% respectively of the administered dose (Brzak, 2001). Goats TRR in excreta (urine, faeces), milk and tissues (kidney, liver, fat) from goats (see Dunsire & Paul, 1995, above) were characterized and/or identified by TLC and HPLC or GC/MS. The major component identified in liver was a conjugated form of quinoxyfen (approximately 10 15% TRR). The only components identified in kidney were quinoxyfen (approximately 3% TRR) and 5,7-dichlorohydroxyquinoline (DCHQ; approximately 2% TRR), with no apparent quinoxyfen conjugates present. In both kidney and liver, major portions of the TRR were characterized as polar based on TLC and HPLC characteristics. The major component present in fat was quinoxyfen (approximately 90% TRR), while milk contained quinoxyfen (approximately 40% TRR) and some very polar material. Small amounts of radioactivity corresponding to 4-fluorophenol, DCHQ, and

8 374 several hydroxy-quinoxyfen metabolites were also present in the liver. Small amount of radioactivity corresponding to 2-oxo quinoxyfen, DCHQ, and isomeric hydroxy quinoxyfens were found in the milk. For both labels, hydroxy metabolites and parent compound were the major components present in faeces, while the urine contained mainly a polar component that was easily hydrolysed to 4-fluorophenol or DCHQ (Dunsire & Paul, 1995). Figure 2. Proposed metabolic pathway for quinoxyfen in rats F Cl O Cl N quinoxyfen Bile Urine B7 F P8; ~70% Cl O O-Conjugate Q13; 24 65% Cl F O 4-Fluorophenol Fluorophenyl-ring-OH-quinoxyfen glucuronide or sulfate conjugate (two isomers) Cl OH and Conjugate N -Conjugate Cl N 5,7-Dichloro-4-hydroxyquinoline (DCHQ) Faeces F F B10 / F6 Approx. 25% Cl O Cl O OH Cl N Cl N quinoxyfen Fluorophenyl-ring-OH -quinoxyfen (two isomers)

9 Toxicological studies 2.1 Acute toxicity (a) General toxicity In a series of GLP-compliant studies, quinoxyfen was shown to be of low acute toxicity in rats and rabbits exposed orally, dermally or by inhalation (Table 4). The only signs of toxicity at the limit test doses in the studies of exposure orally or by inhalation were urinary and/or faecal staining in the perineal area, transient reductions in body weight or decreased activity. No clinical signs were reported in the study of dermal toxicity. Table 4. Results of studies of acute toxicity with quinoxyfen Species Strain Sex Route LD 50 (mg/kg bw) LC 50 (mg/l air) Purity (%) Vehicle Reference Rat F344 M & F Oral > Corn oil Gilbert (1994a) Rabbit NZW M & F Dermal (4-h) > None Gilbert (1994b) Rat Wistar M & F Inhalation (4-h, nose-only) > 3.38(MMAD, 3.6 µm) F, female; M, male; MMAD, mass median aerodynamic diameter; NZW; New Zealand White None Beekman (1994) (b) Ocular irritation, dermal irritation and dermal sensitization Quinoxyfen was not irritating to skin (Gilbert, 1994c), but was slightly irritating to rabbits eyes (Gilbert, 1994d). A study of skin sensitization by the Buehler method gave negative results (Gilbert, 1994e), but a second study using the Magnusson & Kligman maximization method produced clear evidence of skin sensitization (Johnson, 1995). 2.2 Short-term studies of toxicity Oral toxicity with quinoxyfen was investigated in 28-day dietary studies in rats and dogs, 90-day dietary studies in mice, rats and dogs; and in a 52-week dietary study in dogs. Dermal toxicity after repeat doses was investigated in a 28-day study in rats. No studies with repeated doses administered by inhalation had been performed. (a) Oral administration Mice In a GLP-compliant study, groups of 10 male and 10 female CD-1 mice were given diets containing quinoxyfen (purity, 98.7%) at variable concentrations to give nominal doses of 0 (control), 10, 50, 100 or 500 mg/kg bw per day for 13 weeks. The mice were observed for in-life effects and parameters including haematology, clinical chemistry, organ weights, gross and microscopic examination of organs and tissues. Histopathology of all organs and tissues was performed for the control group and for the group at 500 mg/kg bw per day, but the liver with gall bladder, kidneys, lungs and gross lesions only were examined for the groups at lower doses. At the start of treatment, the body weights were g in male mice and g in female mice. The daily intake of quinoxyfen was calculated from weekly assessments of feed consumption. The achieved mean daily intakes were 10, 50, 101 and 507 mg/kg bw per day in males and 10, 52, 105 and 523 mg/kg bw per day in females.

10 376 There were no mortalities or adverse effects on the general health, condition or behaviour of the animals during the study. No statistically significant treatment-related differences in feed consumption were observed compared to controls. Although not statistically significant, the mean body weight of males at 500 mg/kg bw per day was slightly lower than that of controls throughout the study (Table 5). However, body weights in females were not affected. There were no consistent, significant treatment-related differences in clinical chemistry and haematology parameters between treated and control animals. Gross examination at necropsy did not reveal any treatment-related findings. Terminal fasted body weights did not show any significant intergroup differences. There was a significant increase in the relative and absolute liver weight at the 500 mg/kg bw per day. Histopathology revealed slight or moderate centrilobular and midzonal hepatocellular hypertrophy at 500 mg/kg bw per day only. Very slight individual cell hepatocellular necrosis was observed in 3 out of 10 males and 4 out of 10 females at the highest dose (Table 5). The no-observed-adverse-effect level (NOAEL) was 101 mg/kg bw per day on the basis of liver weight increases, hepatocellular hypertrophy, and histopathological changes (including slight necrosis) in the liver at 500 mg/kg bw per day (Grandjean & Szabo, 1992). Table 5. Findings in mice receiving diets containing quinoxyfen for 90 days Finding a Males Nominal dose (mg/kg bw per day) Total leukocyte count ( 10 3 ) 6.9 ± ± ± ± ± 9.0 Albumin (g/dl) Terminal body weight (g) Liver weight, absolute (g) Liver weight, relative (% bw) * Hepatocellular hypertrophy (moderate) (n) * Hepatocellular necrosis (n) Females Total leukocyte count ( 10 3 ) 4.9 ± ± ± ± ± 2.4 Albumin (g/dl) Body weight (g) Liver weight, absolute (g) * Liver weight, relative (% bw) * Hepatocellular hypertrophy (moderate) (n) * Hepatocellular necrosis (n) From Grandjean & Szabo (1992) a n = 10 males and 10 females per group. * p < 0.05

11 377 Rats In a GLP-compliant study, groups of five male and five female Fischer 344 rats were given diets containing quinoxyfen (purity, 97.6%) at variable concentrations, to give target doses of 0 (controls), 250, 500 or 1000 mg/kg bw per day for 4 weeks. The rats were observed for in-life effects and parameters including haematology, clinical chemistry, urine analysis, organ weights, gross and microscopic examinations of organs and tissues. The histopathological examinations were limited to the liver, kidneys and testes. At the start of treatment, the body weight of male rats was g and that of female rats was g. The achieved compound intakes were 272, 549 and 1061 mg/kg bw per day in males and 267, 531 and 977 mg/kg bw per day in females. No mortalities occurred during the study period. There were no overt signs of toxicity. A dose-related reduction in food consumption was observed at all doses when compared with controls. Although the lower feed consumption in rats treated with quinoxyfen was reported by the investigating laboratory to be indicative of the unpalatability of the diet, it is noted that the target doses were achieved. Body-weight gains were statistically significantly reduced at all doses. The terminal body weights were 89%, 83% and 67% of the control values in males and 93%, 89% and 84% of the control values in females at doses of 250, 500 and 1000 mg/kg bw per day, respectively. Clinical chemistry, urine analysis and haematology investigations did not reveal any significant treatment-related changes; only a reduction in plasma concentrations of glucose occurred. Statistically significant changes in organ weights were considered to be probably related to the differences in body weight and, in the absence of any histopathological changes, of limited toxicological significance. Gross examination at necropsy revealed small bilaterally atrophic testes in rats in the group at 1000 mg/kg bw per day (three out of five male rats). Histopathology revealed a moderate to severe decrease in spermatogenesis of the seminiferous epithelium in the testes of four out of five rats. The changes in the morphology of the testes that was associated with reduced testicular weight were noted by the study investigator to be probably related to the body weight changes. These findings in the testes were notably not observed in subsequent studies in rats given lower doses. A NOAEL could not be determined due to reductions in food consumption and body-weight gain (7 11%) at the lowest dose tested, 250 mg/kg bw per day (Szabo & Davis, 1992). In a GLP compliant study, groups of 10 male and 10 female Fischer 344 rats were given diets containing quinoxyfen (purity, 98.7%) at a dose of 0 (control), 10, 100 or 250 mg/kg bw per day for 13 weeks. All animals were killed at the end of the treatment period and underwent necropsy. Additional satellite groups of 10 male and 10 female rats were fed doses of 0 or 250 mg/kg bw per day for 13 weeks and then kept on a quinoxyfen-free diet for an additional 4 weeks in order to investigate recovery from any effects. The achieved daily intakes of quinoxyfen were 10, 102 and 253 mg/kg bw per day) in males and 10, 100 and 249 mg/kg bw per day in females at nominal doses of 10, 100 and 250 mg/kg bw per day, respectively. The parameters investigated included in-life observations, a battery of functional tests, haematology, clinical chemistry, urine analysis, organ weights, gross and histopathological examinations of organs and tissues. Histopathology was limited to the adrenal glands, kidneys, liver, lungs, testes and gross lesions for the groups at 10 and 100 mg/kg bw per day. At the start of the study, the body weights of male rats ranged from to g (group means, g) and that of female rats g (group means, g). No mortalities occurred during the study period. There were no signs of treatment-related effects during regular observations and during the battery of functional tests on day 83. The changes in body-weight gain in males (overall body-weight gain was only 3 6% lower than that of controls

12 378 at doses of 100 mg/kg bw per day) were minimal and not of biological significance. However, reductions in body-weight gain were observed in females at 100 mg/kg bw per day (very slightly but statistically significantly lower than concurrent controls from day 63 to termination) and at 250 mg/kg bw per day (from day 14 onwards and including the recovery period). Terminal body weights in females were reduced by 14% and 16% at 100 and 250 mg/kg bw per day, respectively. There were no clear biologically significant treatment-related differences in the haematology of treated and control animals; reductions in concentrations of haemoglobin (3%) in males at the highest dose achieved statistical significance. Clinical chemistry showed statistically significant reductions in alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in males (11.4%, 19.3% and 19.6% respectively) at 250 mg/kg bw per day compared with controls, but the corresponding changes in females at the same dose (9.3%, 27.8% and 12.8% respectively) were not statistically significant. These observations were considered to be of no biological significance. Increases in blood albumin, total protein and potassium concentrations were observed at doses of 100 mg/kg bw per day in males and at 250 mg/kg bw per day in females compared with controls (Table 6). There was incomplete recovery from the clinical chemistry changes at the end of the recovery period. Urine analysis did not reveal any treatment-related changes. Gross examination at necropsy did not reveal any treatment-related abnormalities. Organ weights showed a significant increase in the mean absolute and relative liver weights at doses of 100 mg/kg bw per day in males (17 33%) and females (9 29%) compared with controls (Table 6). A slight increase in absolute and relative kidney weights in males and in relative kidney weight in females only was observed at 250 mg/kg bw per day compared with controls. At the end of the recovery period, the absolute and relative liver weights were slightly increased in males at 250 mg/kg bw per day compared with controls. Histopathology at 13 weeks revealed slight centrilobular and midzonal hepatocellular hypertrophy with increased basophilia at doses of > 100 mg/kg bw per day in the 90-day group. Panlobular hepatocellular hypertrophy with increased basophilia and very slight individual cell hepatocellular necrosis were observed in males and females at the highest dose only. At the end of the 4-week recovery period, histopathology of the liver showed that 8 out of 10 male rats in the group at the highest dose exhibited very slight centrilobular hepatocellular hypertrophy with increased basophilia, indicating a reduction of the effects on the liver (Table 6). The NOAEL was 10 mg/kg bw per day on the basis of reduction in body-weight gain, a lterations in clinical chemistry, increase in absolute and relative liver weight and changes in the histopathology of the liver (Szabo, Campbell & Davis, 1992). Table 6. Findings in a study in rats given diets containing quinoxyfen for 13 weeks followed by a recovery period Finding Week 13 Dose (mg/kg bw per day) Week 17 (recovery) Males Females Males Females Haemoglobin (g/dl) * * Platelets ( 10 3 ) * * * Albumin (g/dl) * 5.0* * Total protein (g/dl) * 6.7* * * *

13 379 ALP (U/l) * ALT (U/l) * ** AST (U/l) * ** 89 77* Potassium (meq/l) * 4.60* * Body weight (g) * 162* * Liver weight (g) * 9.67* * 5.40* * Liver (% body weight) ** 3.50** * 3.35* * Hypertrophy (centrilobular/ midzonal Hypertrophy (panlobular) Necrosis (hepatocellular) From Szabo, Campbell & Davis (1992) ± ± ± ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; +, slight; ±, very slight. * p < 0.05 by Dunnett s test, two-sided. ** p < 0.05 by Wilcoxon s test, two-sided. Dogs In a GLP-compliant study, conducted as a supplement to the 13-week dietary study, groups of two male and two female beagle dogs were fed diets containing quinoxyfen (purity, 98.7%) at nominal doses of 0 (control) or 250 mg/kg bw per day for 28 days. The parameters assessed were in-life observations, body weight and body-weight gain, feed consumption, haematology, clinical chemistry, urine analysis, organ weights, gross and histopathological evaluations. Blood samples for haematology and clinical chemistry were taken from fasted animals on days 13 and 24. Compound intake was determined from weekly assessments of feed consumption and body weight. Owing to unpalatability of the diet, the concentration in the calculated dietary intake was increased, resulting in increased unpalatability and lower food intake. The calculated weekly mean achieved dose was therefore affected and was mg/kg bw per day for both sexes. However, the calculated mean for individual dogs showed a greater range and was mg/kg bw per day for the 4-week period. Homogeneity and stability of quinoxyfen was shown to be acceptable in a previous 13-week dietary study in the dog (Wood & Szabo, 1992), hence no tests were conducted for this study. There were no deaths or signs of toxicity during the study period. Ocular abnormalities were not observed in ophthalmological examinations. Slight (males) to marked (females) reduction in feed consumption was observed at 250 mg/kg bw per day. Consequently body-weight loss occurred in males (4.2% and 8.2%) and females (16.3% and 21.9%), while the controls gained in body weight. Haematology, clinical chemistry and urine analysis and organ weight parameters did not show any clear treatment-related differences from values for controls. Gross examination of organs and tissues did not reveal any treatment-related changes. Histopathology of the liver showed slight vacuolation of the centrilobular and midzonal hepatocytes in treated animals. The vacuoles were of variable size and had a foamy appearance and were reported by the investigating laboratory to be suggestive of dilated endoplasmic reticulum rather than lipid accumulation. However, differential staining for lipids was not used or reported. This observation in the liver was noted to have been reported in an earlier investigation in dogs at doses of 500 and 1000 mg/kg bw per day (Szabo & Rachunek, 1992). There was no evidence of further treatment-related histopathological changes. A NOAEL could not be determined primarily due to the reduction in food consumption, bodyweight loss and histopathological changes in the liver at the nominal dose of 250 mg/kg bw per day (actual test dose, mg/kg bw per day) (Szabo & Davis, 1993).

14 380 In a GLP-compliant study to determine palatability and potential toxicity, one male and one female beagle dog were given diets containing quinoxyfen (purity, 98.8%) at nominal doses of 0 (control), 100, 500 or 1000 mg/kg bw per day for 30 days. The parameters assessed were in-life observations, body weight and body-weight gain, feed consumption, haematology, clinical chemistry, urine analysis, organ weights, gross examination of organs and tissues and histopathological evaluations of the kidney, liver, testes and gross lesions. Body weights and feed consumption were determined weekly. At the start of treatment, the bodyweights ranged from kg in males and kg in females. The study was performed in accordance with OECD guideline No The study design was based on a previous oral study (via capsules) in dogs (one male and one female per dose) with doses of mg/kg bw per day for 2 weeks (Weaver, 1988). In that study, body weight, feed consumption, electrocardiograms, enzyme induction, clinical chemistry, haematology and pathology of organs and tissues did not show any treatment-related changes. In the present study, the achieved daily intakes of quinoxyfen were 86, 340 and 445 mg/kg bw per day in males and 130, 305 and 481 mg/kg bw per day in females at nominal doses of 100, 500 and 1000 mg/kg bw per day. There were no mortalities or signs of toxicity during the study period. Feed consumption was reduced in all treated males and at doses of 500 mg/kg bw per day in females. The pattern of feed consumption was considered by the reporting laboratory to indicate a high degree of unpalatability. Body-weight loss was observed in males at doses of 500 (11%) and 1000 (35%) mg/kg bw per day and in females at doses of 500 (20%) and 1000 (30%) mg/kg bw per day compared with values for controls. Body-weight gain in males at 100 mg/kg bw per day was reduced (30%) compared with values for controls. There was a very slight statistically non-significant reduction in erythrocyte count, erythrocyte volume fraction and haemoglobin content in females at the highest dose compared with pre-test and control values. Clinical chemistry and urine analysis did not show any significant treatment-related changes. Organ weights were not reported. The testes in the male at the highest dose and the thymus in the male and female at the highest dose appeared to be small/atrophic on gross examination. However, microscopic examination of the testes did not reveal any abnormality. The main histopathological findings were slight multifocal hepatocellular necrosis in females at 500 mg/kg bw per day and slight (500 mg/kg bw per day) or moderate (1000 mg/kg bw per day) centrilobular and midzonal hepatocellular vacuolation in both sexes. Moderate multifocal vacuolation of the proximal convoluted tubule epithelium was observed in both sexes at 1000 mg/kg bw per day. Severe diffuse lymphoid depletion of the thymus was observed in both sexes at 1000 mg/kg bw per day. The NOAEL was mg/kg bw per day (100 mg/kg bw per day nominal dose) on the basis of body-weight loss and histopathological changes in the liver [including slight multifocal hepatocellular necrosis] at mg/kg bw per day (nominal dose, 500 mg/kg bw per day). Impaired body-weight gain and food consumption in the male dog at 100 mg/kg bw per day was considered to result from unpalatability and was therefore of limited toxicological relevance in setting the NOAEL (Szabo & Rachunek, 1992). Groups of four male and four female beagle dogs were fed diets containing quinoxyfen (purity, 98.7%) at nominal doses of 0 (control) 10, 50 or 100 mg/kg bw per day for 90 days. The parameters assessed were in-life observations, body weight and body-weight gain, feed consumption, haematology, clinical chemistry, urine analysis, organ weights, gross and histopathological evaluations. The achieved mean daily compound intakes were 10, 50 and 100 mg/kg bw per day in males and 10, 50 and 101 mg/kg bw per day in females at nominal doses of 10, 50 and 100 mg/kg bw per day. At the start of treatment, body weights ranged from 8.19 to kg in male dogs and kg in female dogs. No mortalities occurred during the study period. There were no adverse effects on animal behaviour. Ophthalmological examinations at the beginning and on day 87 of the study did not reveal any treatment-related ocular defects. A slight transient reduction in food consumption was observed

15 381 in the highest dose males but the animals adapted to the initial unpalatability. However, it was noted that females at the highest dose consumed more feed than did controls. Body-weight gain was not affected. Clinical chemistry, urine analysis and haematology parameters did not show any biologically significant treatment-related changes. The terminal body weights and organ weights did not show any significant treatmentrelated intergroup differences; increased relative liver weights in the groups at 50 mg/kg bw per day were not reproduced at 100 mg/kg bw per day. Gross examination at necropsy did not reveal any significant treatment-related changes. Histopathological examination revealed only a single incidence of slight and midzonal centrilobular hepatocellular hypertrophy, with no associated clinical chemistry or additional histopathology findings, which indicated that the dose of 100 mg/kg bw per day may represent a threshold dose for histopathological effects on the liver (Table 7). The NOAEL was 100 mg/kg bw per day. The single occurrence of hepatocellular hypertrophy, with no associated clinical chemistry or other histopathological findings, was not considered to be adverse (Wood & Szabo, 1992). Table 7. Hepatic findings in dogs fed diets containing quinoxyfen for 13 weeks Finding Male Dose (mg/kg bw per day) Female Body weight (g) Liver weight (g) Liver (% bw) * * 2.98 (range) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) Centrilobular & midzonal hepatocyte hypertrophy From Wood & Szabo (1992) * p < /4 0/4 0/4 1/4 0/4 0/4 0/4 0/4 In a GLP-compliant study, groups of four male and four female beagle dogs were fed diets containing quinoxyfen (purity, 96.2%) at a dose of 0 (control) 5, 20 or 200 mg/kg bw per day for 52 weeks. The parameters assessed were in-life observations, ophthalmological examinations, body weight and body-weight gain, feed consumption, haematology, clinical chemistry, urine analysis, selected organ weights, gross examinations of organs and tissues and histopathological evaluations. The mean achieved compound intakes were 6, 20 and 194 mg/kg bw per day in males and 5, 21 and 203 mg/kg bw per day in females at nominal doses of 5, 20 and 200 mg/kg bw per day respectively. At the start of treatment, the dogs were aged 7 months and body weights ranged from 10.4 to 12.1 kg (group means, kg) in male dogs and from 7.8 to 8.8 kg (group means, kg) in female dogs. One male dog at 200 mg/kg bw per day died on day 62 with symptoms suggestive of aspiration pneumonia, but a severe systemic disease consistent with canine parvovirus infection was also present. Decreased activity, pale mucous membranes, dehydration, weakness and in addition, blood, vomitus, runny and watery stools in the cage were observed before death. A second dog at 200 mg/kg bw per day was humanely killed on day 119 after evidence of treatment-related anaemia and weight loss (approximately 2 kg). Eye examinations performed before the start of treatment and 1 week before termination did not reveal any treatment-related findings. Feed consumption was variable but generally reduced in male and female dogs at 200 mg/kg bw per day compared with dogs in the

16 382 control group. This was observed from the first week of treatment and was considered to be suggestive of very slight unpalatability at the highest dose. It was noted that this did not affect the achievement of target doses. Body-weight gain was affected and the body weights of males and females at the highest dose were lower than those of controls throughout the study. The mean terminal body weights were reduced in males (14.5%) and females (9.25%) at 200 mg/kg bw per day compared with those of dogs in the control group. Haematological tests at 3, 4, 6, 7, 8 and 12 months showed marked reductions in haemoglobin concentration, erythrocyte volume fraction, erythrocyte and leukocyte counts in one male dog at 200 mg/kg bw per day at 3 and 4 months compared with pre-study and inter/intra-group values. Peripheral blood smears from this dog, which was killed and necropsied on day 119, showed marked polychromasia and moderate hypochromasia after 3 months and additionally slight anisocytosis on day 119. A female dog at 200 mg/kg bw per day also showed reductions in haemoglobin concentration, erythrocyte volume fraction and erythrocyte counts from 3 months onwards. Microscopic examination of peripheral blood smears revealed slight hypochromasia. However, there was apparent compensation of anaemia in the female dog and the unscheduled haematological assays were discontinued after 8 months. Extramedullary haematopoiesis was seen in most dogs at 200 mg/kg bw per day. Clinical chemistry showed an increase (greater than twofold) in serum ALP activity in male and female dogs at 200 mg/kg bw per day compared with controls. The magnitude of the increase in ALP exhibited a clear relationship with duration of dosing. Increased serum ALP activity might be indicative of cholestasis (impaired hepatobiliary function), enzyme induction or hepatocellular damage. Transient or non dose-related changes in bilirubin and cholesterol were considered to be not treatment-related. Urine analysis at 6 months and at necropsy did not reveal any significant treatmentrelated changes. The absolute (27.5% in males and 9.4% in females) and relative liver weights of male and female dogs at 200 mg/kg bw per day were increased compared with controls and were considered to be treatment-related (Table 8). The remaining changes in relative organ weights of the brain, kidney and pituitary in dogs at 200 mg/kg bw per day were considered to be related to the lower body weight of this group and, in the absence of any supporting histopathological or functional evidence in these organs, not treatment-related. Histopathology of the liver showed an increase in the incidence of diffuse hepatocyte hypertrophy (three out of four males and three out of four females) and increased bile in bile canaliculi (one out of four males and one out of four females) in both sexes at 200 mg/kg bw per day. No treatment-related changes were observed in the kidneys and testes. The NOAEL was 20 mg/kg bw per day on the basis of reduced food consumption and bodyweight gain, haematological changes consistent with haemolytic anaemia and a compensatory response, increased serum activity of ALP, increased liver weight and histopathological changes in the liver at 200 mg/kg bw per day (Cosse et al., 1995). Table 8. Hepatic findings in dogs a fed diets containing quinoxyfen for 52 weeks Finding Dose (mg/kg bw per day) Male Female b Body weight (g) Liver weight (g) Liver (% bw)