Toxicological evaluations

c Mixture of 3’-hydroxydiflubenzuron, 4-nitrochlorobenzene and N-acetyl-4-chlorophenylurea

The metabolism of 4-chlorophenylurea, a major plant metabolite of diflubenzuron, was studied in four male Fischer 344 rats that received [U-¹⁴C-phenyl]-4-chlorophenylurea (specific activity, 1.9 Ci/mol; radiochemical purity, 97%) in 1% gum tragacanth by gavage at 198 mg/kg bw. Urine and faeces were collected at 20, 48, 72, 96, 120 and 144 h (termination). The total radiolabel in urine, faeces and carcass was determined by liquid scintillation counting after appropriate processing. Pooled 0–20- and 20–48-h faecal and urine samples were examined by HPLC, thin-layer chromatography, liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry and comparison with standards. Metabolites in faeces were identified after sequential extractions with acetonitrile, hexane, acetone and water. Urine samples were also investigated after treatment with glucuronidase or sulfatase. Over 90% of the administered dose was excreted in urine. Four faecal metabolites were identified, all of which were also found in urine. The major faecal metabolite was 4-hydroxyphenylurea (about 3% of the administered dose), with unchanged 4-chlorophenylurea present at 0.6%. Thirteen distinct peaks were obtained from urine samples, the major components being 4-chlorophenylurea-2-sulfate (25% of the administered dose), 4-hydroxyphenylurea (18%), 4-chlorophenylurea-2-glucuronide (17%), 4-chlorophenylurea-3-sulfate (8%) and phenylurea-4-sulfate (7%). 4-Chloroaniline was not detected in urine at a limit of detection of < 0.02 µg/ml. The metabolic profile of 4-chlorophenylurea showed that it undergoes extensive ring hydroxylation and conjugation with either sulfate or glucuronide (Gay et al., 1999).

There is no clear reason for the marked differences between the findings of Cameron et al. (1990) and Wang and Gay (1999). The overall conclusion from these studies is that the primary steps in the metabolism of diflubenzuron are hydrolysis of the anilino ring, cleavage of the ureido bridge and conjugation (mainly to sulfate). The most recent data (Wang & Gay, 1999), and reputedly the most reliable, indicate that two compounds found as plant residues of diflubenzuron, 4-chloroaniline and 4-chlorophenylurea, might not be formed directly in rats. It is possible that hydroxylation of the anilino ring occurs before cleavage of the ureido bridge. Comparison of the results of Wang and Gay (1999) and Gay et al. (1999) suggests that 4-chlorophenylurea is not formed to a significant extent and that cleavage of diflubenzuron gives rise predominantly to 4-chloroaniline derivatives.

The concentrations of methaemoglobin and sulfhaemoglobin and the incidence of Heinz bodies were investigated in animals dosed once with a 25% w/w formulation of diflubenzuron. Groups of 15 male Swiss mice or male Wistar rats received Dimilin at 0 (co-formulants only) or 10 000 mg/kg bw in 1% gum tragacanth by gavage. Samples of blood were taken 4 and 24 h after dosing. In mice, there were no effects on body weight, sulfhaemoglobin concentration or incidence of Heinz bodies. The methaemoglobin concentrations were increased (p < 0.003) at 4 h but not at 24 h. The increase at 4 h was relatively small (2.2% versus 2.6%), but the ranges (1.3–2.5% versus 1.5–3.3%) indicated a treatment-related effect. A similar pattern was found in rats, with no effects on body weight, sulfhaemoglobin concentration or incidence of Heinz bodies. The methaemoglobin concentrations were increased (p < 0.01) at 24 h but not at 4 h. The increase at 24 h was relatively small (1.5% versus 1.9%), but there was a small overlap in the ranges (1.2–1.8% versus 1.6–2.1%), showing a clear treatment-related effect. A single high dose of diflubenzuron (2500 mg/kg bw) can thus increase methaemoglobin concentrations (Keet et al., 1997a,b).

The results of studies if acute toxicity show that diflubenzuron (purity, 99.6%) has little acute toxicity when given by the oral, inhalation or dermal route. The LD₅₀ in mice and rats given diflubenzuron in 1% tragacanth by gavage was > 4600 mg/kg bw (van Eldik, 1973). In rats treated dermally for 24 h, the LD₅₀ was > 10 000 mg/kg bw (Koopman, 1977), and in rats treated by whole-body inhalation of a preparation with a mass median aerodynamic diameter < 5 µm, the LC₅₀ was > 2.9 mg/l of air (Berczy et al., 1973). No clinical signs were seen in these studies, although the level of detail in the study reports was inadequate to permit independent confirmation. No haematological investigations were performed.

Groups of 40 Sprague-Dawley rats of each sex (controls, 90 of each sex) received diets containing diflubenzuron (two lots; purity, 96% and 97.2%) at a concentration of 0, 160, 400, 2000, 10 000 or 50 000 ppm. About half the animals were killed at week 7 and the remainder at 13 weeks. Routine clinical investigations were performed. Samples were taken from 10 animals of each sex per group for haematological, clinical chemical and urinary investigations in weeks 7 and 13. All animals were examined grossly, and extensive histological investigations were performed on controls and animals at the highest dose, with more limited examination (but including liver, spleen and marrow) of other groups.

There were no treatment-related effects on the mortality rate, clinical signs or food consumption. Body-weight gain was reduced by about 20% in females receiving 50 000 ppm and in males receiving > 2000 ppm, but with no clear dose–response relationship. Haematology at week 7 showed a range of dose-related alterations in erythrocyte parameters in animals of each sex receiving doses > 400 ppm, with minimal effects at 160 ppm (Table 4). The haematological findings were reproduced at week 13. Alterations in a number of clinical chemical parameters were seen in animals at the two higher doses, but the findings were sporadic, generally occurred in only a few animals in a group and considered to be not biologically significant. The absolute and relative weights of the spleen were increased in males and females at doses > 400 ppm and in males receiving 160 ppm for 7 weeks (Table 4). At week 13, the absolute weight of the liver was increased (by > 10%) in females at the highest dose, and the relative weights were increased (by > 10%) in males at the highest dose and females at doses > 400 ppm. Pathological lesions related to treatment were chronic hepatitis, haemosiderosis and congestion of the spleen and erythroid hyperplasia of the bone marrow in all treated groups, and haemosiderosis in the liver at doses > 400 ppm. The lesions increased in severity with increasing dose and duration of dosing (Table 4). No NOAEL could be identified, as there were small but statistically significant increases in methaemoglobin concentration, with associated findings in the spleen and bone marrow at the lowest dose, 160 ppm, equivalent to 8 mg/kg bw per day (Burdock et al., 1980; Goodman, 1980).

Groups of 10 Crl:CD BR (VAF/Plus) rats of each sex received diflubenzuron (purity, 96.7%) in 0.25% aqueous gum tragacanth dermally for 6 h/day for 21 days under semi-occlusive dressings at a dose of 0, 20, 500 or 1000 mg/kg bw per day on shaved, unabraded sites representing about 10% of the body area at the two higher doses and 1% at the lowest dose. Routine clinical investigations were performed. Blood samples for haematological and clinical chemical analysis were taken before sacrifice. All animals were examined grossly, and liver, kidney and skin from controls and animals at the highest dose were examined histologically.

One female at 500 mg/kg bw per day and one at the highest dose died on day 9. As these were isolated findings and there were no subsequent deaths, the relationship to exposure to diflubenzuron is uncertain. Body weight, food consumption, clinical signs, clinical chemical end-points and gross and microscopic appearance were unaffected. There were no signs of irritation at the application site. Reductions in erythrocyte parameters were seen in females at 500 mg/kg bw per day and in animals of each sex at 1000 mg/kg bw per day (Table 5), together with increased incidences and severity of polychromasia, hyperchromasia and anisocytosis in animals of each sex at doses > 500 mg/kg bw per day. Statistically significant increases in leukocyte counts were seen in males at > 500 mg/kg bw per day and a nonsignificant increase in females at the highest dose (Table 5). The findings were linked to increased numbers of segmented neutrophils and lymphocytes, but the association with diflubenzuron is unclear. The NOAEL was 20 mg/kg bw per day on the basis of haematological effects at doses > 500 mg/kg bw per day (Goldenthal, 1996).

Groups of 10 Sprague-Dawley rats of each sex were exposed for 6 h/day, 5 days/week for 4 weeks, by nose only, to atmospheres of diflubenzuron (purity, 96.5%), which were generated from a jet mill and contained particles with a mass median aerodynamic diameter of about 2 mm and a gravimetric concentration of 0.2 (control), 12, 34 or 110 mg/m³. The concentrations varied during the study by ± 10%, but homogeneity within the chamber was confirmed. The animals were observed routinely during and after exposure, with a basic neuro-functional assessment. Samples for urinary analysis, haematology and clinical chemistry were taken before sacrifice. All animals were examined grossly. A wide range of tissues from controls and those at the highest concentration were examined microscopically, with kidney, liver and lungs from all other animals.

One animal died during the terminal bleeding. Body weight, food consumption, clinical signs, urinary parameters and gross and microscopic appearance were unaffected. A reduction in ‘grid count’ was evident in the neuro-functional assessment of males and females exposed to 110 mg/m³. Statistically significant (p < 0.05) decreases in some erythrocyte parameters were seen at this concentration, with evidence of a concentration-related effect (Table 6). Slight but statistically significant increases in bilirubin concentrations were seen in both males and females at the highest level of exposure. The NOAEL was 34 mg/m³, approximately 10 mg/kg bw per day, on the basis of effects on behaviour, haematological end-points and bilirubin concentration at 110 mg/m³ (Newton, 1999).

Diflubenzuron (purity unknown) was reported not to be irritating to intact or abraded skin of rabbits, although the level of detail in the report was inadequate to permit independent confirmation (Taylor, 1973).

Diflubenzuron (purity, 99.6%) was slightly irritating to the eyes of New Zealand white rabbits after instillation of 40 mg (0.1 ml). The findings were similar when the eyes were rinsed 5 min or 24 h after administration (Davies & Liggett, 1973).

The skin sensitizing potential of diflubenzuron (purity, 95.6%) was investigated in groups of 10 CRL HA BR guinea-pigs exposed in a ‘maximization’ protocol. For induction, 10% diflubenzuron in maize oil with Freund’s complete adjuvant was injected or 30% diflubenzuron in Vaseline was applied topically. The animals were challenged by application of 10% or 30% diflubenzuron in Vaseline. Mild responses were seen after induction by either route. After challenge, mild reactions were seen in one control and two test animals. Diflubenzuron was not a skin sensitizer in this study (Prinsen, 1992).

Groups of three male and three female pure-bred beagle dogs aged 16–23 weeks received diets (400 g/day) containing diflubenzuron (purity unspecified) at a concentration of 0, 10, 20, 40 or 160 ppm for 13 weeks. The achieved intakes were 0, 0.4, 0.8, 1.6 and 6.4 mg/kg bw per day. The animals underwent routine clinical investigations. Samples for haematology were taken before dosing and at weeks 2, 4, 6 and 12. Clinical chemistry (limited), urine analysis and ophthalmological investigations were performed before dosing and at weeks 6 and 12. All animals were examined grossly at sacrifice, and an extensive range of tissues were examined microscopically. The results were presented for the two sexes combined.

There were no deaths or adverse clinical signs. Body weights and food and water consumption were not affected by treatment. The body weights fluctuated considerably during the study, but this was to be expected, given the spread of ages, and there was no indication of an association with treatment. The results of urine analysis, ophthalmology and gross and histopathological examinations were similar in all groups. Increases in aspartate aminotransferase activity were seen in animals at the highest dose in weeks 6 and 12, but the finding is of dubious significance as it was not reproduced when the samples were re-tested. Haematological end-points were similar in all groups at week 2, but, by week 4, the haemoglobin concentration of animals at 160 ppm was reduced by about 10%. At week 6, both the haemoglobin concentration and erythrocyte count were reduced and the methaemoglobin and free haemoglobin concentrations were increased in animals at 160 ppm. Haematological end-points were similar in all groups at week 12, although there was a clear increase in the myeloid:erythroid ratio in bone marrow in dogs at 160 ppm (1.2 versus 0.7 in controls). There was an indication of an increase in spleen weight (by about 40%) at 160 ppm, but inter-group variation was high and the ranges in treated and control groups showed considerable overlap. The NOAEL was 40 ppm, equal to 1.6 mg/kg bw per day, on the basis of alterations in haematological end-points and bone marrow at 160 ppm (Chesterman et al., 1974).

Groups of six male and six female beagle dogs (controls, 12 of each sex) received gelatin capsules containing diflubenzuron (purity, 97.6%) at a dose of 0, 2, 10, 50 or 250 mg/kg bw per day. The animals underwent routine clinical investigations. Blood samples for haematology and clinical chemistry were taken before dosing and at weeks 4, 7, 13, 26 and 51. Urine was analysed before dosing and at weeks 7, 13, 26 and 51. Ophthalmoscopy was performed before dosing and at weeks 26 and 51. All animals were examined grossly and microscopically.

One female at the highest dose died from liver failure and one female at 50 mg/kg bw per day died from pneumonia, but neither death was clearly related to treatment. No consistent differences were found between control and test animals in respect of clinical signs, food consumption, body weight, water consumption or urinary parameters. A range of effects related to impaired erythrocytes were seen at 50 and 250 mg/kg bw per day from week 13 onwards. At doses > 50 mg/kg bw per day, signs of haemolytic anaemia, destruction of erythrocytes and compensatory regeneration of erythrocytes were observed. Increases in methaemoglobin and sulfhaemoglobin concentrations were evident at doses > 10 mg/kg bw per day (Table 7). The severity of the haematological alterations appeared to increase with duration of dosing, stabilizing at week 26 and with some evidence of adaptation at week 52. Increased sulfhaemoglobin concentrations seen in animals at the lowest dose in week 26 were considered not to be adverse, as they were not statistically significant and were not seen at 52 weeks. Other findings included increased platelet numbers at doses > 10 mg/kg bw per day in females and > 50 mg/kg bw per day in males; increased lactic dehydrogenase activity from week 7 in animals of each sex receiving 250 mg/kg bw per day and increases in liver (10%) and spleen weights (35%) at doses > 50 mg/kg bw per day. The only notable histopathological findings were in the liver, comprising increased pigmentation of Kupfer cells and macrophages (Table 7). The latter effect in males at the lowest dose was considered not biologically significant as it was graded ‘minimal’ and the frequency was within the normal range of background findings. The NOAEL was 2 mg/kg bw per day on the basis of significant effects on methaemoglobin and sulfhaemoglobin concentrations, platelet counts and hepatic pigmentation at 10 mg/kg bw per day (Greenough et al., 1985).

Groups of 52 HC/CFLP mice of each sex (controls, 104 of each sex) received diets containing diflubenzuron (purity, 97.6%) at a concentration of 0, 16, 80, 400, 2000 or 10 000 ppm for up to 91 weeks. Groups of 24 controls and 12 mice receiving diflubenzuron were killed at weeks 26, 52 and 78 for interim investigations. Routine investigations were supplemented by haematology (weeks 26, 52, 78 and 91), clinical chemistry (weeks 24, 50, 76 and 89) and urine analysis (weeks 25, 51, 77 and 90). All animals were examined grossly, and a wide range of tissues from all animals were examined microscopically.

The homogeneity and achieved concentrations in the diet were acceptable, the mean achieved intakes covering an approximately 800-fold range (1.2–960 mg/kg bw per day; Table 8). The mortality rates were similar in all groups, survival to week 78 being > 50% except for females receiving 80 or 2000 ppm. Clinical signs (blue–grey colouration of the extremities) were noted at week 1 in animals receiving doses > 2000 ppm and subsequently in animals in all groups receiving doses > 80 ppm. Body weights and food consumption were similar in all groups. Water consumption was increased at some times for females receiving doses > 2000 ppm, with an associated increase in urine volume. Urine analysis did not indicate any impairment of renal function. Statistically significant, dose-related changes were seen in a number of haematological parameters from week 26 onwards, in animals receiving doses > 80 ppm (Table 8). Statistically significant increases in the concentrations of sulfhaemoglobin were seen in animals of each sex at 16 ppm at 52 weeks and in females at week 78. A dose-related increase in the incidence of Heinz bodies was seen consistently in mice at doses > 400 ppm and sporadically in males at 80 ppm. Increased serum alkaline phosphatase and aspartate aminotransferase activities were seen in males and females at the highest dietary concentration at week 26 and in males at this concentration subsequently. Gross pathological examination ahowed increased liver and spleen weights (Table 8) and cyanotic skin as the only consistent findings. Histopathological examination confirmed that the liver and spleen were the primary target organs for non-neoplastic effects (Table 8). No increase in tumour incidence was associated with administration of diflubenzuron. The increased sulfhaemoglobin concentrations at 16 ppm are of questionable biological significance as they occurred in only a proportion of animals in the group, and the frequency was within the range of fluctuating control values seen during the study. The NOAEL was 16 ppm, equal to 1.2 mg/kg bw per day, on the basis of increased methaemoglobin concentrations and incidences of cyanosis at 80 ppm. The NOAEL for carcinogenicity was the highest concentration tested, 10 000 ppm, equal to 840 mg/kg bw per day (Colley et al., 1984).

Groups of 60 CD rats of each sex were fed diets containing diflubenzuron (purity unspecified) at a concentration of 0, 10, 20, 40 or 160 ppm for 2 years. All animals were investigated for clinical signs, body weight, food consumption and gross and microscopic appearance of a limited range of tissues. Samples from controls and animals at the highest concentration were taken at 3, 6, 12, 18 and 24 months for determinations of haematological end-points (10 of each sex), limited clinical chemistry (five of each sex) and urinary parameters (five of each sex). Ophthalmological investigations were performed on controls and animals at the highest concentration at 3, 6, 12, 18 and 24 months. Haematological investigations were performed on animals at 40 ppm (10 of each sex) at 12, 18 and 24 months.

The achieved dietary concentrations and homogeneity were not confirmed. The survival rate was unaffected by treatment but was low, < 30% of animals surviving in all groups at termination. There were no treatment-related effects on urinary, clinical chemical or ophthalmic end-points, food consumption or body-weight gain. Statistically significant (p < 0.05) increases in methaemoglobin concentrations were seen at 12 and 18 months in males and females receiving 160 ppm but not in those at 40 ppm. Sulfhaemoglobin was unaffected by treatment, but the free haemoglobin concentration was reduced in groups at the highest concentration at 3 and 12 months. No increase in the incidence of tumours was seen in treated animals, although the poor survival and limited range of tissues examined severely limited the power of this study to detect a tumorigenic compound.

The NOAEL was 40 ppm, equivalent to 2 mg/kg bw per day, on the basis of haematological changes at 160 ppm. The NOAEL for carcinogenicity was the highest concentration tested, 160 ppm, equivalent to 8 mg/kg bw per day (Hunter et al., 1976; Colley & Offer, 1977)

Groups of 50 Sprague-Dawley rats of each sex (controls, 100 of each sex) were fed diets containing diflubenzuron (purity, 97.6%) at a concentration of 0, 160, 620, 2500 or 10 000 ppm for 2 years. All animals were investigated for clinical signs, body weight, food consumption and gross and microscopic appearance of a wide range of tissues. Haematological investigations were performed on 10 rats of each sex per group at 12 and 24 months. No clinical chemical, urinary or ophthalmological investigations were performed, but as these parameters were unaffected in rats at 160 ppm in a previous study, this is considered not to be a significant omission.

The achieved dietary concentrations and homogeneity were satisfactory. The survival rate was similar in all groups and was > 50% at termination. Food consumption was similar in all groups, but body-weight gain was reduced by > 10% in females at 2500 and 10 000 ppm from week 4, achieving statistical significance at week 26. A range of erythrocyte parameters were altered in both males and females at concentrations > 620 ppm at 12 and 24 months, although there was no marked progression with duration of dosing (Table 9). The methaemoglobin and sulfhaemoglobin concentrations were increased significantly in males at the lowest concentration at 12 months, but there was no clear dose–response relationship, and the biological significance of the finding is debatable. Increased numbers of reticulocytes and the extent of bone-marrow hyperplasia indicated the rats were attempting to respond to the effects on erythrocytes. The only gross finding was enlarged spleens, and this was confirmed by the data on organ weights. The main treatment-related histopathological findings were pigmented (golden brown) macrophages in the spleen and liver and erythroid hyperplasia of the bone marrow. The background incidence of pigmented macrophages in the spleen was high, however, and there was no clear dose–response relationship. Evidence of irritation in the stomach was seen in males receiving concentrations > 2500 ppm that survived to termination. The overall incidence of tumours was low, with no evidence of atypical findings or any dose–response relationship.