DIFLUBENZURON EXPLANATION Diflubenzuron was reviewed by the Joint Meeting in 1981 (Annex 1, FAO/WHO 1982a), at which time a toxicological monograph was prepared (Annex l, FAO/WHO, 1982b). The dog appeared to be the most sensitive species. However, the brief duration of the dog study precluded use of this study for estimating an ADI. Therefore a temporary acceptable daily intake (TADI) of 0-0.004 mg/kg b.w. was established based on the results of the rat study and on a safety factor of 500. Moreover, a dog study of adequate duration was required. Results of ongoing carcinogenicity studies in rats and mice were also required. These studies have been provided for the Meeting and are evaluated in this monograph addendum. EVALUATION FOR ACCEPTABLE DAILY INTAKE BIOLOGICAL DATA Toxicological studies Special Studies on Carcinogenicity Mouse Groups of HC/CFLP mice (104 males and females in control, and 52 males and females in each treated group) were administered diflubenzuron (purity 97.6%) in the diet at levels of 0, 16, 80, 400, 2000, and 10,000 ppm for 91 weeks. An additional 72 males and females for the control group and 36 males and females in each treated group were used for clinical pathology and pathological investigations for interim sacrifices. Detailed observations for clinical symptoms were performed weekly, as well as food consumption and body-weight determinations. Haematology, blood chemistry, urinalysis and water consumption were examined periodically througout the study. Interim sacrifices were performed at 6, 12 and 18 months and organ weights and macro/micropathological evaluations of selected tissues performed. At terminal sacrifice, remaining animals were subjected to complete gross and histopathological evaluations, along with organ-weight measurements. Clinical observations included blue/gray discoloration of the extremities and dark eyes in animals given > 80 ppm diflubenzuron. There were no treatment-related effects on mortality, body weight gain, food consumption, water intake, or urinalysis. Haematological evaluations demonstrated significant compound-related effects. The mean corpuscular haemoglobin concentration (MCHC) and mean corpuscular haemoglobin (MCH) were consistently increased in the 2000- and 10,000-ppm groups throughout the study. There was a treatment-related increase in platelet count at > 400 ppm, and Heinz bodies were found consistently in samples from mice treated with > 2000 ppm. The mean packed-cell volume (PCV) values were decreased in both sexes given > 2000 ppm for the first 52 weeks, in females only at 78 weeks and in males only at 91 weeks. A dose-related increase in methaemoglobin (6% total haemoglobin (THGB)) and sulfhaemoglobin were determined in both sexes througout the study at > 80 ppm. Significantly-elevated serum alkaline phosphatase (SAP) and serum glutamic-pyruvic transaminase (SGPT) activities, as well as cholesterol values, were observed the first 26 weeks in males given 10,000 ppm diflubenzuron. Significant increases in SAP and SGPT activities for 10,000-ppm males were also observed at most predetermined measurement intervals throughout the study, as well as 2,000-ppm males at termination. There were no treatment-related effects on other blood chemistry parameters during the study (e.g., glucose, protein, and albumin). There were no treatment-related effects or organ weights except for increased liver and spleen weights in both sexes at > 2000 ppm. Gross pathological examination demonstrated an increased incidence of chronic pneumonitis in males at > 80 ppm, but not in females at any level. There was a significant increase in extramedullary haemopoiesis in the spleen of males at > 400 ppm and females at > 2000 ppm; as well as increased siderocytes in males at > 400 ppm and females at > 80 ppm. Effects on the liver were evident predominantly in males, with hepatocyte enlargement, congested/dilated centrilobular sinusoids (> 400 ppm), increased extramedullary haemopoiesis, hepatocyte vacuolation and brown-pigmented Kupffer cells at > 2000 ppm. Females demonstrated increased hepatocyte enlargement at > 2000 ppm, and vacuolation, congested/dilated centrilobular sinusoids, brown-pigmented Kupffer cells, and fat deposition at 10,000 ppm. Neoplastic lesions were sporadically distributed among the treated and control mice of both sexes. There was no significant difference between control and treatment groups regarding neoplastic changes. There was no evidence of carcinogenicity from the ingestion of diflubenzuron by CFLP mice at dose levels up through 10,000 ppm. A NOEL of 16 ppm (2.4 mg/kg b.w.) was demonstrated for methaemoglobinemia and sulfhaemoglobinemia (Colley et al., 1984). Rat Groups of CR-CD Sprague-Dawley rats (50 of each sex per treated group, 100 of each sex in control group) were administered diflubenzuron (97.6% purity) in the diet at levels of 0, 156, 625, 2500, and 10,000 ppm for 104 weeks. All animals were observed daily for mortality and moribundity. Individual body weights, food consumption, and clinical signs were examined weekly througout the test period. Haematological evaluations were performed on 10 rats/sex/ group at 52 and 104 weeks. Clinical chemistry and urinalysis examinations were not conducted. Necropsies were performed on all animals, which included gross and histopathological examination. Organ weights were determined for brain, heart, liver, kidneys, spleen and testes w/epididymides (males) from all sacrificed animals. There were no compound-related effects on body-weight gain or food consumption, except for a decreased body weight for females in the 10,000-ppm group. Mortality in all groups averaged 25-50% at 104 weeks, with most deaths occurring after 80 weeks for females and 91 weeks for males. These deaths were not attributed to compound ingestion, although the confirmed presence of Sendai virus may have contributed. Symptoms of sialodacryoadenitis evident at 40 weeks were transient and generally clear by week 41. There were no compound- related effects noted on cage-side clinical examinations. Mean clinical haematology values for the myeloid:erythroid (M:E) ratio were decreased in both sexes in all treatment groups in comparison to controls. Reticulocyte counts for females were significantly increased throughout the study at > 2500 ppm, and for males at 10,000 ppm during the first 52 weeks. Females also demonstrated a significantly-increased mean-corpuscular volume (MCV) at > 2500 ppm throughout the study. Diflubenzuron produced a dose-related increase (both absolute or g/dl, and relative or % THGB, values; 2% THGB was Met HGB) formation of methaemoglobin and sulfhaemoglobin pigments, in both sexes at > 156 ppm. Heinz bodies, however, were not detected in samples analyzed. There was a significant increase in both absolute and relative spleen weights in animals of both sexes administered > 2500 ppm diflubenzuron. Enlarged spleens were also observed in these animals at gross necropsy. A significant increase in relative liver weights was also determined in females given > 2500 ppm diflubenzuron. Treatment-related histomorphologic changes were observed in the spleen, liver and marrow (sternum) of treated animals. Increased pigmented macrophages (indicative of haemosiderosis) were found in the spleen and liver of all dosed animals (156 through 10,000 ppm). There were also increased incidences of marrow hyperplasia, erythroid hyperplasia and distended marrow space in males given > 2500 ppm diflubenzuron. The finding of erythroid hyperplasia was also increased with dose in females given > 625 ppm diflubenzuron. These results correlated well with the decreased M:E ratio, substantiating the response of the marrow to changes in the peripheral blood (e.g. methaemoglobin and sulfhaemoglobin formation). A NOEL for methaemoblobin and sulfhaemoglobin formation was not determined. There was no evidence of neoplastic change resulting from tratment with diflubenzuron in either sex at doses up to and including 10,000 ppm (Burdock, et al., 1984. Short-term Study Dog Groups of Beagle dogs (6M/6F per dose group, 12M/12F in the control group) were administered diflubenzuron (purity 97.6%) via gelatin capsule 7 days per week in doses equal to 0, 2, 10, 50, and 250 mg/kg b.w. per day for 52 weeks. Food was offered to each dog approximately 1 hour prior to dosing. Water was available ad libitum. All animals were observed daily for mortality, general health and signs of toxicity. Food consumption was determined daily, and body weights were measured weekly. Water intake, haematology, urinalysis and clinical chemistry investigations were conducted routinely throughout the study. All animals were subject to complete gross and histopathological evaluation, as well as to ophthalmological examination (wks. 26 and 51) and organ-weight determinations. There were 2 deaths which were unrelated to treatment. There was no compound-related effect on body weight except for a slight decreased mean body-weight gain in the 250 mg/kg females. There were no effects on food consumption, water consumption, general appearance, ophthalmological evaluations, urinalysis, clinical chemistry determinations, relative organ weights or gross necropsy. Absolute liver and spleen weights were increased in the 50 and 250 mg/kg males. Histopathology was unremarkable except for the observation of pigmented macrophages and Kupffer cells in the liver of both sexes in all groups, including control. Although the incidence was slightly increased over controls at the low doses, the severity was not measurably increased until > 50 mg/kg diflubenzuron was administered to both males and females. Isolated evidence of erythrophagocytosis occurred within pancreatic lymph nodes in both sexes at the higher doses. These findings, suggestive of anaemia, are supported by the haematology parameters wherein both males and females demonstrated decreased HGB and MCHC, with an increased reticulocyte count at > 50 mg/kg. The percent sulfhaemoglobin formed was increased significantly at > 10 mg/kg in both sexes, while the percent methaemoglobin formed was increased at > 10 and 50 mg/kg in males and females, respectively. Both sexes were also positive for the presence of Heinz bodies, but only consistently at > 50 and 250 mg/kg for females and males, respectively. Heinz bodies are believed to be an insoluble denaturation product of haemoglobin, composed of electron-dense units. Erythrocytes containing Heinz bodies are phagocytized primarily in Kupffer cells, where the digestion of erythrocytes occurs within phagocytic vacuoles. The presence of a Heinz body within the erythrocytes reduces deformability of the cell, and this impedes its passage through the microcirculation of the spleen. Sequestration within the spleen may also lead to erythrophagocytosis or haemolysis of the more severely-damaged erythrocytes within the splenic pulp. Females also demonstrated increased mean corpuscular volume (MCV) and platelet count at > 50 mg/kg. Bone marrow examined in all animals, both grossly and microscopically, appeared normal in all groups. The NOEL in this study is 2 mg/kg b.w. based on the increase in methaemoglobin and sulfhaemoglobin (Greenough et al., 1985). COMMENTS Diflubenzuron was reviewed by the Joint Meeting in 1981 and a temporary ADI of 0-0.004 mg/kg body weight was estimated. Additional long-term feeding and carcinogenicity studies were required and these data have been evaluated by the 1985 Meeting. Dogs administered diflubenzuron orally for 1 year demonstrated effects consistent with methaemoglobinamia and sulfhaemoglobinaemia, with a NOEL of 2 mg/kg b.w. These data demonstrate that the Beagle dog is no more sensitive than rat or mouse to the formation of methaemoglobin and sulfhaemoglobin pigments from exposure to diflubenzuron. Carcinogenicity studies in rats and mice were negative for oncogenic effects at 10,000 ppm. Non-neoplastic changes observed were consistent with those associated with methaemoglobin and sulfhaemoglobin formation. While the observation of measurable pathological changes (e.g., hepatoctye enlargement/vacuolation and haemosiderosis of the spleen and liver) were not evident except at higher dietary doses of diflubenzuron, the Meeting nonetheless considered the elicitation of toxic methaemoglobinaemia to be the basis for estimating an ADI. TOXICOLOGICAL EVALUATION LEVEL CAUSING NO TOXICOLOGICAL EFFECT Rat: 40 ppm in the diet, equivalent to 2 mg/kg b.w. Mouse: 16 ppm in the diet, equivalent to 2.4 mg/kg b.w. Dog; 2 mg/kg b.w. ESTIMATION OF ACCEPTABLE DAILY INTAKE FOR MAN 0-0.02 mg/kg b.w. FURTHER WORK OR INFORMATION DESIRED Observations in man REFERENCES Burdock, G.A., Wolfe, G.W., Hepner, K.E., Alsaker, R.D., Koka, M., & (1984) Phipps, R.B. Oncogenicity study in rats. Diflubenzuron. Unpublished report from Hazleton Labs., Project No. 553-122. Submitted to WHO by Duphar. Colley, J., Warren, S., Crome, S.J., Heywood, R., Chanter, D.O., (1984) Street, A.E., Offer, J.M., Gibson, W.A., & Gopinath, C. The effect of diflubenzuron given by oral administration with the feed on toxicity and tumour development in male and female HC/CFLP mice. Unpublished report from Huntingdon Research Centre, Report No. PDR 360/831096/A. Submitted to WHO by Duphar. Greenough, R.J., Goburdhun, R., Hudson, P., & Macnaughton, F. (1985) Diflubenzuron 52 week oral toxicity study in dogs. Unpublished report from Inveresk Research International, Project No. 630146, IRI Report No. 2728. Submitted to WHO by Duphar.
See Also: Toxicological Abbreviations Diflubenzuron (EHC 184, 1996) Diflubenzuron (HSG 99, 1995) Diflubenzuron (Pesticide residues in food: 1981 evaluations) Diflubenzuron (Pesticide residues in food: 1983 evaluations) Diflubenzuron (Pesticide residues in food: 1984 evaluations) Diflubenzuron (JMPR Evaluations 2001 Part II Toxicological)