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.



    Toxicological studies

    Special Studies on Carcinogenicity


         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

         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).


         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


         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).


         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

         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.



         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.


         0-0.02 mg/kg b.w.


         Observations in man


    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

    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)