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.