First draft prepared by
Dr M. Miller
Center for Veterinary Medicine
Food and Drug Administration
Rockville, Maryland, USA
Diminazene is a veterinary drug with a long history of use for
the treatment of trypanosomiasis and babesiosis. Diminazene was
evaluated at the thirty-fourth meeting of the Committee (Annex 1,
reference 85). At that time the Committee was unable to establish an
ADI and suggested that additional genotoxicity and teratogenicity
studies should be performed before bringing the compound to the
Committee for re-evaluation. This monograph addendum, which
supplements the information provided in the original toxicological
monograph (Annex 1, reference 86), summarizes the data that have
become available since the previous evalution.
2. BIOLOGICAL DATA
2.1 Biochemical aspects
The excretion of diminazene into milk was examined in four
lactating cows, each treated with one i.m. dose of diminazine (3.56 mg
diminazene/kg bw). The diminazene content of milk collected at 7.5,
24, 31.5, 48, 55.5, 72, 240, 360, and 480 hours after application was
quantitated by an HPLC method with a detection limit of 50 ng/ml. The
results showed that the diminazene concentration in milk from all 4
cows was below the detection limit of the assay at all time points
(Klatt & Schmidt, 1991a).
Residue levels of diminazene were determined in plasma and
tissues of 14 young cattle following one i.m. administration of 3.56
mg diminazene/kg bw. Blood plasma levels were measured for 35 days
after application and tissues residue analyses were conducted in
liver, kidney, muscle, and injection site muscle at 21, 28, and 35
days post-treatment. The study reported that a plasma level of 1250
ng/ml was acheived one day after drug administration. This value
decreased to 350 ng/ml after 7 days. After 25-28 days, plasma levels
of diminazene were at or below the detection limit of the assay (50
ng/ml). The highest tissue concentration of diminazene occurred at
the injection site followed by liver and kidney. The lowest values
were found in the muscle (Klatt & Schmidt, 1991b).
2.2 Toxicological studies
2.2.1 Special studies on embryotoxicity
The embryotoxicity of diminazene dissolved in distilled water was
studied in mated female Wistar rats. Prior to the start of the study,
the females were mated with fertile males. Pregnant animals
(determined by detection of spermatozoa in the vaginal smear) were
randomly assigned to 4 groups of 22-24 animals each. The test groups
were gavaged daily on the seventh to sixteenth days of pregnancy with
5 ml of distilled water (control), or 200, 400, or 800 mg of
diminazene/kg bw/day. Behaviour and general physical condition were
assessed daily. Feed consumption was monitored continuously and body
weight was checked once weekly. On day 21 of pregnancy, the dams were
killed and delivered by caesarean section. The uteri were opened and
live and dead fetuses, concepta under resorption, placentas, and
corpora lutea were counted and examined microscopically.
Increased salivation was observed sporadically in several treated
animals at all doses. This effect was attributed to local irritation
resulting from the compound's administration. Doses of 200 and 400
mg/kg bw/day did not affect general physical condition, feed
consumption, or body weight development. The 800 mg/kg bw/day dose was
maternotoxic, causing a decrease in food consumption, an increase in
spleen weight, and the death of five dams.
No effects on intrauterine development of the conceptuses were
observed or the low and middle doses. Fetal development in dams
treated with 800 mg diminazene/kg bw/day was retarded, as indicated by
a slight increase in the number of fetuses with body weights under 3
g, poor skeletal ossification and low placental weights. Morphological
examination of fetuses from the treated dams revealed no
malformations. The incidence of minor anomalies and variations in
fetal development in the compound-treated groups were no higher than
incidences observed in the contemporary controls and were also within
the range of historical controls. Because materno- and embryotoxicity
were observed at 800 mg/kg bw/day, the NOEL was 400 mg/kg bw/day
under the study conditions (Baeder et al., 1991).
Diminazene dissolved in deionized water was administered to
pregnant rats daily by gavage on days 8-15 of pregnancy at dose levels
of 0 (vehicle only), 100, 250, 500, or 1000 mg/kg bw/day. On day 21 of
pregnancy, the dams were killed and the number of implants,
resorptions and live fetuses counted. All fetuses were examined by
routine teratological methods. A significant increase in fetal
resorptions and a decrease in fetal body weights were observed at 1000
mg/kg bw/day dose. No significant increase in the incidence of fetal
anomalies was observed in external, skeletal, and internal
examinations even at the maternally toxic dose of 1000 mg/kg. These
data suggest that diminazene is not teratogenic. The NOEL was 500
mg/kg bw/day (Yoshimura, 1990).
2.2.2 Special studies on genotoxicity
Table 1 summarizes the results of genotoxicity assays on
Table 1. Results of genotoxicity assays with diminazene
Test system Test object Concentration Results Reference
Ames test1 S.typhimurium 0-500 µg/ml Negative Muller, 1989
1537, 1538, 98
E. coli WP2uvrA
Micronucleus Mouse bone
test2 marrow 0 and 1500 mg Negative Muller, 1988a
Forward Fetal hamster 10-100 µg/ml Negative Muller,
mutation assay lung V79 (-S9 mix)3 1988b
(HGPRT locus) 10-150 µg/ml Negative
1 Both with and without rat liver S9 fraction.
2 Reviewed at the thirty-fourth Meeting of the Committee.
3 Ethylmethansulfonate was used as the positive control.
4 9,10-dimethyl-1,2-benzanthracene was used as the positive
Diminazene was negative in the micronucleus test and in bacterial
and mammalian genotoxicity assays. These data together with the data
from the subchronic toxicology studies, which showed no lesions
predictive of a carcinogenic response, suggest that there is no
concern for the carcinogenic potential of the compound.
The embryotoxicity of diminazene was examined in female Wistar
rats dosed by gavage with 0, 200, 400, or 800 mg diminazene/kg bw/day
on days 7 to 16 after mating. A dose of 800 mg/kg bw/day was
maternotoxic, causing a decrease in food consumption and increased
spleen weight and death. This dose also caused retarded fetal
development. The NOEL for this study was 400 mg/kg bw/day. In another
embryotoxicity study, diminazene was administered to rats by gavage on
days 8 to 15 of pregnancy at dose levels of 0, 100, 250, 500, or 1000
mg/kg bw/day. The 1000 mg/kg bw/day dose produced maternotoxicity and
a significant increase in the number of fetal resorptions and a
decrease in fetal body weights. The NOEL for this study was 500 mg/kg
bw/day. No fetal malformations were detected in either study.
The previous monograph on diminazene (Annex 1, reference 86)
summarized the toxicity studies in rats, dogs, and various
food-producing animals. At low doses, i.m. administration of
diminazene produced central nervous system lesions in several species
including cows, donkeys, camels, and dogs. In dogs fed diminazene at
20 or 60 mg/kg bw/day for nine months, the higher dose caused brain
damage, testicular atrophy and death. Rats dosed by gavage with 63 or
160 mg/kg bw/day for 3 months showed no signs of toxicity. This
indicates that the rat is not a sensitive model for evaluating the
toxicity of diminazene, including reproductive toxicity. The NOEL in
this study was 20 mg/kg bw/day.
In keeping with general principles for the evaluation of
veterinary drugs with a long history of use described at the fortieth
Meeting of the Committee (Annex 1, reference 104), an ADI of 0-100
µg/kg bw/day was established for diminazene based on the NOEL of 20
mg/kg bw/day in the nine-month study in dogs and using a 200-fold
safety factor. The Committee chose this safety factor to compensate
for the inadequacies in the design of the study.
BAEDER, C., MAYER, D. & LANGER, K.H. (1991). Oral embryotoxicity study
of Berenil in Wistar rats (Segment II). Unpublished report No.91.1190
from Pharma Development Toxicology, Hoechst Aktiengesellschaft,
Frankfurt, Germany. Submitted to WHO by Hoechst AG, Frankfurt,
KLATT, P. & SCHMIDT, D. (1991a) Report on the excretion of diminazene
aceturate in the milk of cows after one intramuscular administration
of the commercial preparation berenil(R) for veterinary use.
Unpublished report No. K1 90/1 from Abt. Forschung Tiergesundheit,
Hoechst AG, Frankfurt, Germany. Submitted to WHO by Hoechst AG,
KLATT, P. & SCHMIDT, D. (1991b) Report on blood plasma level
determinations and residue studies in organs of cattle after one
intramuscular administration of the commercial preparation
berenil(R) for veterinary use. Unpublished report No. K1 90/1 from
Abt. Forschung Tiergesundheit, Hoechst AG, Frankfurt, Germany.
Submitted to WHO by Hoechst AG, Frankfurt, Germany.
MULLER, W. (1988a). Diminazendiaceturat (Berenil) micronucleus test in
male and female NMRI mice after oral administration. Unpublished
report No.88.0248 from Pharma Research Toxicology and Pathology,
Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by
Hoechst AG, Frankfurt, Germany.
MULLER, W. (1988b). Berenil: Detection of gene mutations in somatic
cells in culture HGPRT-test with V79 cells. Unpublished report
No.88.0990 from Pharma Research Toxicology and Pathology, Hoechst
Aktiengesellschaft, Frankfurt, Germany. Submitted to WHO by Hoechst
AG, Frankfurt, Germany.
MULLER, W. (1989). Berenil: Study of the mutagenic potential in
strains of Salmonella typhimurium (Ames test) and Escherichia coli.
Unpublished report No.88.1425 from Pharma Research Toxicology and
Pathology, Hoechst Aktiengesellschaft, Frankfurt, Germany. Submitted
to WHO by Hoechst AG, Frankfurt, Germany.
YOSHIMURA, H. (1990). Teratological assessment of the antiprotozoal,
diminazene diaceturate, in rats. Toxicol Lett., 54: 55-59.