DAMINOZIDE
First draft prepared by Dr. S. Caroldi,
University of Padua,
Padua, Italy
EXPLANATION
Daminozide was previously evaluated by the Joint Meeting in 1977,
1983 and 1989 (Annex I, 28, 40 and 56). In 1989 an ADI was
established, based on a reproduction study in rats, at 0.5 mg/kg bw
daminozide containing less than 30 ppm 1,1-dimethylhydrazine (UDMH),
a contaminant and degradation product of daminozide. At that time,
the Meeting was aware that carcinogenicity bioassays of UDMH were in
progress. Three long term carcinogenicity studies, one in rats and
two in mice, were submitted to the 1991 FAO/WHO Joint Meeting for
evaluation.
EVALUATION FOR ACCEPTABLE INTAKE
Toxicological studies
Long-term/carcinogenicity studies
Mice
Ninety male and 90 female Charles River CD-1 mice (Charles River
Breeding Laboratories, approximately 6 weeks old at the beginning of
the study) were given UDMH at concentrations of 0, 1 or 5 ppm in
deionized tap water (with 25% citrate buffer to neutralize acidic pH)
equal to 0, 0.19, 0.97 mg/kg bw/day for males and 0, 0.27, 1.4 mg/kg
bw/day for females (calculated as average daily intake throughout the
duration of the study) for 24 months. Another 90 mice per group
received UDMH at concentrations of 10 ppm (males) and 20 ppm
(females), equal to 1.9 and 2.7 mg/kg bw/day, respectively. Test
article (100 mg/ml in 1 N HCl) was supplied by the producer in 26
different specimens containing 93 to 115% of the claimed concentration
(range of concentrations after being analyzed by IRDC). Test article
solutions were prepared and offered to the animals three times a week
(stable up to three days at all concentrations). The actual content
of UDMH in the solutions was checked twice monthly and showed 102%,
100%, 100%, 101% of nominal concentrations (mean of 51 determinations)
for 1, 5, 10, 20 ppm, respectively. Food and water were available
ad libitum.
No consistent dose-related differences in body weight were
detected throughout the study in any group which corresponded to
similar daily food and water intakes. At 6, 12, 18, 24 months from
the beginning of the study, the following haematological tests were
performed in 10 animals/sex group; leukocyte count, erythrocyte count,
haemoglobin, haematocrit, MCV, MCH, MCHC, platelets, differential
leukocyte count. At 24 months from the beginning of the study the
following biochemical tests were performed in 10 animals/sex/group:
total bilirubin, alkaline phosphatase, aspartate amino-transferase,
alanine amino-transferase, sorbitol dehydrogenase. Statistically but
not biologically significant differences of some haematological
parameters were detected at the 18 month interval in female mice at 5
and 20 ppm. At 24 months these differences disappeared. There were
no toxic effects detectable on biochemical tests at the 24 month
interval. At the end of treatment the mortality rate was 48%, 54%,
52% and 68% in males in the 0, 1, 5, 10 ppm dose groups and 60%, 50%,
64% and 76% in females in the 0, 1, 5, 20 ppm dose groups. The
mortality rate was significantly increased only in males at 10 ppm.
Pathology was performed at spontaneous death and at 8 and 12 months
(20 mice/sex/group) and at 24 months (remaining animals) from the
beginning of the study. Gross pathology showed an increased incidence
of macroscopic masses/nodules in the lung for the 12 to 24 month
period in males at 5 ppm (47%) but not at 10 ppm (26%) in comparison
to controls (21%) and in females at 20 ppm (43%) in comparison to
controls (12%).
Histopathological examination showed a variety of non-neoplastic
and neoplastic lesions in both sexes across dose levels. A
dose-related increase of brown pigment in the liver was noted in both
sexes. Although special stains were not performed to determine the
specific type of pigment, it appeared to be of the ceroid or
lipofuscin pigment type, sometimes haemosiderin or bile pigment.
Brown pigment was also present in some male mice at the highest dose
at the 8 and 12 month sacrifice but not in other groups. The
incidence of pulmonary neoplasms in females at the 20 ppm
concentration was increased. Alveolar/bronchiolar adenomas were found
in 20/49 dosed mice versus 5/49 in controls and alveolar/bronchiolar
carcinomas were 7/49 versus 1/49. These differences were
statistically significant and a positive statistical trend for
pulmonary neoplasms was found. The incidence of these neoplasms in
female controls was in the range of historical controls at IRDC (for
adenomas 6/69 and for carcinomas 2/69). The incidence of pulmonary
neoplasms was not increased in male mice up to 10 ppm UDMH. The
statistically significant increase of mortality rate in males at 10
ppm may be incidental because other clinical or morphological signs of
toxicity were lacking in these animals. The mortality rate is not
different from that calculated in controls in a subsequent study
performed in the same strain of animals by IRDC. On the basis of an
increased incidence of lung tumours in females at 20 ppm, the NOAEL in
the present study is 5 ppm corresponding to 1.41 mg/kg bw/day
(Goldenthal, 1989).
Ninety male and 90 female Charles River CD-1 mice (Charles River
Breeding Laboratories, approximately 6 weeks old at the beginning of
the study) were given UDMH at concentration of 0, 40 and 80 ppm in
deionized tap water (with 25% citrate buffer to neutralize acidic pH)
equal to 0, 7.34, 13.01 mg/kg bw/day for males and 0, 11.59, 21.77
mg/kg bw/day for females (calculated as average daily intake
throughout the duration of the study) for 24 months. Test article was
the same as supplied for previously cited study. Test article
solutions were prepared and offered to the animals three times a week
and was found to be stable at all concentrations. The actual content
of UDMH in the solutions was checked twice monthly and resulted 103%
and 102% of nominal mean of 54 determinations) for 20 and 40 ppm,
respectively. Food and water were available ad libitum.
The small number of surviving animals in the highest dose groups
and the great variability of data between weeks hampered evaluation of
mean body weight, water and food consumption. No consistent
dose-related differences in body weight were detected throughout the
duration of the study at 40 ppm for either sex nor at 80 ppm in males.
Body weight was reduced approximately 10% in females at 80 ppm during
the last 6 months of UDMH administration. Scattered significant
reductions in daily food intake suggests reduced average food
consumption for both sexes at the highest dose during the last months
of the study. Water consumption was reduced at both UDMH
concentrations for the duration of the study in males and during the
first 13 weeks only in females. A dose-related effect on some
haematological parameters was detected in males, but not in females
(except at 24 months when the small number of survivors hampered
statistical evaluation), starting 6 months (80 ppm) and 12 months (40
ppm) from the beginning of the study. A significant but not
dose-related increase of alanine amino-transferase and sorbitol
dehydrogenase was measured at 12 months in both sexes at both
concentrations. At the end of the treatment the mortality rate was
70%, 76%, 98% in males and 58%, 92%, 92% in females in the 0, 40, 80
ppm dose groups, respectively. The mortality rate was significantly
increased in both sexes at the highest dose. Gross pathology showed
accentuated liver lobulation in male mice but not in females at both
dose levels. Macroscopic nodules in the lungs and nodules/masses in
the liver were increased in males from 8 months onwards and in
females from 12 months onwards at both dose levels. Histopathological
examinations showed several signs of hepatotoxicity as: multifocal
chronic inflammation (in males at both dose levels prevalent in the
12-24 month section of the study), cell hypertrophy and necrosis (in
males at both dose levels detectable in all months of the study),
brown pigment (in both sexes detectable from 0-8 month section up to
the end of the study at both dose levels). Special stains were
performed to identify pigments. Haemosiderin, bile pigments, ceroid
and lipofuscin, the amount of collagen and reticulum were all
increased. Increased extramedullary haematopoiesis in the spleen of
both species occurred from 12 months onwards. At the end of the study
the incidence of vascular neoplasms (haemangioma and haemangiosarcoma)
in the liver was 9%, 67% and 81% at 0, 40, 80 ppm in males and 4%, 26%
and 82% at 0, 40 and 80 ppm in females. In mice sacrificed/dead during
the 8-12 months period of the study, the incidence of alveolar/
bronchiolar neoplasms was 18%, 45% and 55% at 0, 40 and 80 ppm in
males and 14%, 50% and 48% at 0, 40 and 80 ppm in females. At the end
of the study the incidence of these neoplasms was 54%, 73% and 51% at
0, 40 and 80 ppm in males and 31%, 53% and 56% at 0, 40 and 80 ppm in
females. High mortality and severe toxicity observed in treated
animals suggest that MTD was exceeded in both high and low dose group
animals. A NOAEL cannot be drawn from this study (Goldenthal, 1990).
Rats
Seventy male and 70 female Charles River Fischer 344 rats
(Charles River Breeding Laboratories, 6 weeks old at the beginning of
the study) were given UDMH at concentrations of 0, 1, 50, 100 ppm in
deionized tap water (with 25% citrate buffer to neutralize acidic pH)
equal to 0, 0.07, 3.2, 6.2 mg/kg bw/day for males and 0, 0.1, 4.5,
7.9 mg/kg bw/day for females (calculated as average daily intake
throughout the duration of the study) for 24 months. Test article
(100 mg/ml in 1 N HCl) was supplied by the producer in 26 different
specimens containing 93 to 115% of the target concentration. Test
article solutions were prepared and offered to the animals three times
a week (stable up to three days at all concentrations). The actual
content of UDMH in the solutions was checked twice monthly and results
showed 102%, 103% and 103% of nominal concentrations (mean of 50
determinations) for 1, 50, 100 ppm, respectively. Food and water were
available ad libitum.
Statistically significant but trivial reductions in body weight
(range 2-5%) were detected at 100 ppm in males and both at 50 and 100
ppm in females. No differences in food intake were detected.
Scattered reductions in water intake were noted in UDMH treated rats
at all concentrations; reduced water intake was more consistent at 50
and 100 ppm, in males during the last 20 weeks of the study and in
females throughout the entire study. No haematological effects were
detected at any time. No signs of UDMH related toxicity were noted in
any treated group during the study. At the end of the treatment the
mortality rate was 36%, 36%, 28%, 18% (males) and 32%, 24%, 28%, 10%
(females) at 0, 1, 50, 100 ppm UDMH, respectively. At the 12-month
sacrifice, there were no macroscopic or microscopic treatment-related
lesions. At the 24-month sacrifice, gross pathological effects were
comparable between groups except for the incidence of cloudy corneas
(slightly increased in females at 50 and 100 ppm with respect to
controls (37% and 41% instead of 27%)). This macroscopic alteration
corresponded histologically to higher incidence of corneal
mineralisation. There was no morphological evidence of
treatment-related hepatotoxicity. The incidence of pituitary adenoma
was increased in females at 100 ppm (56%) in comparison to that in
controls (32%). The incidence of total hepatocellular neoplasms was
increased in female rats at 50 ppm (10%) and 100 ppm (10%) with
respect to controls (0%) but not in male rats. In female rats the
incidence was 0%, 2%, 4% and 2% for hepatocellular adenoma and 0%, 0%,
6% and 8% for hepatocellular carcinomas at 0, 1, 50 and 100 ppm UDMH,
respectively. The historical incidence of hepatocellular neoplasms in
female Fischer rats at IRDC in 2-year chronic studies is 0.5% (2
adenomas/370 rats) which agrees with the low incidence observed in the
present study. The increase in hepatocellular carcinomas, a rare
neoplasm in female F344 rats at doses almost devoid of other toxic
effects strongly suggests an oncogenic effect of UDMH in female rats.
The NOAEL level in this study is 1 ppm UDMH in drinking water
corresponding to 0.09 mg/kg/day (Goldenthal, 1989a).
COMMENTS
Because of concern over the possible carcinogenic potential of
its contaminant and degradation product unsymmetrical
dimethylhydrazine (UDMH), the 1989 JMPR requested the results of
ongoing carcinogenicity bioassays of UDMH in rats and mice and
quantitative data on the conversion of daminozide to UDMH in
experimental animals, as well as results of observations in humans.
Administration of drinking water containing 0, 1, 5, 10 (male),
20 (female), 40 or 80 ppm UDMH to mice for two years was associated
with an increased incidence of pulmonary neoplasms in female mice at
20 ppm but not at 5 ppm (equal to 1.4 mg/kg bw/day) or in male mice up
to 10 ppm. At higher doses of 40 and 80 ppm, hepatotoxicity and high
mortality confounded interpretation, although pulmonary and hepatic
neoplasms occurred in both sexes.
UDMH similarly administered to rats in drinking water at 0, 1,
50, or 100 ppm induced hepatocellular neoplasms in females at 50 ppm
and 100 ppm but not in males at any concentration. The incidence of
pituitary adenomas was also increased in females at 100 ppm.
Nonetheless, UDMH was without carcinogenic activity to rats at 1 ppm
(equal to 0.09 mg/kg bw/day).
The Meeting noted the lack of carcinogenic potential of
daminozide and UDMH in a novel rapid in vivo bioassay system in F344
male rats (Cabral et al., 1991).
The Meeting concluded that the results of these bioassays were
consistent with its previous evaluation of the carcinogenicity of
daminozide containing up to 30 mg/kg UDMH and confirmed its previous
estimate of the ADI which was based upon the most sensitive
toxicological end-point, effects seen in a rat multigeneration study,
using a 100-fold safety factor.
Quantitative data on the conversion of daminozide to UDMH in
experimental animals, which had been requested, were not submitted.
Comparative in vitro studies of this conversion using appropriate
human and rat systems would be particularly relevant.
REFERENCES
Cabral, R. Hoshiya, T., Hakoi, K., Hasegawa, R., Fukushima, S. & Ito,
N. (1991) A rapid in vivo bioassay for the carcinogenicity of
pesticides. Tumori, 77: 185-188.
Goldenthal, E.I. (1989) Two-year oncogenicity study in mice.
Unpublished report No. 399-063 by IRDC, 500 North Main Street,
Mattawan, Michigan, USA. Submitted to WHO by Uniroyal Chemical
Company, Bethany, Connecticut, USA.
Goldenthal, E.I. (1989a) Two-year oncogenicity study in rats.
Unpublished report No. 399-062 by IRDC, 500 North Main Street,
Mattawan, Michigan, USA. Submitted to WHO by Uniroyal Chemical
Company, Bethany, Connecticut, USA.
Goldenthal, E.I., (1990) Two-year oncogenicity study in mice.
Unpublished report No. 399-065 by IRDC, 500 North Main Street,
Mattawan, Michigan, USA. Submitted to WHO by Uniroyal Chemical
Company, Bethany, Connecticut, USA.
See Also:
Toxicological Abbreviations
Daminozide (Pesticide residues in food: 1977 evaluations)
Daminozide (Pesticide residues in food: 1983 evaluations)
Daminozide (Pesticide residues in food: 1989 evaluations Part II Toxicology)