DAMINOZIDE
EXPLANATION
Daminozide was considered by the Joint Meeting in 1977 and 1983
(Annex 1, FAO/WHO 1978a, 1984). The following information was
required before an ADI could be considered:
1. adequate data to assess the carcinogenicity of daminozide;
2. an adequate teratology study; and
3. information on biotransformation in animals.
Additional data are reviewed in this monograph addendum.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOLOGICAL DATA
Biochemical aspects
Absorption, distribution and excretion
Rat
Groups of two male Sprague-Dawley rats/dose group were given a
single oral dose of 30, 300 or 3000 µg of [14C-methyl]daminozide by
gavage. One additional male served as a control. The primary route
of excretion of radioactivity was the feces: 63-86% (dose-related)
of the dose was recovered in feces in 48 hours. At all doses, about
10% of the administered radioactivity was recovered in urine.
Excretion as CO2 accounted for 2-16% of the dose and was inversely
dose-related. Tissue levels at 48 hours accounted for 1.9-8.8% of
the dose and was inversely dose-related indicating that tissue
accumulation is unlikely. Excretion was rapid: 74-87% of the dose
within 12 hours and 91-96% within 24 hours (Milad et al. 1984).
In another study in which 14C-daminozide was applied dermally
to 24-28 male Sprague-Dawley rats/group at doses of 0.05, 0.5 or
5 mg, 98.6-100% of the administered dose was recovered from the site
of treatment after up to 24 hours exposure. At 5 mg, about 0.75% of
the dose was recovered in the carcass after a 24-hour exposure
period. Carcass residues were negligible at shorter exposure times
and at the lower doses (Chadwick & Silveira, 1987).
Guinea pig
Groups of 15 male Hartley guinea pigs were given 2 ml oral
doses of aqueous solutions of [14CH3]-daminozide at
concentrations of about 13 or 135 ppm. At both dose levels about
10% of the dose was recovered in urine mainly in the first 12 hours
after dosing. Fecal excretion accounted for about 13% of the low
dose and 25% of the high dose, again mainly in the first 12 hours
after dosing. CO2 was collected from two animals/dose level and
accounted for 46% of the low dose and 13% of the high dose
radioactivity. GI tract and contents had high levels of
radioactivity at 6 hours but only 1.5% of the dose at 48 hours.
Liver levels were 4-5% of the dose level, declining slightly from 6
to 48 hours. Blood, lung and kidney levels accounted for less than
1% of the dose at all intervals. Total recovery in this study was
low (Novak & Ambrose, 1983a).
Groups of 15 male Hartley guinea pigs were given 2 ml oral
doses of aqueous solutions of 14C-UDMH at about 5 or 150 ppm
(actually 5.6 or 186 ppm). About 16-19% was recovered in urine
within 48 hours with most recovered in the first 4 hours. Fecal
excretion accounted for 6% of the low dose and 12% of the high dose
and was recovered mainly in the first 24 hours. CO2 was collected
from two animals/dose level and accounted for about 40% of the dose
at both dose levels. GI tract and contents contained about 16% of
the dose at 2 hours but 2-4% of the dose at 48 hours. Liver levels
were 17-19% of the dose at 2 hours declining to 10% of the dose at
48 hours. Blood levels were about 3% at 2 hours and 1% at 48 hours
(Novak et al. 1983b). Autoradiography of the stomach and duodenum
from these animals indicated that in the stomach radiolabel was
localized in epithelial cells and in the duodenum in the lower
mucosa. The muscularis layer in both tissues contained very little
radioactivity (Lengen & Frederick, 1984).
Biotransformation
Tissue analyses indicated that, of the administered dose,
unchanged daminozide comprised 0-30%, dimethyl hydrazine 0-40%,
1,1-dimethylhydrazine 0-3%, and unidentified metabolites 1-30%,
suggesting that 1,1-dimethylhydrazine is a transient metabolite
(Frederick et al. 1984a,b).
Toxicological studies
Short-term study
Dog
Groups of 6 male and 6 female beagle dogs were given diets
containing 0, 300, 3000 or 7500 ppm of daminozide (99% pure) for one
year. Clinical observations, body weight and food consumption were
recorded throughout the study. Physical examinations were made every
3 months and ophthalmoscopic examinations pre-test and at
termination. Blood samples were taken at 0, 6 and 12 months for
analyses of hematological and clinical chemistry parameters. Urine
samples were collected at the same intervals.
One female dog at 7500 ppm died with acute hemorrhagic
enteritis on Day 336. The males at 3000 and 7500 ppm had body
weights 5% and 5-8%, respectively, higher than controls by the end
of the study. The biological significance of these findings is
doubtful. There were no other apparent treatment-related effects at
any dose level. One female at 7500 ppm had a renal cell adenoma but
no other kidney effects were noted in any other animal. There were
a few difficulties in the conduct of the study; achieving
homogeneity of mixing of the diet at the low dose level and
observations of convulsions in a few of the dogs including one
control. However, the NOAEL (considering the body weight effects to
be treatment-related but the renal cell adenoma incidental) was
7500 ppm (equal to 193-209 mg/kg bw/day (males/females) (Johnson
et al. 1988c).
Long-term/carcinogenicity studies
Rats
Groups of 50 male and 50 female Charles River CD-1 mice were
given diets containing 0, 300, 3000, 6000 or 10000 ppm daminozide
(99.8% pure) for 105 weeks. Clinical observations, body weight gain
and food consumption were measured throughout the study. Blood
samples were taken from 10/sex/group for hematological studies at
12, 18 and 24 months. All survivors to 105 weeks were sacrificed.
All animals which died or were sacrificed were given a complete
post-mortem examination. Sections of kidney, liver and lungs from
all animals were examined for histopathology. Sections of about 37
other tissues/organs were examined for the sacrificed control and
high dose animals, and all animals which died or were sacrificed in
extremis from all groups.
Mortality was slightly higher in the high dose males than in
the other groups: 50% mortality was observed at week 85 in the high
dose males compared to weeks 96-103 for other groups of males and
weeks 99-105 for all groups of females. The high dose females had
slightly lower bodyweights (approx. 5%) than controls during weeks
1-7 but all groups were similar for the rest of the study. RBC
counts were lower than controls in the high dose males at 18 months
and the high dose females at 24 months. Platelet counts were very
variable; mean values were lower than controls in females at 3000,
6000, and 10000 ppm but no pattern was obvious in males. In males
at 6000 and 10000 ppm there were increased incidences of brown
pigment (probably hemosiderin and bile pigments) in the liver.
Brown pigment was observed in the adrenal cortex of both sexes at
10000 ppm and increased extramedullary hematopoiesis in spleen was
observed in males at 10000 ppm. Lymph node congestion was increased
in both sexes at 10000 ppm and in males at 6000 ppm. There were
high incidences of lung tumours in all groups. The incidences of
adenomas were slightly higher in treated groups but there was no
dose relationship observed. Hemagiomas and hemangiosarcomas were
observed in all groups of animals, chiefly in liver in males and
liver and uterus in females. The incidences of hemangiosarcomas at
any site was slightly higher in the 10000 ppm males and females than
controls (8 vs 4 in both cases). However, control incidences were
higher than in any other treated groups (0-2/group). The earliest
observation of an hemangiosarcoma (at death of animal) was 477 days
in control males, 415 days in males at 10000 ppm, 530 days in
control females and 585 days in females at 10000 ppm. Mean day of
death of animals dying because of hemangiosarcomas was generally
similar. The increased incidence of hemangiosarcomas was not
statistically significant and, therefore, it is concluded that no
carcinogenic effect was demonstrated. The NOAEL for non-neoplastic
effects in this study was 3000 ppm (equal to 396 mg/kg bw/day)
(Johnson et al. 1988a).
Rats
Groups of 60 male and 60 female Charles River Fischer 344
rats/dose level were given diets containing 0, 100, 500, 5000 or
10000 ppm daminozide (99.8% pure and containing 29 ppm UDMH) for 105
weeks. Clinical observations, body weights and food consumption
were measured throughout the study. Ophthalmoscopic examinations
were made pre-test and at 12 and 24 months. Blood samples were taken
from the same 10 rats/sex/group when possible at 6, 12, 18 and 24
months for hematological and clinical chemistry analyses. Urine was
collected during the fasts preceding blood sampling. At 12 months 10
rats/sex/group were sacrificed; the survivors among the remaining
animals were sacrificed at 24 months. From these animals organ
weights were recorded for adrenal, brain, heart, kidney, liver,
ovary and testes. All animals which died or were sacrificed were
given a complete post-mortem examination and samples of about 36
tissues/organs were saved. All tissues from all control and high
dose animals and all animals which died were examined for
histopathology. Liver, lung, kidney, ovary and gross lesions were
examined for low and mid-dose animals.
In this study there was considerable variability in the weekly
concentrations achieved but the diets were within ± 20% of the
target. Overall mean concentrations were very close to the nominal
concentrations.
There was no effect on survival in this study. Survival at 105
weeks was 46-72% in males (highest in the high dose group) and
70-78% in females. There appeared to be a slight increase in number
of males with withdrawn testes at 10000 ppm during the later weeks
of the study. In females at 10000 ppm heart/body weight ratio was
higher than controls The only other non-neoplastic pathological
change which may have been related to treatment was slightly
increased incidence of atrophy of the ovary in all treated groups.
There were no apparent treatment-related increases in tumour
incidences.
Although some ovarian atrophy appeared to occur at all dose
levels, the degree was slight at dose levels of 5000 ppm and less.
Therefore, 5000 ppm (equal to 243 mg/kg bw/day) appeared to be a
NOAEL in this study (Johnson et al. 1988b).
Reproduction study
Rat
In a two-generation reproduction study, groups of 25 male and
25 female Charles River Crl:CD(SD)BR rats were given diets
containing 0, 100, 1000 or 10000 ppm daminozide (99.3% pure) during
growth, mating, gestation and lactation for one litter per
generation. The rats were mated on a one to one basis after 10
weeks of dietary exposure to produce the F1 litters. From these
litters 25 rats/sex/group were selected to be parents for the F2
litters.
In both generations females given 10000 ppm consumed more food
than controls during the pre-mating stages of the study. There was a
slight tendency toward increased food intakes in the F0 males at
10000 ppm but not in the F1 males. The F1 males had reduced body
weight gains in he latter part of the study period. Fertility was
not affected but there was some indication of an increase in the
pre-coital interval in the 10000 ppm F1 - F2 mating in the
10000 ppm group. Pup weight at weaning was slightly lower than in
other groups in the F1 10000 ppm pups. No pathological lesions
were observed.
The possible effects, although slight, showed some consistency
and in the absence of additional litters, a conservative NOAEL of
1000 ppm (equivalent to about 50 mg/kg bw/day) was established
(MacKenzie et al. 1987).
Special studies on mutagenicity
UDMH was negative in three of four in vitro mutagenicity
studies. in the fourth a weakly positive result was obtained but
there was some concern about the methodology used since HC1 was used
as the diluant. An initial forward gene mutation assay gave
equivocal results but on repetition the assay was determined to be
negative (Table 1).
TABLE 1. RESULTS OF MUTAGENICITY ASSAYS ON UDMH
CONCENTRATION
TEST SYSTEM TEST ORGANISM OF UDMH RESULTS REFERENCE
Ames testa Salmonella typhimurium 25-5000 ug/plate Negative Stankowski & Naismith, 1986
TA1535, TA1537, TA1538
TA98, TA100
Chromosome aberration Chinese hamster ovary cells 5-5000 ug/ml Weakly San Sebastien & Naismith,
analysisa (CHO-K1-BH4, lot #A-12) positiveb 1986
DNA repair test Rat hepatocytes 8.3-250 ug/ml Negative Barfknecht & Naismith, 1986
Forward gene mutation Chinese hamster ovary cells 50-1000 ug/ml Equivocal Stankowski & Naismith, 1987
assaya (CHO-K1-BH4) (study)
repeated
below)
Forward gene mutation Chinese hamster ovary cells 50-1000 ug/ml Negative Stankowski, 1988
a,c (CHO-K1-BH4)
a With and without metabolic activation.
b The appropriateness of the methodology is questions. HC1 was used as the diluant.
c A repeat confirmatory study to replace the study above.
Special study on teratogenicity
Rabbit
In a range-finding study, groups of 5 female virgin
Dutch-Belted rabbits were inseminated artificially. Daminozide (99%
pure) was administered by gavage at a dose of 0, 100, 300, 1000,
2000 or 3000 mg/kg bw/day Days 7-29 of gestation. Day of
insemination was designated Day 0 of gestation. Clinical
observations and body weights were recorded during gestation. On
Day 28 the females were sacrificed and uteri examined.
At doses of 1000, 2000, and 3000 mg/kg bw/day all females died
by Day 25, 15 and 9, respectively. Soft stool and/or diarrhea were
noted prior to death in these animals and also in 3/5 and 4/5
females at 100 and 300 mg/kg bw/day, respectively. One dam at
300 mg/kg bw/day delivered on Day 28 prior to sacrifice. One female
at 300 mg/kg bw/day had non-viable fetuses as well as viable fetuses
in utero (Schardein et al. 1985a).
As a result of the above study the main study was done with
doses of daminozide of 0, 50, 150, or 300 mg/kg bw/day. Groups of
16 artificially inseminated virgin female Dutch Belted rabbits were
dosed by gavage Days 7 through 19 of gestation (Day of insemination
= Day 0 of gestation). Clinical observations, body weights and food
consumption were recorded throughout the study. On Day 28 all
surviving females were sacrificed and the ovary and uterus of each
examined. All fetuses were weighed and examined for external,
visceral and skeletal malformations.
One rabbit at 300 mg/kg bw/day died Day 12. Diarrhea and
absence of stool were noted only in some females at 150 and
300 mg/kg bw/stool. There were no apparent effects on reproductive
performance. There was no evidence of embryotoxicity or
teratogenicity.
Because of the deaths observed in the range-finding study, the
death at 300 mg/kg bw/day might be treatment-related although it
occurred earlier than would be expected. A conservative NOAEL for
maternal toxicity would be 150 mg/kg bw/day. Only some stool
changes were noted at this level. The NOAEL for teratogenicity was
300 mg/kg bw/day (Schardein et al. 1985b).
COMMENTS
When administered orally, daminozide was rapidly excreted by
rats and guinea pigs. Absorption appeared to be dose-related with
larger proportions of the dose being excreted in feces at higher
dose levels. In rats, the primary route of excretion was in the
feces, with lesser amounts in urine and as expired CO2 and tissue
analysis indicated biotransformation to unsymmetrical
dimethylhydrazine (UDMH, 1,1-dimethylhydrazine) and then to
1,1-dimethylhydrazone.
Radiolabelled UDMH administered orally to guinea pigs was
excreted primarily as CO2. The concentration in the liver tended
to be high, with 10% of the dose remaining in this organ after 2
hours.
The NOAEL in a one-year feeding study with daminozide to dogs
was 7500 ppm (equal to 200 mg/kg bw/day).
In a 2-year feeding study in mice, a level of 10000 ppm dietary
daminozide (containing 29 ppm UDMH) resulted in the accumulation of
brown pigment in the liver. Although the incidence of pulmonary
adenomas was slightly higher in all treated groups than in controls,
it was neither statistically significant (except for males at
6000 ppm) nor dose-related. The incidence of hepatic and uterine
hemangiosarcomas were increased in males and females respectively at
10000 ppm, but these increases were also not statistically
significant. The Meeting therefore concluded that daminozide was
not oncogenic in mice. The NOAEL was 3000 ppm (equal to 396 mg/kg
bw/day). Daminozide (containing 29 ppm UDMH) was not oncogenic in a
2-year feeding study in rats. The NOAEL was 5000 ppm (equal to
243 mg/kg bw/day) based on ovarian atrophy.
Daminozide was not embryotoxic or teratogenic in rabbits at
300 mg/kg bw/day, although maternal toxicity was observed at doses
above 150 mg/kg bw/day. In a 2 generation, 1 litter per generation
reproduction study in rats, the NOAEL was 1000 ppm in the diet
(equivalent to 59 mg/kg bw/day).
UDMH was weakly positive in a chromosomal aberration assay but
not in other mutagenicity assays. The Meeting was informed that
carcinogenicity bioassays of UDMH are in progress.
UDMH, which is a contaminant and degradation product of
daminozide, occurs as a trace component of daminozide residues in
food (see Residues Section). Since the daminozide used in the
carcinogenicity bioassays was contaminated with UDMH, and because
daminozide is partially transformed to UDMH in vivo, the Meeting
concluded that both compounds had been adequately bioassayed, and
therefore the available data are relevant to the assessment of
daminozide and its residues.
TOXICOLOGICAL EVALUATION
Level causing no toxicological effect
Mouse: 3000 ppm in the diet, equal to 396 mg/kg bw/day
Rat: 1000 ppm in the diet, equivalent to 50 mg/kg bw/day
(based on reproduction)
Dog: 7500 ppm in the diet, equal to 200 mg/kg bw/day.
Estimate of acceptable daily intake for humans
0-0.5 mg/kg bw *
Studies which will provide information valuable to the continued
evaluation of the compound
1. Results of ongoing carcinogenicity bioassays of UDMH in rats and
mice.
2. Quantitative data on the conversion of daminozide to UDMH in
experimental animals.
3. Observations in humans.
* Daminozide containing less than 30 ppm UDMH.
REFERENCES
Barfnecht, T.R. & Naismith, R.W. (1986) Rat Hepatocyte Primary
Culture/DNA Repair Test - UDMH. Unpublished Report PH 311-UN-001-86
from Pharmakon Research International, Inc. Submitted to WHO by
Uniroyal, Inc.
Chadwick, M. & Silveira, D.M. (1987) Dermal absorption of alar in
rats. Unpublished Report from Arthur D. Little, Inc. Submitted to
WHO by Uniroyal, Inc.
Frederick, C.B., Tortora, N.J., Lengen, M.R., Fitzpatrick, K.C.,
Sullivan, E.A. & Dzialo, D.G. (1984a) Analysis of the residues of
daminozide in treated guinea pigs. Uniroyal Chemical Project.
No. 8308. Submitted to WHO by Uniroyal, Inc.
Frederick, C.B., Abdel-Kader, M.H., Peterson, G.S., Lengen, M.R. &
Tortora, N.J. (1984b) Structure analysis of the principal residues
in the urine of daminozide-dosed guinea pigs. Uniroyal Chemical
Project. No. 8308B. Submitted to WHO by Uniroyal, Inc.
Johnson, D.E., Frith, C.H., Laughlin, K.A. & Blair, M. (1988a) Alar
technical (Daminozide) - Two-year oncogenicity study in mice.
Unpublished Report No. IRDC 399-054 from International Research and
Development Corporation. Submitted to WHO by Uniroyal, Inc.
Johnson, D.E., Rajasekaran, C.H., Frith, C.H., Laughlin, K.A. &
Blair, M. (1988b) Alar technical (Daminozide) - Two-year
oncogenicity study in rats. Unpublished Report No. IRDC 399-055 from
International Research and Development Corporation. Submitted to
WHO by Uniroyal, Inc.
Johnson, D.E., Rajasekaran, C.H., Blair, L.S. & Blair, M. One-year
dietary toxicity study in dogs - Alar technical. Unpublished Report
No. IRDC 399-066 from International Research and Development
Corporation. Submitted to WHO by Uniroyal, Inc.
Longen, M.R. & Frederick, C.B. (1984) Distribution of radioactivity
in stomach and duodenum from guinea pigs dosed with [14C-CH3]-
1,1-dimethylhydrazine, Uniroyal Chemical Co. Project No. 8433.
Unpublished report submitted by Uniroyal, Inc.
MacKenzie, K.M. (1987) Two-generation reproduction study with Alar
in rats (one litter per generation). Final report. Unpublished
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Milad, G., Gottfried, G.J. & Dillon, F. (1984) Balance study of
14C-Labelled daminozide in the rat. Unpublished Report from
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Novak, R.A. & Ambrose, S.M. (1983a) Daminozide distribution and
excretion study in guinea pigs. Unpublished Report No. 2308B from
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Novak, R.A. & Ambrose, S.M. (1983b) 1,1-dimethyl hydrazine.
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San Sebastian, J.R. & Naismith, R.W. (1986) In vitro chromosome
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Schardein, J.L., Aldridge, D. & Blair, M. (1985a) Alar range
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