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    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 g 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
    Report No. (HLA) from Hazleton Laboratories America, Inc.  Submitted
    to WHO by Uniroyal, Inc.

    Milad, G., Gottfried, G.J. & Dillon, F. (1984)  Balance study of
    14C-Labelled daminozide in the rat.  Unpublished Report from
    Biospherics, Inc., Rockland, Md. Submitted to WHO by Uniroyal, Inc.

    Novak, R.A. & Ambrose, S.M. (1983a)  Daminozide distribution and
    excretion study in guinea pigs.  Unpublished Report No. 2308B from
    Borriston Laboratories, Inc. Submitted to WHO by Uniroyal, Inc.

    Novak, R.A. & Ambrose, S.M. (1983b)  1,1-dimethyl hydrazine. 
    Bioavailability study in guinea pigs.  Unpublished Report No. 2308A
    from Borriston Laboratories, Inc.  Submitted to WHO by Uniroyal,
    Inc.

    San Sebastian, J.R. & Naismith, R.W. (1986)   In vitro chromosome
    aberration analysis in Chinese hamster ovary (CHO) cells-UDMH. 
    Unpublished Report No. PH 320-UN-002-86 from Pharmakon Research
    International, Inc.  Submitted to WHO by Uniroyal, Inc.

    Schardein, J.L., Aldridge, D. & Blair, M. (1985a)  Alar range
    finding teratology study in rabbits.  Unpublished Report No. 399-056
    from International Research and Development Corporation (IRDC). 
    Submitted to WHO by Uniroyal, Inc.

    Schardein, J.L., Arnold, K.S. & Blair, M. (1985b)  Alar teratology
    study in rabbits.  Unpublished Report No. 399-059 from International
    Research and Development Corporation (IRDC).  Submitted to WHO by
    Uniroyal, Inc.

    Stankowski, L.F. & Naismith, R.W. (1986)  Ames/ Salmonella plate
    incorporation assay - UDMH.  Unpublished Report PH 301-UN-005-86
    from Pharmakon Research International, Inc.  Submitted to WHO by
    Uniroyal, Inc.

    Stankowski, L.F. (1988)  Unsymmetrical dimethylhydrazine (UDMH) -
    CHO/HPRT mammalian cell forward gene mutationa assay.  Unpublished
    Report PH 314-UN-001-88 from Pharmakon Research International, Inc. 
    Submitted to WHO by Uniroyal, Inc.

    Stankowski, L.F. & Naismith, R.W. (1987)  CHO/HPRT mammalian cell
    forward gene mutation assay - UMDH.  Unpublished Report PH
    314-UN-001-86 from Pharmakon Research International, Inc.  Submitted
    to WHO by Uniroyal, Inc.


    See Also:
       Toxicological Abbreviations
       Daminozide (Pesticide residues in food: 1977 evaluations)
       Daminozide (Pesticide residues in food: 1983 evaluations)
       Daminozide (Pesticide residues in food: 1991 evaluations Part II Toxicology)