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    DAMINOZIDE       JMPR 1977

    IDENTITY

    Chemical name

    1. N-dimethylaminosuccinamic acid.

    2. succinic acid 2,2-dimethylhydrazide

    3. butanedioic acid mono (2,2-dimethylhydrazide).

    Synonyms

    Alar (R); SADH, Kylar, B-Nine (R), B-995, aminocide (obsolete common
    name).

    Structural formula

    CHEMICAL STRUCTURE 3

    Other information on identity and properties

    Daminozide is a white crystalline material of low volatility and
    slight odour. It is soluble in polar solvents (g/100 g a 25C: water
    10; methanol 5; acetone 2.5). It is practically insoluble in aromatic
    and aliphatic hydrocarbon solvents.

    The technical grade product from the primary producer is typically
    >99% daminozide. The manufacturing specifications require 98% minimum
    and a melting point range of 154C to 161C. It is a white powder

    which may contain traces of moisture, succinic acid, succinic
    anhydride and a salt of daminozide and unsymmetrical dimethyl
    hydrazine (UDMH). Volatile impurities are removed by an oven drying
    step in the process. Daminozide was formerly manufactured by a process
    in which dimethylnitrosamine was a starting reactant. The primary
    manufacturer now uses a new process in which dimethylnitrosamine is
    not used.

    Daminozide is now commercially available only in formulations of
    water-soluble powders containing 85% active ingredient and 15% inert
    surfactants and mineral salts. Shelf-life studies show no degradation
    of the formulations over a period of years. Liquid formulations
    formerly produced are no longer commercially available.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

    Daminozide is rapidly excreted and does not bioaccumulate in rats.

    Two male and two female rats were treated with a single oral dose of
    approximately 5 mg/kg unlabelled daminozide and 96 hours later with a
    single 5 mg/kg oral dose of 14C-labelled daminozide (position of 14C
    label unspecified) to study the absorption and excretion of
    daminozide. After 2 days 69% of the administered dose had been
    excreted in the faeces, 24% in the urine and 2.4% expired as 14CO2 
    Rats sacrificed 2 days after dosing contained an average of 0.35% of
    the administered dose in the brain, liver, lung,,heart and spleen with
    the majority of this residue in the liver. The rats sacrificed 4 days
    after dosing contained only 0.03 or 0.12% of the administered dose in
    these organs (Ryer, 1966).

    A cow fed a diet containing 25 ppm daminozide for 4 days excreted 81%
    of the daminozide in the faeces and 1.2% in the urine (St. John, Jr.
    et al., 1969). See also "Fate of residues", "In animals".

    TOXICOLOGICAL STUDIES

    Special study on mutagenicity

    Groups of male mice (20/group) were fed dietary levels of 0, 10, 300
    or 10,000 ppm daminozide for 5 consecutive days to determine dominant
    lethal effects. Following treatment, the males were monogamously
    paired with untreated females, weekly, for 4 consecutive weeks.
    Thirteen days after pairing females were killed and the uterine
    contents examined for implantations, viable embryos and early or later
    deaths.

    No male deaths occurred during the test period and no signs of
    compound effect on behavior were noted. During treatment, weight gain
    at 10,000 ppm was marginally retarded. No effect was observed on
    mating performance or pregnancy rate in any group. Neither
    implantation rate nor viable litter size were significantly affected
    by any treatment level. Inter-group differences in post-implantation
    loss never attained significance (P>0.05) and showed no consistent
    dose relationship (Palmer and Lovell, 1973).

    Special study on teratogenicity

    Groups of female rats (25/control group, 21/test group) were
    administered 0, 250 or 500 mg/kg/day daminozide (12.5% w/v) in corn
    oil, by gavage, from day 6 through 15 of gestation to determine
    teratogenic effects. Daily observations were made and body weights
    recorded on days 1, 9, 12 and 15. Records were kept of corpora lutea,
    resorption sites, viable foetuses, foetal external and internal
    abnormalities and skeletal development. Approximately equal numbers of
    foetuses were examined for internal effects, using the Wilson
    technique, and skeletal effects using clearing and alizarin staining
    techniques.

    Resorption sites, expressed as mean/female, were as follows: control,
    0.4; 250 mg/kg, 1.1; 500 mg/kg, 0.7. Two females with a total of 10
    resorption sites were included in the 250 mg/kg mean. Examination of
    foetuses revealed no compound-related effects and none of the other
    parameters were affected by compound administration (Keplinger et al.,
    1972).

    Special study on reproduction

    Groups of rats (20/sex/group) were fed dietary levels of 0 or 300 ppm
    daminozide, from weaning through three generations. The F0 group was
    part of the 2 year feeding study described under "Long term studies".
    PUP weights, number born alive or dead and the usual indexes of
    reproductive performance were recorded. Data from two litters/3
    generations revealed no significant effect on either fertility or
    reproductive capacity. The pups survived equally well in the control
    and test groups and no effect on growth was evident. Blood and urine
    values were comparable to controls in each generation. Organ weights
    were not affected nor was any compound-induced histopathology observed
    (Oser, 1966).

    Acute toxicity

    Observed signs or oral intoxication were depression, ptosis, ataxia,
    diarrhoea, excessive urination and laboured respiration. Dermal
    application produced mild erythema and oedema.

        TABLE 1. Acute toxicity of daminozide.
                                                                                         
                                                  LD 50'
    Species          Route         Sex            mg/kg              Reference
                                                                                         
    Rat              Oral          M              6,810              Anonymous, 1966

                     Oral          M,F            8,400              Carson, 1963

                     Inhalation    M              147 mg/l           Carson, 1963

    Rabbit           Dermal        M,F            >10,000            Anonymous, 1966

                     Dermal        M,F            16,000             Carson, 1963
                                                                                         
    


    Short term studies

    Rat

    Groups of rats (5 males and 5 females/group; 10 of each sex were
    controls) were fed dietary levels of 0, 26, 80, 240, 720 or 2160
    mg/kg/day daminozide technical for 90 days. No differences were noted
    in body weight gains between any test group and the control gains. No
    significant differences in haematological, blood chemistry or urine
    values were observed and organ weights and ratio were comparable to
    the control group. No gross or microscopic changes occurred in any
    group which could be attributed to daminozide administration (Carson,
    1964).

    Dog

    Groups of dogs (6/sex/control and 4/sex/test groups) were fed dietary
    levels of 0, 300, 1000 or 3000 ppm daminozide technical for two years.
    Body weight gains were within normal limits and comparable to control
    gains. Appearance, behaviour, haematological, blood chemistry and
    urine analyses of the test groups were also comparable to controls.
    Gross necropsies, microscopic examination of tissues and organ weight
    data revealed no compound or dose-related effects (Oser, 1966).

    Long term studies

    Rat

    Groups of rats (37/sex/control and 25/sex/test group) were fed dietary
    levels of 0, 300, 1000 and 3000 ppm diaminozide technical for two
    years.

    No differences in appearance or behaviour were observed between
    control and test rats during the study. Throughout the study the body
    weights gains of the 300 ppm group were slightly less than those of
    the controls, but weight gains of the 1000 and 3000 ppm groups were
    comparable to the control gains. No haematological or blood chemistry
    differences related to compound consumption were noted during the
    study. Survival over two years was not affected and was comparable
    between all groups. Liver/body weight ratios for both sexes in the 300
    and 3000 ppm groups were significantly higher than the control ratio
    at 104 weeks. In the 300 ppm male group the mean liver weight was 21%
    higher than the mean control liver weight. Nothing in the gross or
    microscopic examinations explains this difference. Microscopic
    examination of tissues revealed no dose-related effects. Tumors were
    both benign and malignant, the former being principally mammary
    adenomas or fibromas. In the latter category, reticulum cell sarcomas
    predominated in both the test and control groups, the incidence being
    not significantly higher at the 3000 ppm level than in the control
    group. Several varied types of malignant neoplasms were also noted but
    in no cage was there more than one of each type in any group (Oser,
    1966).

    COMMENTS

    Daminozide is not highly acutely toxic as measured by LD50 values in
    the g/kg range for oral and dermal studies and is only mildly
    irritating to the eyes and skin. It is rapidly excreted and does not
    bioaccumulate in mammals. Daminozide did not produce dominant lethal
    effects in male mice at 10,000 ppm and no teratological or
    reproductive effects were observed at levels of 300 ppm or lower. An
    adequate two-year dog study has been conducted.

    The long-term rat study was inadequate because of internal conflicts
    within the report and because of the inability to evaluate the
    carcinogenic potential of daminozide. Information on the
    biotransformation of daminozide in animals is needed.

    Because of the above concerns no recommendation for an acceptable
    daily intake for man can be made.

    RESIDUES IN FOOD AND THEIR EVALUATION

    USE PATTERN

    Daminozide is a plant growth regulator applied only in foliar sprays
    to various tree fruits, grapes, vegetables, melons and peanuts.
    Depending on the crop and timing of treatment, it is variously
    intended to hasten ripening, increase fruit set, enhance fruit colour,
    retard stem elongation, control fruit drop, delay maturity, reduce
    vine growth or to produce certain other beneficial growth regulating
    effects.

    Introduced on an experimental basis in the early 1960s, it was first
    registered for use on ornamentals. Since that time there have been
    numerous reports in the horticultural journals on the efficacy of the
    compound as a growth regulator. Use was expanded to include food crops
    in 1968 in the U.S.A. Table 2 shows current (1977) registered use
    patterns in the U.S.A. Uses in Canada are similar to the U.S. uses.
    There was other information available to the Meeting that the product
    has been used in Italy, South Africa, Japan, Australia, Federal
    Republic of Germany, the Netherlands and the United Kingdom. The
    directions for use on the product labelling accepted in those
    countries generally conform to the use patterns shown in Table 2.

    RESIDUES RESULTING FROM SUPERVISED TRIALS

    A submission to the Joint Meeting contained data from supervised
    trials in the U.S.A. on fourteen crops and controlled feeding
    experiments with meat and dairy animals (Uniroyal, 1977). The
    government of the Netherlands submitted reports of supervised residue
    trials on apples and pears. Analyses were by the colorimetric
    procedure described below under "Methods of residue analysis".
    Analyses of untreated controls (crop blanks) were reported in each
    experiment, along with % recovery of daminozide from fortified
    controls. Test samples were corrected for crop blank. Recoveries were
    adequate. A brief discussion of the residue finding for each crop
    follows. Data are summarized in tabular form where such presentation
    is feasible.

    Apples

    Field trials were conducted at locations in 10 states representing all
    of the major apple growing areas of the U.S.A. Some 180 harvest
    samples were analyzed in duplicate. The wide range of experimental
    conditions in the various field tests and the resulting wide range of
    residues found, preclude a tabular summary of data. The residues
    ranged from 0.1 mg/kg to as high as 80 mg/kg. If the high values
    resulting from exaggerated treatments are excluded, the data indicate
    that residues would not be likely to exceed 30 mg/kg when the normal
    dosages (Table 2) are applied and the prescribed 60-70 day preharvest
    interval is observed.



        TABLE 2. Registered use patterns for daminozide in the U.S.A. (1977).

                                                                                                                                

    Crop                     Purpose                Spray                 No. of              Timing or pre-harvest
                                                    Application           Applications        limitation
                                                    Rate (a.i.)
                                                                                                                                

    sweet cherries           hasten                 0.1-0.2%              1                   2 weeks after full
                             ripening,                                                        bloom
                             concentrate
                             maturity

    sour cherries            "                      0.4%                  1                   "

    peaches,                 hasten                 0.1-0.2%              1                   13 weeks post-bloom
    nectarines               ripening,                                                        to pit hardening
                             concentrate
                             maturity

    pears                    prevent fruit drop     0.1%                  1                   18 to 24 days before harvest

    apples                   multiple effects(1)    0.075-0.2%                                160 to 70 days before harvest

    prunes                   hasten ripening        0.05-0.1%             1                   1 month before
                                                                                              harvest

    grapes                   increase fruit set,    0.05-0.1%             1                   from 1st bloom to
                             reduce vine growth     (1.4-2.4 kg/ha)                           full bloom

    Brussels                 uniform sprout         1.9-3.75 kg/ha        1                   30 days before
    sprouts                  development                                                      harvest

    cantaloupes              reduce vine growth     1.9 kg/ha             1                   2 to 4 leaf stage

    TABLE 2. (Continued)

                                                                                                                                

    Crop                     Purpose                Spray                 No. of              Timing or pre-harvest
                                                    Application           Applications        limitation
                                                    Rate (a.i.)
                                                                                                                                
    tomatoes                 multiple effects(1)    0.6-2.4 kg/ha         2                   before transplanting

                                                    4.8 kg/ha             1                   after transplant - not
                                                                                              within 7 days
                                                                                              of picking

    peanuts                  multiple effects(1)    1.0 kg/ha             2                   at pegging time
                                                    0.5 kg/ha                                 30 days before
                                                                                              harvest

    peppers                                         0.5-2.3 kg/ha         2                   at 2-4 leaf stage
    (seedlings)                                                                               and 7-10 days
                                                                                              later
                                                                                                                                
    (1)   There are 9 separate beneficial effects claimed for apples alone. Other claims are similarly complex. The directions for use of this
          product are complex because of many factors (plant vigour, variety, moisture stress, etc.) which can not only modify the desired effect
          on the plant but can contribute adverse effects.
    

    The residues from trials in the Netherlands were generally 5 mg/kg or
    less. The rates of application in these experiments were somewhat
    lower than the rates permitted in some other countries.

    Studies designed to measure initial deposits or residue decline rate
    (half-life) were not included. The data clearly show, however, that
    daminozide is extremely persistent in apples. Significant residues
    remain for more than 159 days after the spray application.

    Brussels sprouts

    Four separate trials were conducted at locations in California and New
    York. A single treatment was made at normal and exaggerated rates.
    Crop blanks were reported as zero. Recoveries were 87 and 83% at 1 and
    10 mg/kg respectively. The data are summarized in Table 3. Under good
    agricultural practice the residues would not be likely to exceed 20
    mg/kg.

        TABLE 3. Residues in Brussels sprouts, mg/kg (single application).

                                                                                                

    Application rate,                                Interval, days from treatment
    kg a.i./ha                     30                      33                      46
                                                                                                

    1.9                            6.3, 5.2, 6.8           7.1, 1.4, 0.0

    3.4                                                                            2.8, 1.4

    3.8                            14.6, 15.5, 13.0        2.3, 2-7, 1.5

    6.8*                                                                           4.8, 3.2

    7.7*                           25.4, 24.8, 24.6        5.6, 6.2, 5.2
                                                                                                

    *excessive dosage

    
    Cantaloupes

    Nine residue trials were reported from locations in California,
    Arizona, Texas and Michigan. The registered use patterns on melons
    provide for treatment when the plant is at the 2-4 leaf stage, at
    which point no fruit would be present. The experiments included
    exaggerated treatment rates, and samples taken as early as 44 days
    after treatment. If these samples were mature melons, fruit must have
    been present at the time of treatment (melons take from 85 to 150 days
    from planting to maturity). Even under these conditions the residue

    values of some 20 samples ranged from < 0.1 to 1.6 mg/kg (Table 4)
    compared with apparent daminozide in untreated controls up to 1.1
    mg/kg. It would appear that residues to be expected in cantaloupes
    from good agricultural practice would be of a lower order than those
    found in the tree fruits. It is not likely that residues in excess of
    3 mg/kg would occur in cantaloupes. Data were not available on other
    melon varieties, but the conclusion regarding cantaloupes could be
    extrapolated to other melons.

        TABLE 4. Residues in cantaloupes,mg/kg

                                                                                             

    Interval from            No. of                          Application Rate,
    last treatment           treatments                       kg a.i./ha

    days                                       0.47     0.95    1.9       3.8       7.6
                                                                                             

    44                       2*                <0.1     1.6

    50                       1                                  1.0,1.5   1.5,1.6

    57                       1                                  0.6,0.8   1.2,1.6

    68                       1                                  1.2       1.6

    73                       2*                <0.1     <0.1

    87                       1                                            <0.1      <0.1

    115                      2*                         <0.1    <0.1
                                                                                             

    *Rate given for second treatment only.
    

    Sour cherries

    Trials were carried out at several locations in four states
    representing the important cherry producing areas in the U.S.A. A
    total of 70 test samples and 25 control samples were analyzed. Crop
    blanks ranged from 0.1 to 0.64 mg/kg.

    The dosages applied in the tests ranged from 25% of the manufacturer's
    minimum recommended dosage to twice the maximum recommended rate (See
    Table 2). Samples were taken at periods after treatment ranging from
    56 to 65 days. This roughly corresponds to the label directions which
    call for a single spray 10-14 days after full bloom. The season from
    bloom to harvest in the U.S.A. is 80 to 100 days (Magness, 1971).

    The sampling schedule in all trials was designed to show residues at
    normal harvest. Therefore, no estimates of initial deposit or residue
    decline rate are possible. Overall the residues found on mature
    cherries ranged from 5.3 to 78 mg/kg. Although some correlation could
    be made with application rate, the variation in harvest residues
    appeared to be almost random (the data do not lend themselves to
    tabulation). If the data from trials with excessive or minimal dosages
    were excluded, the residues fall between 10 and 53 mg/kg. It is not
    likely that residues under actual (commercial) conditions would ever
    exceed 60 mg/kg.

    Sweet cherries

    Analyses of 176 test samples and 39 control samples from field trials
    in five states were made available. The experimental design generally
    paralleled that discussed above for sour cherries. That is, the trials
    were designed to show residues on harvested cherries treated
    approximately as prescribed on the product label (2 weeks after full
    bloom). The overall range of residues found (1 to 54 mg/kg) was
    comparable to that reported on sour cherries. If only the data from
    trials following good agricultural practice (See Table 2) are used,
    most samples were in the range of 9-18 mg/kg, with a few higher values
    approaching 30 mg/kg. The difference between the residue load expected
    on sweet and sour cherries is mainly due to the application rate,
    which is 0.2% for sweet and 0.4% for sour cherries. A maximum residue
    limit of 30 mg/kg for sweet cherries would therefore be consistent.

    Grapes

    The residue data are summarized in Table 5. Some 44 samples from field
    trials in 4 states were reported. Analyses were in duplicate,
    including 8 untreated control samples. All controls were reported as
    0.1 mg/kg, apparent daminozide.

    The registered use directions for grapes (Table 2 requires treatment
    during the period from first bloom to full bloom. The period between
    bloom and harvest for grape varieties will differ greatly. In some
    varieties it may be as much as 5-6 months (Magness, 1971). It is
    interesting to note that in the field trials, the intervals between
    spray and harvest were from 102 to 111 days, and probably represent an
    early maturing variety, probably Concord. From the standpoint of
    residues at harvest, it therefore represents the "worst case". Even
    with some exaggerated dosages, all residues were below 10 mg/kg.

    TABLE 5. Residues in grapes, single application, 102-111 days from
    treatment

              Application rate         Residue range,
                 % a.i.              mg/kg   

              0.05                     0.4 - 2.5

              0.10                     1.3 - 5.4

              0.20                     2.6 - 9.5

    Nectarines and peaches

    The use directions for these two fruits are the same. They also incur
    residues to the same degree, and may be discussed together.

    The prescribed cut-off date for sprays is at "pit hardening".
    Presumably, this growth stage is recognizable to experienced fruit
    growers. As near as can be determined, pit hardening occurs 40 to 80
    days before harvest, depending on variety, geographic location and
    other factors.

    The data made available on nectarines and peaches included a total of
    more than 500 analyses including 70 untreated controls. Treatments
    were made at the prescribed rate, at excessive rates, and at less than
    the prescribed rate. The intervals between treatment and sampling were
    variously identified as treatments at "style abscission", pit
    hardening, and in some cases by numerical day before harvest. Owing to
    the variables in the field tests, a wide range of residues was
    reported (1.0 to 93.0 mg/kg).

    Interest is mainly in the residues from applications closely
    approximating the registered use pattern (Table 2). The distribution
    of residues from application of 0.1 to 0.2% sprays in the period from
    style abscission (3 weeks post-bloom) to pit hardening in nectarines
    and peaches was:

    mg/kg               <1   1-5  5-10 10-15     15-20     20-25     >25
    No. of samples      35   140  48   45        3         7         1

    The distribution indicates that residues would not be likely to exceed
    30 mg/kg when uses described in Table 2 are followed.

    Peanuts

    Results of residue trials in Georgia, N. Carolina, Texas, Alabama,
    Florida and Oklahoma were submitted to the Meeting. Separate analyses
    were made on kernels, hay and hulls.

    Kernels

    A total of 166 analyses of test samples and 13 of controls were made.
    All controls samples were zero except two reported at 3.1 and 2.8
    mg/kg. (The analyst noted that contamination was suspected.)

    The manufacturer's recommendations are for one treatment of 0.96 kg
    a.i./ha at pegging time followed by a second treatment of 0.47 kg
    a.i./ha thirty days before harvest. For those field trials in which
    the treatment approximated the recommended pattern, the following
    residue distribution was obtained:

    Range, mg/kg:       <5.0      5-10  10-15    15-20     20-25     25-30
    Number of samples:  31        20    7        4         4         2

    From this skewed distribution pattern it is apparent that maximum
    residue limit of 30 mg/kg for kernels would be adequate to cover all
    residues from good agricultural practice.

    Peanut hay

    62 analyses were submitted, 24 of which corresponded to the normal
    split dosage pattern mentioned above. Residues in the trials
    reflecting recommended usage were mostly in the range of 1-5 mg/kg.
    However, three were in the range of 5-10 mg/kg and three were greater
    than 10 mg/kg. A maximum residue limit of 20 mg/kg would be adequate.

    Peanut hulls

    Very limited data were available on peanut hulls. Residues were 2
    mg/kg or less. Peanut hulls are not an important item of international
    trade. The only interest in this commodity is that the hulls are
    incorporated at low levels into some mixed animal feeds.

    Peanut meal

    Field-treated peanuts which were determined to be carrying 12 mg/kg of
    daminozide were processed in a laboratory experiment simulating a
    commercial process. The meal presscake was found to contain 30-33
    mg/kg. On the basis of this approximately three-fold concentration
    factor, peanut kernels bearing the maximum residues of 20 mg/kg would
    be expected to produce meal containing about 60 mg/kg.

    Pears

    Limited data (14 test samples + 7 controls) from Washington and Oregon
    were available. Seven samples were from trials in which the

    manufacturer's recommended rate was applied (0.1% spray). Residues at
    this dosage ranged from 0.8-17.0 mg/kg, with an average of 10.8 mg/kg.
    Residues from uses according to good agricultural practice would
    probably not exceed 20 mg/kg.

    Peppers

    Field trials were conducted in Texas, Georgia, Connecticut, Delaware
    and Michigan. Samples were collected 77-125 days after treatment.
    Single and double applications were made at the prescribed rate and at
    four times the prescribed rate. The net difference between the
    apparent daminozide in the test samples and the control samples was
    less than 1 mg/kg in all cases, the highest residue reported being
    0.44 mg/kg. A maximum residue limit of 1 mg/kg would be adequate.

    Plums

    Residue trials at 6 locations in California were reported to the
    Meeting. The plums (fresh prunes) were treated at the prescribed rate
    (Table 2) and at rates up to 4 times the prescribed rate. The interval
    between treatment and sampling ranged from 34 to 78 days (as compared
    with the registered label requirement of a 30 day pre-harvest
    interval). Residues from all treatment rates ranged from 3.0 to 71.5
    mg/kg. Residues from the normal rates were 3.0 to 43.7 mg/kg. It is
    unlikely that residues from normal usage would exceed 50 mg/kg.

    Field-treated fresh prunes were analyzed and converted to dried prunes
    by the commercial procedure. This involves a 5-10 minute mechanical
    wash and drying on trays at 170F for 30 hours. The maximum
    concentration factor (mg/kg in dried prunes: mg/kg in fresh) was found
    to be 2.7. On this basis, a prediction can be made that fresh prunes
    bearing maximum residues of 50 mg/kg would produce dried prunes
    containing 135 mg/kg.

    Tomatoes

    The registered uses of daminozide on tomatoes include 2 applications
    prior to transplanting (greenhouse or outdoors) and one application in
    the field no later than 7 days prior to picking (see Table 2). The
    application on plants to be transplanted is for all practical purposes
    a "no-residue" use. Residue data were made available on tomatoes which
    had received both transplant and field treatments at the normal and
    excessive rates. All residue trials (7) were carried out in Florida,
    on green tomatoes. The only field use for daminozide on tomatoes
    permitted in the U.S.A. is on Florida winter tomatoes which are
    harvested green and artificially ripened. These residue trials
    therefore were highly specialized and do not necessarily reflect
    residues which might result from more general usage on tomatoes. The
    data are shown in Table 6.

    TABLE 6. Residues in tomatoes, mg/kg

                                                                    

    Interval from                           Application Rate*
    last treatment                          kg a.i./ha

    Days                                    4.76           9.52
                                                                    

    11                                      13             68.5

    21                                      9              35

    7                                       6              8

    7                                       8              12.5

    18                                      12             20.5

    14                                      20             45.5

    19                                      15             28

    29                                      22             8
                                                                    

    * Field treatment after transplant use.



    Alfalfa

    There are no national tolerances or registrations for daminozide use
    on alfalfa. However, the product has some utility in the culture of
    alfalfa seed, and registration is pending in U.S.A. In growing alfalfa
    for seed there is some utilization of alfalfa forage, hay, and seed
    screenings for animal feeds. The submission to the Meeting therefore
    contained some residue data on alfalfa hay, fresh forage, and seed
    screenings. Residues on fresh alfalfa ranged from 4-12 mg/kg and on
    hay from 2-20 mg/kg. Seed screenings showed a maximum of 1.4 mg/kg. In
    view of the specialized nature of the use it would not appear
    necessary at this time for the meeting to recommend a maximum residue
    limit.

    Meat, milk, poultry and eggs

    Daminozide is used on a number of crops which yield by-products used
    commercially in animal feeds. Primary daminozide sources would be
    peanut meal and peanut hay. Some additional contribution to the diet
    could occur from peanut hulls and the pomaces resulting from
    processing apples, grapes and tomatoes. In recognition of this, the

    basic manufacturer submitted to the Meeting some animal feeding,
    studies (Uniroyal, 1977).

    (a) Cattle were fed at levels of 0, 20, 60 and 200 ppm in the total
    diet for 6 weeks. Milk was collected at intervals and the animals
    slaughtered at the end of the feeding period. No daminozide residues
    were found in any tissues (muscle, fat, liver, kidney) by a method
    with a lower limit of detection estimated at 0.2 mg/kg. Trace residues
    were found in the milk of animals on the 60 and 200 ppm diets at
    levels of 0.05 and 0.07 mg/kg respectively.

    (b) Hogs were fed 0, 60 and 300 ppm in the diet (2 animals at each
    level) for 31 days. No daminozide residues were detected (40.2 mg/kg)
    in muscle or fat. Apparent daminozide residues were found in liver and
    kidney, at 0.3 and 0.8 mg/kg respectively, in animals on the highest
    feeding level.

    (c) Poultry were fed 0, 20, 60 and 200 ppm in the diet (10 hens in
    each group) for 31 days. Eggs were composited weekly and on day 31 all
    birds were sacrificed. On the final sampling, eggs from the 300 ppm
    and 60 ppm feeding levels contained 0.3-0.5 and 0.25 mg/kg
    respectively. No detectable residues were found (< 0.2 mg/kg) in eggs
    from the 20 ppm feeding level. No residues were found in muscle or fat
    of birds on the lower feeding levels. At the 200 ppm level, residues
    of 0.4 and 0.7 mg/kg were found respectively in muscle and fat.
    Residues of 1 and 1.1 mg/kg were found in liver of birds on the
    highest level. Daminozide concentrates in poultry kidney. Values of
    1.3, 1.8 and 7.4 mg/kg were reported in kidney for the 3 feeding
    levels.

    The studies show that residues occur at high feeding levels, but this
    must be related to the actual anticipated intake from peanut meal and
    peanut hay. Peanut hay would be used to the extent of 25% in beef
    cattle diets, 60% in dairy diets, and 10% for pigs. Peanut meal can
    make up 15%-beef; 25%-dairy, 10%-poultry and 10%-pigs (Harris, 1975).
    After adjusting for the proportion of peanut meal and hay in the total
    diet of each species, it is apparent that the residues in milk, eggs
    and tissues (except poultry kidney) Will be below the level of
    detection of the colorimetric method.

    FATE OF RESIDUES

    General

    Daminozide is readily absorbed and translocated from foliar sprays.
    The entrapment of 14CO2 from applications of labelled daminozide
    indicates that, under certain conditions, at least part of the applied
    pesticide is fully degraded. Other data show that daminozide, in free
    or loosely bound form, is the principal residue component and very
    persistent in fruits. Many of the conclusions regarding the nature of
    the terminal residues rest on characterization by selective solvent
    extractions, in vitro experiments and analysis by wet chemical

    procedures for predicted metabolites. In view of the known biological
    activity of some postulated metabolites of the compound, particularly
    nitrosamines, hydrazides and other hydrazine derivatives, further
    identification of the residues in harvested fruits and vegetables
    would be desirable.

    In animals

    Rats were fed 14C-labelled daminozide and the excreta monitored. The
    majority of the administered dose, apparently occurred in the urine as
    unchanged daminozide. As far as can be determined, there has been no
    effort to study metabolic pathways in meat or milk animals with radio-
    labelled daminozide. However, analyses by the chemical method
    (previously discussed) were made for one postulated metabolite (NDMA)
    in milk from cows fed 200 ppm daminozide in the diet. No NDMA was
    found at a minimum detection level of 0.03 mg/kg. See also the
    sections "Biochemical aspects" and "Residues resulting from supervised
    trials".

    In plants

    It has been reported that daminozide decomposes to unsymmetrical
    dimethylhydrazine (UDMH) in the plant and that it is the latter
    compound which is the active principle through its effect on the plant
    enzyme diamine oxidase (Reed, 1963). Another investigator (Dahlgren,
    1963) found that maleic acid dimethyl hydrazide (CO11, a related
    growth regulator) is hydrolysed to UDMH in aqueous solutions. Dahlgren
    noted that daminozide was stable under the same conditions. In
    experiments reported by the manufacturer (Uniroyal, 1967) no free UDMH
    or succinic acid was detected after attempts (in vitro) to enzymically
    hydrolyze daminozide with trypsin, proteinase, papain, ficin and
    urease. No evidence was found of hydrolysis to UDMH and succinic acid
    in macerates of plant leaves by colorimetric tests for UDMH and paper
    chromatographic tests for succinic acid. While the evidence regarding
    the occurrence of UDMH is somewhat contradictory it is likely that,
    even if UDMH occurs as an intermediate metabolite, its presence would
    be fleeting owing to its reactivity and that it would not be present
    per se in harvested crops.

    The gas-chromatographic method of analysis for nitrosodimethylamine
    (NDMA), discussed below ("Methods of residue analysis",) has been used
    in studies intended to show the absence of this possible oxidation
    product in treated crops (Uniroyal, 1967). No NDMA was detected in
    treated apples, tomatoes or peanut foliage by the method, which is
    said to be sensitive to 0.002 mg/kg NDMA.

    Radiotracer studies were conducted by the U.S. Department of
    Agriculture (Uniroyal, 1966) on apple trees in the field and on apple
    seedlings in the greenhouse. Daminozide was labelled in the succinic
    acid moiety and (in a separate test) in the UDMH group. Activity in
    the sprayed seedlings was distributed throughout the plant with

    highest activity in the leaves. 20% of the original activity was
    released as 14CO2 in the first seven weeks after application.

    In the field studies, 15% of the total applied activity remained in
    the tree after 100 days. The activity in the apple fruit after 125
    days was identified as 75-85% unmetabolized daminozide. Trace amounts
    of succinic acid were identified and there was some incorporation of
    14CO2 into plant components.

    14C-labelled daminozide was used in metabolism studies with peanut
    plants (Uniroyal, 1977). Characterization of the activity by selective
    solvent extraction indicated that the majority of the activity was
    present in bound form, probably as sugar complexes. Daminozide per se
    was recoverable from the extracts and it was concluded that residues
    present in peanuts in complexed form would be detected by the
    colorimetric method of analysis. Analyses of peanut oil for 14C
    activity showed residues of about 2-5 mg/kg expressed as daminozide.
    Selective solvent extraction of the oil indicated that the activity
    was not daminozide but was probably in the form of fatty acid
    glycerides.

    In soil

    Residue decline studies were conducted on sandy loam and clay soils
    spiked at 5 and 10 mg/kg. Daminozide per se is not persistent in
    soils. The breakdown is probably through microbial action. In the clay
    soil, < 0.1 mg/kg was present two days after treatment. The sandy
    loam contained 0.1 mg/kg at 2 and 3 weeks respectively at the 5 and 10
    mg/kg fortification levels. The compound is very mobile in soils
    because of its water solubility.

    In processing

    Studies show that in the commercial processing of peanuts there is no
    appreciable concentration of residues in the refined peanut oil. There
    is, however, a 3 fold concentration of residues in peanut meal (e.g.,
    peanut meats bearing 20 mg/kg will produce peanut meal bearing 60
    mg/kg).

    Data on the processing of fresh tomatoes bearing approximately 40
    mg/kg into ketchup and tomato paste show that there is essentially no
    loss of daminozide residues in processing. (The residues concentrate
    to the same degree as the solids do in the concentration process.)
    Residues found in tomato paste ranged from 155 to 197 mg/kg. Analyses
    for nitrosodimethylamine (NDMA) in tomato paste were negative by the
    chemical method with a reported sensitivity of 0.002 mg/kg.

    Experiments in which field-treated plums were processed into dried
    prunes showed that residues were concentrated in the dried fruit by a
    factor of 2.7. See "Residues resulting from supervised trials" for
    details of this work and of the effect on residues of processing
    peanuts to peanut meal.

    There are national tolerances on grapes (10 ppm, U.S.A. and Canada)
    but no data were made available on transfer of residues to wine or
    grape pomace.

    Evidence of residues in food in commerce or at consumption

    As noted under "Methods of residue analysis", daminozide is not
    detected by any of the multi-residue screening methods currently used
    in market basket surveys, monitoring, or government regulatory
    programmes. The "specific" colorimetric method has not been employed
    in the Canadian or the U.S. Food and Drug Administration surveillance
    programme and there is no record of a finding of daminozide in foods
    in commerce. In view of the demonstrated stability of daminozide
    residues in food processing, and the relatively high levels incurred
    in fruits from good agricultural practices, it may be concluded that
    residues are occurring in the diet in those countries where there is
    substantial use.

    METHODS OF RESIDUE ANALYSIS

    Daminozide residues in crops are determined by a colorimetric method
    (PAM II, 1967). The method involves hydrolysis in boiling 50% sodium
    hydroxide to release unsymetrical dimethylhydrazine (UDMH) which is
    distilled and reacted with trisodium pentacyanoamine ferroate (TPF) at
    pH 5.0. The red colour produced is measured spectrophotometrically at
    490 and 600 nm. The difference between the two readings is plotted as
    net absorbance against g of compound in a calibration curve.
    Modifications of this basic method permit determination of daminozide
    in peanuts, animal tissues, milk and eggs.

    The chromogenic agent TPF used for determination of daminozide also
    forms colored complexes with nitroso compounds, primary aromatic
    amines and aliphatic or aromatic hydrazines (Feigl, 1949). Some
    possible metabolites of daminozide would therefore contribute
    absorbance which would be additive to that of the daminozide-TPF
    complex. If present they would be interpreted as daminozide residues
    (but not measured quantitatively).

    The method has been tested in government regulatory laboratories on
    grapes and animal tissues. Because of large and variable crop blanks
    it was concluded that a reliable lower limit of determination for
    daminozide in samples of plant origin was no better than 1 mg/kg for
    regulatory purposes. The estimated sensitivity for animal products is
    0.05 mg/kg for milk and 0.2 mg/kg for tissues and eggs. The government
    analyst noted that 2 hours colour development time was required rather
    than the 1 hour prescribed in the procedure.

    The assumption was made that any free UDMH, or any metabolites which
    would yield UDMH under the rigorous caustic hydrolysis, would also be
    measured as daminozide by this method. There are no experimental data
    to confirm that conclusion. Whether in fact any such metabolites

    actually occur in crops at harvest is uncertain (see discussion under
    "Fate of residues").

    Colorimetric methods are ordinarily not specific. In this case, the
    method was tried in the presence of most pesticides used on the
    subject crops (in 1968) to gauge interference. Only the fungicide
    "Botran" was found to interfere, and this interference can be
    eliminated by a modification of the method.

    A second colorimetric method of analysis for daminozide in plant
    substrates has been published (Edgerton, 1967). This method also
    measures UDMH in a hydrolysate, but colour development is with
    phosphomolybdic acid. Sensitivity is comparable to the colorimetric
    method discussed above.

    As noted above, the colorimetric method for the parent compound would
    also measure any alkylnitrosamines as daminozide. Because of concern
    for the toxic potential of a possible metabolite, nitrosodimethylamine
    (NDMA), a "specific" method for NDMA was developed by the manufacturer
    (Uniroyal, 1967). This is a GC method in which NDMA is separated from
    crop substrate by vacuum distillation, sorbed on polymer beads,
    removed from the beads by baking and determined by micro-coulometric
    gas chromatography (measurement of NH3). Sensitivity was estimated at
    0.002 mg/kg. Recovery experiments at a fortification level of 0.010
    mg/kg were reported to be adequate. This method has not been validated
    in government laboratories.

    Daminozide is not detected by any of the multi-residue screening
    procedures used in government market basket surveys and monitoring
    programs. There is no record of any government regulatory experience
    with the colorimetric method for daminozide. It is questionable
    whether by present standards it would be satisfactory for regulatory
    purposes, particularly for enforcement of the lower tolerance levels.
    As far as can be determined there is no confirmatory method of
    analysis available. It would be desirable to have further information
    on the reliability of this method, particularly with respect to (a)
    whether it actually measures UDMH metabolites in free or conjugated
    forms and (b) actual experience with the method in government
    laboratories. An independent method, preferably a more specific GC or
    HPLC method would be desirable.

    APPRAISAL

    Daminozide is a growth regulator which has been used on a variety of
    fruits and vegetables since 1967. The product produces numerous
    beneficial growth regulating effects. It is said to control premature
    fruit drop, delay maturity, enhance fruit colour, reduce vine growth,
    retard stem elongation and increase fruit set, among other claims.
    Information submitted to the Joint Meeting by the primary manufacturer
    indicates that the principal uses of the product are in the U.S.A.
    Other information made available to the Meeting indicates that
    daminozide is also used in Italy, South Africa, Japan, Australia,


        NATIONAL TOLERANCES REPORTED TO THE MEETING

                                                                                                    

    Commodity                                              Tolerance, mg/kg
                                       U.S.              Canada      Australia      Netherlands
                                                                                                    

    Fruits and vegetables                                                           10
    Sour cherries                      55                55
    Plums                              50                20
    Tomatoes                           40                 0.5
    Apples                             30                30          30
    Nectarines                         30
    Peaches                            30                25          30
    Peanuts                            30                20          30
    Sweet cherries                     30                30
    Brussels sprouts                   20                15
    Peanut hay                         20
    Pears                              20                15          30
    Grapes                             10                10
    Peanut hulls                       10
    Melons                              3
    Peppers                             1

    Meat, fat and meat                  0.2                           0.2
    by-products of cattle,
    goats, horses, poultry
    (except kidney), sheep

    Poultry kidney                      2
    Eggs                                0.2                           0.2
    Milk                                0.02                          0.05
                                                                                                    
    

    Canada, Federal Republic of Germany, the United Kingdom and the
    Netherlands. National tolerances have been established in the U.S.A.,
    Canada, Australia and the Netherlands.

    Daminozide was formerly manufactured by a process in which
    dimethylnitrosamine was a starting reactant, and occurred as impurity
    in the technical grade product. The primary manufacturer informed the
    Meeting that a new process has been adopted in which
    dimethylnitrosamine is not used. The recommended guideline limits
    apply only to daminozide manufactured by the process in which
    dimethylnitrosamine is excluded.

    The compound is readily absorbed and translocated from foliar sprays.
    While there in some evidence of degradation, the parent molecule in
    free or loosely bound forms is persistent, particularly in the fruits.
    The chemical structure of daminozide suggests the possible presence of
    some metabolites of interest, including nitrosamines, hydrazides and
    other hydrazine derivatives. No positive findings of these metabolites
    were reported. The postulated metabolite N-dimethylnitrosamine was not
    detected by a method said to be sensitive to 0.002 mg/kg. Indirect
    evidence was reported of the absence of unsymmetrical
    dimethylhydrazine (UDMH).

    A feeding study with 14C daminozide in rats indicated that the
    majority of the administered dose was excreted in the urine as
    unchanged daminozide. There was no information on the metabolism
    patterns in large animals or on metabolites in meat or milk. In
    general, the information available on the fate of daminozide in plants
    and animals is not very extensive, particularly with respect to the
    nature of the terminal residues.

    The analytical method currently available to enforce national
    tolerances is a colorimetric method which measures unsymmetrical
    dimethyl hydrazine (UDMH) released from daminozide by vigorous caustic
    hydrolysis. The chromogenic agent is not specific for UDMH. Other
    classes of compounds also produce colour complexes, including
    aliphatic hydrazines, nitroso compounds and primary aromatic amines.
    It is theorized that complexed daminozide, free UDMH and any other
    metabolic products which would yield UDMH would be measured as
    daminozide. It has been determined that the method has never been
    applied in the U.S. or Canadian official surveillance programmes and
    there is no information on experience with the method in other
    government laboratories. The method would not appear to be very useful
    for regulatory purposes.

    Studies on the effects of processing show that the compound is not
    destroyed and tends to concentrate in products such as tomato paste
    and peanut meal. It has never been reported in foods in commerce (see
    above comment on the analytical method). It is not persistent in
    soils. Extensive data from supervised residue trials carried out in
    the U.S.A. were available. Data on supervised trials on apples and
    pears from the Netherlands were also reported. All analyses were by

    the colorimetric method discussed above. The data generally showed the
    highest residues in fruits, with considerable variability in harvest
    residues. The residue values on a given crop followed almost a random
    distribution. The experiments on which reports were received were all
    designed to show harvest residues from registered use patterns and
    therefore permitted no estimates of decline rate.

    EVALUATION

    As no ADI or temporary ADI could be allocated, no recommendations for
    maximum residue limits could be made. Guideline levels are recorded
    for the commodities listed below. The usual statements regarding the
    intervals on which the levels are based are omitted in this case
    because of the highly specialized uses of the product which are
    related to stages of growth or maturation.

         Commodity                Guideline level, mg/kg

         Sour cherries            60
         Plums                    50
         Tomatoes                 40

         Apples, nectarines,
         peaches, peanuts
         (kernels), pears,
         sweet cherries           30

         Brussels sprouts         20
         Grapes, peanut hay       10
         Melons                   3
         Peppers                  1
         Eggs, meat               0.2*
         Milk                     0.05*

    *At or about the limit of determination

    FURTHER WORK OR INFORMATION

    Required (before an acceptable daily intake for humans (ADI) and
    maximum limits (MRL can be established)

    1. An adequate long-term rat study.

    2. Information on the biotransformation in animals.

    Desirable

    1. An analytical method more suitable for regulatory purposes than the
    present colorimetric method.

    2. Information on the occurrence of residues in foods in commerce.

    3. Further identification of the terminal residues in crops and in
    foods of animal origin.

    REFERENCES

    Anonymous, (1966) Acute Oral Administration-Male Rats. Unpublished
    report from Hazleton Labs., Inc., submitted to the World Health
    Organization by the Uniroyal Chemical Company.

    Carson S., (1963) Toxicological Examination of Compound B-995.
    Unpublished report from Food and Drug Research Labs., submitted to the
    World Health Organization by Uniroyal Chemical Company.

    Carson S., (1964) Subacute (90 day) Feeding Studies with B-995 in
    Rats. Unpublished report from Food and Drug Research Labs., submitted
    to the World Health Organization by Uniroyal Chemical Division of
    Uniroyal,Corp.

    Dahlgren, Dahlgren and Simmerman, (1963) Science, 148, 485; 1963

    Edgerton, L.J., Rockey, M., Arnold, H., and Lisk, D.J., (1967)
    "Colorimetric Determination of Alar Residues in Apples", J. Agr. Food
    Chem. 15, no. 5, p. 812, 1967

    Feigl, F., (1949) Chemistry of Specific Selective, and Sensitive
    Reactions, p. 377, Academic Press, New York, 1949

    Harris, L.E., (1975) "Guide for estimating toxic residues in animal
    feeds or diets", U.S. National Technical Information Service,
    accession no. PB-243-748-LK

    Keplinger, M.L., Kennedy, G.L., and Haley, S., (1972) Teratogenic
    Study with Alar in Albino Rats. Unpublished report from Industrial
    Bio-Test Labs., submitted to the World Health Organization by the
    Uniroyal Chemical Division of Uniroyal, Corp.

    Magness, J.R., Markle, G.M., Compton, C.C., (1971) Food and Feed Crops
    of the United States, Bull. 828, New Jersey Agricultural Experiment
    Station, 1971

    Oser, B.L., (1966) Chronic (2 year) Feeding Studies with B-995 in Rats
    and Dogs. Unpublished report from Food and Drug Research Labs.,
    submitted to the World Health Organization by Uniroyal Chemical
    Division of Uniroyal, Corp.

    Palmer, A.K. and Lovell, M.P., (1973) Dominant Lethal Assay of Alar in
    the Male Mouse. Unpublished report from Huntingdon Research Center,
    submitted to the World Health Organization by Uniroyal Chemical
    Division of Uniroyal, Corp.

    Pam, II., (1967) Pesticide Analytical Manual, Vol. II, revised; U.S.
    Department of Health, Education and Welfare, Food and Drug
    Administration

    Reed, (1963) Science, 148, 1097; 1963

    Ryer, F.H., (1966) Final Report. Radiotracer Metabolism Study,
    Alar-C14. Unpublished report from Hazleton Labs., Inc., submitted to
    the World Health Organization by the United States Rubber Company.

    St. John, Jr., L.E., Arnold, H. and Lesk, D.J., (1969) Metabolic
    Studies with Alar Growth Regulator in the Dairy Cow.
    J. Agr. Food Chem., 17, No. 11 116.117.

    Uniroyal, (1966) Unpublished information, Uniroyal, Inc.v to U.S. Food
    and Drug Administration

    Uniroyal, (1967) Unpublished information, Uniroyal, Inc., to U.S. Food
    and Drug Administration

    Uniroyal, (1973) Unpublished information, Uniroyal, Inc., to U.S. Food
    and Drug Administration

    Uniroyal, (1977) Submission to Joint Meeting, May 17, 1977, Uniroyal,
    Inc.
    


    See Also:
       Toxicological Abbreviations
       Daminozide (Pesticide residues in food: 1983 evaluations)
       Daminozide (Pesticide residues in food: 1989 evaluations Part II Toxicology)
       Daminozide (Pesticide residues in food: 1991 evaluations Part II Toxicology)