DODINE           JMPR 1976

         Dodine was evaluated at the 1974 Joint Meeting. Temporary
    maximum residue limits were recommended for apples, pears, grapes,
    peaches, strawberries, and cherries. Further work was required on
    metabolism in plants and animals and the Meeting noted that it
    would be desirable to have information on teratogenicity, the fate
    in dairy cows fed with treated apple and grape pomace, supervised
    residue trials in countries other than U.S.A., and further details
    on residues in supervised trials on peaches and grapes, and during
    wine processing.

         Data which have become available since the 1976 meeting are
    evaluated in this monograph addendum.



    Absorption, distribution and excretion

         Twelve male rats received 5.15 mg/kg bw 14C-dodine, marked at
    the quanidine moiety, in a single dose by gavage. Urine and faeces
    were collected daily. Three rats were killed after 24, 48, 96 and
    196 hours, and samples from liver, kidney, muscle, fat and carcass
    were analysed.

         In urine 41.6% and 43.8% of the administered dose were
    excreted through 24- and 192-hour intervals, respectively while in
    the faeces 43.6% and 49.9% of the dose were eliminated in the same
    intervals. Total recovery from all sources (urine, faeces, g.i.
    tract and cage rinse) was 96%.

         Two rats were administered radio-labelled 14C-dodine at a dose
    of 34 mg/kg bw. The expired air, urine and faeces were monitored
    daily for 10 days.

         In expired air of two rats only 0.24% of the administered dose
    of 34 mg/kg was attributed to 14C carbon dioxide through the
    240-hour experimental interval. Therefore oxidation of dodine to
    CO2 is not suggested as primary route of metabolism in the rat.
    The excretion of 14C-material by urine and faeces occurred rapidly.
    After 48 hours about 52% and 38% of the total dose were eliminated
    in the urine and faeces. After 240 hours, the total elimination by
    these routes was only slightly increased. Retention of
    radioactivity (expressed as ppm equivalents of 14C-dodine) in rat
    tissues (blood, liver, kidney, muscle) ranged between 0.01 and 0.21
    mg/kg at the 24-hour interval and less than 0.01 mg/kg at the
    192-hour interval in all tissues studied.

         Residues in fat were 9.5 mg/kg at 24 hours, 2.6 mg/kg at 48
    hours and less than 0.01 mg/kg at 192-hours. The half-life of

    radioactive residues was less than 25 hours in all tissues

         The nature of the radioactivity in urine, faeces and fat was
    studied with the help of thin-layer chromatography and isotope
    dilution analysis (co-crystallization). Among 20 radio-spots in
    urine four accounted for between 60 and 70% of radioactivity and
    more than 90% was due to dodine, suggesting that the urinary
    radioactivity arose from conjugates or other derivatives of dodine.
    Parent dodine represented 99% of the radioactivity in fat and 70%
    in faeces. Three minor faecal metabolites, whose migration values
    were similar to the major urinary metabolites, accounted for 20 to
    30% of the extractable radioactivity (Cox and Eisner, 1976).


         Further studies on absorption, excretion and metabolism have
    been reported. In rats, elimination of an oral dose was rapid,
    predominantly in urine and faeces. Dodine, the major chemical
    residue in fat, decreased to negligible levels within 8 days.
    Concern was expressed on the relevance of the metabolic studies in
    rats only with respect to known differences in accumulation by
    mammalian species. For example, dodine accumulates in guinea pigs
    and mice in contrast to its rapid elimination from rats. The
    experiments performed on rats cannot be regarded as wholly
    sufficient. However, the results of new studies on metabolism in
    rats, augmenting the information previously considered showing
    dietary no-effect levels in rat (200 ppm) and dog (50 ppm) allowed
    an ADI to be recommended.


    Level causing no toxicological effect

         rat: 200 ppm in the diet equivalent to 10 mg/kg bw
         dog:  50 ppm in the diet equivalent to 1.25 mg/kg bw


         0 - 0.01 mg/kg bw


         Country statements were received from the Netherlands, New
    Zealand, and Sweden. The statements only confirm information on use
    patterns and national residue limits available to the 1974 Meeting.
    The U.S. manufacturer submitted the results of metabolism studies
    in rats (discussed above) and on foliage of apple trees. The
    results of residue trials on apples at three locations in Germany
    were also made available. The other information designated as
    "desirable" was not available.


         In 1975, field experiments were conducted on dodine residues
    in apples at three locations in Germany (American Cyanamid, 1976a).
    The results are summarized in Table 1.

    TABLE 1. Residues of dodine in apples treated in Germany


    Location    Variety      Application         Interval     Residue
                                                 weeks        (mg/kg)

    Hove        Jonathan     1.125 kg/ha, 2      16 & 17      <2.0
                             applications of
                             a 65% 
    Jork        Marsh        W formulation       16 & 17      <2.0

    Moorende    Gloster                          17 & 22      <2.0

         Analyses were by a colorimetric method. Controls had apparent
    dodine (crop blanks) equivalent to 0.5 mg/kg. Samples were frozen
    between sampling and analysis. Recoveries from fortified samples
    ranged from 89 to 109%.


    In plants

         The 1974 Meeting (FAO/WHO, 1975) noted a study by Curry (1962)
    on the metabolism of dodine in apple trees using 14C labelled
    dodine. The study was in the nature of a tracer experiment, showing
    only the movement and distribution of total activity in the leaves
    and fruit. No attempt was made to identify any metabolic products,
    except for some speculation that the activity in fruit may have
    been due to protein bound residues of amino acid and guanidine
    moieties. The 1974 Meeting required additional metabolism data,
    presumably with identification of any alteration products.

         A report of a 1976 metabolism study on seedling apple trees
    was made available to the Joint Meeting (American Cyanamid Co.,
    1976b). The plants (greenhouse) were sprayed with 14C-dodine at a
    rate calculated to be equivalent to 2 kg a.i./ha. Total activity
    and the distribution of activity between surface rinses,
    extractable and unextractable deposits were followed for 8 weeks.
    Separation of 8 radioactive metabolites of dodine was obtained by
    two dimensional thin layer chromatography, but the metabolites were
    not identified. The study was of value in that it confirmed the
    earlier conclusions of Curry that (a) there is only minor
    translocation to new growth (1 mg/kg in new leaves) and (b) that
    the major portion of the residue is unchanged dodine.

         Although the metabolites were not identified, the work
    provides some additional basis for assessment of their
    toxicological significance in that it shows their proportion to
    dodine in the total residue. Table 2 shows the total unextractable
    residue in leaves and Table 3 shows the relative proportions of
    metabolites and parent in extracts of leaves for the 8 week period.

    TABLE 2. Total unextractable activity, expressed as mg/kg dodine,
              in apple leaves


                                       Residue radioactivity
    Time (Weeks)                       expressed as dodine, mg/kg

    0                                  60.3

    1                                  60.9

    2                                  60.3

    4                                  51.2

    8                                  43.3

    8 (Translocation to new growth)    1.0

    TABLE 3. Distribution of dodine-derived metabolites found in
    extracts of Apple leaves

    Metabolite     Coordinates   Radioactivity (dodine equivalents,
    Number         of spot       mg/kg) after interval (weeks)
                                 0       1        2        4        8

    1 (dodine)     55-50         7.3     7.9      9.6      6.9      5.8

    2a             50-37                 0.8      0.7      -        -

    2              50-35                 1.0      1.7      2.7      2.1

    3              41-25                 0.5      -        0.9      0.7

    4              38-25                 0.5      <0.1     1.2     

    TABLE 3. (Continued)


    Metabolite     Coordinates   Radioactivity (dodine equivalents,
    Number         of spot       mg/kg) after interval (weeks)
                                 0       1        2        4        8

    5              47-18                 <0.1     1.0      0.6      -

    6              38-14                 0.4      -        1.1      0.7

    7              35-16                 1.1      1.9      1.2      2.4
    Total 14C-
    residues                     7.3     11.8     15.7     14.6     14.0

    In processing and cooking

         Studies on the reduction of dodine residues in cooking and/or
    processing spinach were conducted by General Foods Corporation and
    Del Monte Corporation (EPA, 1975). Field treated spinach bearing
    residues in the range 5.4-18.3 mg/kg was washed before canning and
    freezing. Residues in washed spinach were 26% of those in unwashed.
    Residues in blanched frozen and canned spinach were 16% and 7%
    respectively of residues in the unwashed spinach. Residues in the
    blanched frozen and canned spinach without prior washing were 61%
    and 25% respectively of those in unwashed spinach. In a second
    experiment, washing field treated spinach reduced residues by about
    66% but home type cooking resulted in no further reduction of
    dodine residues.


         The only method previously available for regulatory analyses
    was the colorimetric method of Steller (1960). A new
    gas-chromatographic method by Newsome (1976) offers considerable
    improvement. This method involves methanol extraction, partitioning
    with chloroform, and GC of the hexafluoroacetylacetone derivative
    with EC detection. A modification of this method was validated on
    spinach at 10 and 20 mg/kg fortification levels in U.S. government
    laboratories and is suitable for regulatory analysis.


         The temporary maximum residue limits recommended by the 1974
    Meeting were contingent upon additional information on metabolism
    in plants and animals, teratogenicity studies, fate in dairy cows
    fed with treated apple and grape pomace, supervised trials on

    various crops from countries other than U.S.A., and further details
    on supervised trials on peaches and grapes, and residues in wines.

         The material designated as required by the 1974 Meeting was
    partly provided. The material designated as desirable has not been
    made available (except for a limited residue trial on apples from

         A significant advance in analytical methodology was made with
    the development of the electron-capture GC procedure of Newsome
    which measures a derivative. Useful information was made available
    on the reduction of residues in processing and cooking. There was
    a significant reduction of residues in washing, canning and
    blanching of spinach but no observable reduction in home cooking.


         Since an ADI has now been allocated, the temporary maximum
    residue limits recommended by the 1974 Joint Meeting are converted
    to maximum residue limits. No additional limits are recommended.


    REQUIRED(before further Maximum Residue
     Limits can be recommended)

    1.   A large animal feeding study to determine whether feeding of
         apple pomace and grape pomace contributes residues to meat and

    2.   Identification of metabolites occurring in crops if the levels
         shown in the 1976 study are found to be toxicologically
         significant by WHO.


    1.   Studies of the metabolism of dodine in species other than the
         rat, preferably in guinea-pigs and/or dogs.

    2.   Teratological studies in appropriate animal species.

    3.   Appropriate mutagenicity studies.


    American Cyanamid. Technical Report no. 566, Cyanamid
    1976a               Cyanamid of Great Britain Ltd. May 13, 1976.

    American Cyanamid. Fate of radiolabeled dodine in young apple
    1976b               plants American Cyanamid Co., Agricultural
                        Division, Princeton, N.J. Project no. 0542,
                        E.J. Orloski, June 17, 1976. (Unpublished)

    Cox, G.W., and Eisner, S.K. CL 7521: Absorption, excretion
    1976a               and metabolism of carbon-14 labelled
                        n-dodecylguanidine acetate in rats. PD-M 13-5:
                        1-78. Unpublished report submitted by American
                        Cyanamid Company.

    Cox, G.W., and Eisner, S.K. Dodine: Fate of radiolabeled
    1976b               dodine (n-dodecylguanidine acetate) in young
                        apple plants. PD-M 13-4: 1-36. Unpublished
                        report submitted by American Cyanamid Company.

    Curry, A.M. Translocation and metabolism of dodecylguanidine
    1962                acetate (dodine) fungicide in apple trees,
                        using C14 radiotagged dodine. J. Agr. Food
                        Chem., 10: 13-17.

    EPA                 Unpublished data on reduction of dodine
    1975                residues by cooking and processing.

    FAO/WHO             1974 Evaluations of some pesticide residues in
    1975                food. FAO/AGP: 1974/M/11; WHO Pesticide
                        Residues Series, No. 4.

    Newsome, W.H. A gas-liquid chromatographic method for the
    1976                determination of dodine residues on foods. J.
                        Agr. Food Chem., 24: 997-999

    Steller, W.A., et al. Colorimetric estimation of dodecylguanidine
    1960                acetate residues. J. Agr. Food Chem., 8:


    See Also:
       Toxicological Abbreviations
       Dodine (WHO Pesticide Residues Series 4)
       Dodine (Pesticide residues in food: 1977 evaluations)
       Dodine (JMPR Evaluations 2000 Part II Toxicological)