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.
EVALUATION FOR ACCEPTABLE DAILY INTAKE
BIOCHEMICAL ASPECTS
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
examined.
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).
COMMENTS
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.
TOXICOLOGICAL EVALUATION
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
ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN
0 - 0.01 mg/kg bw
RESIDUES IN FOOD AND THEIR EVALUATION
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.
RESIDUES RESULTING FROM SUPERVISED TRIALS
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%.
FATE OF RESIDUES
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
0.9
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-
extractable
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.
METHODS OF RESIDUE ANALYSIS
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.
APPRAISAL
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
Germany).
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.
RECOMMENDATIONS
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.
FURTHER WORK OR INFORMATION
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
milk.
2. Identification of metabolites occurring in crops if the levels
shown in the 1976 study are found to be toxicologically
significant by WHO.
DESIRABLE
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.
REFERENCES
American Cyanamid. Technical Report no. 566, Cyanamid
International.
1976a Cyanamid of Great Britain Ltd. May 13, 1976.
(Unpublished)
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:
460-464.
See Also:
Toxicological Abbreviations
Dodine (WHO Pesticide Residues Series 4)
Dodine (Pesticide residues in food: 1977 evaluations)
Dodine (JMPR Evaluations 2000 Part II Toxicological)