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    DIPHENYLAMINE        JMPR 1976

    Explanation

         Diphenylamine was previously evaluated by the 1969 Joint
    Meeting (FAO/WHO 1970), at which time an acceptable daily intake
    was allocated and a maximum residue limit for apples of 10 mg/kg
    was recommended.

         The 7th Session of the Codex Committee on Pesticide Residues
    requested that diphenylamine should be reconsidered by the Joint
    Meeting as new toxicological data had become available. These data
    and others on residues in food are evaluated in this addendum.

    EVALUATION FOR ACCEPTABLE DAILY INTAKE

    BIOCHEMICAL ASPECTS

    Absorption, distribution and excretion

         A cow was given 5 ppm diphenylamine in the feed for 4 days. No
    residues of diphenylamine were detected in milk or urine. A small
    amount (1.4% of the dose) was excreted in the faeces. No conjugates
    were detected in the urine. The method used could not detect any
    hydroxylated derivatives. On in vitro incubation of
    diphenylamine with liver fractions about 50% disappeared within 30
    min (Gutenman and Lisk, 1975).

         Diphenylamine was present in manure samples (fresh or aged) at
    concentrations of 10 mg/kg, together with nitrates and nitrites.
    However, no diphenylnitrosamine or other nitrosamines were detected
    (Bergstrom et al., 1972).

    Effects on enzymes and other biochemical parameters

         Diphenylamine given at half the LD50 to rats decreased the
    hemoglobin and oxyhemoglobin content and caused the appearance of
    methemoglobin and Heinz bodies. The blood indexes returned to
    normal 10-14 days after administration (Volodchenko, 1975).

    TOXICOLOGICAL STUDIES

    Special studies on carcinogenicity

         The synthesis of nitrosamines in the stomach of rats was
    demonstrated when the diet was supplemented with NaNO2 and
    diphenylamine (Sander et al., 1968). Oral administration of 5 mg
    diphenylamine together with 15 mg NaNO2 to rats significantly
    decreased the growth of the animals. Nitrosodiphenylamine was found
    in the stomach. Feeding of the combination caused kidney and liver
    toxicities and papillomatous hyperplasia of the bladder (Gales et
    al., 1975).

    Special studies on cystic kidney disease

         A study was carried out to evaluate the cystic kidney disease
    resulting from 2-12 months feeding of diphenylamine in the diet of
    rats (unspecified sex). Histologically a significant change in the
    kidneys was found in rats on 15,000 and 25,000 ppm diphenylamine in
    the diet. These cystic changes showed a relationship to the dose
    level and time an the diets. Occasionally the cystic dilated
    tubules were filled with red cells, hemoglobin or a breakdown
    product of hemoglobin. Addition of sulphur-containing amino acids
    increased the degree of cystic changes. The concentration capacity
    of the kidneys was already reduced after 5 weeks on 25,000 ppm
    diphenylamine. The glomerular filtration rate was reduced under the
    influence of diphenylamine and was correlated roughly with the
    severity of the morphologic lesion. In addition a decrease of the
    urea and sodium concentration in the papillary tip and an increase
    in serum potassium concentration was found (Safouh et al., 1970).

         Oral dosing of rats with diphenylamine results in a
    characteristic gross tubular dilatation and polycystic appearance
    of the cortex and outer medulla. This reaction was greatly reduced
    or eliminated by prior removal of the vulnerable renal papillary
    tip. It is suggested that much of the histological changes in the
    outer zones of the kidney are secondary to the papillary necrosis
    (Hardy, 1974).

         Diphenylamine caused papillary necrosis in kidneys and
    mortality in lambs dehydrated at the time of dosing (dose level not
    known) (Salisbury et al., 1969).

         The effects of diphenylamine on sodium and water transport
    across the toad skin and bladder were studied. The results indicate
    that diphenylamine inhibits both the active sodium transport and
    the anti-diuretic hormone-induced passive water transport in
    vitro. Such actions, when occurring in vivo could play a role in
    renal cyst formation (Hong et al., 1974).

    Short-term studies

    Mouse

         A short-term study with mice was carried out because a
    striking increase in the proportion of erythrocytes containing
    Heinz bodies was found in the long-term study with mice. Groups of
    50-100 mice of each sex were fed diets containing 0, 5, 10, 50,
    100, 250 and 1000 ppm diphenylamine for 6 months. At various times
    5 animals-per group were studied. Heinz bodies were observed in the
    four highest dose groups (50, 100, 250 and 1000 ppm). Maximum
    numbers of affected erythrocytes were reached an days 9, 19, 19 and
    30 respectively for each group in 70-80%, 50-60%, 50-60% and 20% of
    the erythrocytes respectively. Heinz bodies were not observed at

    any time in control mice or mice receiving 5 or 10 ppm during 6
    months.

         Subsequent analyses of mice in the groups at 50-1000 ppm
    showed a clear trend towards decreasing numbers of Heinz bodies,
    suggesting some form of adaptation to the action of diphenylamine.
    The activity of glucose-6-phosphate dehydrogenase in the
    erythrocytes was decreased significantly only in the 1000 ppm group
    after 6-9 days. A random increase in iron was seen morphologically
    in the spleens of treated groups, but this was not dose-related. No
    increase in iron was evident in the liver or kidney. Electron
    microscopy indicated no increase in phagocytosis of erythrocytes
    containing Heinz bodies in spleen, liver or kidney (Coulston et
    al., 1972; Ford et al., 1972).

    Long-term studies

    Mouse

         Groups of 100-200 mice of each sex were fed diets, containing
    0, 50, 100 and 250 ppm diphenylamine (99.5% pure) for periods up to
    92 weeks. No effects were found on growth, clinical condition,
    survival, hematological parameters (especially no anemia nor
    methemoglobinemia) or incidence of histopathological changes. The
    most striking effect was a dose-related increase in the proportion
    of Heinz bodies in the erythrocytes at the end of the experiment.
    Even at 50 ppm Heinz bodies were slightly elevated, while at 250
    ppm a very high proportion was observed. On transfer of some
    animals to the control diet, the number of Heinz bodies decreased
    rapidly, but even after 5 weeks they had not been reduced
    completely to control levels. After 12 and 18 months spleen weight
    was increased in the female animals at 250 ppm, while at the same
    time liver weight was increased in both sexes in this group. After
    6 and 12 months a slight increase in hemosiderosis was found in the
    spleen of the animals at the highest dose level, while the number
    of reticulocytes in the blood was also slightly higher. At the end
    of the experiment, however, these effects were not observed. There
    was no difference in iron content in liver and spleen between the
    control and 250 ppm group. Electron microscopy did not reveal any
    indication of hepatocellular damage, but inclusions of red cell
    origin (Heinz bodies) were found in the reticuloendothelial cells.
    This finding was dose-dependent. The rate of tumour formation and
    tumour incidence were not different from the control values. A very
    low number of spontaneous tumours was observed in this study. No
    bladder tumours were found (Coulston et al., 1971).

    Comments

         In a long-term feeding study with mice, an increased incidence
    of tumours was not observed. However, Heinz body formation was
    increased at all levels of diphenylamine tested in this study.
    Methemoglobinemia was not detected. In a short-term study in mice,

    a no-effect level of 10 ppm was found for the induction of Heinz
    bodies. The effect level in this study was 50 ppm. Previously
    considered dietary studies indicated a no-effect level in the rat
    and dog to be 100 ppm.

    TOXICOLOGICAL EVALUATION

    Level causing no toxicological effect

         Dog:           100 ppm in the diet equivalent to 2.5 mg/kg. bw

         Rat:           100 ppm in the diet equivalent to 5 mg/kg bw

         Mouse:         10 ppm in the diet equivalent to 1.5 mg/kg bw

    ESTIMATE OF ACCEPTABLE DAILY INTAKE FOR MAN

         0 - 0.02 mg/kg bw

    RESIDUES IN FOOD AND THEIR EVALUATION

    RESIDUES RESULTING FROM SUPERVISED TRIALS

         Data from supervised trials in Australia and the Netherlands,
    summarized in Table 1, show no residues above 3 mg/kg.

    FATE OF RESIDUES

    In storage

         New data were obtained from Australia, the Netherlands and New
    Zealand on the behaviour of the residue during cold storage for one
    to five months at 4-5C following a post harvest dip or spray
    treatment or use in wrappers.

         The dosages applied were in general slightly lower than the
    rates considered by the 1969 Joint Meeting. It was indicated from
    New Zealand that high dosages applied as a drench or dip may cause
    phytotoxic effects in some susceptible and important apple
    varieties. There is a tendency in practice to use the lowest
    dosages which adequately prevent apple scald.

    In processing

         It was shown in the Netherlands experiments that the amount in
    the peel was 89.7 - 91.5% of the total apple residue. 90% of the
    residue would therefore be removed by peeling.

    RESIDUES IN FOOD IN COMMERCE OR AT CONSUMPTION

         Information was provided from New Zealand to the effect that
    residues in apples wrapped in diphenylamine-treated paper did not
    exceed 3 mg/kg.


    
    TABLE 1. Residues of diphenylamine in apples resulting from supervised trials

                                                                                                                             
                                                                  Residues, mg/kg, at interval (weeks)
                                       Application                           after application

    Country         Year
                                       Rate      Mode of
                              No.      mg/l      application      4          8           12/13         15/16           20/21
                                                                                                                             

    Australia1      1976      1        1000      dip                                                   <0.5

                              1        1000      dip                                     <0.5

                              1        1500      spray                                                 <0.5

                              1        1500      spray                                   <0.5

                              1        250       dip*                                                  <0.5

                              1        250       dip*                                     <0.5

    Netherlands2    1972      1        800       dip +            1.70       1.37        1.09          0.82            0.78
                                                 wetting          (0.82-     (0.69-      (0.54-        (0.25-          (0.14-
                                                 agent            2.70)      1.88)       1.54)         1.64)           1.62)
                                                                                                                             

    * combined with fumigation with methylbromide 64g/m3.

    References:

    1 Snelson, 1976

    2 ten Brueke and Dornseiffen, 1973

    

    METHODS OF RESIDUE ANALYSIS

         Residue analysis in the Australian supervised trials referred to
    above was by a colorimetric method with a limit of determination of
    0.5 mg/kg. In the Netherlands' trial the gas-chromatographic method of
    Gutenmann et al., 1963, with electron capture (63Ni) detection, was
    used. The limit of determination was 0.005 mg/kg. The mean recovery in
    apple peel at the 1 mg/kg level was 92.9% (sigma  9.5%); in apple
    peel at a level of 0.08 mg/kg, 93% (sigma  15.8%).

    APPRAISAL

         The bulk of the residue data evaluated in 1969 and the new
    information received by the Meeting indicate that residues of
    diphenylamine in apples generally do not exceed 5 mg/kg. However, data
    evaluated in the 1969 monograph indicate that following commercial
    practice a small percentage of samples showed residues up to
    8.6 mg/kg.

         Whilst recognising that a maximum residue limit of 5 mg/kg
    appeared appropriate in the light of the latest information available,
    the Meeting was reluctant to recommend an amendment until countries
    were given a further opportunity to submit data determined by modern
    GLC methods to reflect residues resulting from practices now approved
    and in use in commercial packing houses.

         The Meeting noted that the theoretical potential intake of
    diphenylamine would not exceed the A.D.I. even if it were assumed that
    all apples contained residues at the maximum level recommended.
    However, the following factors ensure that the intake is very much
    lower than the potential.

    1.   Diphenylamine is used on only some varieties of apples
         (occasionally on some pears).

    2.   Only apples that are to be held in cold storage for long periods
         are treated.

    3.   Only some of the treated apples have residues approaching the
         limit.

    4.   Only in some regions is it necessary to use treatments that give
         rise to higher than average residues.

    5.   Diphenylamine-treated apples are available only during limited
         periods of the year.

    6.   The residue is principally in the peel.

    RECOMMENDATIONS

         The Meeting recommends that the existing maximum residue limit of
    10 mg/kg for apples should remain unchanged at present but should be
    lowered to 5 mg/kg in 1978 unless data which may become available on
    the residues resulting from current practices, determined by modern
    GLC methods, indicate that such a reduction is inappropriate.

    FURTHER WORK OR INFORMATION

    DESIRABLE

    1.   Short-term studies with special attention to the formation of
         Heinz bodies.

    2.   Data determined by modern GLC methods to reflect residues
         resulting from practices now approved and in use in packing
         houses.

    REFERENCES

    Bergstrom, P.D., Grant, D.W. and Morrison, S.M. Nitrosation
    1972                in feedlot manure Environ. Lett. 3: 151-157 (C.A.
                        77: 95209)

    ten Brueke, R. and Dornseiffen, J.W. Residues of diphenylamine
    1973                and o-phenylphenol on apples. Unpublished report
                        No. KvW 163, Food Inspection Service, Amsterdam.

    Coulston, F., Golberg, L., Abraham, R. and Benitz, K.F.
    1971                Long-term study of the toxicity of diphenylamine
                        in mice. Final report. Unpub. rept. Inst. Exp.
                        Pathol. Toxicol., Albany Medical College, Albany,
                        New York.

    Coulston, F., Golberg, L., Abraham, R. and Ford, W. Supplementary
    1972                report on diphenylamine. Unpub. rept. Inst. Exp.
                        Pathol. Toxicol., Albany Medical College, Albany,
                        New York.

    FAO/WHO 1969 Evaluation of some pesticide residues in food.
    1970                FAO/PL:1969/M/17/1; WHO/Food Add./70.38.

    Ford, W., Abraham, R., Rockwood, W. and Golberg, L. Observations
    1972                consequent upon a long-term study of diphenylamine
                        in mice. Toxicol. Appl. Pharmacol. 22: 302
                        (Abstract no. 73).

    Galea, V., Preda, N., Pope, L. and Simu, G. Experimental production
    1975                of nitrosamines in vivo. IARC Sci. Publ. 1975:
                        121-122 (C.A. 83:-127211).

    Gutenmann, W. H., and Lisk, D. J. A feeding study with diphenylamine
    1975                in a dairy cow. Bull Environ. Cont. Toxicol. 13:
                        177-180.

    Hardy, T. L. Experimental surgery in the evaluation of drug induced
    1974                renal toxicity in the rat with particular
                        reference to diphenylamine. Proc. Eur. Soc. Study
                        Drug Toxic. 15: 337-344 (C.A. 83: 108513).

    Hong, S. K., Szekerczes, J., Park, Y. S., Kurata, F. K. and
    1974                Gardner, K. Effects of diphenylamine on sodium and
                        water transport across the toad skin and bladder.
                        Toxicol. Appl. Pharmacol. 27: 612-620.

    Safouh, M., Crocker, J. F. S. and Vernier, R. L. Experimental
    1970                cystic disease of the kidney. Sequential,
                        functional and morphological studies. Lab. Invest.
                        23: 392-400.

    Salisbury, R. M., McIntosch, I. G. and Staples, E. L. J.
    1969                Mortality in lambs and cattle following the
                        administration of phenothiazine. N. Z. Vet. J. 17:
                        227-233 (C.A. 78: 119262).

    Sander, J., Schweinsberg, F. and Menz, H. P. Formation of
    1968                carcinogenic nitrosamines in the stomach. Hoppe-
                        Seyler's Z. Physiol. Chem. 349: 1691-1697 (C.A.
                        70: 35968).

    Snelson, J. T. Diphenylamine residues in apples; results
    1976                of supervised trials in Western Australia.
                        Pesticides Section, Department of Primary
                        Industry, Canberra, October 1976.

    Volodchenko, V. A. Toxicological characteristics of diphenylamine
    1975                and certain of its derivatives depending on the
                        chemical structure. Gig. Sanit. 1975: 114-116
                        (C.A. 84: 116482).

    


    See Also:
       Toxicological Abbreviations
       Diphenylamine (ICSC)
       Diphenylamine (FAO/PL:1969/M/17/1)
       Diphenylamine (Pesticide residues in food: 1979 evaluations)
       Diphenylamine (Pesticide residues in food: 1982 evaluations)
       Diphenylamine (Pesticide residues in food: 1984 evaluations)
       Diphenylamine (Pesticide residues in food: 1984 evaluations)