WHO/FOOD ADD./70.38



    Issued jointly by FAO and WHO

    The content of this document is the result of the deliberations of the
    Joint Meeting of the FAO Working Party of Experts and the WHO Expert
    Group on Pesticide Residues, which met in Rome, 8 - 15 December 1969.



    Rome, 1970



    Chemical name




    Structural formula



    Minimum 99.9 per cent by cryoscopic method. Primary amines as aniline,
    not more than 10 parts per million.



    Absorption, distribution and excretion

    Diphenylamine is absorbed from the digestive tract of the rat, rabbit,
    dog and man; however no information is available on the extent of
    absorption (DeEdu, 1961; Alexander et al., 1965).

    The metabolism of diphenylamine has been studied in these four species
    and is essentially the same in all of them. A slight amount of
    unchanged diphenylamine has been detected in the urine of rabbits and
    of man (but not rats) after oral administration of the compound. The
    major metabolites are 4-hydroxydiphenylamine and
    4,4'-dihydroxydiphenylamine. Both of these compounds have been
    identified as conjugates in the urine of human subjects which had
    received a single oral dose of 100 mg of diphenylamine, but no free
    hydroxylated derivatives are excreted in man. In the rat, rabbit and
    dog, 4-hroxydiphenylamine is excreted partly unchanged and partly as
    conjugates with glucuronic acid and sulphuric acid, and these
    conjugates have been isolated from rabbit urine. Hydroxylation in the
    ortho-position occurs only in the rabbit and then only to a slight
    extent. N-hydroxydiphenylamine has not been detected from any species
    and there is no evidence for its formation (Alexander et al., 1965;
    Booth, 1963).

    The N-glucuronide of 4-hydroxydiphenylamine has been reported to have
    been detected in the urine or rats after oral administration of
    diphenylamine. However, the method described for characterizing the

    compound would not have distinguished it from the O-glucuronide
    (DeEds, 1961).

    Studies with carbon14-labelled diphenylamine indicate that the
    compound in rapidly metabolized and excreted by the rat. After 48
    hours, 75 per cent of an intraperitoneal dose appears in the urine. An
    intravenous dose results in the excretion of 25 per cent of the
    radio-activity in bile after six hours (Alexander et al., 1965).

    Hydroxylated derivatives of diphenylamine have been detected in rat
    and dog faeces and represent excretion via the bile, since acid
    hydrolysed bile was found to contain 4-hydroxydiphenylamine. The
    possibility that intestinal bacteria may be also hydroxylate
    unabsorbed diphenylamine has, however, not been ruled out (DeEds.

    No information is available on the relative amounts of urinary or
    faecal metabolites derived from an oral dose of diphenylamine.

    Special studies on carcinogenicity


    Two groups, each containing 40-50 male and 40-50 female mice weighing
    30-35 g, were selected for this study. The animals received
    subcutaneous injections of 0.5 ml of a 25 per cent solution of
    diphenylamine in trioctanoin (approximately 4000 mg/kg body-weight)
    once every two weeks on alternate sides of the body. After two months
    it was found that the compound accumulated when given this frequently
    and the injections were then given once every three weeks until the
    animals had received injections for a total period of 80 weeks. A
    control group of mice received injections of the solvent vehicle
    alone. There was no significant difference in the incidence of
    malignant or benign tumours between the test and the control groups,
    nor was the injection of diphenylamine associated with the development
    of tumours at the injection site. A related study involving oral
    feeding of diphenylamine to mice also involved tissue examination for
    tumours and this study is described under "Long-term studies" (Univ.
    of Birmingham, 1966).

    An 80 week oral feeding study in mice is currently in progress and the
    study will include an evaluation for carcinogenicity. The results from
    a preliminary progress report of this study are given under
    "Short-term studies" (Golberg, 1969).


    It is known that bacteria in the human stomach will reduce nitrates to
    nitrites. Studies in 31 patients showed that these nitrites will react
    with diphenylamine to form diphenylnitrosamine (Sander and Seif,
    1969). However, it has previously been demonstrated with studies in
    the rat that, unlike many nitrosamines, diphenylnitrosamine is not a
    carcinogen (Druckrey et al., 1961).

    Special studies on reproduction


    A two-generation rat reproduction study was conducted with dietary
    levels of 0, 1000, 2500 and 5000 ppm of diphenylamine. Treatment was
    began at weaning. At 100 days of age, 12 females and three males were
    selected from each of these dose level groups and grouped as three
    females and one male per cage. Once a week for three weeks the males
    were isolated among the three groups of females, after which time the
    males were removed and the females placed in individual cages. When
    all the litters were weaned, the females were given a 2-3 week rest
    and then remated. In addition offspring from the first mating were
    mated once, as described above, for a second generation study. Feeding
    of diphenylamine did not influence the number of litters born or the
    incidence of mortality of the offspring. It was uncertain if
    diphenylamine was responsible for the reduced litter-size and weight
    of the young at 21 days which was observed in the group fed 5000 ppm
    of diphenylamine (Thomas et al., 1967a; Booth, 1963).

    Acute toxicity

    The oral LD50 of diphenylamine to male rats is approximately 3,200
    mg/kg body-weight (American Cyanamid Co., 1956).

    Short-term studies


    Groups of four dogs (two males and two females) were fed diets
    containing 0, 100, 1000 and 10,000 ppm of diphenylamine for a period
    of two years. The dogs were about eight months of age at the
    commencement of the study. Decreased body-weight gain occurred in the
    1000 and 10,000 ppm groups although food consumption was normal. A
    pronounced anaemia developed in the 10,000 ppm group and mild anaemia
    in the 1000 ppm group. The bromsulphthalein (BSP) test of liver
    function from day 618 to day 627 indicated a moderate degree of liver
    damage at 10,000 ppm. The phenolsulphonaphthalein (PSP) test of kidney
    function gave normal values, and the urine gave negative tests for
    albumin and glucose. All organ-weight changes and microscopic lesions
    were limited to the 10,000 ppm level. These manifestations consisted
    of peripherolubular fatty change in the liver with a marked increase
    in liver-weight and other-extractable lipids; a mild haemosiderosis of
    the spleen, kidneys and bone marrow, and a slight increase in kidney
    weight (Thomas et al., 1967b).


    A preliminary progress report is available on the feeding of dietary
    levels of 0, 50, 100 and 250 ppm of diphenylamine to groups of mice.
    Over a 2-3 month period there has been no difference in body-weight or
    incidence of mortality between control and test groups. The feeding

    will be continued for a total of 80 weeks and will include
    haematological examinations and histological evaluation at autopsy
    (Golberg, 1969).


    Groups of 10 male rats were fed diets containing 0, 100, 1000, or
    10,000 ppm of diphenylamine for 30 days. There were no deaths in any
    group, and no signs of toxicity at 100 and 1000 ppm. The animals fed
    10,000 ppm made only about one-half the weight gain of the controls
    even though the food intakes of the two groups did not differ. Food
    intake and weight gain at 1000 ppm were significantly higher than
    those of the controls. Autopsy of animals at 100 and 1000 ppm
    disclosed no gross pathologic lesions that could be attributed to
    feeding of diphenylamine. At 10,000 ppm the animals had dark and
    roughened spleens, and three had hyperaemic kidneys. Paleness of the
    extremities in most of the rats fed the highest dose level was noted
    but no chemical analysis was performed to determine if methaemoglobin
    was present in the blood. No histological examination was made of the
    tissues (American Cyanamid Company, 1956).

    Groups of six female rats were fed diets containing 0, 250, 1000,
    5000, 10,000, or 15,000 ppm of diphenylamine for 226 days. Inhibition
    of growth occurred at dietary levels of 5000 ppm or more. At necropsy
    no gross change was noted except for enlargement of the kidneys in
    animals which had received 15,000 ppm. Microscopic examination of
    tissues revealed the formation of foci of dilated renal tubules and
    pigmentation of the liver and kidney suggestive of blood destruction
    at levels of 5000 ppm and above (Thomas et al., 1957; Booth, 1963).

    An unspecified number of rats, including weanling rats of both sexes
    and adult male rats weighing 250 grams, were fed a diet containing 2.5
    per cent of diphenylamine for periods up to 12 months. Morphologic
    alterations of the renal tubules were found in all animals. These
    alterations varied from tubular dilation to overt cyst formation.
    Glomerular filtration rate and maximum urinary osmolality were
    decreased and the degree of decrement corresponded to the degree of
    involvement. The animals with polycystic kidneys had an increased
    susceptibility to pyelonephritis (Kime et al., 1962).

    Long-term studies


    Groups of mice (40-50 animals of each sex) were fed diets containing
    0, 100, 300, 1000, and 5000 ppm of diphenylamine for 80 weeks. The
    study began when the animals were 4-5 weeks of age. Increased
    mortality occurred on diets containing 300 ppm or more of
    diphenylamine. Abnormalities in the liver (chronic inflammatory change
    and iron pigment deposition), kidneys (iron deposition) and spleen
    (iron deposition, fibrosis, lymph follicle hypoplasia) occurred at
    5000 ppm, and also in the spleen at 1000 ppm. Although the total

    tumour incidence was significantly increased in the 100 ppm group,
    this increase appeared incidental and not associated with
    diphenylamine intake. The effect of subcutaneous injections of
    diphenylamine to another group of mice in this study is described
    under "Special studies on carcinogenicity". In this experiment except
    for one isolated period between weeks 30 and 40 with respect to the
    female test group, the incidence of mortality was not different from a
    control group (Univ. of Birmingham, 1966).


    Groups of 40 rats each comprising 20 males and 20 females were fed
    diets containing 0, 10, 100, 1000, 5000, or 10,000 ppm of
    diphenylamine, commencing at weaning and continuing for two years.
    There was no evidence of toxicity among animals at the 100 ppm level
    and below. There was a decreased rate of growth at 5000 and 10,000
    ppm, the effect at the latter level being at least partly due to
    reduced food intake. Doubtful effect on growth occurred at 1000 ppm in
    the females. A moderate degree of anaemia occurred at 10,000 ppm,
    which was reversible upon returning the animals to the control diet.
    Total white cell count and differential white cell count remained
    normal at all dose levels. Kidney damage in the form of dilated
    tubules was produced at levels above 1000 ppm, and to a lesser degree
    at 1000 ppm. The incidence and type of tumours found were unrelated to
    treatment with diphenylamine (Thomas et al., 1967a).


    Dihydroxylated products of metabolism have been identified in the
    urine of laboratory animals and human subjects; N-hydroxylation was
    not observed. Short-term studies with an insufficient number of rats
    have been carried out; at higher concentrations of diphenylamine,
    morphologic alterations of the renal tubules were found. There is
    insufficient information on the possible formation of methaemoglobin
    which might be expected with an aromatic amine. In the long-term
    studies on mice, an increased tumour incidence appeared incidental,
    but subcutaneous injections of diphenylamine in trioctanoin solution
    demonstrated no significant difference between test and control
    groups. In the long-term studies on rats, the incidence and type of
    tumours were unrelated to treatment with diphenylamine. A study on the
    carcinogenicity in mice after oral ingestion is in progress. The
    short-term studies on dogs and the lone-term studies on rats form the
    experimental basis for the established adi.


    Level causing no toxicological effect

    Dog:   100 ppm in the diet, equivalent to 2.5 mg/kg body-weight/day

    Mouse: 100 ppm in the diet, equivalent to 15 mg/kg body-weight/day

    Rat:   100 ppm in the diet, equivalent to 5 mg/kg body-weight/day

    Estimate of acceptable daily intake for man

    0.0-0.025 mg/kg body-weight



    DPA is used to prevent a storage disorder of apples known as scald. It
    is the only known use of DPA. The incidence and severity of the
    disease varies, depending upon locality, seasonal conditions prior to
    and at harvest. Varietal differences in susceptibility and severity of
    the condition are frequent, as well as a requirement for greater
    concentrations of CPA (and higher residues) for them to prevent
    losses. The protective action of DPA is believed to be due to the
    antioxidant effect on alpha-farnesene a sesquiterpene which occurs in
    the natural coatings of apples (Huelin and Murray, 1966).

    Alcohol suspensions have been used in some of the earlier work
    reported here, but it is believed that only wettable powders, oil
    emulsions and impregnated papers are in use now. Data are available
    from Australia, New Zealand, U.S.A. and the U.K. DPA is also used in
    Canada, and probably in many other apple producing countries.

    Pre-harvest treatments

    Mature apples on the trees are sprayed a few days before harvest if
    the first symptoms of the disease are detected at this stage, or if
    weather conditions suggest treatments should be applied. An aqueous
    suspension is applied at concentrations of 500 to 2000 ppm.

    Post-harvest treatments

    Harvest fruit is (a) dipped in aqueous suspension (500 to 3000 ppm),
    (b) sprayed in boxes, pallet loads, bins or conveyors (1000 to 2000
    ppm), boxes immersed in suspensions or emulsions, or (c) individual
    apples wrapped in impregnated paper (1 to 2 mg/wrap).

    Most fruit treated is held in storage for from 80 to 200 days or more
    before marketing.


    Residues in whole fruit from tree sprays are usually comparatively
    low, less than 1 ppm (Bruce et al., 1958; Harvey and Clark, 1959), but
    can commonly be as high as 6 ppm on a particular variety (Gutenmann
    and Lisk, 1963) at the rates outlined above. Initial residues from
    post-harvest sprays and dips have been reported as high as 63 ppm, but
    these decline rapidly in storage. Most post-harvest spray residues
    after storage are in the range of 2 to 6 ppm (Harvey and Clark, 1959;
    Bache et al., 1962), but have been reported as high as 7.7 ppm (Harvey
    and Clark, 1959). Dip deposits from 2000 ppm suspension can have
    initial residues as high as 12 ppm, usually most are above 8 ppm

    (Bruce et al., 1958). In one variety, an initial residue of 62.6 ppm
    declined after 120 days in storage to 10 ppm. 1000 ppm dip residues
    are not likely to be higher than 4 ppm. Residues from impregnated
    paper wraps are less than 4 ppm (Bruce et al., 1958). One-half the
    total DPA content of apples was found in peel (Bruce et al., 1958),
    but 90% can be found in the outer 2 to 4 mm of the fruit (Harvey and
    Clark, 1959). Little migration of DPA occurs, either laterally or into
    the apple flesh (Hall et al., 1961). This has recently been confirmed
    using autoradiography and 14C-DPA solutions (Wilson 1969, unpublished


    The disappearance of DPA, from either physical or chemical standpoint
    does not appear to be accounted for. It is assumed that most losses in
    storage are due to volatilization, but this is not verified by
    available literature.

    Cooking studies have not been reported. The post-storage residues
    noted above can be presumed to be those occurring on fruit at the time
    of consumption.

    Five years of data on commercial scale treatments and holding periods,
    involving six varieties of apples are available from Cornell
    University (Bache et al., 1962) resulting in average residues after
    storage of up to 6 ppm required in order to obtain effective scald
    control. Residues of between 8 to 9 ppm also are associated with the
    most effective treatments. In 1965 Cornell University (Smock, R.M.
    1969, unpublished) analysed commercial lots of treated apples
    collected from eight locations in New York State. Treatments applied
    by box flooding or immersion varied from 1000 to 2000 ppm DPA.
    Variations in residues in individual apples depending upon location
    within the box varied considerably, but no consistent pattern was
    established. The highest residue reported from a sample at the top of
    a box was 6.91 ppm (corrected).


    The earliest development work on the use of DPA in New Zealand
    utilized the method of Yatsu described by Harvey (1958). It involves
    vanadium pentoxide-sulphuric acid oxidation of DPA to produce a blue
    colour measured at 600 nanometers (nm). Sensitivity is approximately
    0.5 ppm for apples. Bruce et al. (1958) developed a method of coupling
    the amino with diazotized 2,4-dinitroaniline, measured at 530 nm. This
    method is sensitive to at least 0.1 ppm in apples. This method is
    likely to be acceptable for use by analysts in regulatory
    laboratories. Recently, a procedure for gas chromatography electron
    capture has been described (Guttenmann and Lisk, 1963). It involves
    bromination of DPA to produce presumptive 2,2', 4,4',
    6.6'-hexabromo-DPA and its subsequent GLC determination. Sensitivity
    of about 0.02 ppm is claimed.

    There is a need to monitor to DPA content of solutions during
    commercial treatments in post-harvest applications in order to
    determine the point in time for renewal of solutions. The GLC
    procedure was not found to be reliable for residue values of 0-25 ppm
    for whole apples. A colorimetric procedure was adapted whereby DPA
    extracted from blended apple tissues with acetone was reacted with
    varnadium pentoxide and quantitatively determined
    spectrophotometrically at 605 mu. This procedure was modified for
    monitoring the DPA content of commercial applicator systems (Wilson,


    Country                      Commodity      Tolerance (ppm)

    Australia                    Apples               10

    Canada                       Apples               10

    United States of America     Apples               10

                                 Milk and meat       zero


    Diphenylamine (DPA) has a minimum purity of 99.9 per cent. Primary
    amines such as aniline are stated as not more than 10 ppm of the
    technical grade material.

    DPA is used to prevent losses from a storage disease of apples known
    as "scald", which varies in severity and incidence due to locality,
    weather and in different varieties of apples. The protective action of
    DPA is attributed to antioxidant effect on alpha-farnesene, a
    sesquiterpene which occurs in the natural coating of apples.
    Pre-harvest emulsion sprays applied to mature apples very close to
    harvest and post-harvest treatments of bulk apples on sorting rollers,
    in boxes, pallets, bins, etc. are applied as emulsions or wettable
    powders. Impregnated paper wraps are also used.

    The residue is believed to be DPA alone. No degradation products or
    metabolites in fruit are identified, nor are they presumed to exist.
    Losses are attributed to volatilization. The amount of residue
    immediately after treatment varies with the technology of treatment,
    but can very from 63 ppm to as low as 1 ppm. All treated apples are
    held in storage from 80 to over 200 days. Residues decline in storage
    to an average range of less than 1 to 8 ppm, with an occasional
    residue in one variety as high as 10 ppm after 120 days, storage
    reported. About half the residue is in the peel, and 90% in the outer
    2 to 4 mm of fruit. Little migration occurs afterwards.

    No results of cooking studies are available. Residues reaching the
    consumer can be assumed to be in the order of those mentioned above.

    Five years of data collected from commercial scale treatments and
    storage holding periods suggest average residues will be 6 ppm, with
    some at 8 to 9 ppm. The most recent commercial sampling from eight
    locations in New York State resulted in the highest residue found as
    6.9 ppm.

    An analytical method based on coupling the amine with diazotized
    2,4-dinitroaniline, measured at 530 nm is sensitive to 0.1 ppm in
    apples. This method is likely to be acceptable for most regulatory
    laboratories. A GLC electron capture method is also available. It
    involves bromination of DPA to produce presumptive 2, 2', 4, 4', 6,
    6'-hexabromo DPA.



    Apples       10 ppm



    1. Experiments to determine if methaemoglobin is formed in animals.

    2. Short-term studies using an adequate number of rats.

    3. Additional metabolic studies in a non-rodent mammalian species.

    4. The results of the carcinogenicity study in mice which is currently
       in progress.


    Alexander, W.E., Ryan, A.J. and Wright, S.E. (1964) Metabolism of
    diphenylamine in the rat and rabbit. Experientia 20:223-4

    Alexander, W.E., Ryan, A.J. and Wright, S.E. (1965) Metabolism of
    diphenylamine in rat, rabbit and man. Food Cosmet. Toxicol. 3:571-9

    American Cyanamid Co. (1956) Diphenylamine : limited release toxicity
    studies. Unpub. rept. submitted by C.B. Shaffer

    Bache, C.A., Smock, R.M., Yatsu, L., Mooney. C., and Lisk, D.J. (1962)
    Diphenylamine residues on apples in relation to scald control.
    Proc. Amer. Soc. Hort. Sci. 81:57-60

    Booth, A.N. (1963) Summary of toxicological data. Chronic toxicity 
    studies on diphenylamine. Food Cosmet. Toxicol 1:331-3

    Bruce, R.B., Howard, J.W., and Zink, J.B. (1958) Determination of
    diphenylamine residues on apples. J. Agr. Food Chem. 6:597-600

    DeEds, F. (1961) Chronic toxicity studies on diphenylamine. Unpub. 
    rept. on data for use in a petition requesting tolerance for
    diphenylamine used in prevention of "scald" of apples during
    storage. Western Utilization R. and D. Div. U.S.D.A., Albany Calif.
    Druckrey, H., Preussmann, R., Schmähl, D. and Miller, M. (1961)
    Chemische Konstitution und Carcinogens Wirkung bei Nitrosaminen.
    Naturwissenschaften 48:134-5

    Golberg, L. (1969) Determination of the toxicity of diphenylamine in mice.
    First progress report. Unpub. rept. Inst. Exp. Pathol. Toxicol., 
    Albany, New York

    Gutenmann, W.H., and Lisk, D.J. (1963) Rapid determination of
    diphenylamine in apples by direct bromination and gas chromatography.
    J. Agr. Food Chem. 11:468-70

    Hall, E.G., Scott, K.J., and Coote, G.G. (1961) Control of superficial
    scald on Granny Smith apples with diphenylamine. Australian J. Agr.
    Res. 12:834-52

    Harvey, H.E. (1958) Determination of diphenylamine residues on apples.
    New Zealand J. Sci. 1:378-82

    Harvey, H.E., and Clerk, P.J. (1959) Diphenylamine residues on apples.
    New Zealand J. Sci. 2.266-72

    Huelin, F.E., and Murray, K.E. (1966) alpha-Farnesene in the natural
    coating of apples. Nature 210.1260-61

    Kime, S.W. Jr., McNamara, J.J., Luse, S., Farmer, S., Silbert, C. and
    Brieker, N.S. (1962) Experimental polyeystic renal disease in rats :
    electron microscopy, function and susceptibility to pyelonephritis. 
    J. Lab. Clin. Med. 60:64-78

    Sander, von J., and Seif, F. (1969) Bakterielle Reduktion von Nitrat
    im Magen des Menschen als Ursache einer Nitrosamin-Bildung.
    Arzneimittel-Forsch. 19:1091-98

    Thomas, J.O., Cox, A.J. Jr. and DeEds, F. (1957) Kidney cysts produced
    by diphenylamine. Stanford Med. Bull. 15:90-93

    Thomas, J.O., Ribelin, W.E., Wilson, R.H., Keppler, D.C. and DeEds, F.
    (1967a)  Chronic toxicity of diphenylamine to albino rats. Toxicol.
    appl. Pharmacol. 10:362-74

    Thomas. J.O., Ribelin. W.E., Woodward, J.R. and DeEds, F. (1967b)
    The chronic toxicity of diphenylamine for dogs. Toxicol. appl.
    Pharmacol. 11:184-94

    University of Birmingham. (1966) Studies on the long-term effect of
    diphenylamine in mice. Unpub. rept. Toxicol. Unit, Dept. Med. Biochem. 
    Pharmacol., Birmingham, England

    Wilson, L.G. (1969) Unpublished Ph.D. thesis. Ann Arbor, Mich. 17 Jan. Univ.
    Microfilms 69-20-958

    See Also:
       Toxicological Abbreviations
       Diphenylamine (ICSC)
       Diphenylamine (Pesticide residues in food: 1976 evaluations)
       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)