364. Diphenylamine (Pesticide residues in food: 1976 evaluations)

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 4-5°C 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

1 250 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)