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    WHO/Food Add./24.65
    FAO Nutrition Meetings
    Report Series No. 38A




    SPECIFICATIONS FOR IDENTITY AND 
    PURITY AND TOXICOLOGICAL EVALUATION 
    OF SOME ANTIMICROBIALS AND 
    ANTIOXIDANTS





    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met 8-17
    December 1964a





                   

    a Eighth Report of the Joint FAO/WHO Expert Committee on Food
    Additives, Wld Hlth Org. techn. Rep. Ser., 1965, 309; FAO
    Nutrition Meetings Report Series 1965, 38.


    DIPHENYL

    CHEMICAL NAMES        Biphenyl; phenylbenzene

    EMPIRICAL FORMULA     C12H10

    STRUCTURAL FORMULA

    MOLECULAR STRUCTURE 10

    MOLECULAR WEIGHT      154.21

    USE                   As a fungistatic agent in wrapping material to
                          inhibit growth of moulds on citrus fruits.

    DESCRIPTION           White or pale yellow to amber, crystalline
                          solid or leaflets having a characteristic
                          odour.

    IDENTIFICATION TESTS

    A.  Solubility:    Water:    Insoluble
                       Ether:    Soluble
                       Ethanol:  Soluble

    B.  Melting point: About 69

    C.  Boiling point: About 225

    PURITY TESTS

    Arsenic:  Not more than 3 mg/kg.

    Lead:  Not more than 10 mg/kg.

    Solidification point:  Not below 68.5

    Distillation range:  It distils completely within a 2.5 range between
    252.5 and 257.5.

    ASSAY

    Required.

    Biological Data

    Biochemical aspects

    Diphenyl is metabolized in rats1,2 and rabbits2 to various phenolic
    compounds, mainly 4-hydroxydiphenyl which is excreted in the urine in
    both free and conjugated form (ethereal sulfate and glucuronate). 
    This demonstration of a common mechanism in 2 species of animals
    suggests that human metabolism would follow similar paths, but no
    report of this has been found in the literature.

    Diphenyl was found to be less toxic when given with a diet containing
    a supplement of 1-cystine or dl-methionine, but diphenyl is not highly
    conjugated with cystine.

    Acute toxicity

                                                                       

    Animal       Route                 LD50             References
                                 (mg/kg body-weight)
                                                                       

    Rat          oral                 3500-5000              3,4
    Rabbit       oral                   2400                  3
                                                                       

    Short-term studies

    Rabbit.  Five rabbits were each given doses of 1 g/kg body-weight by
    stomach tube in a 25% dilution in olive oil 2 or 3 times a week until
    they died.  Deaths occurred within 5 to 20 weeks.  The blood picture
    showed no significant change, but the gain in body-weight was less
    than in the controls, and retention of urea occurred near the terminal
    stage of intoxication.3

    Rat.  Eleven young female rats were fed for 32 days on a diet low in
    casein and containing 1% diphenyl.  A marked growth retardation was
    observed.  Histological examination of 2 animals showed the beginning
    of fatty changes in the liver and mild toxic changes in the kidney.1

    In a small group of weanling rats fed with a diet containing 0.3%
    diphenyl, weight increase was less than in control animals.5

    Administration of diphenyl to young rats for 4 weeks in doses of 2, 20
    and 200 mg/kg body-weight per day induced neither growth retardation
    nor significant change in the blood picture.6

    Oral administration of 0.3 mg/kg body-weight daily to 10 rats for 12
    days did not induce growth retardation.7

    Groups of young rats were fed for 3 months on rations containing 0%,
    0.01% and 0.1% of diphenyl.  No significant differences were observed
    in the growth rates, food efficiency, organ weights, blood urea or
    histology of tissues between treated and control rats.  At the level
    of 0.1% only a slight polyuria was noted.8

    Monkey.  Small groups of monkeys were fed for a year on rations
    containing 0.01%, 0.1% and 1% diphenyl.  The only significant change
    was a slight increase in weight of the liver at the intake level of
    1%.8

    Dog.  Nine dogs divided into 3 groups received diphenyl in corn oil
    daily at the rate of 0, 2.5 and 25 mg/kg body-weight 5 days a week for
    52 weeks.  One dog on 2.5 mg/kg and 1 on 25 mg/kg lost about 1 kg in
    weight each.  The other animals gained in weight.  Blood and urine
    were unchanged.  No gross or microscopic pathological changes were
    found in any of the tissues examined.9

    Long-term studies

    Rat.  A long-term feeding experiment with rats made use of dietary
    levels of 0.01%, 0.1% and 1% diphenyl for a 2-year period.8
    Unfortunately this study failed to supply the desired information for
    two reasons: (a) a severe respiratory infection caused a high
    mortality among the controls; (b) rats receiving 0.1% and 1% diphenyl
    exhibited tubular dilatation of the kidneys, as did two of the control
    rats.

    Groups of 15 weanling rats of each sex were fed 0.001%, 0.005%, 0.01%,
    0.05%, O.1%, O.5% and 1% diphenyl in the diet.  Growth was inhibited
    particularly at the 0.5% and 1% levels, apparently because of
    decreased food consumption daring the first 100 days of the test.
    During 750 days there was a decrease in longevity in rats on the 0.5%
    and 1% diets.  At the other levels there was no significant evidence
    of toxicity.  With the diet containing 1% diphenyl, haemoglobin values
    of male end female rats were lower in comparison with controls when
    measured at 300 and 400 days respectively.  The histopathological
    changes observed in the kidneys of rats on the 0.5% and 1% diets were
    irregular scarring, lymphocytic infiltration, tubular atrophy and
    patchy tubular dilatation to the point of cyst formation in
    association with hydronephrosis and sometimes albuminuria and
    haemoglobinuria.  These phenomena did not occur at a concentration of
    O.1% diphenyl or less.10

    In another long-term study, the onset end reversibility of kidney
    damage in rats on diets containing 0.1%, 0.25% and 0.5% diphenyl were
    investigated, and the findings quoted under reference 10 were
    confirmed.11

    In one experiment on 13 rats fed daily with a diet containing 0.025%
    to 0.05% diphenyl, after 2 months 1 animal had squamous cell carcinoma
    and 2 had papillomas of the forestomach.4

    Comment on experimental studies reported

    Most of the studies reported have been carried out in dogs and rats.
    The value of negative results of short-term studies in groups of 3
    dogs at levels of 2.5 and 25 mg/kg body-weight is limited.9 
    Long-term studies in rats provide a basis for evaluation.10,11

    Metabolic studies on 2 species reveal a common mechanism of
    detoxication, but no data are available on metabolism in man.

    Evaluation

    Level causing no significant toxicological effect in the rat

    From consideration of the long-term studies in rats, the level causing
    no significant toxicological effect when ingested over a period
    approximating to the life span nay be assessed at 0.1%.

    0.05% (= 500 ppm) in the diet is equivalent to 25 mg/kg body-weight
    per day.

    Estimate of acceptable daily intakes for man

                                         mg/kg body-weight

             Unconditional acceptance          0-0.05
             Conditional acceptance           0.05-0.25 

    Comment

    Diphenyl presents certain peculiar problems since it is primarily used
    as a treatment for wrappers for fruit.  Nevertheless, diphenyl
    penetrates into the skin of the fruit and may consequently be included
    in food or drink prepared from it.  Fruit drinks are commonly consumed
    in considerable quantities by children and by sick people.  For these
    reasons caution should be exercised and the pattern of use should be
    closely studied.

    Further Work Considered Desirable

    1. Long-term studies in a species other than the rat, with careful
    evaluation of tumour incidence.

    2. Biochemical studies in man.

    References

    1.  West, H. D. (1940) Proc. Soc. exp. Biol. (N.Y.), 43, 373

    2.  West, H. D., Lawson, J. R., Miller, I. H. & Mathura, R. (1955)
    Fed. Proc., 140, 303

    3.  Deichmann, W. B., Kitzmiller, K. V., Dierker, M. & Witherup, S.
    (1947) J. industr. Hyg., 29, 1

    4.  Pecehiai, L. & Saffiotei, U. (1957) Med. d. lavoro, 48, 247

    5.  West, H. D. & Jefferson, N. C. (1942) J. Nutr., 23, 425

    6.  MacIntosh, F. C. (1945) Analyst 70, 334

    7.  Rogliani, E. & Procaccini, S. (1956) Biochim. appl., 3, 193

    8.  Newell, G. W. (1953) Toxicological study of diphenyl in citrus
    wraps (Report 1953 of the Standford Research Institute)

    9.  Hazleton. L. H., Kundzins, N., Howard, J. W. & Johnson, C. D.
    (1956) Studies on diphenyl in the dog.  In:  XXth International
    Physiological Congress, Brussels, July 29 - August 4, 1956.  Abstracts
    of communications, p. 412

    10. Ambrose, A. M., Booth, A. N., DeEds, F. & Cox, A. J. (1960) Food
    Res., 25, 328

    11. Booth, A. N., Ambrose, A. M. & DeEds, F. (1956) Fed. Proc., 15,
    403
    


    See Also:
       Toxicological Abbreviations
       DIPHENYL (JECFA Evaluation)
       Diphenyl (FAO/PL:CP/15)
       Diphenyl (FAO/PL:1967/M/11/1)