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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.


    o-PHENYLPHENOL

    CHEMICAL NAMES        Orthoxenol; o-hydroxydiphenyl; 
                          o-hydroxydiphenyl

    EMPIRICAL FORMULA     C12H10O

    STRUCTURAL FORMULA

    MOLECULAR STRUCTURE 8

    MOLECULAR WEIGHT      170.21

    DEFINITION            Contains not less then 98% of C12H10O and
                          conforms to the following specifications.

    DESCRIPTION           White, slightly yellow or pink flaky crystals
                          or solid, having a mild characteristic odour.

    USE                   For the post-harvest treatment of fruits and
                          vegetables to protect against microbial damage.

    IDENTIFICATION TESTS

    A.   Solubility:  Water:     Practically insoluble
                      Ethanol:   Very soluble

    B.   Melting point:  About 57

    C.   An ethanolic solution (1 g in 10 ml) produces a green colour upon
         addition of 10% ferric chloride solution.

    PURITY TESTS

    Arsenic:  Not more than 3 mg/kg.

    Lead:  Not more than 10 mg/kg.

    Total ash:  not more than 0.05%.

    ASSAY

    Weigh 2.000 g of o-phenylphenol, dissolve in 10 ml of 10% sodium
    hydroxide solution by warming and dilute to 500.0 ml.  Pipette 25.0 ml
    into a 250-ml iodine flask and add 30.0 ml of 0.1 N bromide-bromate
    TS and 50 ml of anhydrous methanol.  Place the stopper in the flask
    and add 10 ml of dilute (1 to 1) hydrochloric acid to the well.  Raise
    the stopper slightly to allow the acid to flow down the sides inside
    the flask, but retain a small amount of the acid in the well to act as
    a seal.  Mix the contents by swirling and allow it to react for
    exactly 30 seconds at 25  5.  Immediately add 10 ml of 20%
    potassium iodide solution to the well and allow it to drain into the
    assay flask as for the acid.  Mix well, allow the solution to stand
    for 5 minutes, shaking occasionally.  Wash the stopper and the sides
    of the flask with water.  Titrate the liberated iodine with 0.1 N
    sodium thiosulfate adding starch TS as the endpoint is approached. 
    Each ml of 0.1 N bromide-bromate TS consumed is equivalent to 4.255
    mg of C12H10O.

    SODIUM o-PHENYLPHENOL

    CHEMICAL NAME         Sodium o-phenylphenate

    EMPIRICAL FORMULA     C12H9ONa.4H2O

    STRUCTURAL FORMULA

    MOLECULAR STRUCTURE 9

    MOLECULAR WEIGHT      264.26

    DEFINITION            Contains not less then 97.0% of C12H9ONa.4H2O
                          and conforms to the following specifications.

    DESCRIPTION           White powder or flakes.  When exposed to air it
                          absorbs carbon dioxide and releases free
                          o-phenylphenol which slowly sublimes.

    USE                   For the post-harvest treatment of fruits and
                          vegetables to protect against microbial damage.

    IDENTIFICATION TESTS

    A.   Solubility:  Water:    122 g dissolve in 100 ml water
                      Ethanol:  Very soluble
                      Methanol: 138 g dissolve in 100 ml methanol

    B.   An aqueous solution has a pH of about 12.7.  When neutralized, a
         precipitate of o-phenylphenol forms and, when filtered and
         dried, this material melts at about 57 and its ethanolic
         solution (1 g in 10 ml) produces a green colour upon addition of
         10% ferric chloride solution.

    PURITY TESTS

    Arsenic:  Not more than 3 mg/kg.

    Lead:  Not more than 10 mg/kg.

    Excess alkalinity (as NaOH):  Not more than 1.0%.

    Weigh 5.0 g into a 250-ml beaker, dissolve in 50 ml of water and
    titrate with 1 N hydrochloric acid to a pH of 11.0 using a suitable
    pH meter.  Each ml of 1 N hydrochloric acid is equivalent to 40 mg
    of sodium hydroxide.

    ASSAY

    Weigh 3.100 g of sodium o-phenylphenol, dissolve in water, adding a
    few drops of 10% sodium hydroxide solution if necessary to clear any
    turbidity, and dilute to 500.0 ml with water.  Pipette 25.0 ml into a
    250-ml iodine flask, and add 30.0 ml of 0.1 N bromide-bromate TS and
    50 ml of anhydrous methanol.  Place the stopper in the flask and add
    10 ml of dilute (1 to 1) hydrochloric acid to the well.  Raise the
    stopper slightly to allow the acid to flow down the sides of the
    flask, but retain a small amount of the acid in the well to act as a
    seal.  Mix the contents by swirling and allow it to react for exactly
    3O seconds at 25  5.  Immediately add 10 ml of 20% potassium iodide
    solution to the well and allow it to drain into the flask.  Mix well
    and allow the solution to stand for 5 minutes, shaking occasionally.
    Wash the stopper and the sides of the flask with water and titrate the
    liberated iodine with 0.1 N sodium thiosulfate adding starch TS as
    the endpoint is approached.  Each ml of 0.1 N bromide-bromate TS
    consumed is equivalent to 6.608 mg of C12H9ONa.4H2O.

    Biological Data

    Biochemical aspects

    Storage of o-phenylphenol has not been observed in rats.  In feeding
    experiments lasting 2 years, average values of 22 mg/100 g tissue were
    found in the kidneys of rats on a 2% diet, and approximately 1 mg/100
    g tissue in the kidneys of rats on a 0.2% diet.1

    It is known that the o- and p-hydroxydiphenyls are highly
    conjugated with glucuronic acids in the rabbit, but whether they form
    ethereal sulfates is not known.2

    Acute toxicity

                                                                     

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

    Rat          oral            2700-3000 (approx.)       1,3
    Cat          oral               500 (approx.)           3
                                                                     

    Short-term studies

    Rat.  Over a period of 32 days, groups of 15 male rats were fed
    o-phenylphenol in daily doses of 2, 20 and 200 mg/kg body-weight. 
    No harmful effect was demonstrable in any of the groups.3

    Five male and 5 female rats in each group were given by stomach tube
    doses of 50, 100, 200 and 500 mg/kg body-weight for 5 days a week over
    a period of 6 months.  The only abnormality observed was a slight
    increase in average liver and kidney weights in the animals at the 500
    mg/kg dosage.1

    When diets containing 0.1%, 0.3%, 1.0% and 2% of o-phenylphenol were
    fed for 3 months to groups comprising 12 males and 12 females, slight
    retardation of growth was observed in the 2% group.  There was no
    significant difference between the mortality of control and test
    animals.  There were doubtful increases in weight of liver, kidney and
    spleen of certain rats of the 1% and 2% groups.  No tissue changes
    ware observed.

    Dog.  Daily doses of 1000 mg/kg of o-phenylphenol killed 2 dogs
    within a month.  Groups of 2 dogs each were fed o-phenylphenol for a
    period of 1 year in daily amounts of 20, 200 and 500 mg/kg
    body-weight; no effect related to the administration of
    o-phenylphenol was observed. Haematological values, urinary sugar
    and protein values, organ weights and histopathological examination of
    the various tissues did not differ from the normal range.1

    Man.  A 5.0% solution of o-phenylphenol in sesame oil and a 0.1%
    aqueous solution of the sodium salt tested on 200 subjects caused
    neither primary skin irritation nor skin sensitization.1  The sodium
    salt is slightly irritating in 0.5% aqueous solution and decidedly
    irritating in 1.0% and 5% solutions.

    Long-term studies

    Rat.  Male and female rate (25 of each sex per group) maintained for
    2 years on diets containing 0.02% and 0.2% of o-phenylphenol showed
    no adverse effects when compared with a control group, as judged by
    growth, mortality, gross appearance, haematology, urinary sugar and
    protein values, organ weights, tissue content of o-phenylphenol, and
    histopathological examination of various tissues.  A similar group of
    rats maintained for 2 years on a diet containing 2% of
    o-phenylphenol differed from the controls by exhibiting slight
    retardation of growth, histological kidney changes (marked tubular
    dilatation), and the presence of small amounts of o-phenylphenol in
    the kidney tissues.1

    Comment on experimental studies reported

    From experiments on rats and dogs, o-phenylphenol does not seem to
    be a very toxic substance.  The noted difference in acute toxicity
    between these animals and cats might be explained by the known high
    sensitivity of cats to phenolic compounds.  The long-term study in
    rats is taken as a basis for the evaluation.1

    Evaluation

    Level causing no significant toxicological effect in the rat

    0.2% (=2000 ppm) in the diet, equivalent to 100 mg/kg body-weight per
    day.

    Estimate of acceptable daily intakes for man

                                          mg/kg body-weight

              Unconditional acceptance           0-0.2
              Conditional acceptance           0.2-1.0

    Further Work Considered Desirable

    Metabolic studies in experimental animals and man.

    References

    1.  Hodge, H. C., Maynard, E. A., Blanchet, H. J. jr, Spencer, H. C. &
    Rowe, V. K. (1952) J. Pharmcol. exp. Ther., 104, 202

    2.  Williams, R. T. (1959) Detoxication mechanisms, Chapmn & Hall,
    London

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


    See Also:
       Toxicological Abbreviations