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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.


    POTASSIUM NITRITE

    CHEMICAL NAME        Potassium nitrite

    EMPIRICAL FORMULA    KNO2

    MOLECULAR WEIGHT     85.11

    DEFINITION           Potassium nitrite after drying contains not less
                         than 95% of KNO2.  It conforms to the following
                         specifications.

    DESCRIPTION          Small, white or slightly yellow, deliquescent
                         granules or rods.

    USE                  As a colour fixative in fish products and in
                         pickling and curing meat, sometimes in
                         combination with sodium nitrite and with
                         potassium and sodium nitrates.

    IDENTIFICATION TESTS

    A.   Solubility:   Water:     Very soluble
                       Ethanol:   Sparingly soluble

    B.   In neutral, concentrated or moderately concentrated solutions of
         potassium nitrite, sodium bitartrate TS slowly produces a white,
         crystalline precipitate which is soluble in ammonia TS and in
         solutions of alkali hydroxide or carbonates.

    C.   Potassium nitrite yields brownish red fumes when treated with
         diluted mineral acids or acetic acids.

    PURITY TESTS

    Arsenic:  Not more than 3 mg/kg.

    Lead:  Not more than 10 mg/kg.

    Heavy metals:  Not more than 20 mg/kg.

    Dissolve 2 g in water, and dilute to 40 ml.  For the control, add a
    volume of standard lead TS equivalent to 0.02 mg of lead to 10 ml of
    this solution and use the remaining 30 ml for the sample.  Evaporate
    both solutions to dryness on the steam bath.  To each residue add 5 ml
    of hydrochloric acid, and again evaporate to dryness.  Dissolve the
    residues in 20-ml portions of water, adjust each to a pH between 3.0
    and 4.0, dilute to 40 ml and add 10 ml of hydrogen sulfide TS to each.
    The colour in the sample solution, measured in a 50-ml Nessler tube,
    is not darker than the control solution, similarly measured.

    pH of a 5% solution:  Dissolve 10 g in 200 ml of carbon dioxide- and
    ammonia-free water: the pH of this solution is between 6.0 and 9.0.

    ASSAY

    Dissolve 1.000 g, previously dried over silica gel for 4 hours, in
    water and make to 100.0 ml.  Pipette 10 ml of this solution into a
    mixture of 50.0 ml of 0.1 N potassium permanganate, 100 ml of water
    and 5 ml of sulfuric acid, keeping the tip of the pipette well below
    the surface of the liquid.  Warm the solution to 40C, allow it to
    stand for 5 minutes and add 25.0 ml of 0.1 N oxalic acid.  Heat the
    mixture to about 80C and titrate with 0.1 N potassium permanganate.
    Each ml of 0.1 N potassium permanganate is equivalent to 4.256 mg of
    KNO2.

    SODIUM NITRITE

    CHEMICAL NAME         Sodium nitrite

    EMPIRICAL FORMULA     NaNO2

    MOLECULAR WEIGHT      69.00

    DEFINITION            Sodium nitrite after drying contains not less
                          than 97% of NaNO2.  It conforms to the
                          following specifications.

    DESCRIPTION           White or slightly yellow, hygroscopic granules,
                          powder, or opaque, fused masses of sticks.  It
                          is deliquescent in air.

    USE                   As a colour fixative in fish products and in
                          pickling and curing meat, sometimes in
                          combination with potassium nitrite, or
                          potassium and sodium nitrates.

    IDENTIFICATION TESTS

    A.   Solubility:  Water:    1 g dissolves in 1.5 ml
                      Ethanol:  Sparingly soluble

    B.   Sodium nitrite, after conversion to chloride or nitrate, yields
         with uranyl zinc acetate TS a golden-yellow precipitate, which
         forms after several minutes' agitation.

    C.   Sodium nitrite yields brownish red fumes when treated with
         diluted mineral acids or acetic acid.

    PURITY TESTS

    Loss on drying:  Not more than 0.25% by drying over silica gel for 4
    hours.

    pH of a 5% solution:  Dissolve 10g in 200 ml of carbon dioxide- and
    ammonia-free waters: the pH of this solution is between 6.0 and 9.0.

    Arsenic:  Not more than 3 mg/kg.

    Lead:  Not more than 10 mg/kg.

    Heavy metals:  Not more than 20 mg/kg.

    Dissolve 2 g in water and dilute to 40 ml.  For the control, add a
    volume of standard lead solution equivalent to 0.02 mg of lead to 10
    ml of this solution and use the remaining 30 ml for the sample.
    Evaporate both solutions to dryness on the steam bath.  To each
    residue add 5 ml of hydrochloric acid, and again evaporate to dryness.
    Dissolve the residues in 20-ml portions of water, adjust each to a pH
    between 3.0 and 4.0, dilute to 40 ml and add 10 ml of hydrogen sulfide
    TS to each.  The colour in the sample solution, measured in a 50-ml
    Nessler tube, is not darker than the control solution, similarly
    measured.

    ASSAY

    Dissolve 1.000 g, previously dried over silica gel for 4 hours, in
    water and make to 100.0 ml.  Pipette 10 ml of this solution into a
    mixture of 50.0 ml of 0.1 N potassium permanganate, 100 ml of water
    and 5 Ml of sulfuric acid, keeping the tip of the pipette well below
    the surface of the liquid.  Warm the solution to 40C, allow it to
    stand for 5 minutes, and add 25.0 ml of 0.1 N oxalic acid.  Heat the
    mixture to about 80C and titrate with 0.1 N potassium permanganate. 
    Each ml of 0.1 N potassium permanganate is equivalent to 3.450 mg of
    NaNO2.

    Biological Data

    Biochemical Aspects

    Following absorption of nitrites, the most important biochemical
    reaction that occurs is the conversion of haemoglobin to
    methaemoglobin. There is some controversy concerning the molar ratios
    involved in this reaction.  Making an extreme assumption, it may be
    stated that 1 g of sodium nitrite could convert as much as 1855 g of
    haemoglobin to methaemoglobin.1

    The sub-acute hazard of nitrites rests on the amount of methaemoglobin
    formed and on the ability of the body to reconvert this methaemoglobin
    back to haemoglobin.

    Acute toxicity

                                                                    

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

    Mouse              oral              220                 2
    Mouse-female       oral              175                 3
    Rat-female         oral               85                 3
                                                                    

    Many cases have been reported of accidental poisoning resulting from
    the presence of sodium nitrite in food products.  From this
    information it is possible to deduce that the oral lethal dose in man
    varies from 0.18 to 2.5 g, the lower figures being those for children
    and old people.4,5,6,7  Sodium nitrite has been used for therapeutic
    purposes as a vasodilating agent in dosages of 30-120 mg.

    Short-term studies

    Rat and cat.  Rats were fed a sodium nitrite supplement for a period
    up to 168 days.  One rat received a total of 167 mg of sodium nitrite
    in 121 days.  This represents 93 ppm in the daily diet.  No effects on
    growth or on the weights of important organs were noted.  In a similar
    experiment with cats, one animal received a total of about 4100 mg of
    sodium nitrite during a period of 105 days.  This represents
    approximately 390 ppm in the daily diet.  No effects on the growth
    rate or on the weight of important organs were noted.  No
    histopathological examination has been reported on any animal fed with
    nitrite.

    Long-term studies

    Rat.  The continuous administration of sodium nitrite in the
    drinking-water at the rate of 100 mg/kg body-weight daily over the
    whole life span and in 3 successive generations (95 rats) resulted in
    spite of the high dosage (67% of the acute LD50) in only a slight
    inhibition of growth (10-20%) and in a shortening of the median life
    span from 740 to 640 days.  Reproduction was normal.  Neither the
    blood picture nor the organs showed any ill effects.  The number of
    tumours observed in the test group (1 thymoma and 1 hepatoma) was not
    greater than in the control group.  Cumulative toxic effects were not
    observed.9

    Since nitrous acid reacts easily with secondary amines yielding
    dialkylnitrosamines which are highly toxic and produce cancer of the
    liver in rats, additional experiments have been made in which nitrite
    was administered together with diethylamine.  Thirty hybrid rats were
    given sodium nitrite in the drinking-water at a daily dosage of 100
    mg/kg body-weight and diethylamine in the food at the rate of 

    500 mg/kg body-weight daily.  The same slight shortening of the median
    life span (to 625 days) was observed as in the experiments with
    nitrite alone, but there were no tumours.9

    Comments on experimental studies reported

    Though in the long-term studies cited only a slight inhibition of
    growth occurred, the dose causing this effect appears to give the best
    approximation to the threshold dose level.

    Evaluation

    Level causing no significant toxicological effect in the rat

    From consideration of the long-term studies it can be concluded that
    this level will be somewhat below 100 mg/kg body-weight per day.

    Estimate of acceptable daily intakes for man

                                              mg/kg body-weight

               Unconditional acceptance             0-0.4
               Conditional acceptance             0.4-0.8

    Comment

    It is recommended that if possible sodium nitrite should be used mixed
    with common salt, the amount of nitrite in the salt being not more
    than 0.6%.  Food for babies should not contain added nitrite.

    Further Work Considered Desirable

    1.  The estimation of nitrite in vegetables.

    2.  Chronic toxicity experiments in non-rodent species.

    References

    1.  Lehman, A. J. (1958) Quart. Bull. Ass. Food Drug. Off., 22, 136

    2.  Riemann, H. (1950) Acta pharmacol. (Kbh.), 6, 285

    3.  United States Food and Drug Administration (Unpublished data)

    4.  Schrader, G. & Gessner, O. (1943) Samml. Vergiftungsf., 13, 101

    5.  Greenberg, M., Birnkrant, W. B. & Schiftner, J. J. (1945) Amer. J.
    publ. Hlth., 35, 1217

    6.  Schmidt, H., Stick, W. & Kluge, F. (1949) Dtsch. med. Wschr., 74,
    961

    7.  Naidu, S. R. & Venkratrao, P. (1945) Calcutta med. J., 42, 79

    8.  Tarr, H. L. A. & Carter, M. H. (1942) J. Fish. Res. Board, Can.,
    6, 63

    9.  Druckrey, H. (1963) Arzneimittel-Forsch., 13, 320
    


    See Also:
       Toxicological Abbreviations
       Potassium nitrite (ICSC)
       POTASSIUM NITRITE (JECFA Evaluation)