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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.


    NORDIHYDROGUAIARETIC ACID

    CHEMICAL NAMES        Nordihydroguaiaretic acid; 
                          ,gamma-dimethyl-alpha,delta-bis-(3,4-
                          dihydroxyphenyl) butane; 4,4'-(2,3-
                          dimethyltetramethylene) dipyrocatechol

    SYNONYM               NDGA

    EMPIRICAL FORMULA     C18H22O4

    STRUCTURAL FORMULA

    MOLECULAR STRUCTURE 7

    MOLECULAR WEIGHT      302.37

    DESCRIPTION           Nordihydroguaiaretic acid is a white to
                          greyish-white crystalline solid, which may be
                          prepared from an evergreen desert shrub,
                          Larrea divaricata.  1 g is soluble in 4 ml of
                          ethanol.

    USE                   As an antioxidant in oils and foods.

    Biological Data

    Biochemical aspects

    There does not appear to be any information on the metabolism of
    NDGA.1  The effect of NDGA on enzyme systems has been studied.
    Specific inhibition of peroxidase, catalase, and ethyl alcohol
    dehydrogenase occurs with a concentration of 2x10-4 M of the
    antioxidant.  Non-specific inhibition of ascorbic acid oxidase,
    D-amino-acid oxidase, the cyclophorase system and urease at a
    concentration of 2x10-3 M has been described.2

    Acute toxicity

                                                                   

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

    Rat          oral                2000-5000               3
    Mouse        oral                2000-4000               3
    Mouse        intraperitoneal        550                  3
    Guinea-pig   oral                   830                  3
                                                                   

    Short-term studies

    Guinea-pig.  NDGA was found to be among the more strongly reacting
    compounds among several antioxidants tested for their capacity to
    induce skin sensitivity in the guinea-pig.4

    Dog.  NDGA was fed to young mongrel dogs at dietary levels of 0.1%
    (3 dogs), 0.5% (4 dogs) and 1.0% (5 dogs) for 1 year.  Adult dogs were
    fed the same range of dietary concentrations, the numbers in the
    groups being 2, 3 and 2 respectively, with 2 controls.  The growth
    curves of the young dogs showed some impairment in weight gain at the
    0.5% levels but not at the 0.1% and 1.0% levels.  No significant
    pathological changes were found which were attributable to the
    treatment and all the dogs were in good physical condition at the time
    of sacrifice.5

    Long-term studies

    Mouse.  Long-term studies were carried out using 0.25% and 0.5%
    levels in mice.  No deleterious effects on weight gain were observed,
    nor any important histological changes.6

    Rat.  Chronic toxicity experiments were conducted over a period of 2
    years, in which NDGA was compared with phenol, catechol and gum guaiac
    in concentrations of 0.5% in rats.  NDGA had little or no effect on
    growth or food intake, except in the highest concentration, where
    there was a temporary decrease in growth associated with a decreased
    food intake.  Histological study of the liver, spleen and kidneys
    showed no significant effect.  Necrosis of the liver was noted
    occasionally in all groups, including controls.6

    Concentrations of 0.1%, 0.5% and 1.0% of NDGA were used in another
    series of rats.  Haemorrhage into the caecum was observed in 50% of
    the animals.  In a larger series of tests on rats at the same
    concentrations of NDGA, this haemorrhage did not occur.  Cysts in the
    mesentery were found in several rats on the higher concentrations of
    NDGA.

    Two-year toxicity tests on groups of 10 male rats at 0%, 0.1%. 0.25%,
    0.5% and 1% NDGA in the diet showed that 0.5% was the lowest level
    causing inflammatory caecal lesions and slight cystic enlargement of
    lymph nodes near the caecum.  Growth inhibition occurred after the
    first 6 months at levels of 0.5% and 1%.3

    In another experiment lasting 2 years, 0.5% of NDGA in the diet caused
    massive caecal haemorrhages, with single and multiple cysts in the
    mesentery in the angle of the junction between the small and large
    intestines.  During the first 6 months, inhibition of the growth rate
    occurred only at the 1% level.3

    (Work in progress)  Groups of 20 rats (10 male and 10 female) were fed
    the following diets: 0.5% NDGA, 0.5% NDGA plus aspirin, 1% NDGA, 1%
    NDGA plus aspirin, and aspirin for 65 weeks.  Growth has been retarded
    in all test groups compared with the controls.a  Haemoglobin and
    haematocrit determinations were done at 16 and 23 weeks; all groups
    where NDGA was given had lower readings than those of the controls.a
    Two rats, one fed 0.5% NDGA plus aspirin, the other aspirin alone,
    showed some ulceration of the stomach after 42 and 50 weeks on test.
    Examination of faeces for occult blood was done after 49 weeks; the
    rats fed NDGA with no aspirin were all negative, but 5 rats fed
    aspirin alone or aspirin plus NDGA showed traces of occult blood.7

    Hamster.  (Work in progress)  Hamsters (15 male and 15 female) were
    fed diets containing 0.1%, 0.5% and 1% NDGA for 65 weeks.  Some deaths
    which occurred at the 1% level may be attributed to the effect of NDGA
    in the diet.  Ulceration of the stomach was found in 3 hamsters at the
    1% level after 48 weeks.  At 22 weeks a marked decrease in haematocrit
    values was found for females, but not in males, on 1% NDGA.
    Examination of faeces for occult blood was negative in all groups. 
    The investigator points out that 1% NDGA in the diet of hamsters
    produced a definite toxic effect, particularly in the female.  Whether
    the lower level will prove to be the no-effect level will depend on
    the findings upon completion of the test, which is to he carried out
    over the lifetime of the animals.7

    Comment on experimental studies reported

    The acute toxicity studies indicate that the guinea-pig is more
    sensitive than the rat but no long-term studies with the former
    species have been reported.  The short-term studies in the dog suggest
    that a 1% dietary level is well tolerated in this species although
    there was some impairment of growth at the 0.5% level.  The published
    long-term studies in the rat and mouse provide little detailed
    information and seem to have left some of the investigators in doubt


                   

    a No statistical evaluation has yet been done as these experiments
    are not terminated; they are being carried out over the life span of
    the animals.

    about the acceptability of NDGA.  The more recent long-term studies in
    rat and hamster are still in progress and cannot be evaluated until
    their completion.

    Evaluation

    Level causing no significant toxicological effect in the rat

    From the limited information available, it is not possible to evaluate
    the toxicological status of NDGA.  No guidance can therefore be given
    at present on the use of NDGA as an antioxidant in food.

    Further Work Required

    1.  Further long-term studies in the rat and other species, including
    dog.

    2. Metabolic studies in animals and man.

    References

    1.  Dacre. J. C. (1960) J. N.Z. Inst. Chem., 24, 161

    2.  Tappel, A. L. & Marr. A. G. (1954) J. Agr. Food Chem., 2, 554

    3.  Lehman, A. J., Fitzhugh, O. G., Nelson, A. A. & Woodard, G. (1951)
    Advanc. Food Res., 3, 197

    4.  Griepentrog, F. (1961) Arzneimittel-Forsch., 11, 920

    5.  Anderson, W. D., Bell, B. J. & Bieter, R. N. (1955) Unpublished
    report

    6.  Cranston, E. M., Jensen, M. J., Moren, A., Brey, T., Bell, E. T. &
    Bieter, R. N. (1947) Fed. Proc., 6, 318

    7. Mannell, W. A., Canadian Food and Drug Directorate, Ottawa (Letter
    dated 23 September 1964)
    


    See Also:
       Toxicological Abbreviations
       Nordihydroguaiaretic acid (FAO Nutrition Meetings Report Series 46a)
       Nordihydroguaiaretic acid (WHO Food Additives Series 5)
       NORDIHYDROGUAIARETIC ACID (JECFA Evaluation)