FAO Nutrition Meetings Report Series 
    No. 46A WHO/FOOD ADD/70.36

    The content of this document is the result of the deliberations of the
    Joint FAO/WHO Expert Committee on Food Additives which met in Rome,
    27 May - 4 June 19691

    Food and Agriculture Organization of the United Nations

    World Health Organization

    1 Thirteenth report of the Joint FAO/WHO Expert Committee on Food
    Additives, FAO Nutrition Meetings Report Series, in press;
    Wld Hlth Org. techn.  Rep. Ser., in press.


    Biological Data

    Biochemical aspects

    Biochemical studies have shown that NDGA has an inhibiting effect on a
    number of enzyme systems and it has been suggested that in some cases
    this inhibition in enzyme systems may be due to a denaturing action of
    the antioxidant (Tappel & Marr, 1954). Specific inhibition of
    peroxidase, catalase and ethyl alcohol dehydrogenase occurs with a
    concentration of 2 x 10-4M of the antioxidant. Non-specific
    inhibition of ascorbic acid oxidase, D-amino-acid oxidase, the
    cyclophorase system and urease at a concentration of 2 x 10-3M has
    been described (Tappel & Marr, 1954). The serum esterase system was
    markedly inhibited by four fat antioxidants (octyl gallate, ascorbyl
    palmitate, butylhydroxytoluene, and NDGA, resp.) In concentrations of
    0.1-1.0 mm. In the liver, the inhibition induced by octyl gallate was
    significantly smaller than it was by the other three antioxidants. The
    esterase activity in the fatty tissues (perirenal) did not appear to
    be significantly affected by the four antioxidants. It was concluded
    that the antioxidants examined could affect fat metabolism adversely
    by inhibiting the ester-cleaving systems in the serum and liver
    (Placer et al., 1964).

    The metabolism of NDGA has recently been studied in the rat. No free
    NDGA was found in lymph or kidney extracts after long-or short-term
    feeding studies. The first trace of the o-quinone metabolite was found
    in kidney extracts from rats fed two per cent. NDGA for 28 days. After
    80 days on test, 260 g o-quinone/g kidney tissue was extracted.

    The formation of o-quinone in the rat intestine, after single
    administration of 250 mg NDGA directly into the small intestine, was
    determined at 0, 1, 2, 4, 6 and 7.5 hours after dosing. The content of
    o-quinone in the ileum was found to be 0, 0, traces, 6, 480 and 2760
    g respectively. The content of the caecum, after 7.5 hours was 1620

    Formation of o-quinone metabolite in the ileum and caecum of rats
    from a single dose of 250 mg NDGA

                                            Analysis of contents of
                                           Ileum                 Caecum

    Time after dosing (hr)    0    1      2    4     6      7.5     7.5
    o-Quinone (g)            0    0    Trace  6    480   2 760   1 620

    24-hour urine samples collected from rats fed two per cent. NDGA for
    36 days contained no detectable free NDGA but did contain up to 56 g
    of o-quinone (Grice et al., 1968).

    The effect of NDGA in rat kidney has been studied in greater detail in
    rats fed two per cent. NDGA and sacrificed at various times ranging
    from one to 15 months from start of the experiment. It was postulated
    that the o-quinone metabolite of NDGA was taken up by lysosomes of the
    proximal tubules, where it may have affected the permeability of the
    lysosomal membrane, or cause an inhibition of lysosomal enzymes, with
    subsequent destruction of the cells involved (Goodman et al., 1969).

    Acute toxicity


    Animal       Route                   LD50            Reference
                                    (mg/kg body-weight

    Rat          oral               2 000 - 5 500        Lehman et al.
    Mouse        oral               2 000 - 4 000             "
    Mouse        intraperitoneal         550                  "
    Guinea-pig   oral                    830                  "


    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. (Griepentrog, 1961)

    Mouse. No deleterious effects on the rate of growth or food intake
    were noted in mice fed a control diet and diets containing NDGA, gum
    guaiac, phenol and catechol at concentrations of 0.25 per cent. and
    0.5 per cent. respectively. The average number of months on the diets
    varied from 6-1/2 to 7-1/2 for NDGA to 8.4-12.6 for the other
    compounds. Necrosis of the liver and spleen were occasionally found in
    all the groups, including the controls (Cranston et al., 1947a and

    Dog. NDGA was fed to dogs at dietary levels of 0.1 per cent, (three
    dogs), 0.5 per cent. (four dogs) and 1.0 per cent. (five dogs) for one
    year. Adult dogs were fed the same range of dietary concentrations,
    the number in the groups being two, three and two respectively, with
    two controls. The growth curves of the young dogs showed some
    impairment in weight gain at the 0.5 per cent. levels but not at the
    0.1 per cent. and 1.0 per cent. 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
    (Cranston et al., 1947a).

    Long-term studies

    Rat. Chronic toxicity experiments were conducted over a two-year
    period, in which NDGA was compared with phenol, catechol and gum
    guaiac in concentrations of 0 per cent., and 0.5 per cent., in rats
    (10 males/group). All four compounds showed some tendency to decrease
    the rate of growth as compared to the controls, catechol having the
    greatest and phenol the least effect. Massive haemorrhage in the
    caecum was observed in five  rats fed NDGA at months 12, 13, 19, 20
    and 21; one of the rats had a supperative inflammation near the caecum
    and another had a thrombosed artery and ulcer of the caecum. (Cranston
    et al., 1947a and 1947b).

    Concentrations of 0 per cent., 0,1 per cent., 0.5 per cent., and 1.0
    per cent. NDGA were used with another series of rats (18
    females/group). NDGA had little or no effect on growth or food intake
    except in the highest concentration, where there was temporary
    decrease in growth associated with decreased food intake. Histological
    study of the liver, spleen and kidneys allowed no significant
    pathological effect. Single or multiple cysts in the mesentery (in the
    angle of the junction between the small and large intestine) were
    found in 10/12 surviving rats on one per cent. NDGA and in 15/17
    surviving rats an 0.5 per cent. NDGA. Nodules, slightly larger than
    normal and suggestive of the beginning cyst formation, were observed
    in 1/12 surviving rats on 0.1 per cent. NDGA and in 1/13 surviving
    control rats. No haemorrhage in the caecum occurred in any of the
    animals in this experiment (Cranston et al., 1947a and 1947b).

    Two-year toxicity tests were also carried out with groups of 10 male
    rats at 0 per cent., 0.1 per cent., 0.25 per cent., 0.5 per cent., and
    1.0 per cent. NDGA. Inflammatory caecal lesions were noted in 4/20 of
    the animals fed NDGA at levels of 0.5 per cent. and 1.0 per cent., and
    slight cystic enlargement of the lymph nodes near the caecum in half
    these animals. The changes were absent at levels of 0 per cent.
    (controls 0.1 per cent. and 0.25 per cent. NDGA. The gastrointestinal
    tract apart from the caecum was unaffected as were the other
    structures studied (Lehman et al., 1951; Nelson, 1947).

    In a recent study, NDGA fed to weanling rats (10 males and 10
    females/group, Wistar-derived strain), at levels of 0.5 per cent. or
    1.0 per cent. in the diet for 74 weeks, caused cystic
    retinuloendotheliosis of paracaecal lymph nodes and vacuolation of
    kidney tubular epithelium (Grice et al., 1968). The retardation in
    growth and caecal changes confirmed previously reported work (Lehman
    et al., 1951; Cranston et al., 1947a and 1947b) for rats fed NDGA at
    the 0.5 per cent. and 1.0 per cent. levels. The o-quinone derived from
    NDGA was isolated from kidney tissue and identified by thin-layer
    chromatography and ultra-violet spectroscopy. Rats fed two per cent.
    NDGA in the diet for shorter periods of time exhibited similar
    pathological changes (Grice et al., 1968).


    A monograph on nordihydroguaiaretic acid has appeared in the document
    WHO/Food Add./24.65 (page 42). A significant new finding is the
    conversion of nordihydroguaiaretic acid to the corresponding
    orthoquinone which may be the cause of the formation of mesenteric
    cysts. The rat seems to have a special susceptibility to this action.
    Work is in progress on hamsters.


    Cranston. E. M. et al. (1947a) Unpublished report, dated 8 March

    Cranston, E. M. et al. (1947b) Fed. Proc., 6, 318

    Goodman, T., Grice, H. C. Becking, G. C. & Salem, F. A. (1969)
    Laboratory Investigation (In press)

    Grice, H. C. Becking, G. & Goodman, T. (1968) Fd. Cosmet. Toxicol.,
    6, 155

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

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

    Nelson, A. A. (1947) Unpublished summary report of pathology dated 4

    Placer, Z., Veselkova, Z., & Petrasek, R. (1964) Nahrung, 8, 707

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

    See Also:
       Toxicological Abbreviations
       Nordihydroguaiaretic acid (FAO Nutrition Meetings Report Series 38a)
       Nordihydroguaiaretic acid (WHO Food Additives Series 5)