INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
WORLD HEALTH ORGANIZATION
TOXICOLOGICAL EVALUATION OF SOME
FOOD COLOURS, EMULSIFIERS, STABILIZERS,
ANTI-CAKING AGENTS AND CERTAIN
OTHER SUBSTANCES
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.
NORDIHYDROGUAIARETIC ACID
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 µg 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
µg respectively. The content of the caecum, after 7.5 hours was 1620
µg.
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 (µg) 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 µg
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.
(1951)
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
1947b).
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).
Comments
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.
REFERENCES
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
November
Placer, Z., Veselkova, Z., & Petrasek, R. (1964) Nahrung, 8, 707
Tappel, A. L. & Marr, A. G. (1954) J. Agr. Food Chem., 2, 554