Toxicological evaluation of some food
additives including anticaking agents,
antimicrobials, antioxidants, emulsifiers
and thickening agents
WHO FOOD ADDITIVES SERIES NO. 5
The evaluations contained in this publication
were prepared by the Joint FAO/WHO Expert
Committee on Food Additives which met in Geneva,
25 June - 4 July 19731
World Health Organization
Geneva
1974
1 Seventeenth Report of the Joint FAO/WHO Expert Committee on
Food Additives, Wld Hlth Org. techn. Rep. Ser., 1974, No. 539;
FAO Nutrition Meetings Report Series, 1974, No. 53.
NORDIHYDROGUAIARETIC ACID
Explanation
This substance has been evaluated for acceptable daily intake by
the Joint FAO/WHO Expert Committee on Food Additives (see Annex 1,
Refs Nos 6, 8 and 20) in 1961, 1964 and 1969.
Since the previous evaluation no additional data have become
available. Therefore the previous monograph remains unchanged and is
reproduced in its entirety below.
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 × 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 × 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 2% 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 ilium 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 ILIUM AND CAECUM OF RATS FROM
A SINGLE DOSE OF 250 mg NDGA
Analysis of contents of:
Ilium Caecum
Time after dosing (hour) 0 1 2 4 6 7.5 7.5
o-Quinone (ug) 0 0 Trace 6 480 2 760 1 620
Twenty-four hour urine samples collected from rats fed 2% 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 2% NDGA and sacrificed at various times ranging
from one to 15 months from the 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).
TOXICOLOGICAL STUDIES
Acute toxicity
LD50
Animal Route (mg/kg bw) Reference
Rat oral 2 000 - 5 500 Lehman et al. (1951)
Mouse oral 2 000 - 4 000 Lehman et al. (1951)
Mouse i.p. 550 Lehman et al. (1951)
Guinea-pig oral 830 Lehman et al. (1951)
Short-term studies
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% and 0.5%
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).
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).
Dog
NDGA was fed to dogs at dietary levels of 0.1% (three dogs), 0.5%
(four dogs) and 1.0% (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%
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 (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%, and 0.5% in rats (10 males per 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%, 0.5% and 1.0% NDGA were used with another
series of rats (18 females per group). 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 pathological effect. Single or multiple cysts in the
mesentory (in the angle of the junction between the small and large
intestine) were found in 10/12 surviving rats on 1% NDGA and in 15/17
surviving rats on 0.5% NDGA. Nodules, slightly larger than normal and
suggestive of the beginning cyst formation, were observed in 1/12
surviving rats on 0.1% 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%, 0.1%, 0.25%, 0.5% and 1.0% NDGA. Inflammatory caecal
lesions were noted in 4/20 of the animals fed NDGA at levels of 0.5%
and 1.0%, and slight cystic enlargement of the lymph nodes near the
caecum in half these animals. The changes were absent at levels of 0%
(controls), 0.1 and 0.28% 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 per group, Wistar-derived strain), at levels of 0.5% or 1.0%
in the diet for 74 weeks, caused cystic retinoloendotheliosis 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% and 1.0% levels.
The o-quinone derived from NDGA was isolated from kidney tissue and
identified by thin-layer chromatography and ultra-violet spectroscopy.
Rats fed 2% NDGA in the diet for shorter periods of time exhibited
similar pathological changes (Grice et al., 1968).
Comments:
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.
EVALUATION
Not possible with data provided.
REFERENCES
Cranston, E. M. et al. (1947a) Unpublished report, dated 8 March
Cranston, E. M. et al. (1947b) Fed. Proc., 6, 318
Goodman, T. et al. (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. et al. (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